TWI363093B - - Google Patents

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TWI363093B
TWI363093B TW097105759A TW97105759A TWI363093B TW I363093 B TWI363093 B TW I363093B TW 097105759 A TW097105759 A TW 097105759A TW 97105759 A TW97105759 A TW 97105759A TW I363093 B TWI363093 B TW I363093B
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TW
Taiwan
Prior art keywords
galactosidase
raffinose
amino acid
leu
gly
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Application number
TW097105759A
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Chinese (zh)
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TW200846472A (en
Inventor
Shinichi Imazu
Kazunobu Konishi
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Asahi Kasei Chemicals Corp
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Publication of TW200846472A publication Critical patent/TW200846472A/en
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Publication of TWI363093B publication Critical patent/TWI363093B/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Description

丄363093 九、發明說明： 【發明所屬之技術領域】 本發明係關於一種新穎α_半乳糖酶、及使用該等來製造 棉子糖之方法。 【先前技術】 近年來，伴隨飲食生活及社會生活之多樣化，消費者的 健康意識有所提高，故而對於食品或食品原料等之關注逐 曰上升。其中，棉子糖被認為具有改善腸内細菌群落等功 能，作為飲食品4醫藥。口口、香粧品等”戈其原料而倍受關 注。進而’最近發現棉子糖對於免疫賦活作㈣異位 膚炎亦有用。 目前，棉子糖可作為製造甜菜糖時之副產物而回收，但 甜菜中之棉子糖含量僅有〇.附右，生產量亦與作為主產 物之砂糖之生產量相關，故而棉子糖之增產存在極限。 因此’為了廉價且狀地向市場供給具有如此有用之特 性的棉子糖，不僅需要來自天然物質之萃取物，亦需要來 自廉價之原料的合成品。 作為至今為止所嘗試之棉子糖之合成 取万去，可列舉利用 α·半乳糖酶之觸媒作用的方法，報告有如下方法：使用蔗 =作為半乳糖受體，且使用蜜二糖作為半乳糖供體，而^ 付棉子糖的方法(例如’參照專利文^、非專利文獻卜 非專利文獻2)。且報告有：利用對硝基 «丞本-a_D_吡半 苦作為半乳糖供體之方法（例如，參昭丄 363 093 IX. Description of the Invention: [Technical Field of the Invention] The present invention relates to a novel α-galactosidase, and a method of producing the raffinose using the same. [Prior Art] In recent years, with the diversification of dietary life and social life, consumers' health awareness has increased, so the concern for food or food ingredients has increased. Among them, raffinose is considered to have functions such as improving bacterial communities in the intestines, and is a medicine for food and beverage. Mouth and fragrance products have received much attention. In addition, it has recently been found that raffinose is also useful for immunostimulating (4) ectopic skin. Currently, raffinose can be recycled as a by-product of beet sugar production. However, the content of raffinose in sugar beet is only 〇. Attached to the right, the production amount is also related to the production of granulated sugar as the main product. Therefore, there is a limit to the increase in the production of raffinose. Therefore, it is supplied to the market in an inexpensive manner. Such a useful characteristic of raffinose requires not only an extract derived from a natural substance but also a synthetic product derived from a cheap raw material. As a result of the synthesis of raffinose which has hitherto been tried, the use of α-galactose The method of the enzyme action of the enzyme reports the following methods: using cane = as a galactose receptor, and using melibiose as a galactose donor, and ^ method of paying raffinose (for example, 'refer to the patent text ^, non The patent document is non-patent document 2). It is reported that a method using a nitro group «丞本-a_D_pyrazine" as a galactose donor is used (for example,

Bn ^ , *’导利文獻3)、利用 肌醇半乳糖苷作為半乳糖供體之方法（ 例如，參照專利文 129208-1000713.doc 1363093 獻2)、利用半乳二糖作為半乳糖供體之方法（例如，參照專 利文獻3)等》 又’亦報告有幾種可利用低價之蔗糖及半乳糖作為原料 之例。例如揭示有：使用來自朱紅栓菌（Pycn〇p〇rus cinnabarinus)之α-半乳糖酶而獲得棉子糖的方法（例如，參 照非專利文獻4)、或利用來自葡酒色被抱徵（M〇rtierella vinacea)之α-半乳糖酶而合成棉子糖的方法（例如，參照非 專利文獻5)。 [專利文獻1]曰本專利第2688854號 [專利文獻2]曰本專利特開平10-84973 [專利文獻3]日本專利特公平8-24592 [非專利文獻 1] Agric· Biol. Chem.，52(9)，2305-2311， 1988 [非專利文獻 2] Biosci. Biotech. Biochem·，59(4)，619-623, 1995 [非專利文獻 3] Phytochemistry, 18，35-38.，1979 [非專利文獻 4] Nippon Shokuhin Kogyo Gakkaishi， 38(8)，722-728, 1991 [非專利文獻 5] Carbohydr. Res·，185; 139-146，1989 【發明内容】 [發明所欲解決之問題] 由於利用α-半乳糖酶之棉子糖生成效率良好，故而報告 有利用蜜二糖、對硝基苯_α·〇- β比喃半乳糖苷、肌醇半乳 糖苦或半乳二糖作為棉子糖合成原料之例子，但於使用該 等方法之情形時’所使用之原料價格高，且難以於工業水 129208-1000713.d< 平製造棉子糖。 另方面，使用來自朱紅栓菌之α-半乳糖酶而獲得棉子 糖的方法、或利用來自葡酒色被孢黴之…半乳糖酶而合成 子糖的方法’可利用作為廉價原料之ϋ及半乳糖因 此’自卫業性方面考慮，上述兩種方法係非常有利之方 法。然而，於利用該等α_半乳糖酶之情形時，於產物中會 生成目標棉子糖以外之夾雜三糖，因此必須自最終產品中 分離夾雜三糖’進而產生浪費原料之問題。於將來自朱紅 栓菌之半乳糖酶用於棉子糖合成反應之情形時，生成寡 糖中之棉子糖含有率為4G%;於將來自葡酒色被抱徵之α· 半乳糖酶用於料料成反應之情料，生成寡糖中之棉 子糖含有率為60%，含有率均較低，故先前之技術無法藉 由酶法而選擇性製造棉子糖。 基於上述狀況，本發明係提供一種可使用廉價原料，選 擇性合成棉子糖之新穎。半乳糖酶，且提供一種使用有上 述新穎α·半乳糖酶之創新的棉子糖製造方法。 [解決問題之技術手段] 本發明者等人為解決該等課題而反覆進行潛心研究，結 果發現一種可選擇性合成棉子糖之新穎酶，且發現一種抑 制目標棉子糖以外之夾雜募糖之生成的棉子糖製造方法， 從而完成了本發明。 即，本發明係關於以下Π]〜[20]所示之新穎心半乳糖酶 及棉子糖之製造方法等。 Π]—種α·半乳糖酶，其具有下述特性： 129208-1000713.doc (1) 作用：於以蔗糖及半乳糖為原料之脫水縮合反應中，生 成寡糖中之棉子糖含有率為0.5%以上時，α-半乳糖酶具有 棉子糖合成選擇率為65%以上之性質； (2) 最佳pH值範圍：3.5〜5.0 ; (3) 穩定pH值範圍：3.5〜10.0 ; (4) 分子量：約80,000。 [2] 如[1]之α-半乳糖酶，其係來自屬於凝結芽胞桿菌 (Bacillus coagulans)之微生物0 [3] 如[2]之α-半乳糖酶，其中凝結芽胞桿菌係凝結芽胞 桿菌 AKC003 株、AKC004 株（FERM-ABP10948)(台灣寄存 編號：BCRC 910388)、AKC005 株、AKC006株中之任一 者。 [4] 一種凝結芽胞桿菌及其突變體，該凝結芽胞桿菌屬 於凝結芽胞桿菌AKC003株、AKC004株（FERM-ABP10948)(台灣寄存編號：BCRC 910388)、AKC005 株、 AKC006株中之任一者。 [5] —種α-半乳糖酶，其包含下述（a)、（b)或（c)中之任一 個胺基酸序列： (a) 序列編號2所表示之胺基酸序列； (b) 序列編號2所表示之胺基酸序列中，一個或數個胺基酸 經缺失、置換及/或附加，且具有α-半乳糖酶活性之胺基酸 序列； (c) 與序列編號2所表示之胺基酸序列具有60%以上之同源 性，且具有α-半乳糖酶活性的胺基酸序列。 129208-1000713.doc [6] —種α-半乳糖酶基因，其對包含下述（3)、（b)或（幻中 之任一個胺基酸序列之。半乳糖酶進行編碼： U)序列編號2所表示之胺基酸序列； (b) 序列編號2所表示之胺基酸序列中’一個或數個胺基酸 經缺失、置換及/或附加，且具有α-半乳糖酶活性之胺基酸 序列； (c) 與序列編號2所表示之胺基酸序列具有6〇%以上之同源 性’且具有α-半乳糖酶活性之胺基酸序列。 [7] —種α-半乳糖酶基因，其包含以下（幻或（1))之鹼基序 列： (a) 序列編號1所表示之鹼基序列； (b) 序列編號1所表示之鹼基序列中，一個或數個鹼基經缺 失、置換及/或附加，且編碼具有α-半乳糖酶活性之蛋白質 之驗基序列。 [8] —種重組載體，其含有如[6]或[7]之α_半乳糖酶基 因。 [9] 一種轉形體，其導入有如[6]或[7]之α_半乳糖酶基因 或如[8]之重組載體。 [1 〇] —種α-半乳糖酶，其係培養如[9]之轉形體而獲得。 [η] 一種酶組合物，其含有如[1]至[3]、[5]、或[10]中任 一項之α-半乳糖酶。 [12]如[11]之酶組合物，其進而含有選自α葡萄糖苷 酶、β-葡萄糖苷酶、β-半乳糖酶、纖維素酶、木聚糖酶、 蛋白酶、半乳聚糖酶、***聚糖酶、甘露聚糖酶、鼠李 129208-1000713.doc •10- 1363093 半乳糖醒酸酶、聚半乳糖磐酸酶、果廢甲醋酶、㈣解離 酶、及聚半乳㈣酸解離酶中之至少—種以上之成分。 [13]-種棉子糖合成試劑，其含有如⑴]或叫中任一項 之酶組合物。 Π4]-種棉子糖之製造方法，其特徵在於·其係、使用如 Π]至m、m、或[1G]中任—項之α·半乳糖酶，如[u]或 [12]之酶組合物’或者如[13]之棉子糖合成試劑。 [15]-種棉子糖之製造方法，其特徵在於：其㈣用培 養屬於凝結芽胞桿菌之微生物而獲得之微生物觸媒。 Π6] —種棉子糖之製造方法，其特徵在於：其係利用培 養屬於凝結芽胞桿菌AKC〇〇3株、AKC〇〇4株（FERM_ ΑΒΡ1〇948)(台灣寄存編號：BCRC 9刪8)、akc祕株、 AKC006株中之任一者的凝結芽胞桿菌及/或其突變體而獲 得的微生物觸媒。 [17] —種棉子糖之製造方法，其特徵在於：其係利用培 養如[9]之轉形體而獲得之微生物觸媒。 [18] 如[14]至[17]中任一項之棉子糖之製造方法，其中生 成寡糖中之棉子糖含有率為65%以上。 [19] 如[14]至[18]中任一項之棉子糖之製造方法，其係使 用蔗糖及半乳糖作為原料。 [20] 如[19]之棉子糖之製造方法，其中原料中之蔗糖濃 度為30% (Wv)〜90% (w/v)，原料中之半乳糖濃度為2% (w/v)〜45% (w/v)。 [發明之效果] 129208-1000713.doc 11 1363093 藉由使用本發明，可選擇性製造棉子糖。 【實施方式】 以下，對本發明進行具體說明。 本發明之α-半乳糖酶係來自屬於凝結芽胞桿菌之微生 物，作為該微生物，若為屬於凝結芽胞桿菌之微生物，則 可使用任意者，可使用表現出可選擇性合成棉子糖之α-半 乳糖酶之任意微生物。可較好地列舉：凝結芽胞桿菌 AKC-003 株、AKC-004 株、AKC-005 株、AKC-006 株。 又，本發明之微生物，亦可為以屬於凝結芽胞桿菌之微生 物為母株而獲得之突變株》凝結芽胞桿菌AKC-003株、 AKC-004株、AKC-005株、AKC-006株分別於 2006年（平成 18年）11月14日，寄存於獨立行政法人產業技術综合研究 所專利生物寄存中心（日本茨城縣築波市東1 丁目1番地1 中央第6)。寄存編號如下所述。再者，AKC-004株於2008 年（平成20年）1月30曰以受領編號FERM-ABP10948、及寄 存編號FERM-BP10948交由國際寄存管理（台灣寄存編號： BCRC 910388)。 AKC-003株（FERM P-21091) AKC-004株（FERM P-21092 ； FERM-ABP10948 ； FERM-BP 10948)(台灣寄存編號：BCRC 910388) AKC-005株（FERM P-21093) AKC-006株（FERM P-21094) 本發明之α-半乳糖酶具有下述特性。 (1)作用：於以蔗糖與半乳糖為原料之脫水縮合反應中，生 129208-1000713.doc 12 1363093 成寡糖中之棉子糖含有率為0·5%以上時，本發明之α_半乳 糖酶具有棉子糖合成選擇率為65%以上的性質。 (2) 最佳ΡΗ值範圍·· 3.5〜5.0 (3) 穩定pH值範圍：3.5〜1〇.〇 (4) 分子量：约80,〇〇〇 對於本發明之酶的具體例，可列舉：作為受質特異性及 金屬離子之影響方面而具有以下性質者。 又質特異性.於以對石肖基苯_a_D_〇比脅半乳糖普作為受 質之情形時，分解活性最高，其次是蜜二糖，再次是棉子 糖。另-方面’對喊基苯·Ρ如比喃半乳糖皆、乳糖、蔑 糖不會分解。 金屬離子之影響：於分別添加H鎮、絡、猛、 鉛、鐵（11)、鐵（111)離子之情形時，未發現活性下降。另 一方面，於分別添㈣、鋅離子之情形時，可見活性下 降，於添加銅離子之情形時活性下降最多。 進而，作為本發明之酶之具體例，可為具有下述特性 者。 (5) (正反應）最佳溫度範圍：35〜5〇它 (6) 穩定溫度範圍：直至45。〇為穩定。 友作為用於本發明之微生物之培養方法，可使用通常之通 氣攪拌培養或固體培養，可適 货J迥用通*進行之微生物培養方 …作為培養基，可列舉：該微生物良好生長，且含有微 2中之a·半乳糖酶可順利生產所必需之碳源、氮源、益 機孤’必需之營養源等的合成培養基或者天然培養基。例 J29208-1000713.doc -13· 1363093 如’作為碳源，可使用：葡萄糖、甘油、蔗糖、半乳糖、 礼糖、蜜二糖、棉子糖、水蘇糖、纖維雙糖' erl〇Se、有 機酸、澱粉、撖欖油、大豆油等。作為氮源，例如可列 舉.硫酸銨、硝酸銨、尿素、胺基酸、胺類、氨、各種無 機酸或有機酸之銨鹽、其他含氮化合物 '蛋白腺、騰蛋白 腺聚蛋白腺、肉汁、酵母萃取物、棉籽粕、玉米漿、及 且餅等。又，作為無機鹽類，可使用：磷酸二氫鉀、磷酸 氫一鉀、磷酸鎂、硫酸鎂、氣化鈉、硫酸錳、硫酸鋼、硫 酸鐵、碳酸鈣等》就微生物之生長性方面而言，培養溫度 較好的是25〜8〇t，更好的是4〇〜6π ’更好的 4 0〜5 S 〇广 〜 L。又，培養基之pH值可於較大之範圍内選擇，就 微生物之生長性方面而言，pH值較好的是3.0〜9.0，更好 的疋pH值為3.5〜8.5，更好的是pH值為4 • 0〜8.0 〇 本發明之α-半乳糖酶之分離/純化例如可藉由如下方式實 施。 於上述培養基中培養凝結芽胞桿菌akc_〇〇4株，藉由離 心分離、過料$所周知之方法，將所得培養液分離為菌 體與濾液。 如此獲得之菌體t含有半乳糖酶，可使用溶菌酶或超 音波粉碎機'法式高I破碎機等對菌體進行粉碎，藉此獲 得《_半乳_之粗萃取液。因培養條件等而使培養上清液 3有α半乳糖酶之活性之情形時，亦可直接將培養上清 液、或將經過魏等㈣後之料上清㈣^半乳糖酶 之粗萃取液而用於之後的純化中。 129208-1000713.doc \4 1363093Bn ^ , * 'Guidelines 3), methods using galactose galactoside as a galactose donor (for example, refer to Patent Document 129208-1000713.doc 1363093 2), using galactose as a galactose donor The method (for example, refer to Patent Document 3), etc., also reports that there are several examples in which low-priced sucrose and galactose can be used as raw materials. For example, there is disclosed a method of obtaining raffinose using α-galactosidase derived from Pycn〇p〇rus cinnabarinus (for example, refer to Non-Patent Document 4), or using a flag from Portuguese wine (M) A method of synthesizing raffinose by α-galactosidase of 〇rtierella vinacea) (for example, refer to Non-Patent Document 5). [Patent Document 1] Japanese Patent No. 2688854 [Patent Document 2] Japanese Patent Laid-Open No. Hei 10-84973 [Patent Document 3] Japanese Patent Laid-Open No. Hei 8-24592 [Non-Patent Document 1] Agric Biol. Chem., 52 (9), 2305-2311, 1988 [Non-Patent Document 2] Biosci. Biotech. Biochem., 59(4), 619-623, 1995 [Non-Patent Document 3] Phytochemistry, 18, 35-38., 1979 [Non Patent Document 4] Nippon Shokuhin Kogyo Gakkaishi, 38(8), 722-728, 1991 [Non-Patent Document 5] Carbohydr. Res., 185; 139-146, 1989 [Summary of the Invention] [Problems to be Solved by the Invention] The production efficiency of raffinose using α-galactosidase is good, so it is reported that using melibiose, p-nitrophenyl _α·〇-β galactofuranoside, inositol galactose or galactobiose as cotton An example of a raw material for synthesizing a sugar, but in the case of using these methods, the raw material used is expensive, and it is difficult to produce raffinose in industrial water 129208-1000713.d < On the other hand, a method of obtaining raffinose using α-galactosidase from T. rubrum or a method of synthesizing a saccharide using a galactose from a genus of genus Mortierella can be utilized as an inexpensive raw material. Galactose is therefore considered to be a very advantageous method in terms of self-defense. However, in the case of using such α-galactosidase, inclusion trisaccharides other than the target raffinose are formed in the product, and therefore it is necessary to separate the inclusion of trisaccharide from the final product, thereby causing a problem of wasted raw materials. When the galactosylase from T. rubrum is used for the raffinose synthesis reaction, the raffinose content in the oligosaccharide is 4 G%; and the α-galactosidase from the Portuguese wine color is used for In the reaction of the material, the content of raffinose in the oligosaccharide is 60%, and the content rate is low. Therefore, the prior art cannot selectively produce raffinose by enzymatic method. Based on the above circumstances, the present invention provides a novel alternative to the selective synthesis of raffinose using inexpensive raw materials. Galactase, and an innovative method of producing raffinose using the novel α-galactosidase described above. [Means for Solving the Problems] The inventors of the present invention have repeatedly conducted intensive studies to solve these problems, and as a result, have found a novel enzyme capable of selectively synthesizing raffinose, and have found that it inhibits the inclusion of sugar other than the target raffinose. The resulting method for producing raffinose, thereby completing the present invention. That is, the present invention relates to a novel method for producing a heart galactosidase and raffinose represented by the following Π] to [20]. α]-α-galactosidase, which has the following characteristics: 129208-1000713.doc (1) Role: in the dehydration condensation reaction of sucrose and galactose as raw materials, the production of raffinose content in oligosaccharides When it is 0.5% or more, α-galactosidase has a carnosamine synthesis selectivity of 65% or more; (2) optimum pH range: 3.5 to 5.0; (3) stable pH range: 3.5 to 10.0; (4) Molecular weight: about 80,000. [2] The α-galactosidase of [1], which is derived from a microorganism belonging to Bacillus coagulans, [3] such as [α]-galactosidase, wherein Bacillus coagulans is Bacillus coagulans Any of AKC003 strain, AKC004 strain (FERM-ABP10948) (Taiwan registration number: BCRC 910388), AKC005 strain, and AKC006 strain. [4] A Bacillus coagulans and a mutant thereof, the Bacillus coagulans belonging to any one of Bacillus coagulans AKC003 strain, AKC004 strain (FERM-ABP10948) (Taiwan registration number: BCRC 910388), AKC005 strain, and AKC006 strain. [5] an α-galactosidase comprising the amino acid sequence of any one of the following (a), (b) or (c): (a) an amino acid sequence represented by SEQ ID NO: 2; b) an amino acid sequence having one or several amino acids deleted, substituted and/or added, and having α-galactosidase activity in the amino acid sequence represented by SEQ ID NO: 2; (c) and sequence number The amino acid sequence represented by 2 has an amino acid sequence having a homology of 60% or more and having α-galactosidase activity. 129208-1000713.doc [6] An α-galactosidase gene comprising the following (3), (b) or (any amino acid sequence of the illusion: galactosidase encoding: U) The amino acid sequence represented by SEQ ID NO: 2; (b) one or several amino acids in the amino acid sequence represented by SEQ ID NO: 2 are deleted, substituted and/or added, and have α-galactosidase activity. The amino acid sequence; (c) an amino acid sequence having 6% or more homology to the amino acid sequence represented by SEQ ID NO: 2 and having α-galactosidase activity. [7] An α-galactosidase gene comprising the following (phantom or (1)) base sequence: (a) a base sequence represented by SEQ ID NO: 1; (b) a base represented by SEQ ID NO: In the base sequence, one or several bases are deleted, substituted and/or affixed, and the sequence of the protein having alpha-galactosidase activity is encoded. [8] A recombinant vector containing the α-galactosidase gene of [6] or [7]. [9] A transformant into which an α-galactosidase gene of [6] or [7] or a recombinant vector such as [8] is introduced. [1 〇] An α-galactosidase obtained by culturing a transformant such as [9]. [η] An enzyme composition containing the α-galactosidase according to any one of [1] to [3], [5], or [10]. [12] The enzyme composition according to [11], which further comprises an α-glucosidase, β-glucosidase, β-galactosidase, cellulase, xylanase, protease, galactanase , arabinase, mannanase, buckthorn 129208-1000713.doc •10-1363093 galactose hydrase, polygalactosidase, fruit waste acetaminogenase, (iv) dissociation enzyme, and polygalactone (4) At least one or more of the acid dissociating enzymes. [13] A raffinose synthesizing agent comprising the enzyme composition according to any one of (1) or any one of them. Π4]- A method for producing a raffinose, characterized in that it uses, for example, α] to m, m, or [1G] any of the α-galactosidase, such as [u] or [12] The enzyme composition 'or the raffinose synthesis reagent as in [13]. [15] A method for producing a raffinose, characterized in that: (4) a microbial catalyst obtained by cultivating a microorganism belonging to Bacillus coagulans. Π6] A method for producing a raffinose, characterized in that it is cultured to belong to Bacillus coagulans AKC〇〇3 strain, AKC〇〇4 strain (FERM_ ΑΒΡ1〇948) (Taiwan registered number: BCRC 9 deleted 8) A microbial catalyst obtained by coagulizing Bacillus coagulans and/or a mutant thereof of any of the akc secret strain and the AKC006 strain. [17] A method for producing a raffinose, which is characterized in that it is a microbial catalyst obtained by cultivating a transformant such as [9]. [18] The method for producing raffinose according to any one of [14] to [17] wherein the raffinose content in the oligosaccharide is 65% or more. [19] The method for producing raffinose according to any one of [14] to [18], which uses sucrose and galactose as a raw material. [20] The method for producing raffinose according to [19], wherein the sucrose concentration in the raw material is 30% (Wv) to 90% (w/v), and the galactose concentration in the raw material is 2% (w/v). ~45% (w/v). [Effect of the Invention] 129208-1000713.doc 11 1363093 By using the present invention, raffinose can be selectively produced. [Embodiment] Hereinafter, the present invention will be specifically described. The α-galactosidase of the present invention is derived from a microorganism belonging to Bacillus coagulans, and as the microorganism, if it is a microorganism belonging to Bacillus coagulans, any one may be used, and α-which exhibits selective synthesis of raffinose may be used. Any microorganism of the galactosidase. Preferably, Bacillus coagulans AKC-003 strain, AKC-004 strain, AKC-005 strain, and AKC-006 strain are listed. Further, the microorganism of the present invention may be a mutant strain obtained by using a microorganism belonging to the bacterium of Bacillus coagulans as the parent strain, Bacillus coagulans AKC-003 strain, AKC-004 strain, AKC-005 strain, and AKC-006 strain, respectively. In the year of November, 2006, the company was deposited in the Patent Bio-Reservation Center of the Industrial and Technological Research Institute of the National Institute of Industrial Science and Technology (the sixth in the center of the 1st, 1st, 1st, Tsukuba, Ibaraki, Japan). The registration number is as follows. Furthermore, the AKC-004 strain was handed over to the International Depository Management (Taiwan Registration Number: BCRC 910388) on January 30, 2008 (2008) with the recipient number FERM-ABP10948 and the deposit number FERM-BP10948. AKC-003 strain (FERM P-21091) AKC-004 strain (FERM P-21092; FERM-ABP10948; FERM-BP 10948) (Taiwan registration number: BCRC 910388) AKC-005 strain (FERM P-21093) AKC-006 Strain (FERM P-21094) The α-galactosidase of the present invention has the following characteristics. (1) Action: In the dehydration condensation reaction using sucrose and galactose as raw materials, when the content of raffinose in the oligosaccharide is 0.5% or more, the α_ of the present invention is used in the dehydration condensation reaction using sucrose and galactose as raw materials. The galactosidase has a property that the raffinose synthesis selectivity is 65% or more. (2) Optimum ΡΗ value range·· 3.5~5.0 (3) Stable pH range: 3.5 to 1 〇. 〇 (4) Molecular weight: about 80, 〇〇〇 For specific examples of the enzyme of the present invention, mention may be made of: It has the following properties in terms of the influence of the substrate specificity and the metal ion. It is also more specific. In the case of schwitzyl benzene _a_D_〇 than galactose, the decomposition activity is highest, followed by melibiose, and again raffinose. The other-sides are not decomposed, such as galactose, lactose, and sucrose. Effect of metal ions: When H, com, sb, lead, iron (11), and iron (111) ions were added, respectively, no decrease in activity was observed. On the other hand, in the case of adding (four) and zinc ions, respectively, it was found that the activity decreased, and the activity decreased most when copper ions were added. Further, as a specific example of the enzyme of the present invention, it may have the following characteristics. (5) (Positive reaction) Optimum temperature range: 35~5 〇 It (6) Stable temperature range: up to 45. It is stable. As a culture method of the microorganism used in the present invention, a normal aeration agitation culture or a solid culture can be used, and the microorganism can be cultured as a medium. As a medium, the microorganism can be well grown and contains The a-galactosidase in Micro 2 can be used to produce a synthetic medium or a natural medium such as a carbon source, a nitrogen source, a nutrient source necessary for the benefit of the machine. Example J29208-1000713.doc -13· 1363093 If used as a carbon source, use: glucose, glycerin, sucrose, galactose, sugar, melibiose, raffinose, stachyose, cellobiose erl〇Se , organic acids, starch, eucalyptus oil, soybean oil and so on. Examples of the nitrogen source include ammonium sulfate, ammonium nitrate, urea, amino acid, amines, ammonia, ammonium salts of various inorganic acids or organic acids, other nitrogen-containing compounds, protein glands, and gonadotropin, Gravy, yeast extract, cottonseed meal, corn syrup, and cakes. Further, as the inorganic salt, potassium dihydrogen phosphate, monopotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium carbonate, manganese sulfate, sulfuric acid steel, iron sulfate, calcium carbonate, or the like can be used. The culture temperature is preferably 25~8〇t, and more preferably 4〇~6π' better 4 0~5 S 〇 Guang ~ L. Further, the pH of the medium can be selected within a larger range. In terms of the growth of the microorganism, the pH is preferably 3.0 to 9.0, and the pH of the medium is preferably 3.5 to 8.5, more preferably pH. The value is 4 • 0 to 8.0. The separation/purification of the α-galactosidase of the present invention can be carried out, for example, by the following method. The Bacillus coagulans akk_〇〇4 strain was cultured in the above medium, and the obtained culture solution was separated into a fungus and a filtrate by a method known from centrifugation and over-feeding. The bacterial cell t thus obtained contains galactosidase, and the bacterial cell can be pulverized using a lysozyme or an ultrasonic pulverizer, a French high I crusher, thereby obtaining a crude extract of _half milk. When the culture supernatant 3 has the activity of α-galactosidase due to the culture conditions, etc., the culture supernatant may be directly subjected to the crude extraction of the supernatant (4) galactosidase after the fermentation of Wei et al. The solution was used in the subsequent purification. 129208-1000713.doc \4 1363093

可組合通常之蛋白之純化法，例如離子交換層析法疏 水層析法、凝膠過濾層析法、羥基磷灰石層析法、睡1L 等’對以上述方式而獲得之粗萃取液進行分離，藉 α-半乳糖酶。 上述操作之順序並無特別限制，各操作可進行丨次或2次 以上。又’較理想的是，於將樣品通入各管柱前，藉由： 析等使樣品液與適當之緩衝液進行交換。進而， 段濃縮樣品液。 ' & 較理想的是’於純化之各階段，測定所分離之各成分中 所含之α-半乳糖酶活性，收集活性較高之成分，供給至下 個階段試驗。 作為測定α-半乳糖酶活性之方法，例如有如下方法：於 含有6 mM之對硝基苯_a_D_吡喃半乳糖苷ipH值為$ 〇的 100 mM醋酸鈉緩衝液450 0中，混合酶液15〇 ，於4〇t： 下使其反應5〜30分鐘左右後，將其添加至丨M之碳酸鈉水 溶液1 mL中，使酶失活，而停止反應。對所得溶液之著色 度測疋於波長420 nm處之吸收峰，使用以各濃度之對硝基 苯酴製作之校正曲線算出濃度。又，酶活性之單位係將上 述條件下於1分鐘内使1 μΓη〇1之對硝基苯酚游離的酶量表 示為1 U。 確認經純化之α-半乳糖酶之純化度及測定分子量，可藉 由電泳或凝谬過遽層析法等而進行。又，酶學性質可藉由 如下方法進行研究：改變反應溫度或反應pH值，而測定酶 活性；或者於反應液中添加各種酶抑制劑或金屬離子等， 129208-1000713.doc -15- =測㈣留活性。進而’將α_半乳糖酶於各pH值條件下或 皿度條件下暴路-定時間後，測定酶活性，藉此可研究穩 定pH值範圍及穩定溫度範圍。又’藉由改變受質濃度而進 仃反應，彳求出α_半乳糖酶相對於各受質之米氏常數 (Km)、最大速度（Vmax)。 本發明之α-半礼糖酶基因之獲取例如可藉由以下方式進 行0 於上述培養基中培養凝結芽胞桿菌AKC-004株’藉由離 心分離、過料眾所周知之方法將所得培養液分離為菌體 與遽液。使用溶菌酶或超音波粉碎機、法式高壓破碎機 等對以上述方式獲得之菌體進行粉碎，使染色體⑽Α離 析以各種制限酶，请化以上述方式獲得之染色體， 獲知DNA片|又。使用以上述方式獲得之染色體刪入片 段，藉由利用散彈搶選殖法或反向pCR法等之眾所周知的 方法’獲取包含α-半乳糖酶基因之全長或其一部分之騰 片段。藉由利用DNA測序儀等之眾所周知的方法對此處 所得之包含α_半乳㈣基因之DNA片段之驗基序列進行分 析，而明確其驗基序列。又’於僅明確-半乳糖酶基因之 部分驗基序列之情料，亦可以該驗基序列為基礎再次 獲取包含半乳糖酶基因之DNA片段。又，藉由反覆進行 該操作，亦可明確α-半乳糖酶基因全長之鹼基序列。將以 上述方式方式解碼之(X -半乳糖酶基因之鹼基序列作為基 礎，藉由使用PCR法或制限酶之眾所周知的方法可獲^ 包含《•半乳糖酶基因全長之DNA片段β自以上述方式解碼 129208-l〇〇〇7i3.d, -16 - 1363093 之&半乳糖酶基因的驗基序列，可決定《-半乳糖酶之胺基 酸序列。 於序列編號2中例示有本發明之α半乳糖酶之胺基酸序 列，但只要包含該胺基酸序列之蛋白質具有α-半乳糖酶活 性，則於該胺基酸序列中亦可產生至少一個胺基酸缺失、 置換、附加等之突變，或者可為與該胺基酸序列具有60% ^上之同源性的胺基酸序列。即，本發明之半乳糖酶， 係包3下述（a)、（b)或（c)中之任一個胺基酸序列的α_半乳 糖酶： (a) 序列編號2所表示之胺基酸序列； (b) 於序列編號2所表示之胺基酸序列中，一個或數個胺基 西文經缺失、置換及/或附加，且具有α-半乳糖酶活性的胺基 酸序列； (c) 與序列編號2所表示之胺基酸序列具有6〇%以上之同源 性’且具有心半乳糖酶活性的胺基酸序列。 進而’本發明提供一種α-半乳糖酶基因’其係對包含下 述（a)、（b)或（c)中之任一個胺基酸序列之α-半乳糖酶進行 編碼： (a) 序列編號2所表示之胺基酸序列； (b) 於序列編號2所表示之胺基酸序列中’一個或數個胺基 酸經缺失、置換及/或附加，且具有α-半乳糖酶活性的胺基 酸序列； (c) 與序列編號2所表示之胺基酸序列具有6〇%以上之同源 性，且具有α_半乳糖酶活性的胺基酸序列。 129208-10007J3.doc -17- 1363093 上述（b)中之「一個或數個胺基酸」’通常係指丨〜5 〇個胺 基酸’較好的是1~30個胺基酸，更好的是1〜2〇個胺基酸， 更好的是1〜10個胺基酸，尤其好的是1〜5個胺基酸。 上述（c)中之「具有60%以上之同源性之胺基酸序列」， 通常係指具有60%以上，較好的是70%以上，更好的是 80%以上，更好的是9〇%以上，更好的是95%以上，尤其 好的是99%以上之同源性的胺基酸序列。 作為本發明之α_半乳糖酶基因之具體例，可列舉具有以 下（a)或（b)之鹼基序列之α-半乳糖酶基因。 (a) 序列編號1所表示之驗基序列； (b) 序列編號丨所表示之鹼基序列中，一個或數個鹼基經缺 失、置換及/或附加，且具有α·半乳糖酶活性之編碼蛋白質 的驗基序列。 上述（b)中之「一個或數個鹼基」，通常係指卜15〇個鹼 基，較好的是卜卯個鹼基，更好的是卜6〇個鹼基，更好的 是1〜30個驗基，更好較卜2請驗基，更好的是卜μ個驗 基，更好的是1〜10個鹼基，尤其好的是丨〜5個鹼基。 本發明之α-半乳糖酶基因，可藉由眾所周知之基因操作 方法’於無損本來之催化反應之性質的情況下實現胜肽之 突變，如此之突變體基因，係指藉由基因工程方法，由本 發明之-半乳糖酶基因所製作之人工突變基因，該人工突 變基因可藉由使用定點突變法、或將目標基因之特定DNA 片段置換為人工突變舰等之各種基因工程方法而獲得。 即’作為使《半乳㈣中之胺基酸產生缺失、置換、附加 129208-1000713.doc 1363093 等突變之方法’可利用PCR法、易鉼 勿錯PCR法、DNa重 法、或製造嵌合酶之方法等眾所周知之方法。 可將以上述方式獲得之完整地包含㈠乳糖酶之社縣 因之丽片段，***、連接在大腸桿菌用表現載體例: pBluescriptn KS⑴之多選殖位點上，而構建新的重^ 體。該質體載體令導入有可高效地表現大勝桿菌内作為^卜 來基因而連接之基因的Ue啟動子’藉由培養將重組質體導 入大腸桿菌而獲得之轉形體，可表現出大量〇1•半乳糖酶。 又’上述之外，亦可利用各種宿主微生物、載體而表現 大量α-半乳糖酶，例如藉由培養銚子短芽孢桿菌 (Brevibacillus choshinensis)之轉形體，可表現出大量以半 乳糖酶。更詳細而言’作為組人本發明半乳糖酶基因 之載體，較好的是可於宿主微生物體内自主增殖之噬菌體 或由質體構建之用於基因重組者，作為噬菌體載體，例如 於將屬於大腸桿菌之微生物作為宿主微生物之情形時，可 使用Xgt · λ(：、Xgt · λΒ等。又，作為質體載體，例如將大 腸桿菌作為宿主微生物之情形時，可使用質體pET 3a、 pET-lla、pET_32a 等 pET 載體（N〇vagen)，或 pBR322、 PBR325、pACYC184、pUC12、pUC13、pUcl8、pUCi9、 PUC118、pINI、Bluescript KS+ ;於將枯草桿菌作為宿主 之情形時，可使用 PWH1520 ' PUB110、PKH300PLK ;於 將放線菌作為宿主之情形時，可使用pIJ68〇、pIJ7〇2 ;於 將酵母尤其是啤酒酵母作為宿主之情形時，可使用 YRp7、pYCl、YEpl3等。利用生成有與藉由用於切斷本A purification method in which the usual protein can be combined, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, hydroxyapatite chromatography, sleeping 1 L, etc. Isolation, by alpha-galactosidase. The order of the above operations is not particularly limited, and each operation may be performed once or twice or more. Further, it is preferable to exchange the sample liquid with an appropriate buffer by means of precipitation or the like before passing the sample into each column. Further, the sample liquid is concentrated in the stage. ' & Preferably, the α-galactosidase activity contained in each component to be separated is measured at each stage of purification, and the component having higher activity is collected and supplied to the next stage of the test. As a method for measuring the activity of α-galactosidase, for example, a method of mixing 100 mM sodium acetate buffer 450 0 containing 6 mM of p-nitrobenzene _a_D_galactopyranoside having an ipH value of $ 〇 is used. The enzyme solution was 15 Torr, and after reacting for 5 to 30 minutes at 4 Torr:, it was added to 1 mL of a sodium carbonate aqueous solution of 丨M to inactivate the enzyme, and the reaction was stopped. The color of the resulting solution was measured at an absorption peak at a wavelength of 420 nm, and the concentration was calculated using a calibration curve prepared for each concentration of p-nitrophenyl hydrazine. Further, the unit of the enzyme activity is expressed as 1 U by the amount of the enzyme which frees 1 μΓη〇1 of p-nitrophenol in 1 minute under the above conditions. The degree of purification of the purified α-galactosidase and the measurement of the molecular weight can be confirmed by electrophoresis, gel chromatography or the like. Further, the enzymatic properties can be studied by changing the reaction temperature or the reaction pH to determine the enzyme activity; or adding various enzyme inhibitors or metal ions to the reaction solution, 129208-1000713.doc -15- = Test (four) to stay active. Further, the α-galactosidase is subjected to an expiratory time at each pH condition or under a condition of a dish for a predetermined period of time, and then the enzyme activity is measured, whereby the stable pH range and the stable temperature range can be studied. Further, the reaction was carried out by changing the concentration of the substrate, and the Michaelis constant (Km) and the maximum velocity (Vmax) of α-galactosidase with respect to each substrate were determined. The α-semi-luciferase gene of the present invention can be obtained, for example, by culturing the Bacillus coagulans AKC-004 strain in the above medium. The obtained culture solution is separated into cells by a method known by centrifugation and passing. With sputum. The bacterium which was obtained in the above manner was pulverized by using a lysozyme, an ultrasonic pulverizer, a French high-pressure crusher, etc., and the chromosome (10) was separated by various kinds of enzymes, and the chromosome obtained in the above manner was obtained, and the DNA piece was obtained. Using the chromosome deletion fragment obtained in the above manner, a fragment containing the entire length of the α-galactosidase gene or a part thereof is obtained by a well-known method such as a shotgun sorting method or a reverse pCR method. The nucleotide sequence of the DNA fragment containing the α-galactose (tetra) gene obtained herein is analyzed by a well-known method such as a DNA sequencer, and the nucleotide sequence thereof is clarified. Further, the DNA fragment containing the galactosidase gene can be re-obtained based on the basis of the sequence of the galactosidase gene alone. Further, by repeating this operation, the base sequence of the full length of the α-galactosidase gene can be confirmed. The base sequence of the X-galactosidase gene is decoded in the above manner, and the DNA fragment containing the full length of the galactosidase gene can be obtained by a well-known method using a PCR method or a restriction enzyme. In the above manner, the amino acid sequence of the <galactosidase gene of 129208-l〇〇〇7i3.d, -16 - 1363093 can be decoded, and the amino acid sequence of -galactosidase can be determined. The amino acid sequence of the α-galactosidase of the invention, but as long as the protein comprising the amino acid sequence has α-galactosidase activity, at least one amino acid deletion or substitution may be produced in the amino acid sequence. Additional mutations, or may be amino acid sequences having 60% homology to the amino acid sequence. That is, the galactosidase of the present invention, the following 3 (a), (b) Or the α-galactosidase of any one of the amino acid sequences of (c): (a) the amino acid sequence represented by SEQ ID NO: 2; (b) one of the amino acid sequences represented by SEQ ID NO: 2 Or several amino-based deletions, substitutions, and/or additions, and having alpha-galactosidase activity (c) an amino acid sequence having 6% or more homology to the amino acid sequence represented by SEQ ID NO: 2 and having cardiac galactosidase activity. Further, the present invention provides an α a galactosidase gene which encodes an alpha-galactosidase comprising any one of the following amino acid sequences (a), (b) or (c): (a) an amine represented by SEQ ID NO: 2 (b) an amino acid sequence having one or several amino acids deleted, substituted and/or appended in the amino acid sequence represented by SEQ ID NO: 2 and having α-galactosidase activity; (c) an amino acid sequence having 6 to 30% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having α-galactosidase activity. 129208-10007J3.doc -17- 1363093 "One or several amino acids" generally means 丨~5 胺 amino acid', preferably 1 to 30 amino acids, more preferably 1 to 2 胺 amino acids, More preferably, it is 1 to 10 amino acids, particularly preferably 1 to 5 amino acids. The "amino acid sequence having 60% or more homology" in the above (c) is usually It means 60% or more, preferably 70% or more, more preferably 80% or more, more preferably 9% by weight or more, more preferably 95% or more, and particularly preferably 99% or more homology. The amino acid sequence of the α-galactosidase gene of the present invention includes the α-galactosidase gene having the base sequence of the following (a) or (b). (a) SEQ ID NO: 1 (b) the sequence of the encoded protein of the base sequence represented by the sequence number 丨, one or several bases deleted, substituted and/or appended, and having α-galactosidase activity . The "one or several bases" in the above (b) usually means 15 bases, preferably a ruthenium base, more preferably a ruthenium base, more preferably 1 to 30 test bases, better than 2 check the base, better is a μ test base, more preferably 1 to 10 bases, especially good 丨 ~ 5 bases. The α-galactosidase gene of the present invention can realize the mutation of the peptide by a well-known gene manipulation method without degrading the nature of the catalytic reaction, such a mutant gene means that by genetic engineering methods, The artificial mutant gene produced by the galactosidase gene of the present invention can be obtained by using a site-directed mutagenesis method or a genetic engineering method in which a specific DNA fragment of a target gene is replaced with an artificial mutant ship or the like. That is, 'as a method of deleting, replacing, or adding a mutation such as 129208-1000713.doc 1363093 in the semi-milk (4)', the PCR method, the easy-to-error PCR method, the DNa heavy method, or the chimerization can be used. A well-known method such as an enzyme method. The fragment obtained by the above-described method comprising the (1) lactase can be inserted and ligated into the expression vector of Escherichia coli: pBluescriptn KS (1) to construct a new recombinant. The plastid vector introduces a Ue promoter which can efficiently express a gene linked to the gene in the Phytophthora bacillus, and can display a large amount of 〇1 by culturing a transformant obtained by introducing a recombinant plastid into Escherichia coli. • Galactase. Further, in addition to the above, a large amount of α-galactosidase can be expressed by various host microorganisms and vectors, and for example, a large amount of galactosidase can be expressed by culturing a transformant of Brevibacillus choshinensis. More specifically, the carrier of the galactosidase gene of the present invention is preferably a phage which can be independently propagated in a host microorganism or a plastid for gene recombination, as a phage vector, for example, When a microorganism belonging to Escherichia coli is used as a host microorganism, Xgt · λ (:, Xgt · λ Β, etc. can be used. Further, when a plastid vector, for example, Escherichia coli is used as a host microorganism, plastid pET 3a can be used. pET vector (N〇vagen) such as pET-lla or pET_32a, or pBR322, PBR325, pACYC184, pUC12, pUC13, pUcl8, pUCi9, PUC118, pINI, Bluescript KS+; when using Bacillus subtilis as a host, PWH1520 can be used. PUB110, PKH300PLK; pIJ68〇 and pIJ7〇2 can be used when actinomycetes are used as hosts; YRp7, pYCl, YEpl3, etc. can be used when yeast, especially brewer's yeast, is used as a host. Used to cut this

129208-1000713.doc -19- S 1363093 發明之α-半乳糖酶基因之制限酶所生成之DNA末端相同的 制限酶，切斷上述載體而製作載體片段，藉由DNA連接 酶’依據常法使本發明之α-半乳糖酶基因片段與載體片段 結合’藉此可將編碼本發明之α-半乳糖酶基因之DN Α組入 目標載體中。 作為植入質體之宿主微生物，若重組DNA可穩定且自主 增殖即可，例如於宿主微生物為屬於大腸桿菌之微生物之 情形時’可利用大腸桿菌BL21、大腸桿菌BL21(DE3)、大 腸桿菌BL21trxB、大腸桿菌R〇setta(DE3)、大腸桿菌 Rosetta、大腸桿菌 R〇setta(DE3)pLysS、大腸桿菌 R〇setta(DE3)pLaCl、大腸桿菌 R0settaBlue、大腸桿菌 Rosetta-gami、大腸桿菌0rigami、大腸桿菌〇rigami、大腸 桿菌TUner、大腸桿菌Dm、大腸桿菌jM1〇9、大腸桿菌 W3110、大腸桿菌C6〇〇等。又，於微生物宿主為屬於桿菌 屬之微生物之情形時，可使用枯草桿菌、巨大芽孢桿菌 等，於微生物宿主為屬於放線菌之微生物之情形時，可使 用變鉛青鏈黴菌TK24等；於微生物宿主為屬於啤酒酵母 之微生物之情形時’可使用啤酒酵母INVSC1等。 又，於藉由轉形微生物製造本發明之α_半乳糖酶時，將 3玄轉形微生物培養於f養培養基巾，使㈣内或培養液中 成本發月之半乳糖酶，培養完畢後，藉由對所得培養 物進行過渡或離心分離等方法而採集g體’繼而以機械方 法或溶菌酶等酶法破壞該菌體，又，視需要添加EDTA及/ 或適當之界面活性料而將本發明之《•半乳_之水溶液 129208-1000713.doc •20· 1363093 加以很縮’或不/辰縮’繼而藉由硫酸錄分級分離、凝谬過 濾、、親和層析等吸附層析、離子交換層析法而加以處理， 藉此可獲得純度較高之本發明之α•半乳糖酶。 轉形微生物之培養條件，可考慮到其營養生理性質而選 擇培養條件，大多，m於液體行培養，但工業上 進行深部通㈣拌培養較為_ 1為培養基之營養源， 可廣泛使用微生物之培養中通常使用者。培養溫度可於微 生物發育且生產本發明之„ & 半礼糖酶之範圍内進行適當改 變’於大腸桿菌之情形時，較好的是10至45。(：左右’更好 的是20至3Gt左右。培養條件根據條件而多少有些不同， 只要估算本發明之α_半乳糖酶達到最高產量之時刻而於適 當時候結束培養即可，於大腸桿菌之情形時通常為12至48 小時左右。培養基ΡΗ值可於細菌發育且生產本發明之&半 乳糖酶之範圍内進行適宜變更，於大腸桿菌之情形時，較 好的是pH值為6至8左右。 根據本㈣’於制上述酶時，㈣會抑制本發明之酶 的作用，職於純化程度等並無㈣限定，除了所純化之 本發明之酶以外，亦可使用含有該酶之物質。 根據本發明，可提供+古μ .+. ‘ & 3有上述本發明之α-半乳糖酶之酶 組合物。上述酶組合物中 物中了進而含有選自例如α-葡萄糖苦 轉、^葡萄糖苦酶、β•半乳糖酶、纖維素酶、木聚糖酶、 蛋白鉍、+礼聚糖酶、***聚糖酶、甘露聚糖酶、鼠李 半乳㈣酸酶、聚半乳糖越酸酶、果膠甲酿酶、果膠解離 酶、及聚半乳糖醛酸解離酶中之至少一種以上之成分。 129208-1000713.doc •21 · 1363093 根據本發明，可提供含有上述酶組合物之棉子糖合成試 劑0 於本發明中，作為微生物觸媒，除了凝結芽胞桿菌 AKC-004株以外，亦可使用上述轉形體。於本發明之棉子 糖製造中所利用之微生物觸媒，可利用藉由通常之培養方 法而獲得之微生物本身’無需自微生物純化α_半乳糖酶。 又’視情形，亦可使用微生物培養液、微生物培養上清 液。另一方面，藉由培養法而獲得之微生物，亦可視需要 以水或緩衝液等清洗後使用。例如，可使用經培養之微生 物之培養⑨，或藉由離心分離、緩衝進行清洗等而獲得之 微生物懸浮液，使微生物或微生物之處理物（例如微生物 之粉碎物等）懸浮或溶解的水溶液，或者將微生物或微生 物處理物藉由包埋法、交聯法、或載體結合法 binding)進行固定化而成者。作為固定化時之固定化載體 的例子，可列舉：玻难珠、石夕膠、聚胺基甲酸乙醋、聚丙 稀醯胺、聚乙烯醇、卡拉膠、海蕩酸，但並不限定於該 根據本發明，可提供使用上述本發明之α_半乳糖酶或包 ^半乳糖酶之酶組合物、棉子糖合成㈣或微生物觸媒 糖子糖之裝每方法。作為原料，例如可使用嚴糖及半乳 t =使職糖及半浦料原料時，❹，自利用《-半 二酶之脫水縮合反應之性質上來看，較好的是原料濃度 =，但若半乳糖濃度過高，則由於半乳糖分子間會產生 I ’故而會抑㈣糖與半乳糖間的脫水縮合反應，因此 129208-1000713^0^ -22· 1363093 蔗糠濃度較好的是30% (w/v)〜90°/。（w/v)，更好的是35% (w/v)~80°/。（w/v) ’ 更好的是40% (w/v)〜70°/。（w/v)。半乳糖 濃度較好的是2% (w/v)〜45% (w/v)，更好的是5% (w/v)〜35% (w/v) ’ 更好的是 7% (w/v)〜30% (w/v)。又，較 好的是調製為原料中之蔗糖與半乳糖均可溶之濃度。 使用本發明之α-半乳糖酶或包含心半乳糖酶之酶組合物 或包含酶組合物之棉子糖合成試劑或微生物觸媒製造棉子 糖時的反應溫度，自反應速度或酶之穩定性方面而言，較 好的是10〜90°C，更好的是20〜70。(：，更好的是30〜6(rc。 反應pH值可於廣範圍内進行調整，自酶之穩定性方面而 言，較好的是pH值為2_0〜1〇.〇，更好的是？11值為3 〇〜7 5， 更好的是3.5〜6.0。反應時間亦根據酶之使用量而有所不 同，考慮到工業利用時，通常較好的是2〇分鐘〜2〇〇小時， 更好的是6〜80小時。然而，本發明並不限定於以上之反應 條件或反應形態，可進行適當選擇。 於本發明_，於使用α-半乳糖酶或包含α-半乳糖酶之酶 組合物或包含酶組合物之棉子糖合成試劑的棉子糖合成反 應中，棉子糖於反應溶液中累積至〇5%以上時，生 中=棉子糖含有率可提高至65%以上，於更好之條件下可 提南至80%以上。本發明中之較高之生成寡糖中之棉子糖 含有率，亦可於棉子糖於反應溶液中累積至〇75%以上， 進而累積至】.〇%以上時實現。 又，一般而言，於使用微生物本身之棉子糖合成反應 中’由於夾雜酶之影響而導致生成募糖中之棉子糖含有率 129208-10007l3.doc -23- 1363093 較低’相對於此’使用本發明之微生物觸媒之棉子糖合成 反應中’生成寡糖中之棉子糖含有率較高。 於本發明中所得之生成寡糖中之棉子糖含有率可藉由以 下方法測定。 棉子糖合成反應結束後’藉由將反應液稀釋25倍，於 99 C下保持1〇分鐘，而使反應停止。反應停止後，藉由離 心分離除去微生物，使用高效液相層析儀（HPLC)對所得反 應溶液進行定量。測定係使用Thermoelectron公司製造之 Hypercarb管柱，檢測器係使用RIe生成寡糖中之棉子糖 含有率，可由於HPLC分析圖中檢測出之各個峰面積比， 根據（棉子糖之峰面積）/(生成寡糖之峰面積）><1〇〇算出。 作為將藉由本發明之方法而製造之棉子糖進行純化、分 離之方法，可利用通常所使用之純化處理方法。即，例 如，藉由離心分離、利用MF膜或UF膜等之膜處理、壓濾 等除去微生物觸媒，藉由陽離子交換層析法或陰離子交換 層析法等層析處理或透析等脫鹽處理，除去自緩衝液或培 養基等帶入之鹽類等’進而’利用陽離子交換層析法、陰 離子父換層析法、高效液相層析法、活性碳層析法等層析 處理或利用溶解度之差等之結晶化處理、其他常法，^此 可對棉子糖進行分離、純化。層析處料單獨❹該等方 法，亦可組合使用’可適當利用移動層式或模擬移動層 式、多成分分離模擬移動層式、多成分分離循環式等。該 等棉子糖之純化、分離處理方法’可分批進行，亦可利用/ 管柱等而連續進行。 129208-1000713.doc •24- 1363093 以下，藉由實施例進行具體說明，但本發明並不受該等 實施例之任何限制。 [實施例] 實施例1 將凝結芽胞桿菌AKC-004株（寄存編號FERM P-21092(以 FERM-ABP10948作為編號而移交管理）（台灣寄存編號： BCRC 9103 88);寄存機關：獨立行政法人產業技術综合研 究所專利生物寄存中心）於姨蛋白企璦脂基礎（Tryptose Blood Agar Base，TBAB)平板培養基（Difco)上，於 55°C 下 培養1天，而形成菌落。 將pH值調整為7.2之表1所示的培養基30 mL，加入至150 mL容量之三角燒瓶中，以轴耳於上述平板培養基上植入 菌落，以50°C、25小時、180 rpm之條件進行旋轉振盪培 養，將其作為瓶中培養之種子細菌。 [表1] 表1 :培養基組成 培養基組成 微量金屬元素溶液組成129208-1000713.doc -19-S 1363093 The restriction enzyme of the same DNA end generated by the restriction enzyme of the α-galactosidase gene of the invention, the vector is cleaved to prepare a vector fragment, and the DNA ligase is made according to the conventional method. The α-galactosidase gene fragment of the present invention binds to a vector fragment 'by this, a DN 编码 encoding the α-galactosidase gene of the present invention can be incorporated into a target vector. As a host microorganism to be implanted into a plastid, if the recombinant DNA can be stably and autonomously propagated, for example, when the host microorganism is a microorganism belonging to Escherichia coli, Escherichia coli BL21, Escherichia coli BL21 (DE3), and Escherichia coli BL21trxB can be used. Escherichia coli R〇setta (DE3), Escherichia coli Rosetta, Escherichia coli R〇setta (DE3) pLysS, Escherichia coli R〇setta (DE3) pLaCl, Escherichia coli R0settaBlue, Escherichia coli Rosetta-gami, Escherichia coli 0rigami, Escherichia coli 〇rigami, Escherichia coli TUner, Escherichia coli Dm, Escherichia coli jM1〇9, Escherichia coli W3110, Escherichia coli C6〇〇, and the like. Further, when the microbial host is a microorganism belonging to the genus Bacillus, Bacillus subtilis, Bacillus megaterium, or the like may be used, and when the microbial host is a microorganism belonging to an actinomycete, a strain of Streptomyces lividans TK24 or the like may be used; When the host is a microorganism belonging to the brewer's yeast, beer yeast INVSC1 or the like can be used. Further, when the α-galactosidase of the present invention is produced by a transforming microorganism, the 3 metamorphic microorganism is cultured in a f-culture medium towel, and the galactosidase is produced in (4) or in the culture solution, after the culture is completed. The g body is collected by a method such as transition or centrifugation of the obtained culture, and then the cell is destroyed by an enzymatic method such as mechanical method or lysozyme, and if necessary, EDTA and/or an appropriate interface active material is added. The "aqueous solution of _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ It is treated by ion exchange chromatography, whereby the α-galactosidase of the present invention having a higher purity can be obtained. The culture conditions of the transformed microorganisms can be selected in consideration of their nutritional and physiological properties. Most of them are cultured in liquid, but industrially, deep-through (four) mixed culture is more important than the nutrient source of the medium, and the microorganisms can be widely used. Usually used in culture. The culture temperature can be suitably changed from 10 to 45 in the case where the microorganism is developed and produced in the range of the & semi-glycosidase of the present invention, in the case of Escherichia coli. (: Left and right 'better is 20 to The culture conditions are somewhat different depending on the conditions, and it is only necessary to terminate the culture at the appropriate time as long as the α-galactosidase of the present invention reaches the highest yield, and it is usually about 12 to 48 hours in the case of Escherichia coli. The enthalpy of the culture medium can be appropriately changed within the range of bacterial development and production of the & galactosidase of the present invention, and in the case of Escherichia coli, the pH is preferably about 6 to 8. According to the present (4) In the case of an enzyme, (4) inhibits the action of the enzyme of the present invention, and the degree of purification is not limited to (4), and in addition to the purified enzyme of the present invention, a substance containing the enzyme may be used. According to the present invention, μ .+. ' & 3 has the above-described α-galactosidase enzyme composition of the present invention. The above enzyme composition further contains, for example, α-glucose, glucosinolate, β•half milk Enzyme, cellulase, xylanase, peptone, +-glycanase, arabinase, mannanase, rhamno-galactosylase, polygalactose-acidase, pectin a component of at least one of a pectin dissociation enzyme and a polygalacturonan dissociating enzyme. 129208-1000713.doc • 21 · 1363093 According to the present invention, a raffinose synthesis reagent containing the above enzyme composition can be provided. In the present invention, as the microbial catalyst, the above-mentioned transforming body may be used in addition to the Bacillus coagulans AKC-004 strain. The microbial catalyst used in the production of the raffinose of the present invention can be utilized by a usual culture method. The obtained microorganism itself does not need to purify the α-galactosidase from the microorganism. Further, depending on the case, the microbial culture solution or the microbial culture supernatant can also be used. On the other hand, the microorganism obtained by the culture method can also be used as needed. It can be used after washing with water, buffer, etc. For example, culture of microorganisms 9 can be used, or a suspension of microorganisms obtained by centrifugation, buffering, or the like can be used to make microorganisms or An aqueous solution in which a biological treatment (for example, a pulverized microorganism such as a microorganism) is suspended or dissolved, or a microorganism or a microorganism treatment is immobilized by an embedding method, a crosslinking method, or a carrier binding method. Examples of the immobilized carrier at the time of catalysis include, but not limited to, glassy beads, shijiao, urethane, polyacrylamide, polyvinyl alcohol, carrageenan, and salicylic acid. The present invention can provide an enzyme composition using the above-described α-galactosidase or galactose-containing enzyme of the present invention, a raffinose synthesis (4) or a microbial catalyst saccharide, and as a raw material, for example, can be used. Sugar and galactose t = when making sugar and semi-precipitated raw materials, ❹, from the use of - semi-two enzyme dehydration condensation reaction, it is better to concentrate the raw material =, but if the galactose concentration is too high, Then, due to the fact that I will produce I's between the galactose molecules, it will inhibit the dehydration condensation reaction between the sugar and the galactose. Therefore, the concentration of the cane toad is 129208-1000713^0^ -22· 1363093, which is preferably 30% (w/v)~ 90°/. (w/v), more preferably 35% (w/v) ~ 80 ° /. (w/v) ‘More preferably 40% (w/v)~70°/. (w/v). The galactose concentration is preferably 2% (w/v) to 45% (w/v), more preferably 5% (w/v) to 35% (w/v) 'more preferably 7% ( w/v) ~30% (w/v). Further, it is preferred to prepare a concentration in which the sucrose and galactose in the raw material are soluble. Reaction temperature, self-reaction rate or enzyme stability when using the α-galactosidase of the present invention or the enzyme composition containing cardiac galactosidase or the raffinose synthesis reagent or the microbial catalyst comprising the enzyme composition In terms of sex, it is preferably 10 to 90 ° C, more preferably 20 to 70. (:, more preferably 30~6 (rc. The pH of the reaction can be adjusted in a wide range. From the aspect of the stability of the enzyme, the pH is preferably 2_0~1〇.〇, better. Yes, the value of 11 is 3 〇 to 7 5, and more preferably 3.5 to 6.0. The reaction time varies depending on the amount of enzyme used. In consideration of industrial use, it is usually 2 minutes to 2 〇〇. In the hour, it is more preferably 6 to 80 hours. However, the present invention is not limited to the above reaction conditions or reaction forms, and may be appropriately selected. In the present invention, α-galactosidase or α-galactose is used. In the raffinose synthesis reaction of the enzyme composition of the enzyme or the raffinose synthesis reagent containing the enzyme composition, when the raffinose is accumulated in the reaction solution to 5% or more, the raw meal = raffinose content can be increased to 65% or more, under better conditions, can be increased to more than 80%. In the present invention, the higher the content of raffinose in the oligosaccharide, or the accumulation of raffinose in the reaction solution to 〇75 % or more, and then accumulate to 〇% or more. Also, in general, the use of the microorganism itself is the synthesis of raffinose The content of raffinose in the sugar-producing sugar produced by the influence of the inclusion enzyme should be 129208-10007l3.doc -23- 1363093 lower than the relative use of the microbial catalyst of the present invention in the raffinose synthesis reaction The content of raffinose in the produced oligosaccharide is high. The raffinose content in the oligosaccharide produced in the present invention can be determined by the following method. After the raffinose synthesis reaction is finished, 'by the reaction solution The reaction mixture was diluted 25 times and kept at 99 C for 1 minute to stop the reaction. After the reaction was stopped, the microorganisms were removed by centrifugation, and the obtained reaction solution was quantified using a high performance liquid chromatography (HPLC). The measurement was performed using Thermoelectron. The Hypercarb column manufactured by the company uses the R elsewhere to produce the raffinose content in the oligosaccharide, which can be determined by the peak area ratio detected in the HPLC analysis chart, according to (the area of the peak of raffinose) / The peak area of the sugar is calculated as a method of purifying and separating the raffinose produced by the method of the present invention, and a purification treatment method generally used can be used. The microbial catalyst is removed by centrifugation, membrane treatment using a MF membrane or a UF membrane, pressure filtration, or the like, and is subjected to a desalting treatment such as cation exchange chromatography or anion exchange chromatography or dialysis. a salt such as a buffer or a medium, and the like, and further, by cation exchange chromatography, anion-parent chromatography, high-performance liquid chromatography, activated carbon chromatography, or the like, or the difference in solubility, etc. Crystallization treatment, other common methods, this can be used to separate and purify raffinose. The chromatographic materials can be used separately or in combination, and can be combined with the use of mobile layer or simulated mobile layer, multi-component Separate simulated moving layer type, multi-component separation cycle type, and the like. The method for purifying and separating the raffinose can be carried out in batches or continuously using a column or the like. 129208-1000713.doc • 24- 1363093 The following is specifically described by way of examples, but the invention is not limited by the examples. [Examples] Example 1 Bacillus coagulans AKC-004 strain (registration number FERM P-21092 (transferred with FERM-ABP10948 as a number) (Taiwan registration number: BCRC 9103 88); depository authority: independent administrative corporation industry The Institute of Technology's Patent Bioburden Center was cultured on a Tryptose Blood Agar Base (TBAB) plate medium (Difco) at 55 ° C for 1 day to form colonies. Adjust the pH to 30 mL of the medium shown in Table 1 of 7.2, add to a 150 mL capacity Erlenmeyer flask, and implant the colonies on the above-mentioned plate medium with axons at 50 ° C, 25 hours, 180 rpm. Spinning culture was carried out as a seed bacterium cultured in a bottle. [Table 1] Table 1: Composition of medium Composition of medium Composition of trace metal elements

棉子糖 l〇g CH3COONa 12.8 g 甘油 l〇g FeS04.7H20 1 g 酵母萃取物 l〇g MnCl2-4H20 0.5 g 蛋白腺 l〇g CoC12-6H20 0.3 g MgS04-7H20 0.5 g ZnS04-7H20 0.4 g CaCb 0.04 g CuC12.2H20 50 mg 0.01 Μ之檸檬酸 0.5 mL NaMo04-2H20 50 mg 醋酸鈉三水合物 0.132 g H3BO4 20 mg ΚΗ2Ρ〇4 0.544 g NiCl2-6H20 20 mg Na2HP04-12H20 2.14 g 蒸餾水 1 L 微量金屬元素溶液 0.5 mL 蒸餾水 1 L -25 129208-1000713.doc 1363093 將pH值調整為7.2之表 繼而 至10 L容量之缸式醱酵槽中 細菌15 mL，以50°C、48小時 行通氣攪拌培養。 1所示之培養基6 L，加入 ’移植上述瓶中所培養之種子 200 rpm、0.2 vvm之條件進 繼而，於4°C下，對該讲晨、-*、谷v 培養液進行10,000 gx30分鐘之離 心分離，以除去上清液，而0妝益触 ， 叩口收菌體。測定所得菌體之α_ 半乳糖酶之活性，結果為261 υ。 使該菌體懸浮於含有50 mMiTrisHa、2〇瓜河之 EDTA、50 mM之葡萄糖的Lysis緩衝液（pH值8〇)中而成 為150 mL。於4°CT，對該懸浮液進行1〇，_ gx3〇分鐘之 離心分離，以除去上清液，而回收菌體。 將經Lysis緩衝液清洗後之菌體再次懸浮於上述卜…緩 衝液中，而成為150 mL。於其中添加〇〇2%之溶菌酶 (Sigma公司製造，來自印白），以37它、13小時、12〇印爪 之條件進行振盪’而進行菌體粉碎。 於4C下，對菌體粉碎後之溶液進行1〇〇〇〇 gx3〇分鐘之 離心分離，以除去菌體殘渣，而回收上清液。 於該上清液中添加硫酸銨，以形成37 5%之飽和溶液， 於4°C下放置一晚而產生沈澱。於4。(：下，進行1〇〇〇〇以3〇 分鐘之離心分離，以除去該沈澱，而回收上清液。 於該上清液中添加硫酸銨，以形成54 5%之飽和溶液， 於4°C下放置一晚而產生沈殿。於4°C下，進行10,〇〇〇 gx3〇 分鐘之離心分離，以回收該沈澱，再將其溶解於pH值為 7.0之20 mM磷酸緩衝液中，於4°C下放置一晚，對與上述 129208-1000713.doc -26· 1363093 相同之填酸缓衝液進行透析。 使藉由透析而獲得之上清液吸附於以與上述相同之磷酸 緩衝液進行平衡化之「DEAE Sepharose FF」（GE HEALTHCARE. BIO-SCIENCES股份有限公司）上，之後藉 由含有0〜0.4 Μ之氣化鈉之pH值為7.0的20 mM磷酸緩衝液 之濃度梯度法而使酶溶出。Raffinose l〇g CH3COONa 12.8 g Glycerol l〇g FeS04.7H20 1 g Yeast extract l〇g MnCl2-4H20 0.5 g Protein gland l〇g CoC12-6H20 0.3 g MgS04-7H20 0.5 g ZnS04-7H20 0.4 g CaCb 0.04 g CuC12.2H20 50 mg 0.01 citric acid 0.5 mL NaMo04-2H20 50 mg sodium acetate trihydrate 0.132 g H3BO4 20 mg ΚΗ2Ρ〇4 0.544 g NiCl2-6H20 20 mg Na2HP04-12H20 2.14 g distilled water 1 L trace metal elements Solution 0.5 mL Distilled water 1 L -25 129208-1000713.doc 1363093 The pH was adjusted to 7.2 and then 15 mL of bacteria in a 10 L-capacity tank fermentation tank was aerated at 50 ° C for 48 hours. 6 L of the medium shown in Fig. 1 was added to the condition of transplanting the seeds cultured in the above bottle at 200 rpm and 0.2 vvm, and the morning, -*, and valley v culture medium was subjected to 10,000 g x 30 minutes at 4 °C. Centrifugal separation to remove the supernatant, while 0 makeup touch, sputum collection. The activity of α-galactosidase of the obtained cells was measured and found to be 261 υ. The cells were suspended in Lysis buffer (pH 8 Torr) containing 50 mMiTrisHa, 2 guana EDTA, 50 mM glucose to obtain 150 mL. The suspension was subjected to centrifugation at 4 ° CT for 1 〇, g x 3 〇 minutes to remove the supernatant, and the cells were recovered. The cells washed with the Lysis buffer were resuspended in the above buffer to become 150 mL. To the mixture, lysozyme (manufactured by Sigma, manufactured by Imprint) was added thereto, and the cells were pulverized under the conditions of 37, 13 hours, and 12 〇 paws, and the cells were pulverized. The solution after the pulverization of the cells was centrifuged at 1 〇〇〇〇 gx for 3 minutes at 4 C to remove the bacterial residue, and the supernatant was recovered. Ammonium sulfate was added to the supernatant to form a 375% saturated solution, which was allowed to stand overnight at 4 ° C to cause precipitation. At 4. (:, the next one is centrifuged for 3 minutes to remove the precipitate, and the supernatant is recovered. Ammonium sulfate is added to the supernatant to form a 54 5% saturated solution, at 4 Place a night at °C to produce a shoal. At 4 ° C, perform 10, 〇〇〇gx3 〇 centrifugation to recover the precipitate, and then dissolve it in 20 mM phosphate buffer at pH 7.0. , placed at 4 ° C for one night, and dialyzed against the same acid buffer as the above 129208-1000713.doc -26· 1363093. The supernatant obtained by dialysis was adsorbed to the same phosphate buffer as above. The solution was equilibrated with "DEAE Sepharose FF" (GE HEALTHCARE. BIO-SCIENCES Co., Ltd.), and then subjected to a concentration gradient method of 20 mM phosphate buffer having a pH of 7.0 of 0 to 0.4 Μ. The enzyme is dissolved.

收集上述所溶出之活性成分，使用平均截留分子量為 10,000之超濾膜進行濃縮，使其吸附於以含有2 Μ之氣化 鈉之pH值為7.0的20 mM磷酸緩衝液進行平衡化之「HiTrap Phenyl FF(high sub)」（GE HEALTHCARE. BIO-SCIENCES 股份有限公司）上，之後藉由含有2.0~0 M之氣化鈉之pH值 為7.0的20 mM磷酸緩衝液之濃度梯度法而使酶溶出。The above-dissolved active ingredient was collected, concentrated using an ultrafiltration membrane having an average molecular weight cut off of 10,000, and adsorbed to a "HiTrap" which was equilibrated with 20 mM phosphate buffer having a pH of 7.0 containing 2 Μ of sodium hydride. Phenyl FF (high sub)" (GE HEALTHCARE. BIO-SCIENCES Co., Ltd.), followed by a concentration gradient method of 20 mM phosphate buffer containing a pH of 7.0 to 2.0 M of sodium methoxide Dissolution.

收集上述所溶出之活性成分，使用平均截留分子量為 10,000之超濾膜進行濃縮，使其吸附於以pH值為7.0之20 mM磷酸緩衝液進行平衡化之「MonoQ 5/50 GL」（GE HEALTHCARE. BIO-SCIENCES股份有限公司）上，之後藉 由含有0〜0.4 Μ之氣化鈉之pH值為7.0的20 mM磷酸緩衝液 之濃度梯度法而使酶溶出。 收集上述所溶出之活性成分，使用平均截留分子量為 10,000之超濾膜進行濃縮，將其填充至以pH值為7.0之20 mM填酸緩衝液進行平衡化之「「HiLoad 16/60 Superdex 200」（GE HEALTHCARE. BIO-SCIENCES股份有限公司） 上，之後以同樣之缓衝液進行溶出。 收集上述所溶出之活性成分，使用平均截留分子量為 129208-1000713.doc -27- 1363093 10,000之超濾膜進行濃縮，而製成純化〇[_半乳糖酶。活性 產率為15°/。，活性為270 U/mL。 實施例2 使用實施例1所得之純化α_半乳糖酶，進行關於其作用 之實驗。 (1) 最佳pH值範圍 於溶解在各PH值之1〇〇 mM緩衝液中的6 mM之對硝基苯_ (χ-D-吡喃半乳糖苷150 ^^中，混合稀釋1〇〇倍的本發明之 純化酶液50 pL，於40t下使其反應5分鐘。反應後，對游 離之對硝基苯酚進行定量，藉此測定活性，求出將最大活 性設為100時之相對活性。將該結果示於圖i。再者，所使 用之緩衝液為甘胺酸-HC1緩衝液（pH值為2.5〜3.5)、醋酸鈉 緩衝液（pH值為3_5〜6.0)、磷酸鈉緩衝液（pH值為6 〇〜8 5)、 甘胺酸-NaOH緩衝液（pH值為8.5〜10.0)。 (2) 穩定pH值範圍 於pH值為4.0〜1〇.〇之範圍内使用之緩衝液，於各 pH值下進行45°C、140分鐘之加熱處理，再測定其殘留活 性’與上述同樣地求出將最大活性設為1 〇〇時之相對活 性。將其結果示於圖2。再者，所使用之各pH值之緩衝液 的種類係與上述相同。 (3) 最佳溫度範圍 於溶解在pH值為4‘5之醋酸鈉緩衝液中之4·7 mM之對硝 基苯-α-D-吡喃半乳糖苦190吣中，混合稀釋2〇倍的本發明 之純化酶液10 ，於20〜6(TC之範圍内使其反應5分鐘。 129208-1000713td〇, •28· 1363093 反應後，對游離之對硝基苯酚進行定量，藉此測定活性， 求出將取大活性設為100時之相對活性。將其結果示於圖 3 ° (4) 穩定溫度範圍 使用pH值為4.5之100 mM醋酸鈉緩衝液，於3〇〜55°C之 各溫度下進行20分鐘之加熱處理，測定其殘留活性，與上 述同樣地求出將最大活性設為1 〇〇時之相對活性。將其結 果示於圖4。 (5) 分子量 藉由使用分離凝膠濃度為1 〇%之「Ready Gels J」（日本 Bio-Rad Laboratories股份有限公司）之SDS_聚丙烯醯胺凝 膠電泳，求出分子量。將其結果示於圖5。分子量約為 80,000 ’與由胺基酸序列推算之分子量83，122基本一致。 (7)受質特異性 於含有10 mM之各種受質的pH值為4.5之1〇〇 mM醋酸鈉 緩衝液150 gL中，混合稀釋1〇〇倍的本發明之純化酶液5〇 KL ’於40°C下使其反應2〇分鐘》其中，將對硝基苯_a_D_ °比喃半乳糖苷用於受質之反應中，將反應時間設為5分 鐘。反應後，對游離之對硝基苯酚進行定量，或根據藉由 使用「Shodex SUGAR」（昭和電工股份公司）管柱之高效液 相層析儀之分析，對分解之受質進行定量，求出將最大活 性設為100時各受質之相對活性。將其結果示於表2。又， 其中，對形成受質者測定反應速度，將結果示於表3。 129208-1000713.doc -29- 1363093 [表2] 表2 : α-半乳糖酶之受質特異性 基質 相對活性 pNP-a-D比鳴半乳糖苦 100 ρΝΡ-β-吡喃半乳糖苷 0 棉子糖 10 蜜二糖 38 乳糖 0 蔗糖 0 [表3] 表3 : α-半乳糖酶之反應速度常數 基質 Km(mM) Vmax(U/mg) ρΝΡ-α-吡喃半乳糖苷 0.53 82 棉子糖 40 48 蜜二糖 8.4 106 (8)抑制因素 於溶解在pH值為4.5之醋酸鈉緩衝液中之6 mM之對硝基 苯-α-D-吡喃半乳糖苷中，以最終濃度達到1 mM之方式添 加各種金屬離子，而成為150 pL，再混合稀釋100倍的本 發明之純化酶液50 μί，於40°C下使其反應20分鐘。反應 後，對游離之對硝基苯酚進行定量，藉此測定活性，而求 出將最大活性設為100時之相對活性。將其結果示於表4。 [表4] 表4 :金屬鹽對α-半乳糖酶之抑制 金屬離子 相對活性 對照 100 鉀 97 鈣 97 129208-1000713.doc -30- 鎂 95 鉻 99 錳 95 鐵(Π) 98 鐵(III) 99 鈷 95 鎳 88 銅 33 鋅 80 1363093 實施例3 分別取出實施例1中記載之純化α-半乳糖酶0.5 U，將其 添加至裝入1.5 mL容量之聚丙烯製試管中的200 gL之糖液 (pH值5.0之100 mM醋酸鈉緩衝液中含有蔗糖73%、半乳糖 12%)中，並進行混合，以60°C ' 1，200 rpm之條件使其反 應。自反應開始經過45小時後，自反應液中取出20 μι， 以480 gL蒸餾水進行稀釋後，於99°C下處理10分鐘，藉此 使酶熱失活。使熱失活後之稀釋糖液恢復至常溫後，藉由 使用「Hypercarb」（Thermoelectron公司製造）管柱之高效 液相層析儀進行分析。將其結果示於圖6。生成寡糖中之 棉子糖含有率為82%，反應液中之棉子糖濃度為1.67重量 %。 實施例4 (1)染色體DNA之製備 依據常法，製備凝結芽胞桿菌AKC-004株之染色體 DNA。對藉由實施例1中所記載之方法而獲得之凝結芽胞 桿菌AKC-004株之培養液60 mL進行離心分離，回收菌 體。使所得菌體懸浮於Lysis緩衝液（50 mM Tris-HCl(pH值 129208-1000713.doc -31 - 1363093 為8.0)、20 mM之EDTA、50 mM葡萄糖）中，再充分清洗。 進行離心分離，而回收菌體後，再次將其懸浮於Lysis緩衝 液中，於其中添加溶菌酶，於37°C下培養45分鐘。繼而， 添加SDS及RNase，於37°C下培養45分鐘。其後添加蛋白 酶K(Proteinase K)，於50°C下穩定振遺60分鐘。以苯紛-氣 仿、氣仿，對此處所獲得之溶液進行處理後，進行乙醇沈 澱，將所析出之核酸纏繞在玻璃吸管上，而加以回收。以 70%乙醇對該核酸進行清洗後，加以乾燥，再將其溶解於 TE中。藉由該操作，製備約1 mg之染色體DNA。 (2)α-半乳糖酶基因之離析 由上述（1)中所製備之染色體DNA，設計、合成用以增 幅α-半乳糖酶基因片段之PCR引子。引子之設計係以眾所 周知之來自數種微生物之α-半乳糖酶基因之比對結果為基 礎而進行，同義引子合成具有 5’-GAAGTITACGGITTYAGYYTTGTITACAGYGG-3,（序列編號 3)之序列的寡聚DNA ，反義引子合成具有5’-CCAAACCAICCRTCRTCIARIACRAA-3丨（序列編號 4)之序歹丨J 的寡聚DNA ^再者，序列中，I表示肌苷，Υ表示C或Τ，R 表示Α或G。使用此處所得之PCR引子，以上述（1)中所製 備之染色體DNA作為模板，藉由PCR法進行α-半乳糖酶基 因片段之增幅，而獲得含有3 80個鹼基對之α-半乳糖酶基 因片段。使用DNA測序儀對此處所得之基因片段之鹼基序 列加以分析。 為了獲取含有α-半乳糖酶基因全長之DNA片段，以由分 129208-1000713.doc •32· 1363093 析所得之α-半乳糖酶基因片段之鹼基序列作為基礎，而設 計、合成反向PCR用PCR引子。同義引子合成具有5'_ TGATCAACAACTGGGAAAGCGACCT-3'(序列編號 5)之序 列的寡聚DNA ， 反義引子合成具有5ι_ GAACTGGTCCTGCTGCACAATTCC-3’（序列編號 6)之序列 的寡聚DNA。繼而，製備用於反向PCR之模板。以制限酶 Hindlll，將上述（1)中所製備之染色體DNA消化後，使用 T4 DNA連接酶（T4 DNA Ligase)使所得DNA片段自身環 化’而形成反向PCR之模板。針對該模板，使用如上述般 合成之反向PCR用引子，藉由PCR法對包含α-半乳糖酶基 因之DNA片段進行增幅，獲取含有α-半乳糖酶基因之全部 序列的包含4500個鹼基對的PCR產物。 (3)驗基序列之分析 使用DNA測序儀，由（2)中所得之PCR產物決定α-半乳糖 酶基因之鹼基序列。其結果為，解碼出具有自序列編號1 所示之5'末端起之DNA鹼基序列之2190個鹼基對的α-半乳 糖酶之結構基因。該序列係從未發現之新穎基因。又，由 該DNA鹼基序列類推之凝結芽胞桿菌AKC-004株所生產之 α-半乳糖酶包含730個胺基酸，具有序列編號2所示之自Ν 末端起之胺基酸序列《檢索資料庫，結果發現，該胺基酸 序列與來自熱桿菌芽孢（Geobacillus stearothermophilus) 之α-半乳糖酶（AgaN)具有57%之同源性，與來自嗜熱桿菌 芽孢之α-半乳糖酶（AgaA)具有56%之同源性，與來自嗜熱 桿菌芽孢之a半乳糖酶（AgaB)具有56%之同源性，與來自棉 129208-1000713.doc •33- 1363093 子糖乳球菌（Lactococcus raffinolactis)之α-半乳糖酶具有 56%之同源性，明確其係編碼新穎之α-半乳糖酶。 (4) α-半乳糖酶基因之表現質體載體之構建及轉形 以（3)中所得之α-半乳糖酶基因之序列為基礎，設計、合 成用以增幅包含α-半乳糖酶基因之SD序列、結構基因、終 止密碼子之區域的PCR引子。PCR引子之設計係以（2)中所 得之包含4500個鹼基對之PCR產物的鹼基序列為基礎而進 行，同義引子合成具有 5'-TAAGGTAAAGCAGATGTGCCATT-3· (序列編號7)之序列的寡聚DNA，反義引子合成具有5’-TTACTCGTACACCGCCTC-3'(序列編號8)之序列的寡聚 DNA。以（2)中所得之包含4500個鹼基對之PCR產物為模 板，使用合成之PCR引子，藉由PCR法進行增幅，而獲取 包含2325個鹼基對之PCR產物。將此處所得之PCR產物末 端平滑化、磷酸化。於藉由制限酶EcoRV進行消化後，再 進行脫磷酸化而成的pBluescriptll KS(+)載體上，連接上 述所得之經末端平滑化、磷酸化之PCR產物，構建新的質 體載體pBlue/agaA。該質體載體中導入有可有效轉錄在大 腸桿菌内作為外來基因而連接之基因的lac啟動子，可有效 表現/製造α-半乳糖酶》將所得質體載體於以氯化鈣法製備 之大腸桿菌JM109株之勝任細胞中，藉由熱震法進行轉轉 形，而製作重組微生物。 (5) 轉形體之培養及α-半乳糖酶之表現 將（4)中所製作之重組大腸桿菌JM109-pBlue/agaA，於含 129208-1000713.doc • 34- 1363093 有100 mg/L之安比西林的30 mLiLB培養基中於37。〇下 振盪培養24小時。培養後’對重組大腸桿菌進行離心分 離，而將其回收。以100 mM醋酸鈉緩衝液（pH值為5 〇)對 菌體進行清洗後，再次將其懸浮於相同之缓衝液中，藉由 超音波粉碎機進行粉碎。藉由上述方法測定該粉碎液之& 半乳糖酶活性，結果為19.9 U/培養液（mL)。 實施例5 分別取出培養實施例4中所記載之重組體而獲得之心半 乳糖酶1.0U，添加至裝入UmL容量之聚丙烯製試管中之 200 μί之糖液（pH值為5.0之1〇〇 mv[醋酸鈉緩衝液中含有蔗 糖73%、半乳糖12%)中，並進行混合，以6〇。〇、Lao卬瓜 之條件進行反應。自反應開始經過丨6小時後，自反應液中 取出20 pL，以480 μί蒸餾水進行稀釋後，於99t：下處理1〇 分鐘，藉此使酶熱失活。使熱失活後之稀釋糖液恢復至常 /皿後，藉由使用「Hypercarb」（Thermoelectron公司製造） 官柱之高效液相層析儀進行分析。將其結果示於圖7。生 成券糖中之棉子糖含有率為82〇/0，反應液中之棉子糖濃度 為1·20重量％。 實施例6 將凝結芽胞桿菌AKC-004株（寄存編號FERM Ρ-21092(以 FERM-ABP10948之編號移交管理）（台灣寄存編號：bcrc 9103 88);寄存機關：獨立行政法人產業技術综合研究所 專利生物寄存中心）於胰蛋白血瓊脂基礎（Tryptose BloodThe above-dissolved active ingredient was collected, concentrated using an ultrafiltration membrane having an average molecular weight cut off of 10,000, and adsorbed to "MonoQ 5/50 GL" (GE HEALTHCARE) which was equilibrated with a pH buffer of 7.0 mM phosphate buffer. The BIO-SCIENCES Co., Ltd. was then dissolved by a concentration gradient method of 20 mM phosphate buffer having a pH of 7.0 of 0 to 0.4 Μ. The above-dissolved active ingredient was collected, concentrated using an ultrafiltration membrane having an average molecular weight cut off of 10,000, and filled into "HiLoad 16/60 Superdex 200" which was equilibrated with a pH of 7.0 mM acid buffer. (GE HEALTHCARE. BIO-SCIENCES Co., Ltd.), then eluted with the same buffer. The above-dissolved active ingredient was collected and concentrated using an ultrafiltration membrane having an average molecular weight cut off of 129208-1000713.doc -27 - 1363093 10,000 to prepare purified quinone [_galactosidase. The activity yield was 15 ° /. The activity is 270 U/mL. Example 2 Using the purified α-galactosidase obtained in Example 1, an experiment was conducted on the action thereof. (1) The optimum pH range is 6 mM p-nitrobenzene _ (χ-D-galactopyranoside 150 ^^ dissolved in 1 mM buffer of each pH value, mixed and diluted 1 〇 50 pL of the purified enzyme solution of the present invention was reacted for 5 minutes at 40 t. After the reaction, the free p-nitrophenol was quantified to measure the activity, and the relative activity when the maximum activity was set to 100 was determined. The activity is shown in Figure i. Further, the buffer used is glycine-HC1 buffer (pH 2.5 to 3.5), sodium acetate buffer (pH 3_5 to 6.0), sodium phosphate. Buffer (pH 6 〇~8 5), glycine-NaOH buffer (pH 8.5~10.0). (2) Stable pH range is used within the range of pH 4.0~1〇.〇 The buffer solution was heat-treated at 45 ° C for 140 minutes at each pH value, and the residual activity was measured. The relative activity when the maximum activity was 1 〇〇 was determined in the same manner as above. The results are shown in Figure 2. Furthermore, the type of buffer used for each pH value is the same as above. (3) The optimum temperature range is 4 in sodium acetate buffer dissolved in pH 4'5. 7 mM of p-nitrophenyl-α-D-galactopyranoside 190 ,, mixed and diluted 2 times the purified enzyme solution 10 of the present invention, and reacted for 5 minutes in the range of 20 to 6 (TC). 129208-1000713td〇, •28·1363093 After the reaction, the free p-nitrophenol was quantified to measure the activity, and the relative activity when the large activity was set to 100 was determined. The results are shown in Fig. 3 ° ( 4) In a stable temperature range, a 100 mM sodium acetate buffer having a pH of 4.5 was used, and heat treatment was performed at a temperature of 3 to 55 ° C for 20 minutes, and the residual activity was measured, and the maximum activity was determined in the same manner as above. The relative activity was set to 1 。. The results are shown in Fig. 4. (5) The molecular weight was obtained by using "Ready Gels J" (Japan Bio-Rad Laboratories Co., Ltd.) having a separation gel concentration of 1%. The molecular weight was determined by SDS_polyacrylamide gel electrophoresis, and the results are shown in Fig. 5. The molecular weight of about 80,000 ' is substantially the same as the molecular weight of 83,122 estimated from the amino acid sequence. (7) The specificity of the substrate is 1 〇〇 mM sodium acetate buffer 150 with a pH of 4.5 with various contents of 10 mM In gL, 5 〇KL ' of the purified enzyme solution of the present invention diluted and diluted 1 〇〇 is reacted at 40 ° C for 2 》 minutes, wherein p-nitrobenzene _a_D_ ° is used for galactoside In the reaction, the reaction time is set to 5 minutes. After the reaction, the free p-nitrophenol is quantified or according to a high performance liquid chromatography using a column of "Shodex SUGAR" (Showa Denko Co., Ltd.). In the analysis, the amount of decomposition was quantified, and the relative activity of each substrate when the maximum activity was set to 100 was determined. The results are shown in Table 2. Further, among them, the reaction rate was measured for the subject who formed the subject, and the results are shown in Table 3. 129208-1000713.doc -29- 1363093 [Table 2] Table 2: 受-galactosidase-specific matrix relative activity pNP-aD than galactose bitter 100 ρΝΡ-β-galactopyranoside 0 cottonseed Sugar 10 Honey disaccharide 38 Lactose 0 Sucrose 0 [Table 3] Table 3: Reaction rate constant of α-galactosidase Matrix Km (mM) Vmax (U/mg) ρΝΡ-α-galactopyranoside 0.53 82 Cottonseed Sugar 40 48 melibiose 8.4 106 (8) Inhibitory factor in 6 mM p-nitrophenyl-α-D-galactopyranoside dissolved in sodium acetate buffer at pH 4.5, at the final concentration Various metal ions were added in a manner of 1 mM to obtain 150 pL, and 50 μL of the purified enzyme solution of the present invention diluted by 100 times was mixed and reacted at 40 ° C for 20 minutes. After the reaction, the free p-nitrophenol was quantified to measure the activity, and the relative activity at which the maximum activity was set to 100 was determined. The results are shown in Table 4. [Table 4] Table 4: Inhibition of α-galactosidase by metal salt Relative activity of metal ion 100 Potassium 97 Calcium 97 129208-1000713.doc -30- Magnesium 95 Chromium 99 Manganese 95 Iron (Π) 98 Iron (III) 99 Cobalt 95 Nickel 88 Copper 33 Zinc 80 1363093 Example 3 The purified α-galactosidase 0.5 U described in Example 1 was separately taken and added to 200 g of sugar in a 1.5 mL-capacity polypropylene test tube. The solution (containing 73% sucrose and 12% galactose in 100 mM sodium acetate buffer at pH 5.0) was mixed and reacted at 60 ° C '1,200 rpm. After 45 hours from the start of the reaction, 20 μm was taken out from the reaction liquid, diluted with 480 g of distilled water, and treated at 99 ° C for 10 minutes to inactivate the enzyme heat. After the heat-inactivated diluted sugar solution was returned to normal temperature, it was analyzed by a high performance liquid chromatography using a "Hypercarb" (manufactured by Thermoelectron) column. The result is shown in Fig. 6. The raffinose content in the oligosaccharide was 82%, and the raffinose concentration in the reaction solution was 1.67 wt%. Example 4 (1) Preparation of chromosomal DNA Chromosomal DNA of Bacillus coagulans AKC-004 strain was prepared according to a usual method. 60 mL of the culture solution of the B. coagulans AKC-004 strain obtained by the method described in Example 1 was centrifuged to collect the cells. The obtained cells were suspended in Lysis buffer (50 mM Tris-HCl (pH 129208-1000713.doc -31 - 1363093, 8.0), 20 mM EDTA, 50 mM glucose), and washed thoroughly. After centrifugation, the cells were collected, suspended again in Lysis buffer, and lysozyme was added thereto, followed by incubation at 37 ° C for 45 minutes. Then, SDS and RNase were added and cultured at 37 ° C for 45 minutes. Thereafter, proteinase K (Proteinase K) was added, and the vibration was stabilized at 50 ° C for 60 minutes. After the solution obtained here was treated with benzene-gas-like, gas-like, ethanol precipitation was carried out, and the deposited nucleic acid was wound on a glass pipette and recovered. The nucleic acid was washed with 70% ethanol, dried, and dissolved in TE. By this operation, about 1 mg of chromosomal DNA was prepared. (2) Isolation of α-galactosidase gene From the chromosomal DNA prepared in the above (1), a PCR primer for amplifying the α-galactosidase gene fragment was designed and synthesized. The design of the primer is based on the well-known results of the α-galactosidase gene from several microorganisms. Synthetic primers are used to synthesize oligo DNA with the sequence of 5'-GAAGTITACGGITTYAGYYTTGTITACAGYGG-3, (SEQ ID NO: 3). An antisense primer synthesizes an oligo DNA having the sequence 5J of 5'-CCAAACCAICCRTCRTCIARIACRAA-3丨 (SEQ ID NO: 4). In the sequence, I represents inosine, Υ represents C or Τ, and R represents Α or G. Using the PCR primer obtained here, the chromosomal DNA prepared in the above (1) was used as a template, and the α-galactosidase gene fragment was amplified by a PCR method to obtain an α-half containing 380 base pairs. Lactase gene fragment. The base sequence of the gene fragment obtained here was analyzed using a DNA sequencer. In order to obtain a DNA fragment containing the full length of the α-galactosidase gene, reverse PCR was designed based on the base sequence of the α-galactosidase gene fragment obtained from 129208-1000713.doc •32·1363093. Use PCR primers. Synonymous primers were used to synthesize oligo DNA having a sequence of 5'_ TGATCAACAACTGGGAAAGCGACCT-3' (SEQ ID NO: 5), and an antisense primer was used to synthesize an oligo DNA having a sequence of 5ι_GAACTGGTCCTGCTGCACAATTCC-3' (SEQ ID NO: 6). Then, a template for reverse PCR was prepared. The chromosomal DNA prepared in the above (1) was digested with the restriction enzyme Hindlll, and the resulting DNA fragment was itself cyclized using T4 DNA ligase (T4 DNA Ligase) to form a template for reverse PCR. For the template, the DNA fragment containing the α-galactosidase gene was amplified by PCR using the primer for reverse PCR synthesized as described above, and 4,500 bases containing the entire sequence of the α-galactosidase gene were obtained. Base pair PCR product. (3) Analysis of the sequence of the test sequence The base sequence of the α-galactosidase gene was determined from the PCR product obtained in (2) using a DNA sequencer. As a result, a structural gene of 2,190 base pairs of α-galactosidase having a DNA base sequence from the 5' end shown in SEQ ID NO: 1 was decoded. This sequence is a novel gene that has never been discovered. Further, the α-galactosidase produced by the Bacillus coagulans AKC-004 strain derived from the DNA base sequence contains 730 amino acids, and has the amino acid sequence from the Ν end shown in SEQ ID NO: 2 The database revealed that the amino acid sequence was 57% homologous to alpha-galactosidase (AgaN) from Geobacillus stearothermophilus, and alpha-galactosidase from thermophilic spores ( AgaA) has 56% homology and 56% homology with a galactosidase (AgaB) from thermophilic spores, and from cotton 129208-1000713.doc •33-1363093 Lactococcus lactis (Lactococcus) The alpha-galactosidase of raffinolactis has 56% homology, which is clearly encoded by the novel alpha-galactosidase. (4) Expression of α-galactosidase gene Construction and transformation of the plastid vector based on the sequence of the α-galactosidase gene obtained in (3), designed and synthesized to increase the alpha-galactosidase gene PCR primers for the region of the SD sequence, structural gene, and stop codon. The PCR primer was designed based on the base sequence of the PCR product containing 4,500 base pairs obtained in (2), and the synonymous primer was used to synthesize the sequence having the sequence of 5'-TAAGGTAAAGCAGATGTGCCATT-3· (SEQ ID NO: 7). Poly DNA, antisense primers were used to synthesize oligo DNA with the sequence of 5'-TTACTCGTACACCGCCTC-3' (SEQ ID NO: 8). The PCR product containing 4,500 base pairs obtained in (2) was used as a template, and the PCR product was amplified by PCR using a synthetic PCR primer to obtain a PCR product containing 2325 base pairs. The PCR product obtained at the end of the PCR product was smoothed and phosphorylated. The pBluescriptll KS(+) vector which was digested by the restriction enzyme EcoRV and then dephosphorylated, was ligated to the terminal smoothed and phosphorylated PCR product to construct a new plastid vector pBlue/agaA. . A lac promoter capable of efficiently transcribed a gene linked as a foreign gene in Escherichia coli, which can efficiently express/manufacture α-galactosidase, is prepared in the plastid vector, and the obtained plastid vector is prepared by a calcium chloride method. A recombinant microorganism was produced by transforming into a competent cell of Escherichia coli JM109 strain by thermal shock method. (5) Culture of the transformant and expression of α-galactosidase The recombinant Escherichia coli JM109-pBlue/agaA prepared in (4) has an antibacterial ratio of 100 mg/L in 129208-1000713.doc • 34-1363093. Xilin's 30 mL iLB medium was at 37. Shake the flask for 24 hours. After the cultivation, the recombinant Escherichia coli was centrifuged and recovered. After the cells were washed with 100 mM sodium acetate buffer (pH 5 〇), they were again suspended in the same buffer and pulverized by an ultrasonic pulverizer. The galactose activity of the pulverized liquid was measured by the above method and found to be 19.9 U/culture liquid (mL). Example 5 The cardiac galactosidase 1.0 U obtained by culturing the recombinant body described in Example 4 was taken out, and added to a 200 μί sugar liquid (pH of 5.0) in a polypropylene tube filled with a UmL capacity. 〇〇mv [sodium acetate buffer containing 73% sucrose, galactose 12%), and mixed, 6 〇. The conditions of 〇 and Lao 卬 melon are reacted. After 6 hours from the start of the reaction, 20 pL was taken out from the reaction solution, diluted with 480 μL of distilled water, and treated at 99 t: for 1 minute to inactivate the enzyme. After the heat-inactivated diluted sugar solution was returned to the usual dish, the analysis was carried out by using a high performance liquid chromatograph of "Hypercarb" (manufactured by Thermoelectron). The result is shown in Fig. 7. The raffinose content in the raw sugar was 82 〇 / 0, and the raffinose concentration in the reaction liquid was 1 · 20% by weight. Example 6 Bacillus coagulans AKC-004 strain (registration number FERM Ρ-21092 (transferred by the number of FERM-ABP10948) (Taiwan registration number: bcrc 9103 88); depository: patent of the Institute of Industrial Technology, Independent Administrative Corporation Biohosting Center) on Trypose Blood Agar (Tryptose Blood)

Agar Base，TBAB)平板培養基（Difco)上，於55。〇下培養1 129208-1000713.doc ·35· 1363093 天，形成菌落。將其1鉑耳接種至將表！所示之培養基3〇 mL分注於150 mL容量之三角燒瓶而成者上，以55r、15〇 rpm之條件培養2天。Agar Base, TBAB) plate medium (Difco), at 55. Underarm cultivation 1 129208-1000713.doc · 35 · 1363093 days, colonies formed. Inoculate 1 platinum ear to the watch! 3 μ mL of the indicated medium was dispensed into a 150 mL capacity flask, and cultured at 55 rpm and 15 rpm for 2 days.

本培養進行2天後，將1〇 mL量的培養菌體回收至15 容量之試管中。對回收有培養液之15 mL容量之試管，以 10000 rpm進行離心後，除去上清液。繼而，添加1〇() mM 醋酸納緩衝液（pH值為5.0)1 mL，使之再懸浮後，將懸浮 液移至2 mL容量之聚丙烯製試管中。再次對試管進行離 心，除去上清液後，添加300吣之糖液（pH值為5 〇之1〇〇 mM醋酸鈉緩衝液中含有蔗糖62 5%、半乳糖125%)，藉由 旋渦混合器使菌體充分懸浮，而使糖合成反應開始進行。 本糖合成反應之反應溫度為60°c，旋轉速度為12〇〇 rpm。 糖合成反應開始16小時後，回收反應液40 pL，將其與960 μι蒸餾水充分混合，於991下進行10分鐘之酶的熱失活。 使本稀釋糖液恢復至常溫後，藉由使用r Hypercarb」 (Thermoelectron公司製造）管柱之高效液相層析儀進行分 析，將結果示於圖8»生成寡糖中之棉子糖含有率為 80% (反應液中之棉子糖濃度為〇 70重量％)。 實施例7 將凝結芽胞桿菌AKC-003株（寄存編號FERM P-21091 ; 寄存機關：獨立行政法人產業技術综合研究所專利生物寄 存中心）於胰蛋白血瓊脂基礎（Tryptose Blood Agar Base, TBAB)平板培養基（Difc〇)上，於μι下培養1天，而形成 菌落。將其1鉑耳接種至將表1所示之培養基3〇 mL分注於 129208-10007l3.doc •36- 1363093 150 mL容量之三角燒瓶而成者上，以55°c、150 rprn之條 件培養2天。 本培養進行2天後，將1〇 mL量的培養菌體回收至15 mL 容量之試管中。對回收有培養液之15 mL容量之試管以 10000 rpm進行離心後，除去上清液。繼而，添加100 醋酸鈉缓衝液（pH值為5.0)1 mL，使之再懸浮後，將懸浮 液移至2 mL容量之聚丙烯製試管中。再次對試管進行離 心’除去上清液後，添加3〇〇 pL之糖液（PH值為5.0之100 mM醋酸鈉緩衝液中含有蔗糖62 5%、半乳糖12.5%)，藉由 旋渦混合器使菌體充分懸浮’而使糖合成反應開始進行。 本糖合成反應之反應溫度為6〇°C，旋轉速度為1200 rpm。 糖合成反應開始3 8小時後’回收反應液4〇 ，將其與960 KL蒸餾水充分混合，於99°c下進行1〇分鐘之酶的熱失活。 使本稀釋糖液恢復至常溫後，藉由使用「Hypercarb」 (Thermoelectron公司製造）管柱之高效液相層析儀進行分 析’結果為，生成寡糖中之棉子糖含有率為8〇0/〇(反應液中 之棉子糖濃度為0.76重量％)。 實施例8 將凝結芽胞桿菌AKC-005株（寄存編號FERM P-21093 ; 寄存機關·獨立行政法人產業技術综合研究所專利生物寄 存中〜）於胰蛋白血瓊脂基礎（Trypt〇se Bi〇〇d Agar Base， TBAB)平板培養基（Difc〇)上，於55它下培養i天而形成 菌落。將其1鉑耳接種至將表丨所示之培養基3〇 mL分注於 15〇 mL容量之三角燒瓶而成者上，以55t、150 rpm之條 J29208-1000713.doc -37· 1363093 件培養2天。 本培養進行2天後，將1〇 mL量的培養菌體回收至15 mL 谷量之試管中》對回收有培養液之15 mL容量之試管以 loooo rpm進行離心後，除去上清液。繼而，添加1〇〇 mM 醋酸鈉緩衝液（pH值5 ·0) 1 mL，使之再懸浮後，將懸浮液 移至2 mL容量之聚丙烯製試管中。再次對試管進行離心， 除去上清液後’添加300 μΐ^之糖液（pH值為5 〇之1〇〇 mM醋 酸鈉緩衝液中含有蔗糖62.5%、半乳糖12.5%)，藉由旋渦 混合器使菌體充分懸浮，而使糖合成反應開始進行。本糖 合成反應之反應溫度為60°C，旋轉速度為12〇〇 rpm。糠合 成反應開始38小時後，回收反應液4〇 ，將其與96〇 蒸餾水充分混合，於991：下進行1 〇分鐘之酶的熱失活◦使 本稀釋糖液恢復至常溫後，藉由使用「Hypercarb」 (Therm〇electron公司製造）管柱之高效液相層析儀進行分 析’結杲為’生成寡糖中之棉子糖含有率為91 %(反應液中 之棉子糖濃度為0.32重量％)。 實施例9 將凝結芽胞桿菌AKC-006株（寄存編號FERM P-21094 ; 寄存機關：獨立行政法人產業技術綜合研究所專利生物寄 存中心）於膜蛋白血環脂基礎（Trypt〇se Blood Agar Base, TBAB)平板培養基（Difco)上，於5rc下培養1天，而形成 菌落。將其1翻耳接種至將表1所示之培養基30 mL分注於 150 mL容量之三角燒瓶而成者上，以55t：、15〇 rpm之條 件培養2天。 129208-1000713.doc -38 - 1363093 本培養進行2天後，將〗〇 mL量的培養菌體回收至】$ 容量之試管中。對回收有培養液之15 mL容量之試管以 10000 rpm進行離心後，除去上清液。繼而，添加ι〇〇^Μ 醋酸鈉緩衝液（pH值5.0)1 mL，使之再懸浮後，將懸浮液 移至2 mL容量之聚丙烯製試管中。再次對試管進行離心， 除去上清液後，添加3〇〇 pL之糖液（PH值為5.〇之1〇〇 111河醋 酸鈉緩衝液中含有蔗糖62.5%、半乳糖125%)，藉由旋渦 混合器使菌體充分懸浮，而使糖合成反應開始進行。本糖 合成反應之反應溫度為6(TC，旋轉速度為12〇〇 rpm。糖合 成反應開始38小時後，回收反應液4〇 ，將其與96〇 蒸餾水充分混合，於99t下進行1〇分鐘之酶的熱失活。使 本稀釋糖液恢復至常溫後，藉由使用「Hyperearb」 (Thermoelectron公司製造）管柱之高效液相層析儀進行分 析，結果為，生成寡糖中之棉子糖含有率為76%(反應液中 之棉子糖濃度為0·86重量％)。 [產業上之可利用性] 藉由使用本發明，可提供一種可使用廉價原料，而選擇 性製造棉子糖之方法。 【圖式簡單說明】 圖1係表示自凝結芽胞桿菌AKC-004菌體純化之α·半乳 糖酶之最佳pH值範圍（實施例2)。 圖2係表示自凝結芽胞桿菌AKC-004菌體純化之α-半乳 糖酶之穩定pH值範圍（實施例2)。 圖3係表示自凝結芽胞桿菌AKC-004菌體純化之α-半乳 129208-1000713.doc -39· 1363093 糖酶之最佳溫度範圍（實施例2)。 圖4係表示自凝結芽胞桿菌AKC-004菌體純化之α-半乳 糖酶之穩定溫度範圍（實施例2)。 圖5係表示自凝結芽胞桿菌AKC-004菌體純化之α-半乳 糖酶之SDS-PAGE(實施例2)之結果。 圖6係表示對使用自凝結芽胞桿菌AKC-004株純化之α-半乳糖酶的糖合成（實施例3)之反應液進行HPLC分析的結 果。 圖7係表示對使用自重組大腸桿菌JM1 09株獲得之α-半乳 糖酶的糖合成（實施例5)之反應液進行HPLC分析的結果。 圖8係表示對使用培養凝結芽胞桿菌AKC-004株而獲得 之微生物觸媒的糖合成（實施例3)之反應液進行HPLC分析 的結果。 129208-1000713.doc -40- 1363093Two days after the culture was carried out, the cultured cells in an amount of 1 mL were recovered into a 15-capacity test tube. The supernatant was removed by centrifugation at 10,000 rpm in a test tube having a volume of 15 mL of the culture solution. Then, 1 mL of 1 mM sodium acetate buffer (pH 5.0) was added, and after resuspending, the suspension was transferred to a 2 mL polypropylene tube. The tube was centrifuged again, and after removing the supernatant, 300 ounces of sugar solution (pH 51 〇〇 mM sodium acetate buffer containing 62% sucrose and galactose 125%) was added by vortex mixing. The device allows the cells to be fully suspended, and the sugar synthesis reaction begins. The reaction temperature of the present sugar synthesis reaction was 60 ° C, and the rotation speed was 12 rpm. Sixteen hours after the start of the sugar synthesis reaction, 40 pL of the reaction solution was recovered, and it was thoroughly mixed with 960 μm of distilled water, and the enzyme was thermally deactivated at 991 for 10 minutes. After the diluted sugar solution was returned to normal temperature, it was analyzed by a high performance liquid chromatograph using a column of Hypercarb (manufactured by Thermo Electron Co., Ltd.), and the results are shown in Fig. 8»The raffinose content in the oligosaccharide produced. It is 80% (the raffinose concentration in the reaction liquid is 〇70% by weight). Example 7 Bacillus coagulans AKC-003 (Accession No. FERM P-21091; Depository: Independent Patented Industrial Technology Research Institute Patent Biological Deposit Center) on Tryptose Blood Agar Base (TBAB) plate The culture medium (Difc〇) was cultured for 1 day under μιη to form colonies. 1 platinum ear was inoculated to a flask containing 3 mL of the medium shown in Table 1 and placed in a flask of 129208-10007 l3.doc • 36-1363093 150 mL, and cultured at 55 ° C, 150 rprn. 2 days. Two days after the culture was carried out, the cultured cells in an amount of 1 mL were recovered into a 15 mL-capacity test tube. The supernatant was removed by centrifugation at 10,000 rpm on a 15 mL tube in which the culture solution was recovered. Then, 1 mL of 100 sodium acetate buffer (pH 5.0) was added, and after resuspending, the suspension was transferred to a 2 mL polypropylene test tube. Centrifuge the tube again. After removing the supernatant, add 3 〇〇pL of sugar solution (pH ≥ 100 mM sodium acetate buffer containing 62 5% sucrose, galactose 12.5%) by vortex mixer The bacteria are sufficiently suspended to start the sugar synthesis reaction. The reaction temperature of the present sugar synthesis reaction was 6 ° C and the rotation speed was 1200 rpm. After the start of the sugar synthesis reaction, the reaction solution was recovered for 4 hours, and the mixture was thoroughly mixed with 960 KL of distilled water, and heat-deactivated of the enzyme was carried out at 99 ° C for 1 minute. After the diluted sugar liquid was returned to normal temperature, it was analyzed by a high performance liquid chromatograph using a "Hypercarb" (manufactured by Thermoelectron) column, and the result was that the raffinose content in the oligosaccharide was 8 〇 0. /〇 (The raffinose concentration in the reaction liquid was 0.76% by weight). Example 8 The Bacillus coagulans AKC-005 strain (registered number FERM P-21093; the depository organization, the independent administrative corporation, the Industrial Technology Research Institute, the patent bio-hosting ~) on the tryptone blood agar base (Trypt〇se Bi〇〇d) Agar Base, TBAB) Plate medium (Difc(R)) was cultured at 55 for i days to form colonies. 1 platinum ear was inoculated to a flask containing 3 mL of the medium shown in Table 于 in a 15 mL mL flask, and cultured at 55t, 150 rpm, J29208-1000713.doc -37·1363093 2 days. Two days after the culture was carried out, the cultured cells in an amount of 1 mL were recovered into a 15 mL test tube. The supernatant was removed by centrifugation at a loooo rpm on a 15 mL test tube in which the culture solution was recovered. Then, 1 mL of 1 mM sodium acetate buffer (pH 5 · 0) was added, and after resuspending, the suspension was transferred to a 2 mL-capacity polypropylene test tube. The tube was centrifuged again, and after removing the supernatant, '300 μΐ of the sugar solution was added (pH 值为1〇〇 mM sodium acetate buffer containing 62.5% sucrose, galactose 12.5%), mixed by vortex The device allows the cells to be fully suspended, and the sugar synthesis reaction begins. The reaction temperature of the present sugar synthesis reaction was 60 ° C and the rotation speed was 12 rpm. 38 hours after the start of the hydrazine synthesis reaction, the reaction solution was recovered for 4 Torr, and it was thoroughly mixed with 96 Torr of distilled water, and subjected to heat inactivation of the enzyme for 1 minute at 991:, after the diluted sugar solution was returned to normal temperature, by The high-performance liquid chromatograph of "Hypercarb" (manufactured by Therm〇electron) was used to analyze the content of raffinose in the sucrose-forming oligosaccharide was 91% (the raffinose concentration in the reaction solution was 0.32% by weight). Example 9 The Bacillus coagulans AKC-006 strain (registered number: FERM P-21094; the depository: the patented bio-registration center of the Institute of Industrial Technology and Technology, the independent administrative agency) is based on the Trypt〇se Blood Agar Base. TBAB) Plate medium (Difco) was cultured for 1 day at 5 rc to form colonies. One of them was inoculated to a flask containing 30 mL of the medium shown in Table 1 and placed in a 150 mL capacity flask, and cultured at 55 t:, 15 rpm for 2 days. 129208-1000713.doc -38 - 1363093 After the culture was carried out for 2 days, the cultured cells in the amount of 〇 〇 mL were recovered into a test tube of the capacity. The supernatant was removed by centrifugation at 10,000 rpm on a 15 mL tube in which the culture solution was recovered. Then, 1 mL of ι〇〇^Μ sodium acetate buffer (pH 5.0) was added, and after resuspending, the suspension was transferred to a 2 mL-capacity polypropylene test tube. The tube was centrifuged again, and after removing the supernatant, 3 〇〇pL of sugar solution was added (pH value of 5. 〇1〇〇111 River sodium acetate buffer containing 62.5% sucrose, galactose 125%), borrowed The cells are sufficiently suspended by a vortex mixer to start the sugar synthesis reaction. The reaction temperature of the present sugar synthesis reaction was 6 (TC, the rotation speed was 12 rpm. After 38 hours from the start of the sugar synthesis reaction, the reaction liquid was recovered for 4 Torr, and it was thoroughly mixed with 96 Torr of distilled water, and subjected to 1 Torr at 99 Torr. Thermal inactivation of the enzyme. After the diluted sugar solution was returned to normal temperature, it was analyzed by a high performance liquid chromatography using a column of "Hyperearb" (manufactured by Thermoelectron Co., Ltd.), and as a result, cottonseed in the oligosaccharide was produced. The sugar content is 76% (the raffinose concentration in the reaction liquid is 0. 86% by weight). [Industrial Applicability] By using the present invention, it is possible to provide a cotton which can be selectively produced using inexpensive raw materials. The method of the sugar is shown in Fig. 1 is the optimum pH range of α-galactosidase purified from Bacillus coagulans AKC-004 (Example 2). Fig. 2 shows the self-coagulated spores. Stable pH range of α-galactosidase purified from Bacillus AKC-004 (Example 2). Figure 3 shows α-half 129208-1000713.doc -39 purified from Bacillus coagulans AKC-004 · 1363093 The optimal temperature range for carbohydrase (Example 2). Figure 4 The stable temperature range of α-galactosidase purified from Bacillus coagulans AKC-004 (Example 2). Figure 5 shows the SDS-α-galactosidase purified from Bacillus coagulans AKC-004. The results of PAGE (Example 2) Fig. 6 shows the results of HPLC analysis of the reaction solution of the sugar synthesis (Example 3) using α-galactosidase purified from Bacillus coagulans AKC-004 strain. The results of HPLC analysis of the reaction solution of the sugar synthesis (Example 5) using α-galactosylase obtained from the recombinant Escherichia coli JM1 09 strain are shown. Fig. 8 shows the results obtained by using the cultured Bacillus coagulans AKC-004 strain. The result of HPLC analysis of the reaction solution of the sugar synthesis of the microbial catalyst (Example 3). 129208-1000713.doc -40- 1363093

序列表 <110>日商旭化成化學股份有限公司 <120>新穎α-半乳糖酶 <130> F108020-WO-00 <140 097105759 <141> 2008-02-19 <150> 特願2007-037504 ;特願2007-037505 <151> 2007-02-19 ； 2007-02-19 <160〉 8 <170〉Patentln 3.1 版 <210> 1 <211〉 2193 <212〉 DNA <213〉凝結芽胞桿菌 <220〉 <221〉 CDS <222〉（1)..(2190) <223〉 <400> 1 atg att aca ttt gat gaa caa acg aaa act ttc cat ttg caa aat gat Met lie Thr Phe Asp Glu Gin Thr Lys Thr Phe His Leu Gin Asn Asp 15 i〇 15 gaa gtg agt tac etc tta caa gtg gat gca ttc ggc tgc gtt gaa cat Glu Val Ser Tyr Leu Leu Gin Val Asp Ala Phe Gly Cys Val Glu His 20 25 30 ctg tat tgg gga geg ccg gtc agg gcc tac cac ggg ggg ege gcc tat Leu Tyr Trp Gly Ala Pro Val Arg Ala Tyr His Gly Gly Arg Ala Tyr 35 40 45 ccc ege ate gcc ege agt ttt tee ccg aac ccg ccc ggt gca aaa gac Pro Arg lie Ala Arg Ser Phe Ser Pro Asn Pro Pro Gly Ala Lys Asp 50 55 60 ege aaa ttt tea ctg gat acg gtg ctg cag gaa ttc ccg gga tac ggc Arg Lys Phe Ser Leu Asp Thr Val Leu Gin Glu Phe Pro Gly Tyr Gly 65 70 75 80 48 96 144 192 129208.doc 240 2881363093Sequence Listing <110> Nissho Asahi Kasei Chemical Co., Ltd. <120> Novel α-galactosidase <130> F108020-WO-00 <140 097105759 <141> 2008-02-19 <150> I wish 2007-037504; I wish 2007-037505 <151>2007-02-19; 2007-02-19 <160〉 8 <170>Patentln 3.1 <210> 1 <211> 2193 <212 〉DNA <213>Bacillus coagulans<220> <221> CDS <222>(1)..(2190) <223><400> 1 atg att aca ttt gat gaa caa acg aaa act ttc Cat ttg caa aat gat Met lie Thr Phe Asp Glu Gin Thr Lys Thr Phe His Leu Gin Asn Asp 15 i〇15 gaa gtg agt tac etc tta caa gtg gat gca ttc ggc tgc gtt gaa cat Glu Val Ser Tyr Leu Leu Gin Val Asp Ala Phe Gly Cys Val Glu His 20 25 30 ctg tat tgg gga geg ccg gtc agg gcc tac cac ggg ggg ege gcc tat Leu Tyr Trp Gly Ala Pro Val Arg Ala Tyr His Gly Gly Arg Ala Tyr 35 40 45 ccc ege ate gcc ege Agt ttt tee ccg aac ccg ccc ggt gca aaa gac Pro Arg lie Ala Arg Ser Phe Ser Pro Asn Pro Pro Gly Ala Lys Asp 50 55 60 ege aaa ttt t Ea ctg gat acg gtg ctg cag gaa ttc ccg gga tac ggc Arg Lys Phe Ser Leu Asp Thr Val Leu Gin Glu Phe Pro Gly Tyr Gly 65 70 75 80 48 96 144 192 129208.doc 240 2881363093

aac ggc gat ttc cgt gag ccg gcg cat gtt ate egg cat geg gac ggg Asn Gly Asp Phe Arg Glu Pro Ala His Val lie Arg His Ala Asp Gly 85 90 95 tea aeg gtc aeg gac ttc ege tac egg gcg tat gac att tta aaa ggc Ser Thr Val Thr Asp Phe Arg Tyr Arg Ala Tyr Asp lie Leu Lys Gly 100 105 110 aag ccg gcg ett ccc ggg ett ccc get act ttt gee aat cca aat gaa Lys Pro Ala Leu Pro Gly Leu Pro Ala Thr Phe Ala Asn Pro Asn Glu 115 120 125 gee gaa aca ttg aaa ate atg ctg gaa gac aaa etc aca ggc ett caa Ala Glu Thr Leu Lys lie Met Leu Glu Asp Lys Leu Thr Gly Leu Gin 130 135 140 tta aeg ett tta tac acc ate ttt ege gac ttg ccg gcg att tee ege Leu Thr Leu Leu Tyr Thr He Phe Arg Asp Leu Pro Ala lie Ser Arg 145 150 155 160 gcg get gtg ctg aca aac ggc gga aac aag cct gtt egg att gaa cgt Ala Ala Val Leu Thr Asn Gly Gly Asn Lys Pro Val Arg lie Glu Arg 165 170 175 ttg atg age ctg aat gtc gat ttc ccg gee gga cgt ttt gag ctg ttg Leu Met Ser Leu Asn Val Asp Phe Pro Ala Gly Arg Phe Glu Leu Leu 180 185 190 cat etc ccc ggc gee cat aaa ege gaa egg caa ate aaa aga gaa aeg His Leu Pro Gly Ala His Lys Arg Glu Arg Gin He Lys Arg Glu Thr 195 200 205 gta aeg gac ggg ate agg cgt gta gac age aaa ege ggc gee age age Val Thr Asp Gly lie Arg Arg Val Asp Ser Lys Arg Gly Ala Ser Ser 210 215 220 cac cag gaa aat cct ttt ttg gcg ett gtc egg cct gaa aca aeg gaa His Gin Glu Asn Pro Phe Leu Ala Leu Val Arg Pro Glu Thr Thr Glu 225 230 235 240 336 384 432 480 528 576 624 672 (S ) 129208.doc -2- 720 7681363093Aac ggc gat ttc cgt gag ccg gcg cat gtt ate egg cat geg gac ggg Asn Gly Asp Phe Arg Glu Pro Ala His Val lie Arg His Ala Asp Gly 85 90 95 tea aeg gtc aeg gac ttc ege tac egg gcg tat gac att tta aaa Ggc Ser Thr Val Thr Asp Phe Arg Tyr Arg Ala Tyr Asp lie Leu Lys Gly 100 105 110 aag ccg gcg ett ccc ggg ett ccc get act ttt gee aat cca aat gaa Lys Pro Ala Leu Pro Gly Leu Pro Ala Thr Phe Ala Asn Pro Asn Glu 115 120 125 gee gaa aca ttg aaa ate atg ctg gaa gac aaa etc aca ggc ett caa Ala Glu Thr Leu Lys lie Met Leu Glu Asp Lys Leu Thr Gly Leu Gin 130 135 140 tta aeg ett tta tac acc ate ttt ege gac Ttg ccg gcg att tee ege Leu Thr Leu Leu Tyr Thr He Phe Arg Asp Leu Pro Ala lie Ser Arg 145 150 155 160 gcg get gtg ctg aca aac ggc gga aac aag cct gtt egg att gaa cgt Ala Ala Val Leu Thr Asn Gly Gly Asn Lys Pro Val Arg lie Glu Arg 165 170 175 ttg atg age ctg aat gtc gat ttc ccg gee gga cgt ttt gag ctg ttg Leu Met Ser Leu Asn Val Asp Phe Pro Ala Gly Arg Phe Glu Leu Leu 180 185 190 cat etc ccc ggc Gee Cat aaa ege gaa egg caa ate aaa aga gaa aeg His Leu Pro Gly Ala His Lys Arg Glu Arg Gin He Lys Arg Glu Thr 195 200 205 gta aeg gac ggg ate agg cgt gta gac age aaa ege ggc gee age age Val Thr Asp Gly Lie Arg Arg Val Asp Ser Lys Arg Gly Ala Ser Ser 210 215 220 cac cag gaa aat cct ttt ttg gcg ett gtc egg cct gaa aca aeg gaa His Gin Glu Asn Pro Phe Leu Ala Leu Val Arg Pro Glu Thr Thr Glu 225 230 235 240 336 384 432 480 528 576 624 672 (S ) 129208.doc -2- 720 7681363093

ttc caa ggt gaa gca tat gcg gtg aac etc gtt tac age ggc aat ttt Phe Gin Gly Glu Ala Tyr Ala Val Asn Leu Val Tyr Ser Gly Asn Phe 245 250 255 gcc gga att gtg cag cag gac cag ttc ggc cag gtg cgt etc ggg ate Ala Gly lie Val Gin Gin Asp Gin Phe Gly Gin Val Arg Leu Gly lie 260 265 270 ggg ctg aat gat ttc ggg ttc age tgg gcg ttg cag ccg ggt gac act Gly Leu Asn Asp Phe Gly Phe Ser Trp Ala Leu Gin Pro Gly Asp Thr 275 280 285 ttt tat tcc cct gaa gca gtg atg gca tac age aga gac ggg ctg aac Phe Tyr Ser Pro Glu Ala Val Met Ala Tyr Ser Arg Asp Gly Leu Asn 290 295 300 ggg atg teg caa aeg ttt cac aeg etc tac ege ege cat ett ttg ege Gly Met Ser Gin Thr Phe His Thr Leu Tyr Arg Arg His Leu Leu Arg 305 310 315 320 ggc aaa cat aaa gat gcg gaa ege ccg gtg ttg ate aac aac tgg gaa Gly Lys His Lys Asp Ala Glu Arg Pro Val Leu He Asn Asn Trp Glu 325 330 335 gcg acc tat ttc egg ttt cat gat caa aaa ttg ett gaa etc gcc gac Ala Thr Tyr Phe Arg Phe His Asp Gin Lys Leu Leu Glu Leu Ala Asp 340 345 350 gag gcg caa aag etc ggc att gag ctg ttt gtg ctg gat gac ggc tgg Glu Ala Gin Lys Leu Gly He Glu Leu Phe Val Leu Asp Asp Gly Trp 355 360 365 ttc ggc cac egg gac aat gac ege agt tet tta ggg gac tgg tat gaa Phe Gly His Arg Asp Asn Asp Arg Ser Ser Leu Gly Asp Trp Tyr Glu 370 375 380 tac gcc gga aaa ate egg atg ggg ate aaa aat ctg gcg gaa gaa ate Tyr Ala Gly Lys lie Arg Met Gly lie Lys Asn Leu Ala Glu Glu lie 385 390 395 400 816 864 912 960 1008 1056 1104 1152 129208.doc 1200 12481363093Ttc caa ggt gaa gca tat gcg gtg aac etc gtt tac age ggc aat ttt Phe Gin Gly Glu Ala Tyr Ala Val Asn Leu Val Tyr Ser Gly Asn Phe 245 250 255 gcc gga att gtg cag cag gac cag ttc ggc cag gtg cgt etc ggg Ate Ala Gly lie Val Gin Gin Asp Gin Phe Gly Gin Val Arg Leu Gly lie 260 265 270 ggg ctg aat gat ttc ggg ttc age tgg gcg ttg cag ccg ggt gac act Gly Leu Asn Asp Phe Gly Phe Ser Trp Ala Leu Gin Pro Gly Asp Thr 275 280 285 ttt tat tcc cct gaa gca gtg atg gca tac age aga gac ggg ctg aac Phe Tyr Ser Pro Glu Ala Val Met Ala Tyr Ser Arg Asp Gly Leu Asn 290 295 300 ggg atg teg caa aeg ttt cac aeg etc tac Gege ge ge ge ge g g g g g g g g g g g g g g g g g g g g g g g g Pro Val Leu He Asn Asn Trp Glu 325 330 335 gcg acc tat ttc egg ttt cat gat caa aaa ttg ett gaa etc gcc gac Ala Thr Tyr Phe Arg Phe His Asp Gin Lys Leu Leu Glu Leu Ala Asp 340 345 350 gag gcg caa aag e Tc ggc att gag ctg ttt gtg ctg gat gac ggc tgg Glu Ala Gin Lys Leu Gly He Glu Leu Phe Val Leu Asp Asp Gly Trp 355 360 365 ttc ggc cac egg gac aat gac ege agt tet tta ggg gac tgg tat gaa Phe Gly His Arg Asp Asn Asp Arg Ser Ser Leu Gly Asp Trp Tyr Glu 370 375 380 tac gcc gga aaa ate egg atg ggg ate aaa aat ctg gcg gaa ga ate Tyr Ala Gly Lys lie Arg Met Gly lie Lys Asn Leu Ala Glu Glu lie 385 390 395 400 816 864 912 960 1008 1056 1104 1152 129208.doc 1200 12481363093

cat aaa cgc ggc ttg aaa ttc ggg etc tgg ttt gaa ccg gaa atg gtt His Lys Arg Gly Leu Lys Phe Gly Leu Trp Phe Glu Pro Glu Met Val 405 410 415 teg egg gac agt gac ett ttc cgt gaa cat ccg gac tgg gca ttg caa Ser Arg Asp Ser Asp Leu Phe Arg Glu His Pro Asp Trp Ala Leu Gin 420 425 430 att ccg ggc cgc ggg att tcc gaa ggg cgc tcc caa tat gtg etc gat lie Pro Gly Arg Gly lie Ser Glu Gly Arg Ser Gin Tyr Val Leu Asp 435 440 445 ttt age cgc ggg gat gtg cgc gaa aat att ttc egg cag atg aeg gat Phe Ser Arg Gly Asp Val Arg Glu Asn lie Phe Arg Gin Met Thr Asp 450 455 460 att ctg gat cat gtc cat gtc gat tat ate aaa tgg gat atg aac cgc He Leu Asp His Val His Val Asp Tyr lie Lys Trp Asp Met Asn Arg 465 470 475 480 cat atg aca gaa gtc cat tcc gcc ctg ctg gaa ccg gaa aac cag gga His Met Thr Glu Val His Ser Ala Leu Leu Glu Pro Glu Asn Gin Gly 485 490 495 gaa acc gcc cac cgc tat atg etc ggg ctg tat gac ttt tta gaa aag Glu Thr Ala His Arg Tyr Met Leu Gly Leu Tyr Asp Phe Leu Glu Lys 500 505 510 ttg aeg teg cgt tac cct gac att ttg ttt gaa age tgt tcc ggc ggc Leu Thr Ser Arg Tyr Pro Asp lie Leu Phe Glu Ser Cys Ser Gly Gly 515 520 525 ggc ggg cgt ttt gat ccg ggc atg ctg tat tat atg ccg caa aeg tgg Gly Gly Arg Phe Asp Pro Gly Met Leu Tyr Tyr Met Pro Gin Thr Trp 530 535 540 aeg age gat aat acc gat geg gcc gac egg ctg aaa ate cag tac ggc Thr Ser Asp Asn Thr Asp Ala Ala Asp Arg Leu Lys lie Gin Tyr Gly 545 550 555 560 1296 1344 1392 1440 1488 1536 1584 1632 1680 129208.doc -4- 1363093Cat aaa cgc ggc ttg aaa ttc ggg etc tgg ttt gaa ccg gaa atg gtt His Lys Arg Gly Leu Lys Phe Gly Leu Trp Phe Glu Pro Glu Met Val 405 410 415 teg egg gac agt gac ett ttc cgt gaa cat ccg gac tgg gca ttg Caa Ser Arg Asp Ser Asp Leu Phe Arg Glu His Pro Asp Trp Ala Leu Gin 420 425 430 att ccg ggc cgc ggg att tcc gaa ggg cgc tcc caa tat gtg etc gat lie Pro Gly Arg Gly lie Ser Glu Gly Arg Ser Gin Tyr Val Leu Asp 435 440 445 ttt age cgc ggg gat gtg cgc gaa aat att ttc egg cag atg aeg gat Phe Ser Arg Gly Asp Val Arg Glu Asn lie Phe Arg Gin Met Thr Asp 450 455 460 att ctg gat cat gtc cat gtc gat tat ate Aaa tgg gat atg aac cgc He Leu Asp His Val His Val Asp Tyr lie Lys Trp Asp Met Asn Arg 465 470 475 480 cat atg aca gaa gtc cat tcc gcc ctg ctg gaa ccg gaa aac cag gga His Met Thr Glu Val His Ser Ala Leu Leu Glu Pro Glu Asn Gin Gly 485 490 495 gaa acc gcc cac cgc tat atg etc ggg ctg tat gac ttt tta gaa aag Glu Thr Ala His Arg Tyr Met Leu Gly Leu Tyr Asp Phe Leu Glu Lys 500 505 510 ttg aeg teg cgt t Ac cct gac att ttg ttt gaa age tgt tcc ggc ggc Leu Thr Ser Arg Tyr Pro Asp lie Leu Phe Glu Ser Cys Ser Gly Gly 515 520 525 ggc ggg cgt ttt gat ccg ggc atg ctg tat tat atg ccg caa aeg tgg Gly Gly Arg Phe Asp Pro Gly Met Leu Tyr Tyr Met Pro Gin Thr Trp 530 535 540 aeg age gat aat acc gat geg gcc gac egg ctg aaa ate cag tac ggc Thr Ser Asp Asn Thr Asp Ala Ala Asp Arg Leu Lys lie Gin Tyr Gly 545 550 555 560 1296 1344 1392 1440 1488 1536 1584 1632 1680 129208.doc -4- 1363093

aca age etc gee tat ccg ccg gtt acc atg ggc geg cat gtt teg gee Thr Ser Leu Ala Tyr Pro Pro Val Thr Met Gly Ala His Val Ser Ala 565 570 575 gtt ccg aac cac caa acc ggc egg gtt aeg ccg eta teg aeg ege ggt Val Pro Asn His Gin Thr Gly Arg Val Thr Pro Leu Ser Thr Arg Gly 580 585 590 gac gtg gca atg ggc ggc aat ttc ggc tat gag ttg gat ttg aca aaa Asp Val Ala Met Gly Gly Asn Phe Gly Tyr Glu Leu Asp Leu Thr Lys 595 600 605 tgc aeg gaa gat gaa aaa gca gtg ate egg gag cag att gca ttt tac Cys Thr Glu Asp Glu Lys Ala Val lie Arg Glu Gin He Ala Phe Tyr 610 615 620 aaa gca cac cgt caa ttg ttc cag ttt ggc cag ttt tac ege ctg ata Lys Ala His Arg Gin Leu Phe Gin Phe Gly Gin Phe Tyr Arg Leu lie 625 630 635 640 1728 1776 1824 1872 1920Aca age etc gee tat ccg ccg gtt acc atg ggc geg cat gtt teg gee Thr Ser Leu Ala Tyr Pro Pro Val Thr Met Gly Ala His Val Ser Ala 565 570 575 gtt ccg aac cac caa acc ggc egg gtt aeg ccg eta teg aeg ege Ggt Val Pro Asn His Gin Thr Gly Arg Val Thr Pro Leu Ser Thr Arg Gly 580 585 590 gac gtg gca atg ggc ggc aat ttc ggc tat gag ttg gat ttg aca aaa Asp Val Ala Met Gly Gly Asn Phe Gly Tyr Glu Leu Asp Leu Thr Lys 595 600 605 tgc aeg gaa gat gaa aaa gca gtg ate egg gag cag att gca ttt tac Cys Thr Glu Asp Glu Lys Ala Val lie Arg Glu Gin He Ala Phe Tyr 610 615 620 aaa gca cac cgt caa ttg ttc cag ttt ggc Cag ttt tac ege ctg ata Lys Ala His Arg Gin Leu Phe Gin Phe Gly Gin Phe Tyr Arg Leu lie 625 630 635 640 1728 1776 1824 1872 1920

age ccg ttt gaa ggc aac age gee gee tgg caa ttt gtc teg ccg gac Ser Pro Phe Glu Gly Asn Ser Ala Ala Trp Gin Phe Val Ser Pro Asp 645 650 655 egg aaa cat acc att gee tat ttt ttc aac gta ctg tet gaa gca gee Arg Lys His Thr lie Ala Tyr Phe Phe Asn Val Leu Ser Glu Ala Ala 660 665 670 ggc ccg gtg aaa ata tta aaa ctg gca ggc ett gac cca ggc aaa aac Gly Pro Val Lys He Leu Lys Leu Ala Gly Leu Asp Pro Gly Lys Asn 675 680 685 tac egg cat att gaa aca ggc aac att tac ggc ggc gat gaa ctg atg Tyr Arg His He Glu Thr Gly Asn lie Tyr Gly Gly Asp Glu Leu Met 690 695 700 aac ate ggg ctg tat ttg cca ttg ttt aac aaa cag gac ttt gca age Asn He Gly Leu Tyr Leu Pro Leu Phe Asn Lys Gin Asp Phe Ala Ser 1968 2016 2064 2112 2160 120208.doc 1363093 705 710 715 720 2193 tac aaa gca gag ttt gag gcg gtg tac gag taa Tyr Lys Ala Glu Phe Glu Ala Val Tyr Glu 725 730 <210〉 2 <211〉 730 <212> PRT <213〉凝結芽胞桿菌 <400> 2Age ccg ttt gaa ggc aac age gee gee tgg caa ttt gtc teg ccg gac Ser Pro Phe Glu Gly Asn Ser Ala Ala Trp Gin Phe Val Ser Pro Asp 645 650 655 egg aaa cat acc att gee tat ttt ttc aac gta ctg tet gaa gca Gee Arg Lys His Thr lie Ala Tyr Phe Phe Asn Val Leu Ser Glu Ala Ala 660 665 670 ggc ccg gtg aaa ata tta aaa ctg gca ggc ett gac cca ggc aaa aac Gly Pro Val Lys He Leu Lys Leu Ala Gly Leu Asp Pro Gly Lys Asn 675 680 685 tac egg cat att gaa aca ggc aac att tac ggc ggc gat gaa ctg atg Tyr Arg His He Glu Thr Gly Asn lie Tyr Gly Gly Asp Glu Leu Met 690 695 700 aac ate ggg ctg tat ttg cca ttg ttt aac Aaa cag gac ttt gca age Asn He Gly Leu Tyr Leu Pro Leu Phe Asn Lys Gin Asp Phe Ala Ser 1968 2016 2064 2112 2160 120208.doc 1363093 705 710 715 720 2193 tac aaa gca gag ttt gag gcg gtg tac gag taa Tyr Lys Ala Glu Phe Glu Ala Val Tyr Glu 725 730 <210〉 2 <211> 730 <212> PRT <213> Bacillus coagulans <400> 2

Met lie Thr Phe Asp Glu Gin Thr Lys Thr Phe His Leu Gin Asn Asp 15 10 15Met lie Thr Phe Asp Glu Gin Thr Lys Thr Phe His Leu Gin Asn Asp 15 10 15

Glu Val Ser Tyr Leu Leu Gin Val Asp Ala Phe Gly Cys Val Glu His 20 25 30Glu Val Ser Tyr Leu Leu Gin Val Asp Ala Phe Gly Cys Val Glu His 20 25 30

Leu Tyr Trp Gly Ala Pro Val Arg Ala Tyr His Gly Gly Arg Ala Tyr 35 40 45Leu Tyr Trp Gly Ala Pro Val Arg Ala Tyr His Gly Gly Arg Ala Tyr 35 40 45

Pro Arg He Ala Arg Ser Phe Ser Pro Asn Pro Pro Gly Ala Lys Asp 50 55 60Pro Arg He Ala Arg Ser Phe Ser Pro Asn Pro Pro Gly Ala Lys Asp 50 55 60

Arg Lys Phe Ser Leu Asp Thr Val Leu Gin Glu Phe Pro Gly Tyr Gly 65 70 75 80Arg Lys Phe Ser Leu Asp Thr Val Leu Gin Glu Phe Pro Gly Tyr Gly 65 70 75 80

Asn Gly Asp Phe Arg Glu Pro Ala His Val lie Arg His Ala Asp Gly 85 90 95Asn Gly Asp Phe Arg Glu Pro Ala His Val lie Arg His Ala Asp Gly 85 90 95

Ser Thr Val Thr Asp Phe Arg Tyr Arg Ala Tyr Asp He Leu Lys Gly 100 105 110Ser Thr Val Thr Asp Phe Arg Tyr Arg Ala Tyr Asp He Leu Lys Gly 100 105 110

Lys Pro Ala Leu Pro Gly Leu Pro Ala Thr Phe Ala Asn Pro Asn Glu 115 120 125Lys Pro Ala Leu Pro Gly Leu Pro Ala Thr Phe Ala Asn Pro Asn Glu 115 120 125

Ala Glu Thr Leu Lys lie Met Leu Glu Asp Lys Leu Thr Gly Leu Gin 130 135 140Ala Glu Thr Leu Lys lie Met Leu Glu Asp Lys Leu Thr Gly Leu Gin 130 135 140

Leu Thr Leu Leu Tyr Thr lie Phe Arg Asp Leu Pro Ala lie Ser Arg 145 150 155 160Leu Thr Leu Leu Tyr Thr lie Phe Arg Asp Leu Pro Ala lie Ser Arg 145 150 155 160

Ala Ala Val Leu Thr Asn Gly Gly Asn Lys Pro Val Arg lie Glu Arg 129208.doc • 6 - 1363093 165 170 175Ala Ala Val Leu Thr Asn Gly Gly Asn Lys Pro Val Arg lie Glu Arg 129208.doc • 6 - 1363093 165 170 175

Leu Met Ser Leu Asn Val Asp Phe Pro Ala Gly Arg Phe Glu Leu Leu 180 185 190Leu Met Ser Leu Asn Val Asp Phe Pro Ala Gly Arg Phe Glu Leu Leu 180 185 190

His Leu Pro Gly Ala His Lys Arg Glu Arg Gin He Lys Arg Glu Thr 195 200 205His Leu Pro Gly Ala His Lys Arg Glu Arg Gin He Lys Arg Glu Thr 195 200 205

Val Thr Asp Gly lie Arg Arg Val Asp Ser Lys Arg Gly Ala Ser Ser 210 215 220Val Thr Asp Gly lie Arg Arg Val Asp Ser Lys Arg Gly Ala Ser Ser 210 215 220

His Gin Glu Asn Pro Phe Leu Ala Leu Val Arg Fro Glu Thr Thr Glu 225 230 235 240His Gin Glu Asn Pro Phe Leu Ala Leu Val Arg Fro Glu Thr Thr Glu 225 230 235 240

Phe Gin Gly Glu Ala Tyr Ala Val Asn Leu Val Tyr Ser Gly Asn PhePhe Gin Gly Glu Ala Tyr Ala Val Asn Leu Val Tyr Ser Gly Asn Phe

245 250 255245 250 255

Ala Gly lie Val Gin Gin Asp Gin Phe Gly Gin Val Arg Leu Gly He 260 265 270Ala Gly lie Val Gin Gin Asp Gin Phe Gly Gin Val Arg Leu Gly He 260 265 270

Gly Leu Asn Asp Phe Gly Phe Ser Trp Ala Leu Gin Pro Gly Asp Thr 275 280 285Gly Leu Asn Asp Phe Gly Phe Ser Trp Ala Leu Gin Pro Gly Asp Thr 275 280 285

Phe Tyr Ser Pro Glu Ala Val Met Ala Tyr Ser Arg Asp Gly Leu Asn 290 295 300Phe Tyr Ser Pro Glu Ala Val Met Ala Tyr Ser Arg Asp Gly Leu Asn 290 295 300

Gly Met Ser Gin Thr Phe His Thr Leu Tyr Arg Arg His Leu Leu Arg 305 310 315 320Gly Met Ser Gin Thr Phe His Thr Leu Tyr Arg Arg His Leu Leu Arg 305 310 315 320

Gly Lys His Lys Asp Ala Glu Arg Pro Val Leu lie Asn Asn Trp Glu 325 330 335Gly Lys His Lys Asp Ala Glu Arg Pro Val Leu lie Asn Asn Trp Glu 325 330 335

Ala Thr Tyr Phe Arg Phe His Asp Gin Lys Leu Leu Glu Leu Ala Asp 340 345 350Ala Thr Tyr Phe Arg Phe His Asp Gin Lys Leu Leu Glu Leu Ala Asp 340 345 350

Glu Ala Gin Lys Leu Gly lie Glu Leu Phe Val Leu Asp Asp Gly Trp 355 360 365Glu Ala Gin Lys Leu Gly lie Glu Leu Phe Val Leu Asp Asp Gly Trp 355 360 365

Phe Gly His Arg Asp Asn Asp Arg Ser Ser Leu Gly Asp Trp Tyr Glu 370 375 380Phe Gly His Arg Asp Asn Asp Arg Ser Ser Leu Gly Asp Trp Tyr Glu 370 375 380

Tyr Ala Gly Lys lie Arg Met Gly lie Lys Asn Leu Ala Glu Glu lie 385 390 395 400Tyr Ala Gly Lys lie Arg Met Gly lie Lys Asn Leu Ala Glu Glu lie 385 390 395 400

His Lys Arg Gly Leu Lys Phe Gly Leu Trp Phe Glu Pro Glu Met Val 129208.doc 1363093 405 410 415His Lys Arg Gly Leu Lys Phe Gly Leu Trp Phe Glu Pro Glu Met Val 129208.doc 1363093 405 410 415

Ser Arg Asp Ser Asp Leu Phe Arg Glu His Pro Asp Trp Ala Leu Gin 420 425 430Ser Arg Asp Ser Asp Leu Phe Arg Glu His Pro Asp Trp Ala Leu Gin 420 425 430

He Pro Gly Arg Gly lie Ser Glu Gly Arg Ser Gin Tyr Val Leu Asp 435 440 445He Pro Gly Arg Gly lie Ser Glu Gly Arg Ser Gin Tyr Val Leu Asp 435 440 445

Phe Ser Arg Gly Asp Val Arg Glu Asn He Phe Arg Gin Met Thr Asp 450 455 460Phe Ser Arg Gly Asp Val Arg Glu Asn He Phe Arg Gin Met Thr Asp 450 455 460

He Leu Asp His Val His Val Asp Tyr He Lys Trp Asp Met Asn Arg 465 470 475 480He Leu Asp His Val His Val Asp Tyr He Lys Trp Asp Met Asn Arg 465 470 475 480

His Met Thr Glu Val His Ser Ala Leu Leu Glu Pro Glu Asn Gin Gly 485 490 495His Met Thr Glu Val His Ser Ala Leu Leu Glu Pro Glu Asn Gin Gly 485 490 495

Glu Thr Ala His Arg Tyr Met Leu Gly Leu Tyr Asp Phe Leu Glu Lys 500 505 510Glu Thr Ala His Arg Tyr Met Leu Gly Leu Tyr Asp Phe Leu Glu Lys 500 505 510

Leu Thr Ser Arg Tyr Pro Asp He Leu Phe Glu Ser Cys Ser Gly Gly 515 520 525Leu Thr Ser Arg Tyr Pro Asp He Leu Phe Glu Ser Cys Ser Gly Gly 515 520 525

Gly Gly Arg Phe Asp Pro Gly Met Leu Tyr Tyr Met Pro Gin Thr Trp 530 535 540Gly Gly Arg Phe Asp Pro Gly Met Leu Tyr Tyr Met Pro Gin Thr Trp 530 535 540

Thr Ser Asp Asn Thr Asp Ala Ala Asp Arg Leu Lys lie Gin Tyr Gly 545 550 555 560Thr Ser Asp Asn Thr Asp Ala Ala Asp Arg Leu Lys lie Gin Tyr Gly 545 550 555 560

Thr Ser Leu Ala Tyr Pro Pro Val Thr Met Gly Ala His Val Ser Ala 565 570 575Thr Ser Leu Ala Tyr Pro Pro Val Thr Met Gly Ala His Val Ser Ala 565 570 575

Val Pro Asn His Gin Thr Gly Arg Val Thr Pro Leu Ser Thr Arg Gly 580 585 590Val Pro Asn His Gin Thr Gly Arg Val Thr Pro Leu Ser Thr Arg Gly 580 585 590

Asp Val Ala Met Gly Gly Asn Phe Gly Tyr Glu Leu Asp Leu Thr Lys 595 600 605Asp Val Ala Met Gly Gly Asn Phe Gly Tyr Glu Leu Asp Leu Thr Lys 595 600 605

Cys Thr Glu Asp Glu Lys Ala Val lie Arg Glu Gin lie Ala Phe Tyr 610 615 620Cys Thr Glu Asp Glu Lys Ala Val lie Arg Glu Gin lie Ala Phe Tyr 610 615 620

Lys Ala His Arg Gin Leu Phe Gin Phe Gly Gin Phe Tyr Arg Leu lie 625 630 635 640Lys Ala His Arg Gin Leu Phe Gin Phe Gly Gin Phe Tyr Arg Leu lie 625 630 635 640

Ser Pro Phe Glu Gly Asn Ser Ala Ala Trp Gin Phe Val Ser Pro Asp 129208.doc 1363093 645 650 655Ser Pro Phe Glu Gly Asn Ser Ala Ala Trp Gin Phe Val Ser Pro Asp 129208.doc 1363093 645 650 655

Arg Lys His Thr lie Ala Tyr Phe Phe Asn Val Leu Ser Glu Ala Ala 660 665 670Arg Lys His Thr lie Ala Tyr Phe Phe Asn Val Leu Ser Glu Ala Ala 660 665 670

Gly Pro Val Lys He Leu Lys Leu Ala Gly Leu Asp Pro Gly Lys Asn 675 680 685Gly Pro Val Lys He Leu Lys Leu Ala Gly Leu Asp Pro Gly Lys Asn 675 680 685

Tyr Arg His lie Glu Thr Gly Asn lie Tyr Gly Gly Asp Glu Leu Met 690 695 700Tyr Arg His lie Glu Thr Gly Asn lie Tyr Gly Gly Asp Glu Leu Met 690 695 700

Asn lie Gly Leu Tyr Leu Pro Leu Phe Asn Lys Gin Asp Phe Ala Ser 705 710 715 720Asn lie Gly Leu Tyr Leu Pro Leu Phe Asn Lys Gin Asp Phe Ala Ser 705 710 715 720

Tyr Lys Ala Glu Phe Glu Ala Val Tyr Glu 725 730 <210〉 3 <211> 32 <212> DNA <213〉人工序列 <220〉 <223〉人工序列之表現：合成核酸 <400> 3 gaagtitacg gittyagyyt tgtitacagy gg 32 <210〉 4 <211〉 26 <212〉 DNA <213〉人工序列 <220〉 <223〉人工序列之表現：合成核酸 <400〉 4 ccaaaccaic crtcrtciar iacraa 26Tyr Lys Ala Glu Phe Glu Ala Val Tyr Glu 725 730 <210> 3 <211> 32 <212> DNA <213>Artificial Sequence <220><223> 223>Expression of Artificial Sequence: Synthetic Nucleic Acid<400> 3 gaagtitacg gittyagyyt tgtitacagy gg 32 <210> 4 <211> 26 <212> DNA <213>Artificial sequence <220><223> 223>Expression of artificial sequence: synthetic nucleic acid <400> 4 ccaaaccaic Crtcrtciar iacraa 26

<210〉 5 <211〉 25 <212〉 DNA -9- 129208.doc 251363093<210> 5 <211> 25 <212> DNA -9- 129208.doc 251363093

<213〉人工序列 <220> <223〉人工序列之表現：合成核酸 <400〉 5 tgatcaacaa ctgggaaagc gacct <210> 6 <211> 24 <212> DNA <213〉人工序列 <220> <223〉人工序列之表現：合成核酸 <400〉 6 gaactggtcc tgctgcacaa ttcc <210> 7 <211〉 23 <212> DNA <213〉人工序列 <220> <223〉人工序列之表現：合成核酸 <400> 7 taaggtaaag cagatgtgcc att <210> 8 <211〉 18 <212〉 DNA <213〉人工序列 <220> <223〉人工序列之表現：合成核酸 <400〉 8 ttactcgtac accgcctc 24 23 18 129208.doc -10-<213>Artificial sequence <220><223> Expression of artificial sequence: synthetic nucleic acid <400> 5 tgatcaacaa ctgggaaagc gacct <210> 6 <211> 24 <212> DNA <213><220><223> Expression of artificial sequence: synthetic nucleic acid <400> 6 gaactggtcc tgctgcacaa ttcc <210> 7 <211> 23 <212> DNA <213>Artificial sequence <220> 223>Expression of artificial sequence: synthetic nucleic acid <400> 7 taaggtaaag cagatgtgcc att <210> 8 <211> 18 <212> DNA <213>artificial sequence <220><223> : synthetic nucleic acid <400〉 8 ttactcgtac accgcctc 24 23 18 129208.doc -10-

Claims (1)

1363093 第097105759號專利申請案 中文申請專利範圍替換本(100年9 十、申請專利範圍： 1. 一種α-半乳糖酶’其包含下述（a)、（b)或（c)中之任一個 胺基酸序列： (a) 序列編號2所表示之胺基酸序列； (b) 序列編號2所表示之胺基酸序列中，丨〜⑼個胺基酸 經缺失、置換及/或附加，且具有^半乳糖酶活性及下述 特性的胺基酸序列： (1) 作用：於以蔗糖與半乳糖為原料之脫水縮合反 應中’棉子糖於反應溶液中累積至〇 5〇/〇以上時，該α_ 半乳糖酶具有生成寡糖中之棉子糖含有率為65%以上 的性質； (2) 最佳pH值範圍：3.5〜5.0 ; (3) 穩定pH值範圍：3.5〜1〇.〇 ; (4) 分子量：約80,000 ; (c) 與序列編號2所表示之胺基酸序列具有8〇%以上之 同源性，且具有α-半乳糖酶活性及下述特性的胺基酸序 列： (1) 作用.於以蔗糖與半乳糖為原料之脫水縮合反 應中，棉子糖於反應溶液中累積至〇 5%以上時，該α_ 半乳糖酶具有生成寡糖中之棉子糖含有率為65%以上 的性質； (2) 最佳pH值範圍：3.5〜5.〇 ; (3) 穩定pH值範圍：3.5〜1〇.〇; (4) 分子量··約80,000。 129208-1000930.doc 2. 如請求項1之α-半乳糖酶，其係來自屬於凝結芽胞桿菌 (Bacillus coagu!ans)之微 ± 物 〇 3. 如請求項2之α-半乳糖酶，其中凝結芽胞桿菌為凝結芽胞桿 菌 AKC004 株（FERM-ABP10948)(台灣寄存編號：BCRC 910388)。 4. 一種凝結芽胞桿菌，其係屬於凝結芽胞桿菌akc〇04株 (FERM-ABP10948)(台灣寄存編號：BCRC 910388)。 5. 一種半乳糖酶基因’其對包含下述（a)、（b)或（c)中之 任一個胺基酸序列之α_半乳糖酶進行編碼： (a) 序列編號2所表示之胺基酸序列； (b) 序列編號2所表示之胺基酸序列中，i〜2〇個胺基酸 經缺失、置換及/或附加，且具有〇_半乳糖酶活性及下述 特性的胺基酸序列： (1) 作用：於以蔗糖與半乳糖為原料之脫水縮合反 應中’棉子糖於反應溶液中累積至〇 .5〇/〇以上時，該α_ 半乳糖酶具有生成募糖中之棉子糖含有率為65%以上 的性質； (2) 最佳pH值範圍：3.5〜5.〇 ; (3) 穩定pH值範圍：3.5〜10.〇 ; (4) 分子量：約80,〇〇〇 ; (c) 與序列編號2所表示之胺基酸序列具有8〇()/。以上之 同源性’且具有α-半乳糖酶活性及下述特性的胺基酸序 列： (1)作用：於以蔗糖與半乳糖為原料之脫水縮合反 129208-1000930.doc 應中’棉子糖於反應溶液中累積至0.5%以上時，該α- 半乳糖酶具有生成寡糖中之棉子糖含有率為65%以上 的性質； (2) 最佳pH值範圍：3.5〜5.0 ; (3) 穩定pH值範圍：3.5〜1〇.〇; (4) 分子量：約8〇,〇〇〇。 6· 一種半乳糖酶基因，其包含以下（3)或（1))之驗基序列： (a) 序列編號1所表示之鹼基序列； (b) 序列編號！所表示之鹼基序列中，卜6〇個鹼基經缺 失、置換及/或附加，且編碼具有α_半乳糖酶活性及下述 特性之蛋白質的鹼基序列： 〇)作用：於以蔬糖與半乳糖為原料之脫水縮合反 應中’棉子糖於反應溶液中累積至〇 5%以上時，該α_ 半乳糖酶具有生成寡糖中之棉子糖含有率為65%以上 的性質； (2) 最佳pH值範圍：3.5〜5.0 ; (3) 穩定pH值範圍：3.5〜1〇.〇; (4) 分子量：約80,000。 或6之α_半乳糖酶基 —種重組載體，其含有如請求項5 因。 —種轉形體，其導入有如請求項5弋& 項5或6之α-半乳糖酶基因 或如請求項7之重組載體。 一種α-半乳糖酶，其係培養如諳龙担。 芽承項8之轉形體而獲得。 一種酶組合物，其含有如請求項丨至3、 、或9中任一項之α· 1292〇8-l〇〇〇93〇.doc 1363093 半乳糖酶》 u·如請求項10之酶組合物’其進而含有選自α—葡萄糖苷 酶、β-葡萄糖苷酶、β-半乳糖酶、纖維素酶、木聚糖 酶、蛋白酶、半乳聚糖酶、***聚糖酶、甘露聚糖 酶、鼠李半乳糖醛酸酶 '聚半乳糖醛酸酶、果膠甲酯 酶、果膠解離酶、及聚半乳糖醛酸解離酶中之至少一種 以上之成分。 12. —種棉子糖合成試劑，其含有如請求項⑺或丨丨中任一項 之§§組合物。 一種棉子糖之製造方法’其特徵在於：其係使用如請求 項1至3、或9中任一項之α-半乳糖酶、如請求項1〇或^之 酶組合物、或者如請求項12之棉子糖合成試劑。 14. 一種棉子糖之製造方法，其特徵在於：其係利用培養屬 於凝結芽胞桿菌之微生物而獲得之微生物觸媒。 15. —種棉子糖之製造方法，其特徵在於：其係利用培養屬 於凝結芽胞桿菌AKC004株（FERM-ABP10948)(台灣寄存 編旒.BCRC 910388)的凝結芽胞桿菌而獲得之微生物觸 媒》 16. —種棉子糖之製造方法，其特徵在於：其係利用培養如 請求項8之轉形體而獲得之微生物觸媒。 17. 如請求項13至16中任一項之棉子糖之製造方法，其中生 成寡糖中之棉子糖含有率為65〇/〇以上。 18. 如請求項13至16中任一項之棉子糠之製造方法其係使 用蔗糖及半乳糖作為原料。 19. 如請求項17之棉子糖之製造方法，其係使用蔗糖及半乳 129208-1000930.doc 1363093 糖作為原料。 20. 如請求項18之棉子糖之製造方法，其中原料中之蔗糖濃 度為30% (w/v)〜90% (w/v)，原料中之半乳糖濃度為2% (w/v)〜45% (w/v)。 21. 如請求項19之棉子糖之製造方法，其中原料中之蔗糖濃 度為30% (w/v)〜90% (w/v)，原料中之半乳糖濃度為2% (w/v)〜45% (w/v)。1363093 Patent Application No. 097105759, the Chinese patent application scope replacement (100 years, 90, patent application scope: 1. an α-galactosidase) which comprises any of the following (a), (b) or (c) An amino acid sequence: (a) an amino acid sequence represented by SEQ ID NO: 2; (b) in the amino acid sequence represented by SEQ ID NO: 2, 丨~(9) amino acids are deleted, substituted and/or attached The amino acid sequence having galactosidase activity and the following characteristics: (1) Action: In the dehydration condensation reaction using sucrose and galactose as raw materials, 'raffinose is accumulated in the reaction solution to 〇5〇/ When 〇 or more, the α-galactosidase has a property that the raffinose content in the oligosaccharide is 65% or more; (2) the optimum pH range: 3.5 to 5.0; (3) the stable pH range: 3.5~ (4) Molecular weight: about 80,000; (c) having more than 8% homology with the amino acid sequence represented by SEQ ID NO: 2, and having α-galactosidase activity and the following characteristics Amino acid sequence: (1) Function. In the dehydration condensation reaction of sucrose and galactose as raw materials, raffinose When accumulating to 5% or more in the solution, the α-galactosidase has a property that the content of raffinose in the oligosaccharide is 65% or more; (2) The optimum pH range: 3.5 to 5. 〇; 3) Stable pH range: 3.5~1〇.〇; (4) Molecular weight··about 80,000. 129208-1000930.doc 2. The α-galactosidase of claim 1 is derived from Bacillus coagulans (Bacillus) Coagu!ans) Micro ± substance 3. The α-galactosidase of claim 2, wherein Bacillus coagulans is a strain of Bacillus coagulans AKC004 (FERM-ABP10948) (Taiwan registration number: BCRC 910388). 4. A coagulation Bacillus, which belongs to the bacterium Bacillus coagulidae akk〇04 (FERM-ABP10948) (Taiwan accession number: BCRC 910388). 5. A galactosidase gene whose pair comprises the following (a), (b) or (c) The α-galactosidase of any one of the amino acid sequences is encoded by: (a) the amino acid sequence represented by SEQ ID NO: 2; (b) the amino acid sequence represented by SEQ ID NO: 2, i~2 An amino acid which is deficient, substituted and/or attached, and which has 〇-galactosidase activity and the following characteristics Column: (1) Action: In the dehydration condensation reaction using sucrose and galactose as raw materials, when the raffinose is accumulated in the reaction solution to above 〇.5〇/〇, the α-galactosidase has the effect of generating sugar. The raffinose content is 65% or more; (2) The optimum pH range: 3.5~5. 〇; (3) The stable pH range: 3.5~10. 〇; (4) Molecular weight: about 80, 〇 (c) has 8 〇 () / with the amino acid sequence represented by SEQ ID NO: 2. The amino acid sequence having the above homology' and having α-galactosidase activity and the following characteristics: (1) Action: Dehydration condensation with sucrose and galactose as raw material anti-129208-1000930.doc When the daughter sugar is accumulated to 0.5% or more in the reaction solution, the α-galactosidase has a property that the content of raffinose in the oligosaccharide is 65% or more; (2) the optimum pH range: 3.5 to 5.0; (3) Stable pH range: 3.5~1〇.〇; (4) Molecular weight: about 8〇, 〇〇〇. 6. A galactosidase gene comprising the following (3) or (1)) sequence: (a) the base sequence represented by SEQ ID NO: 1; (b) SEQ ID NO: In the indicated base sequence, 6 bases are deleted, substituted and/or affixed, and encode a base sequence of a protein having α-galactosidase activity and the following characteristics: 〇) Effect: When the raffinose is accumulated in the reaction solution to 5% or more in the dehydration condensation reaction of sugar and galactose as a raw material, the α-galactosidase has a property that the raffinose content in the oligosaccharide is 65% or more; (2) Optimum pH range: 3.5~5.0; (3) Stable pH range: 3.5~1〇.〇; (4) Molecular weight: about 80,000. Or an alpha-galactosyl-recombinant vector of 6 which contains the cause according to claim 5. A transformant introduced with the α-galactosidase gene of claim 5 弋 & item 5 or 6, or the recombinant vector of claim 7. An α-galactosidase which is cultured, such as a dragon. The bud is obtained by the transformation of the item 8. An enzyme composition comprising α·1292〇8-l〇〇〇93〇.doc 1363093 galactosidase as claimed in any one of claims 3 to 3, or 9; a substance which further comprises a selected from the group consisting of α-glucosidase, β-glucosidase, β-galactosidase, cellulase, xylanase, protease, galactanase, arabinase, mannan At least one or more of an enzyme, a rhamnogalacturonasase polygalacturonase, a pectin methylesterase, a pectin dissociation enzyme, and a polygalacturonan dissociating enzyme. A raffinose synthesizing agent comprising the §§ composition according to any one of the claims (7) or hydrazine. A method for producing raffinose, characterized in that it is an α-galactosase according to any one of claims 1 to 3, or 9, an enzyme composition as claimed in claim 1 or ^, or as requested Item 12 of raffinose synthesis reagent. A method for producing raffinose, which is characterized in that it is a microbial catalyst obtained by culturing a microorganism belonging to Bacillus coagulans. A method for producing a raffinose, which is characterized in that the microbial catalyst obtained by culturing a Bacillus coagulans belonging to the bacterium Bacillus coagulans AKC004 (FERM-ABP10948) (Taiwan Depository Co., BCRC 910388) A method for producing a raffinose, which is characterized in that it is a microbial catalyst obtained by cultivating a transformant as claimed in claim 8. 17. The method for producing raffinose according to any one of claims 13 to 16, wherein the raffinose content in the oligosaccharide is 65 〇/〇 or more. 18. The method of producing cottonseed meal according to any one of claims 13 to 16, which uses sucrose and galactose as raw materials. 19. The method for producing raffinose according to claim 17, which comprises using sucrose and galacto 129208-1000930.doc 1363093 sugar as a raw material. 20. The method for producing raffinose according to claim 18, wherein the sucrose concentration in the raw material is 30% (w/v) to 90% (w/v), and the galactose concentration in the raw material is 2% (w/v) ) ~45% (w/v). 21. The method for producing raffinose according to claim 19, wherein the sucrose concentration in the raw material is 30% (w/v) to 90% (w/v), and the galactose concentration in the raw material is 2% (w/v) ) ~45% (w/v). 129208-1000930.doc129208-1000930.doc
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