TW521088B - A production method of Α-hydroxy acid by using a microorganism - Google Patents

Abstract

This invention provides a production method of Α-hydroxy acids [II]: RCH(OH)COOH (wherein, R represents hydrogen atom, substituted C1-C6 alkyl, substituted C1-C6 alkenyl, substituted C1-C6 alkoxyl, substituted aryl, substituted aryloxy or substituted heterocyclic groups), which uses a microorganism to hydrolyze Α-hydroxynitrile [I]: RCH(OH)CN (wherein, R is as defined as before), so as to convert said compounds into Α-hydroxy acids [II]. Preferably, it is related to a production method of Α-hydroxy acids [II], which makes Α-hydroxy acid [II] be generated and accumulated in an aqueous solvent in the presence of cyanide by using a microorganism having concentration-resistance against Α-hydroxynitrile [I] and/or Α-hydroxy acid [II] as well as durable activity. More specifically, this invention enables to accumulate Α-hydroxy acid [II] in a high concentration by using a microorganism, which has concentration-resistance against Α-hydroxynitrile [I] and/or Α-hydroxy acid [II] as well as long-time-lasted durable activity, and using said microorganism repeatedly, so as to produce Α-hydroxy acid [II] efficiently. Moreover, addition of cyanide into the reaction system makes the production of Α-hydroxy acid [II] more efficient.

Description

經濟部中央標準局員工消費合作社印製 521088 A7 B7 五、發明説明（1 ) 技術領域： 本發明偽關於利用微生物水解α -羥基腈，製造α -羥 基酸之製法，:，α -羥基酸中乳酸可作食品、鑛造用及工業 用，又2 -羥基-4 _ 丁酸為有用之家畜飼料添加物。 抟術背景： 藉由微生物由α -羥基腈[I]來製造α -羥基酸[II]之 方法已知有例如： (1)由擇舗屬（Bacillus》，無穿孢桿菌靥（Bacterium) ，徹球 ® 靨（Micrococcus)及短桿 @ 屬（Brevibacteriuin) 之撤生物製造乳酸，羥基乙酸等之製法〔特公昭5 8 -15120]， (2 )由棒桿_屬（C 〇 r y n e b a c t e r i ϋ m )之擻生物製造乳酸， 羥基乙酸及2 -羥基異丁酸之製法〔特開昭6卜5 6 0 8 6〕， (3 )由假單胞菌屬（P s e u d 〇 m ο n a s ),關節爭孢桿菌屬 (Arthrobater)。麴菌屬（:Aspergillus)，青徽菌屬 (P e n i c丨Π i u in ),旋孢腔菌屬（C 〇 c h 1丨〇 b 〇卜j s )，鐮孢 菌鼷（F u s a r i U m )之微生物製造乳酸，2 -羥基異丁酸， 2 -羥基-2 -羥基苯基丙酸及扁桃酸之製法〔特開昭5 2 -2 2 2 6 9 6 ]， (4)由關節芽孢捍菌屬，麴菌屬，桿菌屬，無芽孢捍菌 屬，短捍菌屬，施孢腔菌屬，棒捍薗屬，微球菌屬， 土壤絲菌屬（N 〇 c a r d i a >，青黴菌屬，假單胞鐘屬，以 及鐮孢蘭靨之微生物，製造2 -羥基-3,3 -二甲基-4-丁 基内酯之製法〔待開昭6 4 - 1 0 9 9 6〕， - 3 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁)Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 521088 A7 B7 V. Description of the Invention (1) Technical Field: The present invention relates to a method for producing α-hydroxyacid by hydrolyzing α-hydroxynitrile with microorganisms. Lactic acid can be used for food, mining and industrial purposes, and 2-hydroxy-4_butyric acid is a useful animal feed additive. Technological background: The method of producing α-hydroxy acid [II] from α-hydroxynitrile [I] by microorganisms is known, for example: (1) from Bacillus, Bacterium ， Cruciol® 靥 （Micrococcus) and short rod @ Brevibacteriuin production method of manufacturing lactic acid, glycolic acid, etc. [Kokusho 5 8 -15120], (2) by rod _ genus (C 〇rynebacteri ϋ m ) Production of lactic acid, glycolic acid, and 2-hydroxyisobutyric acid by 擞 擞 Bio [Japanese Patent Application Laid-Open No. 6B 5 6 0 8 6], (3) Pseudomonas (Pseud om nas), joints Arthrobater. Arthrobater: Aspergillus, Penicillium (Penic 丨 Π iu in), Spirulina (Cocch 1 丨 〇b 〇bjs), Fusarium A method for the production of lactic acid, 2-hydroxyisobutyric acid, 2-hydroxy-2-hydroxyphenylpropionic acid and mandelic acid by microorganisms of Fusari U m [Japanese Patent Application Laid-Open No. 5 2 -2 2 2 6 9 6], (4) By Arthrobacillus, Aspergillus, Bacillus, Aspergillus, Brevibacterium, Sporocystis, Corynebacterium, Micrococcus, Soil Mycelia (Nocardia >, a method for producing 2-hydroxy-3,3-dimethyl-4-butyllactone by microorganisms of Penicillium, Pseudomonas, and Fusarium spp. [To be opened 6 4-1 0 9 9 6],-3-This paper size applies Chinese National Standard (CNS) A4 (210X297 mm) (Please read the precautions on the back before filling this page)

經濟部中央標準局員工消費合作社印製 521088 Α7 Β7 五、發明説明（2 ) (5 )由红球菌屬（R h 〇 d 〇 c 〇 c c u s ),假單胞_ _，關節芽 孢捍菌靨，以及短捍菌屬之微生物製造2 -羥基異丁酸 之製法〔待開平4 - 4 0 8 9 7〕， (6 )酪鐘屬（C a s e 〇 b a c t r e Γ)，假單胞菌屬，産鹼捍菌屬 (A U a丨U e n e s ),棒桿菌屬，短桿菌屬，土壤絲菌屬， 红球菌屬，以及關節芽孢桿菌鼷之微生物製造α -羥 基-4 -甲硫基丁酸之製法〔待開平4-40898]， (7 )産鹼稈鏡屬，紅球菌屬及哥登屬（G 〇 r d ο n a )之微生 物製造4 -甲硫基丁酸之製法〔WO 96/09403〕， (8 )潘妥靨（P a n t o e a ),微球菌屬及無芽孢桿菌屬之微 生物製造α_羥基-4-甲硫基丁酸之製法〔特開平8-1 7 3 1 7 5 ]等。 但是，此等〇(-羥基酸之製造並未必滿足得以高濃度生 成蓄積物質之目的，，例如，乳酸，可確認之蓄積量為來 自，棒捍菌屬之微生物者只為9 . 8重量％ 〔特開昭6 1 -56086〕，來自假屋胞菌靥之徼生物只為10重量％ 〔待 開昭6 3 - 2 2 2 6 9 6〕，來自關節芽孢桿菌靨之微生物只為 0 · 1 5重量％ 〔特開昭6 3 - 2 2 2 6 9 6〕。又，各別地α -羥基 異丁酸可確認之蓄積量來自假單胞菌屬之微生物只為 0.8重量％ 〔特開昭63-222696〕， cc-羥基-4-甲硫丁酸 可確認之蓄積量來自略_屬之微生物只為1 8 8 m Μ ( 2 . 8重 量％ )〔特開平4 - 4 0 8 9 8〕，來自關節穿捍菌屬之徼生物 只為5 5 m Μ ( 0 . 8重量％ )〔特開平4 - 4 0 8 9 8〕，來自産鹼捍 蘭屬之微生物只為9 4 0 m Μ (1 4重量％ )〔 W 0 9 6 ./ 0 9 4 0 3〕。 -4 - 本紙張尺度適用中國國家標準（CNS ) Α4規格（210Χ 297公釐） (請先閲讀背面之注意事項再填寫本頁)Printed by the Employees' Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 521088 Α7 Β7 V. Description of the invention (2) (5) Rhodococcus (Rh 〇d 〇c 〇ccus), Pseudomonas _ _, Arthrosporium spp., And the production method of 2-hydroxyisobutyric acid by microorganisms of Brevibacterium [To be Kaiping 4-4 0 8 9 7], (6) Casease (bacteria), Pseudomonas, alkali-producing Method for producing α-hydroxy-4 -methylthiobutyric acid by microorganisms of genus AU a 丨 U enes, Corynebacterium, Brevibacterium, Agrobacterium, Rhodococcus, and Arthrobacillus To be Kaiping 4-40898], (7) Production method of 4-methylthiobutyric acid produced by microorganisms of the genus Alkali, Rhodococcus and Gordona [WO 96/09403], ( 8) Pantoea, a method for producing α-hydroxy-4-methylthiobutyric acid by microorganisms of the genus Micrococcus and non-bacillus [Japanese Patent Laid-Open No. 8-1 7 3 1 7 5] and the like. However, the production of these 〇 (-hydroxy acids) does not necessarily meet the purpose of generating accumulation substances at high concentrations. For example, lactic acid can be confirmed to be from 9.8% by weight of microorganisms belonging to the genus Corynebacterium. [Japanese Patent Application Publication No. 6 1 -56086], only 10% by weight of pupae organisms derived from Pseudomonas spp. [To be opened 6 3-2 2 2 6 9 6], microorganisms derived from Arthrobacillus pupae were only 0.1% 5% by weight [Japanese Patent Laid-Open No. 6 3-2 2 2 6 9 6]. Also, the amount of α-hydroxyisobutyric acid that can be confirmed separately is only 0.8% by weight from microorganisms of the genus Pseudomonas [Japanese Patent Laid-Open] Sho 63-222696], cc-hydroxy-4-methylthiobutyric acid can be confirmed that the accumulation amount comes from microorganisms of the genus only 1 8 m Μ (2.8% by weight) [Japanese Patent Laid-Open No. 4-4 0 8 9 8], only 5.5 m MU (0.8% by weight) from the genus Pleurotus spp. [Japanese Patent Laid-Open No. 4-4 0 8 9 8], and only 9 40 m Μ (14% by weight) [W 0 9 6 ./ 0 9 4 0 3]. -4-This paper size applies to China National Standard (CNS) Α4 size (210 × 297 mm) (Please read the note on the back first Matters then fill out this page)

經濟部中央標準局員工消費合作社印製 521088 Α7 Β7 五、發明説明（3 ) 其生成物之蓄積濃度低之理由例如為α -羥基睛於水 溶液中對應之醛解離成酮及氡酸〔化學總覽（C h e in i c a 1 R e v i e w s )第4 2卷，1 3 9頁，1 9 4 8年〕，氡酸會阻害酵素 活性〔農業生物化學（/Uricultural Biological Chemistry )第46卷，1165頁，1 982年〕。又，所指出解離醛之作用 造成酵素短時間下失活之可能性，其解決之方法可提出 添加酸性亞硫酸離子或亞二硫酸離子之方法〔特開平5 -1 9 2 1 8 9 ]，添加亞礤酸離子或次亞磷酸離子之方法〔特 開平7-213296〕。又，使用此等添加物〇(-羥基酸之生成 蓄積濃度並不高。 一般於生成物蓄積濃度低的倩況下，此等業者詳知大 規模製造用之設備複雜。於此點依此方法工業地製造 α-羥基酸具有製造效率之問題。本發明之目的係在提 供使用徹生物以高濃度畜積α -羥基酸之效率來製造α -羥基酸之製法。 發明掲示 本案發明者偽探求關於由通式〔I〕 = R C H ( 0 H ) C Ν (式 中，R為氫原子，具取代基之C 1 - C s烷基，具取代基 之C 2 - C s烯基，具取代基之C i - C 3烷氯基，具取代基 之芳基，具取代基之芳氯基，或具取代基之雜環基）所 表示之α-羥基腈[I]，生成通式[II]:RCH(0H)C00H (式 中，R表示前述相同意味）所表示之α-羥基酸[II]之徹 生物，於高濃度之α-羥基腈[I]或α-羥基酸[II]下活 性少受抑制，活性持續良久具耐久性，而且具有蓄積高 本紙張尺度適用中國國家標準（CNS ) Α4規格（210Χ297公釐） (請先閲讀背面之注意事項再填寫本頁)Printed by the Employees' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 521088 Α7 B7 V. Explanation of the invention (3) The reason for the low concentration of its products is, for example, the dissociation of the corresponding aldehydes in the aqueous solution by α-hydroxy groups into ketones and acetic acid [Chemical Overview (C he in ica 1 Reviews) Vol. 42, p. 139, p. 189), acetic acid will inhibit enzyme activity [/ Uricultural Biological Chemistry (vol. 46, p. 1165, 1) 982]. In addition, the possibility of inactivating the enzyme in a short time due to the action of dissociating aldehyde is pointed out. The solution can be proposed by adding an acidic sulfite ion or a disulfite ion [Japanese Patent Laid-Open No. 5 -1 9 2 1 8 9], Method for adding arsenite ion or hypophosphite ion [Japanese Patent Application Laid-Open No. 7-213296]. In addition, the use of these additives 〇 (-hydroxy acid production accumulation concentration is not high. Generally, under conditions where the product accumulation concentration is low, these manufacturers know that the equipment for large-scale manufacturing is complex. At this point The method of industrially producing α-hydroxy acid has a problem of production efficiency. The object of the present invention is to provide a method for producing α-hydroxy acid by using the efficiency of a high-concentration livestock product of α-hydroxy acid. The invention shows that the inventor of the present invention is false To find the general formula [I] = RCH (0 H) C Ν (where R is a hydrogen atom, a C 1 -C s alkyl group having a substituent, a C 2-C s alkenyl group having a substituent, Α-hydroxynitrile [I] represented by C i-C 3 alkylchloro group of substituent, aryl group having substituent, aryl chloride group having substituent, or heterocyclic group having substituent) to form the general formula [II]: RCH (0H) C00H (wherein, R represents the same meaning as above), a complete organism of α-hydroxy acid [II], at a high concentration of α-hydroxynitrile [I] or α-hydroxy acid [ II] The activity is less inhibited, the activity lasts for a long time and has durability, and it has a high accumulation. The paper size is applicable to Chinese National Standard (CNS) Α4 regulations. (210Χ297 mm) (Please read the back of the precautions to fill out this page)

n - ~、1Tn 521088 A7 _B7_ Λ·泠明説明（4 ) 濃度α -羥基酸[I I ]能力之工業上有用之徹生物。 其結果可於V a r i 〇 v 〇 r a X屬及關節芽孢捍菌屬之微生物 發琨目標活性，進而於該反應条中，藉由添加以通式 [III]: Mm(CN)n (式中，Μ為氫、銨或金屬離子，mw為 1 - 3之整數）表示之氡化物，顯現出令酵素活性改善，完 成本發明。 PJI，本發明偽以微生物之作用來水解α -羥基腈[I ]， -成α-羥基酸[II]之α-羥基酸[II]之製法，以對α-羥 % 基晴[1]及/或α -羥基酸[II]具有濃度耐性及耐久性之 徹生物，於水性溶媒中生成蓄積α -羥基酸[I I ]，同時 採取於該反應糸添加氛化物以為特徵之a -羥基酸[I I ] 之製法。 以下，詳細說明本發明。 太發明所使用之微生物並無特別限制，凡可達到本發 明目的，對於α -羥基腈[I ]或α -羥基酸具濃度耐性以 及具有持缠長時間活性之耐久性者均可。微生物舉洌為 Variovorax paradoxus ΙΑΜ12374以及關節穿孢桿菌（n-~, 1Tn 521088 A7 _B7_ Λ · Ling Ming Description (4) The concentration of α-hydroxy acid [I I] ability is an industrially useful organism. As a result, the target activity of the microorganisms of the genus V ari ovavra X and Arthrosporum spp. Can be targeted, and then in the reaction bar, by adding the general formula [III]: Mm (CN) n (wherein Where M is hydrogen, ammonium or metal ions, and mw is an integer of 1 to 3), which shows that the enzyme activity is improved, and the present invention has been completed. PJI, the present invention uses a microorganism to hydrolyze α-hydroxynitrile [I] and α-hydroxyacid [II] to form α-hydroxyacid [II]. / Or α-hydroxy acid [II] has a concentration tolerance and durability of the organism, generates α-hydroxy acid [II] in an aqueous solvent and accumulates. At the same time, it adopts a-hydroxy acid [characterized by adding an odorant in the reaction.] II]. Hereinafter, the present invention will be described in detail. There is no particular limitation on the microorganisms used in the invention. Anyone who can achieve the purpose of the present invention, has a concentration tolerance to α-hydroxynitrile [I] or α-hydroxy acid, and a durability that has long-term activity. Examples of microorganisms are Variovorax paradoxus ΙΑΜ12374 and Arthrobacillus

Arthrobacter)NSSC104(CCRC 910077，FERM P-15424)。 經濟部中央標準局員工消費合作社印'^ (請先閲讀背面之注意事項再填寫本頁) 此等徼生物中，Vario'/orax paradoxus IAM 12374 可由 東京大舉分子細胞生物學研究所（I A Μ )容易取得。又關 節芽孢捍菌N S S C 1 0 4由本案發明者從自然界分離後之樣 本業已寄存。 (台灣） 寄存編號：CCRC910077 -δ - 本紙張尺度適用中國國家標準（CNS ) Α4規格（210X297公釐） 521088 多形性桿菌 離胺酸 好氣的 經濟部中央標準局員Μ消費合作社印製 A7 B7 五、發明説明（5 ) 寄存日：1997年4月8日 (曰本） 寄存编號：F E R Μ B P - 5 3 2 9 (自微工研菌寄第P - 1 5 4 2 4號移 交） 寄存曰：1996年2月6曰在國内寄存、1997年2月20曰 移交國際寄存 寄存場所：日本國茨城縣津久發市東1 丁目1番3號 寄託機關：通商產業省工業技術院生命工學工業技術5幵 究所 又關節穿孢桿菌N S S C 1 0 4之_學的性質如下。 形態 茧蘭氏染色性 r 〇 d - c 〇 c c u s 循環 穿胞 蓮動性 細胞壁二胺基 對氯態度 氯化酶 過氯化氫酶 DNA分解 角蛋白液化 澱粉分解 酪蛋白分解 維生素要求性 本纸張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁)Arthrobacter) NSSC104 (CCRC 910077, FERM P-15424). Printed by the Consumer Standards Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page) Among these creatures, Vario '/ orax paradoxus IAM 12374 can be obtained by the Tokyo Institute of Molecular Cell Biology (IA Μ) easy to get. In addition, the sample of Bacillus sp. N S S C 104 was isolated from nature by the inventor of the present case and has been deposited. (Taiwan) Deposit Number: CCRC910077-δ-This paper size is applicable to Chinese National Standard (CNS) A4 size (210X297 mm) 521088 Polymorphic bacillus lysine aerobic member of the Central Standards Bureau of the Ministry of Economic Affairs M Consumer Cooperatives printed A7 B7 5 5. Description of the invention (5) Deposit date: April 8, 1997 (Japanese version) Deposit number: FER Μ BP-5 3 2 9 (Transferred from Micro-Tech Research Biology P-1 5 4 2 4) Deposit Date: February 6, 1996, Domestic deposit, February 20, 1997, International depository place: Japan's East 1-Chome, Tsukuhatsu, Ibaraki, Japan The industrial properties of the Institute of Industrial Technology 5 Research and Arthrobacillus NSSC 1 0 4 are as follows. Morphology Cocoon orchid staining r 〇d-occcus Cyclic transcellular lotus Motile cell wall diamine group Chlorine attitude Chlorase perhydrogenase DNA degradation Keratin liquefaction starch degradation casein degradation Vitamin requirements This paper size applies China National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling this page)

經濟部中央標準局員工消費合作社印製 521088 A7 B7 五、發明説明（6 ) 藉乙Si基試驗 -ί乙醯型） m 条 MK-9 ( H2) 細胞壁耱組成 半乳耱 + 葡萄糖 + 以上細_學之性質，基於B e r g e y ’ s M a n ii a 1 〇 fPrinted by the Employees' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 521088 A7 B7 V. Description of the Invention (6) Borrowing the Si-based test-the type of ethyl acetate) m pieces of MK-9 (H2) cell wall nuclei constitute galactophore + glucose + above _The nature of learning, based on Bergy's Man an ii a 1 〇 f

Systematic Bacteriology(1986)檢索結果，鑑定 NSSC 1 0 4蘭株為關節芽孢桿菌屬之菌株。 以下，描述本發明之具體實施態樣。 本發明所使用微生物之培養，偽在一般含有酵素誘導 物質.、徹生物可資化之磺素，氮素，無機離子，以及可 能必要之有機營養源之培養基上進行。酵素誘導物質偽 使用異丁晴等晴化合物，ε -己内蘿胺等之環狀醯胺化 合物。碳素源為葡萄溏等磺化合物，乙醇等醇類，其他 適用之銨鹽。無機離子為磷酸根離子，鈣離子，鎂離子 ，硫酸根離子，鐵離子，其他必要地應使用者。有機營 養源為維生素。胺基酸等以及含有此等物質之玉米濃計 ,酵母萃，聚蛋白賺，肉萃，其他適用者。培養宜在好 氣條件下，控制於Ρ Η 6至9 ,溫度2 5至3 7 °C之適當範圍 内進行。 由本發明所用之微生物之水解反應，偽採取上示之培 養蘭體，必要時固定化菌體，調製粗酵素，固定化酵素 之舗·處理物，於水性溶劑中令α-羥基腈[I]與相接鐲 來進行。於固定菌體或酵素之情況下，適用撑體結合法 -3 - 本紙張尺度適用中國國家標準（CNS ) Α4規格（210X 297公釐） (請先閱讀背面之注意事項再填寫本頁)The results of Systematic Bacteriology (1986) search revealed that NSSC 104 strain was identified as a strain of Arthrobacillus. Hereinafter, specific embodiments of the present invention will be described. The microorganisms used in the present invention are cultured on a medium that generally contains enzyme-inducing substances, bio-available sulfonates, nitrogen, inorganic ions, and possibly organic nutrient sources. The enzyme-inducing substance is pseudo-isocyanate and cyclic amidine compounds such as ε-caprolactam. The carbon source is sulfonic compounds such as grapevine, alcohols such as ethanol, and other applicable ammonium salts. Inorganic ions are phosphate ions, calcium ions, magnesium ions, sulfate ions, iron ions, and others should be used as necessary. Organic nutrition is vitamins. Amino acids, etc. and corn concentrates containing these substances, yeast extract, protein extract, meat extract, and others as applicable. Cultivation should be carried out under aerobic conditions in a suitable range of P Η 6 to 9 and temperature 25 to 37 ° C. According to the hydrolysis reaction of the microorganism used in the present invention, the culture blue body shown above is pseudo-immobilized, if necessary, the bacterial body is immobilized, the crude enzyme is prepared, and the enzyme-immobilized enzyme-treated product is treated with α-hydroxynitrile in an aqueous solvent [I] Connect with a matching bracelet. In the case of immobilizing bacteria or enzymes, the support binding method is applicable. -3-This paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) (Please read the precautions on the back before filling this page)

經濟部中央標準局員工消費合作社印製 521088 A7 B7 五、發明説明（7 ) ，包括法等常用之固定化技術。於調製精酵素酵素之情 況下，適用以超音波，高壓均質機來破碎菌體以後，以 硫酸銨鹽析，餍析等常用之酵素精製技術。使用以乾燥 電最換算為0.0 ]. -10重量％之濃度之菌體，於反應终了 後過濾，以離心分離或範圍外過濾膜濃縮回收來進行重 覆之水解反應。水性溶劑可舉例為水，含有緩衝劑等鹽 類或有機溶劑之水溶液，此以兩相分離為宜。 本發明所用之通式〔I〕 ： R C Η ( 0 H ) C N所表示之α -羥 基腈「1],其中R為氫原子，具取代基之C：l-C6烷基， 具取代基之C 2 - C s烯基，具取代基之C jl - C 3烷氯基， 具取代基之芳基，具取代基之芳氣基或具取代基之雜環 基。 具體地，舉例有氫原子，甲基，乙基，丙基，異丙基 ，丁基，異丁基，第二丁基，第三丁基，戊基，己基等 C 1 - C β 院基 ϊ 甲硫基甲基，1-甲硫基乙基，2 -甲硫基乙基，1-甲硫 基丙基，2 -甲硫基丙基，3 -甲硫基丙基，1-甲硫基丁基 ,甲硫基丁基，3_甲硫基丁基，4-甲硫基丁基，6-甲 硫基己基，乙硫基甲基，1-乙硫基乙基，2 -乙硫基乙基 ，1-乙硫基丙基，2 -乙硫基丙基，3 -乙硫基丙基，1-乙 硫基丁基，2 -乙硫基丁基，3 -乙硫基丁基，4 -乙硫基丁 基，丙硫基甲基，1-丙硫基乙基，2 -丙硫基丙基，3-丙 硫基丁基，1-甲硫基異丙基，2 -乙硫基異丙基，1-丙硫 基丁基，2 -丙硫基丁基，3 -丙硫基丁基，4 -丙硫基丁基， -9 - 本紙張尺度適用中國國家標準（CNS ) Α4規格（210X 297公釐） (請先閱讀背面之注意事項再填寫本頁)Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 521088 A7 B7 V. Description of Invention (7), including common fixed technologies such as law. In the case of preparing fermented enzymes, it is suitable to use ultrasonic and high-pressure homogenizers to break the bacterial cells, and then use ammonium sulfate salting out, decanting and other commonly used enzyme purification techniques. The bacterial cells having a concentration of 0.0 to 10% by weight based on the dry electricity were filtered after the reaction, and then subjected to a repeated hydrolysis reaction by centrifugation or concentration recovery of an out-of-range filter membrane. Examples of the aqueous solvent include water, an aqueous solution containing a salt such as a buffer or an organic solvent, and it is preferable to separate the two phases. The general formula [I] used in the present invention: α-hydroxynitrile "1" represented by RC Η (0 H) CN, where R is a hydrogen atom, a substituent C: 1-C6 alkyl, and a substituent C 2-C s alkenyl, C jl-C 3 alkylchloro with substituents, aryl with substituents, aryl with substituents or heterocyclic with substituents. Specific examples include hydrogen Atoms, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, second butyl, third butyl, pentyl, hexyl, etc. C 1-C β Rhenylmethylthiomethyl , 1-methylthioethyl, 2-methylthioethyl, 1-methylthiopropyl, 2-methylthiopropyl, 3-methylthiopropyl, 1-methylthiobutyl, methyl Thiobutyl, 3-methylthiobutyl, 4-methylthiobutyl, 6-methylthiohexyl, ethylthiomethyl, 1-ethylthioethyl, 2-ethylthioethyl, 1-ethylthiopropyl, 2-ethylthiopropyl, 3-ethylthiopropyl, 1-ethylthiobutyl, 2-ethylthiobutyl, 3-ethylthiobutyl, 4- Ethylthiobutyl, propylthiomethyl, 1-propylthioethyl, 2-propylthiopropyl, 3-propylthiobutyl, 1-methylthioisopropyl, 2-ethylthio Propyl, 1-propylthiobutyl, 2-propylthiobutyl, 3-propylthiobutyl, 4-propylthiobutyl, -9-This paper size applies to China National Standard (CNS) A4 specifications (210X 297mm) (Please read the notes on the back before filling this page)

521088 A7 B7 五、發明説明（8 基 ，， 乙基基 基丙丁 硫基基 6 丙硫硫CR 卜丙丙1** ，異異C1 基 2 2 之 甲 ，，等 基基基基 硫丙丁丁 丙基基基 基 硫 烷521088 A7 B7 V. Description of the invention (8-based ,, ethylpropylbutanyl-6, thiothione CR, propylpropyl 1 **, isoiso-C1 group 2 2 dimethyl, isopropylthiopropyl butyl butyl Alkylsulfane

硫硫硫 丙丙丙 異異異 I I - 11- 4 基基基 乙丙丁； 基基基基 硫硫硫烷 丙丙丙 S 2-異異-C 基 3 丙 ， 羥基 2 丁 ，羥 基2- 丙 ， 羥基 卜丙 , 異 基基 乙羥 經1-- 2 , ，基 基丁 乙羥 經1- I il , ，基 基丙 甲羥 釋3- 基 羥 之 等 基 丁 羥 基 烷 基 Ρ 乙丙 院 乙 Α 基基 δ 錢CRi! ϋ 0^0 II ; I - - 1 甲甲，CI胺胺 基基卜2-6Ϊ基基基 乙羧 ，，06乙_乙 羧之基基1~巯之胍 2-等甲丙C.1-等1-，基基基基 ，基 ， 基丙醯 _ 醯基丙基 甲 羧甲甲 甲甲· 甲 羧卜胺胺胺巯3-胍 * 1—I 之 Ϊ 基 烷 基 丙 羧 基 丙 羧 基 基基 基 基^基 烷 乙 乙 (請先閱讀背面之注意事項再填寫本頁)Thiothiopropane isopropyl isopropyl II-11- 4 Ethyl Ethyl Propene Butyl; Thiothiopropane Propyl Isopropyl Sulfuryl Propionyl S 2-Iso-C 3 Propyl, Hydroxy 2 Butyl, Hydroxy 2- Propyl ， Hydroxybutyrate, isopropylethoxyl via 1-2, ethoxybutyryl vial 1-I il, ethoxybutyryl 3-hydroxyl, etc. Ethyl Α δ CRCRi! Ϋ 0 ^ 0 II; I--1 methyl form, CI amine amino group 2-6 fluorenyl ethyl carboxylate, 06 ethyl _ ethyl carboxyl group 1 ~ thioguanidine 2-Isomethylpropyl C.1-Iso1-, propyl, phenyl, propyl, propyl, hydrazino-methyl, propylmethylcarboxyl, methylformyl, methylcarboxamidine, sulfanil 3-guanidine * 1-I Ϊ Alkyl propyl carboxyl propyl carboxyl yl yl ^ alkane ethyl ethyl (Please read the precautions on the back before filling this page)

，等 基基 乙丙 基基 醯醯 甲甲 胺 胺 基 丙 巯 基 丙 胍 基 丙 巯 基 丙 胍 、y5 丙基硝二 脈甲 3 - α 3 苯 ， ， ， 基 a 基 基 經濟部中央標準局員工消費合作社印製 基甲基 基 基 a C ί 2 甲 ， CN3 ， 苯基CI,基 卩基甲之基甲 6^甲苯代甲基 C64-基取基Ϊ-5 \ ,羥具呤吲 C1基4-等吲2-基甲 ，基 2 , 胍苯基甲 ，基 之氯甲苯基乙 等2-苯基甲丨 ，唑 基眯 乙 4 \—/ /( 基 2 呤 ， 吲基 •乙 唑 咪 2 基 甲 唑 咪 甲 苯 基 氧 甲 ，基 甲；} 基 苯基基呤 基烷»5吲 甲芳吲 Λ 基 甲 唑 咪 環 雜 代 取 經 之 等 基 乙 烷 基 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 521088 /Ψ 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（9 ) 乙烯基，丙烯基，異丙烯基，烯丙基，1-氯烯丙基， 2 -氛烯丙基，丁烯基可具取代之C 2 - C 3烯基； 甲氯基，乙氧基，丙氧基，三氣甲氧基等具取代基之 C 1 - C 6院氯基； 笨基，2 -氯苯基，對-甲苯基，3 -硝苯基，4 -氰苯基， α-苯基，_基等具有取代基之芳基； 茱氯基，2 -氛苯氯基，對-甲苯氯基，3 -硝基苯氧基 ，α-萃氧基，/3-萘氧基等具有置換基之芳氧基； 2 -吡啶基，3 -吼啶基，4 - 1%啶基，5 -氯-3 - Utt啶基， 2-喀吩基，3-瞎盼基，2 -吼咯基，3 -吼咯基，2-呋喃基 ，3_呋喃基導至少含有一種氮、氣、硫原子以為雜原子 之3〜7員之雜環基。 具體地α -羥基腈[I ]為例如乳腈，扁桃睛，2 -羥基-4 -甲硫基丁晴等。 此α -羥基腈[I ]反應時以0 . 1〜5 0重量％之濃度來使 用，必要時亦可於反應之間逐次添加。反應液之Ρ Η值依 滴當之緩衝劑藉酸及鹼宜保持於5〜11之間。反應溫度 宜保持為4〜5 0 °C ,以2 0〜4 0 υ為佳。 本發明所用通式[I I I ]表示之氮化物可舉例氡化氫， 氡化鈉，氡化鉀，氰化鈣，氰化纟1 ,氡化鉈，氰化銨等 。氣化物，一般以0.4〜lOOOmM，較佳以4〜500mM之範 圍夾使用，必要時可於反應之間逐次添加。 因此在6小時至1 2 0小時間添加α -羥基腈[I ]下，令 〇(-羥基酸[I I ]銨鹽蓄積1 5重量％以上之高濃度。反應 >11- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁), Etc. Ethyl ethylpropyl methanomethamine amino propyl mercaptopropanyl propyl mercaptopropanyl guanidine, y5 propyl nitrodimethyl 3-α 3 benzene, ketones, employees of the Central Standards Bureau of the Ministry of Economic Affairs Consumer co-operative printed methylmethyl group a C ί 2 methyl, CN3, phenyl CI, methyl amidyl methyl 6 ^ tolyl methyl C64-based radical Ϊ-5 \, hydroxy withindyl C1 group 4-equivalent indyl-2-ylmethyl, guanidiphenylmethyl, chlorotolylethyl, etc. 2-phenylmethyl, oxazolylethyl 4 \ — / / (radicals 2, indylacetazol Imidyl 2-methylcarbazolyl tolyloxymethyl, methylformate;} phenylphenyl methinyl alkane »5 indylaryl indyl yl methazolium ring heterocyclic substitution and other basic ethane basic paper size applicable Chinese national standards ( CNS) A4 specification (210X297 mm) 521088 / Ψ Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (9) Vinyl, propenyl, isopropenyl, allyl, 1-chloroallyl , 2-aminoallyl, butenyl may have substituted C 2 -C 3 alkenyl; methyl chloride, ethoxy, propoxy, trifluoromethyl C 1-C 6 chlorinyl with substituents; benzyl, 2-chlorophenyl, p-tolyl, 3-nitrophenyl, 4-cyanophenyl, α-phenyl, _yl, etc. Substituted aryl groups; rudolyl, 2-phenylphenylchloro, p-toluylchloro, 3-nitrophenoxy, α-extractoxy, / 3-naphthyloxy, etc. -2-pyridyl, 3-pyrimidinyl, 4-1% pyridyl, 5-chloro-3-Uttyl, 2-carpentyl, 3-pyridyl, 2-pyrrolyl, 3- Hexyl, 2-furanyl, 3-furanyl is a heterocyclic group containing 3 to 7 members of nitrogen, gas, and sulfur atoms as heteroatoms. Specifically, α-hydroxynitrile [I] is, for example, lactonitrile, Almond, 2-hydroxy-4-methylthiobutyrene, etc. This α-hydroxynitrile [I] is used at a concentration of 0.1 to 50% by weight during the reaction, and it can also be added one by one between the reactions if necessary. The pH value of the reaction solution should be kept between 5 ~ 11 depending on the acid and alkali of the buffer. The reaction temperature should be kept at 4 ~ 50 ° C, preferably 20 ~ 4 0 υ. The present invention Examples of the nitride represented by the general formula [III] include hydrogen halide, sodium halide, potassium halide, calcium cyanide, and cyanide. 1. Tritiated tritium, ammonium cyanide, etc. Gases are generally used in the range of 0.4 to 1000 mM, preferably 4 to 500 mM, and can be added one by one between the reactions if necessary. Therefore, it is 6 hours to 120 hours When α-hydroxynitrile [I] is added over time, the 0 (-hydroxyacid [II] ammonium salt is accumulated at a high concentration of 15% by weight or more. Response > 11- This paper size applies Chinese National Standard (CNS) A4 (210X297 mm) (Please read the precautions on the back before filling this page)

經濟部中央標準局員工消費合作社印製 521088 A7 B7 五、發明説明（10 ) 用之_體，於實質活性低下後，重覆水解來使用。 生成物藉由濃縮、萃取之常用方法來分離精製，必要 時於_性條件下藉有機溶劑萃取，熱分解等分離銨。為 α -羥基酸[I I ]之生成物可舉例有乳酸，扁桃酸，2 -羥 基-4 -甲硫基丁酸等。 奮施發明之最佳形態： 以下藉實施例具體描述本發明。 (實施例1 ) 將含有0.3%肉汁，0.5%蛋白陳及0,5%食鹽之培養 基2 0 m 1之試管，於容量1 0 0 m 1具備擋板之三角燒瓶中裝 入如下組成之培養基2 0 0 m 1後，各於1 2 1 °C下滅_ 1 5分鐘 。於2 m丨之試管内以白金接種環（白金耳）植入V a r i 〇 v 〇「a X paradoxus IAM 12374菌株，於301下振鹽培養一晚後， 繼而棺入0 . 2 m 1至具備擋板之三角燒瓶中，再於3 0 °C下 振燙培養3天。以離心分離此培養液所得之菌體於生理 食鹽水中洗淨，以乾燥重量換算為1 . 0重量％之菌體於 0 . 1 Μ礤酸緩衝液（p Η 7 . 5 )中懸浮。其次添加2 -羥基-4 -甲 硫基丁腈使終濃度為1 6 0 m Μ ,於3 0 °C緩慢振盪進行水解 反應。添加後每1 2小時追加同份量2 -羥基-4 -甲硫基丁 睛重覆9次，總共進行1 2 0小時之反應。反應終了後， 離心分離反應液除去鐘體，離心上清液中含有2 -羥基 -4-甲硫基丁酸之濃度使用高速液體層析（管柱：TS1(凝 膠0 D S - 8 0 T Μ，載劑：乙腈/水/三氣乙酸=2 0 / 8 0 / 0，Γ) 定量、結果、確認蓄積2 5重量％之2 -羥基-4 -甲硫丁酸 -12- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210Χ297公釐） (請先閱讀背面之注意事項再填寫本頁)Printed by the Employees' Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 521088 A7 B7 V. Description of the invention (10) The body is used after repeated low hydrolysis, after repeated hydrolysis. The product is separated and purified by the usual methods of concentration and extraction, and if necessary, it is extracted by organic solvent under thermal conditions, and thermal decomposition is used to separate ammonium. Examples of the product of α-hydroxy acid [I I] include lactic acid, mandelic acid, 2-hydroxy-4-methylthiobutanoic acid, and the like. The best form of Fenshi invention: The present invention will be described in detail by the following examples. (Example 1) A test tube of 20 m 1 containing 0.3% gravy, 0.5% protein, and 0.5% common salt was placed in a conical flask with a baffle capacity of 100 m 1 and a culture medium having the following composition After 2 0 m 1, each goes off at 1 2 1 ° C for _ 15 minutes. Vari ○ OV 『a X paradoxus IAM 12374 strain was implanted with a platinum inoculation ring (platinum ears) in a test tube of 2 m 丨, and cultured overnight at 301 under shaking with salt, and then 0.2 m 1 to In a baffled conical flask, the cells were cultured by shaking at 30 ° C for 3 days. The cells obtained by centrifuging the culture solution were washed in physiological saline, and the dry cells were converted into 1.0% by weight of cells. Suspension in 0.1 M acetic acid buffer (p Η 7.5). Secondly add 2-hydroxy-4-methylthiobutyronitrile to a final concentration of 160 m Μ, and slowly shake at 30 ° C Hydrolysis reaction. The same amount of 2-hydroxy-4-methylthiobutane is added 9 times every 12 hours after the addition, and the reaction is performed for a total of 120 hours. After the reaction is completed, the reaction solution is centrifuged to remove the bell body and centrifuged. The concentration of 2-hydroxy-4-methylthiobutanoic acid in the supernatant was determined by high-speed liquid chromatography (column: TS1 (gel 0 DS-8 0 T M, vehicle: acetonitrile / water / triacetic acid = 2 0/8 0/0, Γ) Quantitative, results, and confirmed accumulation of 2 -5% by weight of 2-hydroxy-4 -methylthiobutyric acid-12- This paper size applies Chinese National Standard (CNS) Α4 Specifications (210 × 297 mm) (Please read the precautions on the back before filling this page)

521088 經濟部中央標準局員工消費合作社印製 0.5% 0,5% 0,1% 0,1% 0.02¾ 0.02¾ 0.5% 7 . 2 (以2 Ν苛性鹼調整） A7 B7 五、發明説明（U) $安鹽（産率9 8 % )。 酵母萃 甘油 礤酸二氯鉀 礤酸氫二鉀 食鹽521088 Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 0.5% 0,5% 0,1% 0,1% 0.02¾ 0.02¾ 0.5% 7.2 (adjusted with 2 Ν caustic) A7 B7 V. Description of the invention (U ) $ A salt (yield 98%). Yeast Extract Glycerol Dichloropotassium Dipotrate Dipotassium Hydrogen Potassium Salt

硫酸鎂7水合物 ε -己内醯胺 pH (實施例2 ) 將含有0 . 3 %肉汁，0 . 5 %蛋白觫及0 . 5 %食鹽之培養 基2 0 m 1之試管，於容量1 0 0 m 1具備擋板之三角燒瓶中裝 入如下組成之培養基2 0 0 m 1後，各於1 2 1 °C下滅菌1 5分鐘 。於2 m 1之試管内以白金接種環白金接種環植入關節芽 孢桿® N S S C 1 0 4菌株（台灣寄存編號為C C R C 9 1 0 0 7 7 ), 於3 0 t下振盪培養一晚後，繼而植入〇 . 2 m 1至具備擋板 之三角燒瓶中，再於3 0 υ下振培養5天。以離心分離 此培養液所得之菌體於生理食鹽水中洗淨，以乾燥重量 換算為0 . 6重量％之_體於0 . 1 Μ磷酸緩衝液（ρ Η 7 . 5 )中懸 浮。其次添加乳晴使终濃度為1 2 4 m Μ ,於3 0 °C缓慢振盪 進行水解反應添加後每5小時追加同份量乳腈重覆2 0 次，總共進行1 0 0小時之反應。反應终了後，離心分離 反應液除去_體，離心上清液中含有乳酸之濃度使用高 速液體層析（管柱：T S K凝膠0 D S - 8 0 T Μ ,載劑：乙腈/水 -13- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210 X 297公釐） (請先閲讀背面之注意事項再填寫本頁)Magnesium sulfate heptahydrate ε-caprolactam pH (Example 2) A test tube containing 20% of a medium containing 0.3% gravy, 0.5% peptone and 0.5% common salt, in a capacity of 10 A conical flask with a baffle of 0 m 1 was filled with a medium having the following composition 2 0 m 1, and each was sterilized at 12 1 ° C for 15 minutes. In a 2 m 1 test tube, a platinum inoculation ring with a platinum inoculation ring was implanted into the joint spore rod® NSSC 104 strain (Taiwan deposit number CCRC 9 1 0 0 7 7), and cultured overnight at 30 t with shaking. Then implant 0.2 ml into a conical flask equipped with a baffle plate, and incubate at 30 υ for 5 days. The bacterial cells obtained by centrifuging the culture solution were washed in physiological saline, and 0.6% by weight of the _ cells were suspended in a 0.1 M phosphate buffer solution (ρΗ7.5) in terms of dry weight. Next, add lucid so that the final concentration is 124 m, and slowly shake at 30 ° C for hydrolysis reaction. After the addition, the same amount of lactonitrile is added 20 times every 5 hours to repeat the reaction for a total of 100 hours. After completion of the reaction, the reaction solution was centrifuged to remove the body, and the concentration of lactic acid in the centrifuged supernatant was subjected to high-speed liquid chromatography (column: TSK gel 0 DS-8 0 T Μ, vehicle: acetonitrile / water-13- This paper size applies to Chinese National Standard (CNS) Α4 size (210 X 297 mm) (Please read the precautions on the back before filling this page)

玉米濃汁 蔴糖 礤酸二氯鉀 礤酸氯二鉀 食鹽 硫酸鎂7水合物 硫酸鐵 ε -己内醯胺 pH 經濟部中央標準局員工消費合作社印製 521088 Α7 Β7 五、發明説明（12 ) /三氣乙酸= 5/ 95/0.1)定量、結果、確認蓄積23重 景％之乳酸鞍鹽（産率9 3 % 。 1, 〇 % (別滅 a ) 1 · 〇 % (別滅菌） 0.1% 0.1% 0.02% 0.02% 0 . 0 0 1 % (別滅菌） 0.5% 7 , 2 (以2 N苛性鈉諝整） (實施例3 ) 以實施例2同法所製之關節芽孢捍菌N S S C 1 0 4 Μ株（ 寄存號碼：CCRC 910077)菌體以乾燥重量換算為4重量％ 之量懸浮於〇,1Μ礤酸緩衝液（ΡΗ7.5)中。其次添加2-羥 基-4 -甲硫基丁羥使終濃度為2 0 0 m Μ，於3 0 C下緩慢振盪 進行水解。添加後每1小時追加2 -羥基-4 -甲硫基丁腈 重覆7次，再以每1 . 5小時追加8次，總共健行1 9小時 。反應终了後，離心分離反應液除去菌體，離心上清液 中含2 -羥基-4 _甲硫基丁酸之濃度以實施例1同樣方法 定景結果，確認蓄積49重量％之2 -羥基-4-甲硫基丁酸 $安鹽（産率9 6 % 。 (實施例4) 以實施例2同法所製之關節芽孢捍_ N S S C 1 0 4菌株（ 14- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210Χ297公釐） (請先閲讀背面之注意事項再填寫本頁)Corn Juice Dipotassium Magnesium Dichloro Potassium Dipotassium Chloride Dipotassium Chloride Salt Magnesium Sulfate 7 Hydrate Iron Sulfate ε -Caprolactam pH Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 521088 Α7 Β7 V. Description of the Invention (12) / Trigas acetic acid = 5/95 / 0.1) Quantitative, results, and confirm the accumulation of 23% of lactic acid saddle salt (yield 93%. 1, 0% (do not destroy a) 1 · 〇% (do not sterilize) 0.1 % 0.1% 0.02% 0.02% 0. 0 0 1% (Do not sterilize) 0.5% 7, 2 (Adjusted with 2 N caustic soda) (Example 3) Arthrobacter sp. NSSC prepared in the same way as in Example 2 Cells of 1 0 4 M strain (registered number: CCRC 910077) were suspended in an amount of 4% by weight in terms of dry weight in 0,1 M acid buffer (P7.5), followed by addition of 2-hydroxy-4-methylsulfide Butyl hydroxy was used to make the final concentration of 200 μM, and was slowly shaken to hydrolyze at 30 C. After the addition, 2-hydroxy-4-methylthiobutyronitrile was added 7 times every 1 hour, and then every 1. Add 8 times in 5 hours for a total of 19 hours of hiking. After the reaction, the reaction solution was centrifuged to remove bacteria, and the concentration of 2-hydroxy-4_methylthiobutyric acid in the supernatant was the same as in Example 1. According to the results of the scene setting in this method, it was confirmed that 49% by weight of 2-hydroxy-4-methylthiobutyric acid $ an salt was accumulated (yield 96%.) (Example 4) The joint spores prepared by the same method as in Example 2 _ NSSC 1 0 4 strain (14- This paper size applies to the Chinese National Standard (CNS) A4 specification (210 × 297 mm) (Please read the precautions on the back before filling in this page)

經濟部中央標準局員工消費合作社印製 521088 A7 B7 五、發明説明（l3 ) 寄存號碼：CCRC 910077)菌體以乾燥重量換算為3. 2重 景％之暈懸浮於蒸餾水中。其次，於菌體乾燥重量為1 g 時，以0 . 4 6 g / h r之添加速度連續地添加2 -羥基-4 -甲硫 基丁晴，以0.5M氨水調整pH值為pH7,4〜7.6，於30^下 進行水解2 0小時。反應約了後，離心分離反應液回收鐘 體，、回收之菌體以40倍重量之蒸餾水洗淨3次後，於第 1 _[中以同量蒸餾水再懸浮，用於第2回反應中，以第 1回同樣方式進行_體回收及菌體洗淨。此種反應重覆 淮行10次，於各重覆回次中各離心上清液内所含有2 -羥 棊-4-甲硫丁酸之濃度以實施例1同法來定量。結果示 於下。 反應重覆 (回次） 2 -羥基-4-甲硫基丁酸 (重量％ ) 産率 (% ) 1 36.1 96 3 36.3 97 5 36.4 97 7 38.4 97 10 36.0 96 (實施例5 ) 以實施例2所製之關節芽孢捍菌H S S C 1 0 4菌株（ 寄存號碼：C C R C 9 1 0 0 7 7 )菌體以乾燥重量換算為5重量％ -15- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210Χ297公釐） (請先閲讀背面之注意事項再填寫本頁)Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 521088 A7 B7 V. Description of the Invention (l3) Deposit Number: CCRC 910077) The bacterial cells were converted to 3.2 weight by dry weight and suspended in distilled water. Next, when the dry weight of the bacteria is 1 g, 2-hydroxy-4-methylthiobutyrene is continuously added at a rate of 0.46 g / hr, and the pH value is adjusted to pH 7, 4 ~ with 0.5M ammonia water. 7.6, hydrolyze at 30 ^ for 20 hours. After the reaction has been completed, the reaction solution is centrifuged to recover the bell body. The recovered bacteria are washed three times with 40 times the weight of distilled water, and then resuspended in the same amount of distilled water in the first _ [for the second reaction. In the same manner as the first round, _-body recovery and bacterial cell washing were performed. This reaction was repeated 10 times, and the concentration of 2-hydroxypyrene-4-methylthiobutyric acid in each centrifugal supernatant was quantified in the same manner as in Example 1. The results are shown below. Repeated reaction (times) 2-hydroxy-4-methylthiobutyric acid (% by weight) Yield (%) 1 36.1 96 3 36.3 97 5 36.4 97 7 38.4 97 10 36.0 96 (Example 5) Example Arthrobacillus HSSC 1 0 4 strain produced by 2 (Reservation number: CCRC 9 1 0 0 7 7) The bacterial cells are converted to 5% by weight based on dry weight. -15- This paper size applies to China National Standard (CNS) Α4 specifications (210 × 297 mm) (Please read the notes on the back before filling in this page)

經濟部中央標準局員工消費合作社印製 521088 A7 B7 五、發明説明（14) 之最懸浮於0 . 1 Μ氮化鈉水溶液。其次連續添加2 -羥基-4 -甲硫基丁睛，以ρ Η控制器調整ρ Η值為7 , 4〜7 . 6 ,於3 0 I 下_行水解1 0小時。於反蘑终了後，離心分離反應液， 除去蘭體，於離心上清液中所含乳酸之濃度同實施例2 定鼋。比較未添加氡化鈉之情況下之反應結果示於下。 乳酸銨鹽生成量（重量％ ) 無添加 20.5 0. 1 M NaCN 40.0 (實施例6 ) 以實施例2所製得關節穿孢桿菌N S S C 1 0 4菌洙（寄存 編號：C C R C 9 1 0 0 7 7 )麵體以乾燥重量換算為5重量％ 之鼉懸浮於0 . 1 Μ氰化鉀水溶液，其次連續添加2 -羥基-4 -甲硫基丁腈，以ρ Η調節器調整ρ Η值為ρ Η 7 . 4 - 7 . 6，於3 0 下進行水解1 0小時。反應終了後，離心分離反應液除去菌 體，離心上清液中所含2 -羥基_ 4 -甲硫基丁酸之濃度同實 施例1之方法來定量，比較無添加氰化鉀之情況之反應， 結果示於下。 -16、- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁)Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 521088 A7 B7 V. Description of the invention (14) The most suspended in 0.1 M sodium nitride aqueous solution. Secondly, 2-hydroxy-4-methylthiobutane was continuously added, and the ρ 7 value was adjusted by the ρ Η controller to 7, 4 ~ 7.6, and hydrolyzed at 30 I for 10 hours. After the end of the reverse mushroom, the reaction solution was centrifuged to remove the blue body. The concentration of lactic acid in the centrifuged supernatant was the same as that in Example 2. The comparison of the reaction results when sodium sulfide is not added is shown below. Production amount of ammonium lactate (% by weight) without adding 20.5 0. 1 M NaCN 40.0 (Example 6) The P. acnes NSSC 1 0 4 fungus prepared in Example 2 (registered number: CCRC 9 1 0 0 7 7) The noodle is suspended in a 0.1 M potassium cyanide aqueous solution in terms of dry weight and converted to 5% by weight, followed by the continuous addition of 2-hydroxy-4-methylthiobutyronitrile, and the value of ρ is adjusted by the ρ Η regulator. ρ Η 7.4-7.6, hydrolyzed at 30 for 10 hours. After the reaction was completed, the reaction solution was centrifuged to remove bacteria, and the concentration of 2-hydroxy-4-methylthiobutyric acid in the centrifuged supernatant was quantified in the same manner as in Example 1. Compared with the case where potassium cyanide was not added The results are shown below. -16.- This paper size applies to China National Standard (CNS) A4 (210X297 mm) (Please read the precautions on the back before filling this page)

經濟部中央標準局員工消費合作社印製 521088 Α7 Β7 五、發明説明（15) 2 -羥基-4-甲硫基丁酸銨 鹽生成量（重量％ ) 無添加 26.6 0,1M KCN 41.7 (實施例7 ) 以奮施例2所製得關節芽孢捍菌H S S C 1 0 4菌株（寄存 編號：CCRC 910077)之菌體以乾燥重量換算為5重量％ 之量懸浮於4 0 m H之氰化氫水溶液，其次連續添加2 -羥基 -4 -甲硫丁腈，以p Η調節器調整p Η值為p Η 7 . 4 - 7 · 6 ,於3 0 1C 下進行水解1 0小時。反應终了後，離心分離反應液除去 蘭體，於離心上清液中2 -羥基-4 -甲硫基丁酸之濃度同實 施例丨之方法來定量，比較無添加氡化氫情況之反應， 結果示於下，、 2-羥基-4-甲硫基丁酸銨 鹽生成量（重量％ ) 無添加 27.2 40mM HCN 44 . 5 - 17- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210 X 297公釐） (請先閱讀背面之注意事項再填寫本頁)Printed by the Employees' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 521088 Α7 Β7 V. Description of the invention (15) Production amount of ammonium salt of 2-hydroxy-4-methylthiobutyric acid (% by weight) No addition 26.6 0,1M KCN 41.7 (Example 7) The bacterial cells of Arthrobacillus sp. HSSC 104 strain (registration number: CCRC 910077) prepared in Fen Example 2 were suspended in a 40 m H hydrogen cyanide aqueous solution in an amount of 5% by weight in terms of dry weight. Then, 2-hydroxy-4-methylthiobutyronitrile was continuously added, and the p Η value was adjusted to Η 7.4-7 · 6 by the p Η regulator, and the hydrolysis was performed at 30 1C for 10 hours. After the end of the reaction, the reaction solution was centrifuged to remove the blue body, and the concentration of 2-hydroxy-4-methylthiobutyric acid in the centrifuged supernatant was quantified in the same manner as in Example 丨. The reaction without the addition of tritiated hydrogen was compared. The results are shown below. The amount of 2-hydroxy-4-methylthiobutyric acid ammonium salt produced (% by weight) without the addition of 27.2 40mM HCN 44. 5-17- This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) (Please read the notes on the back before filling this page)

經濟部中央標準局員工消費合作社印製 521088 Α7 Β7 五、發明説明（） (參考例） 蕻由於特開平-4 0 8 9 8號公報記載之培養方法及水解方 法，培養關節芽孢捍鐘N S S C 1 0 4菌株進行觸媒反應。 (i )培養基（單位：W / V ) 甘 油 2 % 酵 母 萃 1 • 3% 酸 二氫鉀 0 ♦ 68% m 酸 氫二鉀 0 .71% 鈉 0 .28¾ 氯 化 m 0 ,04¾ 氛 化 鈣 0 .0 0 4% 硫 Μ 錳 4 X 10-4¾ 氯 化 m 6 X 10-5% 硫 酸 鋅 〇 X 10-5%· 洋 菜 1 .8% a -氣丙睛 0 .5% pH 7 . 5 (2 )培養條件 # 由斜面培養基採取1白金接種環之菌體，於蒸氣洋菜 平板培養基上塗佈，於3 0 1C下好氣培養48小時。 (3 )水解反應： 從洋菜平板培養基上採取菌體，藉離心分離菌體於 0 . 0 5 Μ礤酸緩衝液（p Η 7 . 5 )中洗淨三次。沈澱菌體於0 0630 值為2 5時以1 . 5 m L相同緩衝液再懸浮，添加终濃度1 〇 〇 m Μ '18' 本紙張尺度適用中國國家標準（CNS ) Α4規格（210X 297公釐） (請先閱讀背面之注意事項再填寫本頁)Printed by the Employees' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 521088 Α7 Β7 V. Description of the invention () (Reference example) 蕻 Due to the cultivation method and hydrolysis method described in JP-A-4 0 8 98, the joint spore culture NSSC 1 The 0.4 strain was subjected to a catalyst reaction. (i) Culture medium (unit: W / V) Glycerol 2% Yeast extract 1 • 3% potassium dihydrogen acid 0 ♦ 68% m dipotassium hydrogen acid 0.71% sodium 0 .28¾ chloride m 0,04¾ aerated calcium 0. 0 0 4% sulphur manganese 4 X 10-4¾ m 6 X 10-5% zinc sulfate 0 X 10-5% agar 1. 8% a-acetonitrile 0.5% pH 7. 5 (2) Cultivation conditions # Take 1 platinum inoculation fungus from slant culture medium, spread on steam agar plate culture medium, and incubate at 30 ° C for 48 hours under aerobic conditions. (3) Hydrolysis reaction: The bacterial cells were taken from the agar plate culture medium, and the bacterial cells were separated by centrifugation and washed three times in a 0.05 M acetic acid buffer solution (p Η 7.5). The precipitated bacteria were resuspended in 1.5 m L of the same buffer when the value of 0 0630 was 25, and the final concentration was added to 1000 m Μ '18'. The paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X 297) Li) (Please read the notes on the back before filling in this page)

經濟部中央標準局員工消費合作社印製 521088 Α7 Β7 五、發明説明（17 ) 之2 -羥基-4 -甲硫基丁腈，於2 5 3C下進行振盪反應2 0小 時。反應终了後，離心分離反應液除去菌體，於離心上 清液中所含2 -羥基-4 -甲硫基丁酸之濃度以高速液相層 析（管柱：T S Κ凝膠 0 D S - 8 Ο Τ Μ ,載劑：乙腈/水/三氟 丁酸= 20/80/0.1)定量。2 -羥基-4-甲硫基丁酸之濃度 為 0 . 0 1 m Μ。 於特開平4 - 4 0 8 9 8號記載，關節芽孢捍菌-H R 4菌株， 以上記培養方法及水解反應可蓄積5 5 πι Μ之2 -羥基-4 -甲 硫基丁 。因此，很明顯地關節芽孢桿菌N S S C 1 0 4菌株 與關節芽孢捍蘭H R 4舗株為不同菌株。 (發明之效果） 根據本發明，藉由使用對於α -羥基睛[I ]及/或α -經 基酸「U]具濃度耐性且具活性長時間持續之耐久性之微 生物，來以高濃度蓄積α -羥基酸[I I ],在重謾使用此 等蘭體不可以有效率地製造α -羥基酸[I I ]，又，藉由 於反應条統添加氯化物可以有效地製造α -羥基酸[II]。 産業上可利用性： 根據以上説明，本發明偽藉由使用具有對α -羥基腈 [I] 及/或α -羥基酸[II]具濃度耐性且具活性長時間持 續之耐久性之微生物，得到由cc -羥基腈製造α -羥基酸 [II] 之工業上有利之製法。因此其産業之意義極大。 -19- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210X 297公釐） (請先閲讀背面之注意事項再填寫本頁)Printed by the Employees' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 521088 Α7 Β7 V. Description of the invention (17) 2-hydroxy-4-methylthiobutyronitrile, the oscillating reaction was performed at 2 3 3C for 20 hours. After completion of the reaction, the reaction solution was centrifuged to remove bacteria, and the concentration of 2-hydroxy-4-methylthiobutyric acid in the centrifuged supernatant was subjected to high-speed liquid chromatography (column: TS KK gel 0 DS- 8 Ο Μ, vehicle: acetonitrile / water / trifluorobutyric acid = 20/80 / 0.1) quantitative. The concentration of 2-hydroxy-4-methylthiobutyric acid was 0.01 m. According to Japanese Patent Application No. 4-4088, the bacillus sclerotiorum-HR 4 strain, the culture method and hydrolysis reaction described above, can accumulate 2 -hydroxy-4-methylthiobutane of 5 5 μm. Therefore, it is clear that the bacillus arthritis N S S C 104 strain is different from the bacillus arthritis H R 4 strain. (Effects of the Invention) According to the present invention, by using microorganisms that are resistant to the concentration of α-hydroxyl [I] and / or α-acid base "U" and have durability that is active for a long period of time, they can be used at a high concentration. Accumulates α-hydroxy acid [II]. It is not possible to efficiently produce α-hydroxy acid [II] by using these blue bodies in heavy weight. Moreover, the addition of chloride due to the reaction system can efficiently produce α-hydroxy acid [II]. II]. Industrial Applicability: According to the above description, the present invention uses the durability that has concentration resistance to α-hydroxynitrile [I] and / or α-hydroxy acid [II] and has long-lasting activity. Microorganisms have obtained an industrially advantageous method for producing α-hydroxyacid [II] from cc-hydroxynitrile. Therefore, its industrial significance is of great significance. -19- This paper size applies the Chinese National Standard (CNS) A4 specification (210X 297) Li) (Please read the notes on the back before filling in this page)

修正丨補充丨 971-3721 ^112, 〇3 ¥請日i 1997/05/02th 案 號 86105867 類 別 (以上各欄由 <本 >填註） A4 C4 521088 |||專利説明書 中 文 利用微生物之α -羥酸之製法 （91年12月3日修正） 發明 新型 名稱 英 文Amendment 丨 Supplement 丨 971-3721 ^ 112, 〇3 ¥ Pleasei 1997/05 / 02th Case No. 86105867 Category (the above columns are filled by < 本 >) A4 C4 521088 ||| Patent Specification Chinese Use of Microorganisms Method for the production of α-hydroxy acid (as amended on December 3, 91)

A PRODUCTION METHOD 〇F α—HYDROXY ACID BY USING A MICROORGANISM 姓 名 國 籍 一 人一 洋健真公 林部平川 小渡大早 發明 創作 人 本本本本 曰B曰曰 12 3 4 裝· 住、居所 内 所 究 45研Λ3 柳田 田4 高内 市社 原會 田式 小株 縣達 川曹 奈本上上上 神日同同同 訂 姓 名 (名稱） 日本曹達股份有限公司 (曰本曹逹洙式會社） 線 經濟部中央標準局員工消費合作社印製 國 住、居所 (事務所） 代表人 姓 名 曰本 東京部千代田區大手町二丁目2番1號 下村達 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）A PRODUCTION METHOD 〇F α—HYDROXY ACID BY USING A MICROORGANISM Yanagida 4 Takachi City Co., Ltd., Hara-style, Dazhu, Cao, Xiaozhu County, Shangshang, Shenshen, and the same name (name) Nippon Soda Co., Ltd. Consumption cooperative prints national residence and residence (office) Name of representative: Benmoto, Tokyo, Chiyoda-ku, Otemachi, 2-chome, No.1, Shimomura. The paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm).

Claims (1)

年月日 91.12,0 3補无丨 公告不 六、申請專利範圍 第86 1 05 867號「利用微生物之α -羥酸之製法」專利案 (91年12月3日修正） 六申請專利範圍： 1 . 一種式[H ]化合物之製法， RCH ( OH) C00H [ Π ] 其中R爲氫原子，Ci-C6烷基硫烷基，羥烷基或 苯基， 該製法藉由微生物之作用水解式[I ]之化合物（式[ I ]爲RCH ( OH) CN，其中R同前述意義），轉變成式 [Π ]所示之化合物，其特徵在於藉由對式[I ]所示之 化合物及/或式[Π ]所示化合物具濃縮耐性及耐久性 之微生物，於水溶性溶劑中生成蓄積式[Π ]所示之化 合 物，該微生物爲 Variovorax paradoxus I AM 12374 或關節芽孢桿菌NSSC 104菌株（CCRC 910077)。 如申請專利範圍第1項之式[II]RCH(0H)C00H所示化 合物之製法，其中式[I ] : RCH(0H)CN所示化合物係 選自乳腈，扁桃腈，或2 -羥基-4 -甲硫基丁腈。 一種式[II]RCH(0H)C00H所示化合物之製法，其特徵 在於藉微生物作用水解式[I] : RCH(0H)CN(式中，R 同前述定義）所示化合物，製造式[II] : RCH(0H)C00H 所示化合物（式中，R同前述意義）之時該反應系統存 在通式[III] : Mm (CN)n(式中，Μ爲氫，銨或金屬離 子，m，η爲1-3整數）所示之氰化物。 521088 六、申請專利範圍 4·如申請專利範圍第3項之式[II]RCH(0H) C00H所示 化合物之製法，其中R爲氫原子，烷基硫烷基 ，（:厂匕羥烷基或苯基。 5·如申請專利範圍第3項之式[II]RCH(0H)C00H所示化 合物之製法’其中式[I ] : RCH(0H)CN(式中，R同前 述定義）所示化合物係選自乳腈，扁桃腈，或2 -羥基 -4 -甲硫基丁腈。 6 .如申請專利範圍第3項之式[11 ] RCH ( OH ) C00H所示化 合物之製法，其中微生物爲屬於 Variovorax paradoxus I AMI 2374 〇 7 ·如申請專利範圍第3項之式[11 ] RCH ( OH ) C00H所示化 合物之製法，其中微生物爲關節芽孢桿菌NSSC 104 菌株（CCRC 910077)。Date: 91.12,0 3, No. 丨 Announcement No. 6, Patent Application Range No. 86 1 05 867 "Method for the Preparation of α-Hydroxy Acids Using Microorganisms" (Amended on December 3, 91) Six Patent Application Scope: 1. A method for producing a compound of the formula [H], RCH (OH) C00H [Π] where R is a hydrogen atom, Ci-C6 alkylsulfanyl, hydroxyalkyl or phenyl, and the production method hydrolyzes the formula by the action of microorganisms The compound of [I] (formula [I] is RCH (OH) CN, where R has the same meaning as above), is transformed into a compound represented by formula [Π], which is characterized in that the compound represented by formula [I] and / Or the microorganism represented by the formula [Π] has concentrated tolerance and durability, and the compound represented by the formula [Π] is accumulated in a water-soluble solvent, and the microorganism is Variovorax paradoxus I AM 12374 or the bacillus NSSC 104 strain ( CCRC 910077). For example, the method for preparing a compound represented by formula [II] RCH (0H) C00H according to item 1 of the scope of patent application, wherein the compound represented by formula [I]: RCH (0H) CN is selected from lactonitrile, mandelonitrile, or 2-hydroxy -4 -methylthiobutyronitrile. A method for preparing a compound represented by formula [II] RCH (0H) C00H, which is characterized in that a compound represented by formula [I]: RCH (0H) CN (where R is the same as defined above) is hydrolyzed by a microorganism to produce formula [II ]: When the compound represented by RCH (0H) C00H (wherein R has the same meaning as above), the reaction system has the general formula [III]: Mm (CN) n (where M is hydrogen, ammonium or metal ion, m Η is an integer of 1 to 3). 521088 6. Scope of patent application 4. The method for preparing the compound represented by formula [II] RCH (0H) C00H according to item 3 of the scope of patent application, wherein R is a hydrogen atom, an alkylsulfanyl group, and (: a hydroxyalkyl group) Or a phenyl group. 5 · A method for preparing a compound represented by formula [II] RCH (0H) C00H according to item 3 of the scope of the patent application, wherein the formula [I]: RCH (0H) CN (where R is the same as defined above) The compound shown is selected from lactonitrile, mandelonitrile, or 2-hydroxy-4-methylthiobutyronitrile. 6. The method for preparing a compound represented by the formula [11] RCH (OH) C00H according to item 3 of the patent application scope, wherein The microorganism is a method belonging to Variovorax paradoxus I AMI 2374 〇7. The method for preparing a compound represented by formula [11] RCH (OH) C00H according to item 3 of the patent application scope, wherein the microorganism is Bacillus sp. NSSC 104 strain (CCRC 910077).