200932911 九、發明說明： 【發明所屬之技術領域】 本發明是有關於一種聚麩胺酸生產菌株及利用聚麩 胺酸生產菌株生產聚麩胺酸的方法，且特別是有關於一種 可生產聚麩胺酸的枯草桿菌菌株及γ-聚麩胺酸的生產方 法.。 【先前技術】 ® 聚麩胺酸(poly-Y-glutamic acid; PGA)為一種不尋常陰 離子多胜肽（unusualanionic polypeptide)，其係由D型及L 型麩胺酸或全由L型麩胺酸經由〇:-胺基及γ-羥基鍵結聚合 而成。此物質最早於5. 之英膜（capsule)中發現， 陸續之研究發現芽孢桿菌屬的特定菌種如5. var. natto反B. licheniformis亦會支轰此物質。 聚麩胺酸之合成與PGA操縱元（PGA operon)有關， PGA操縱元會表現出聚麩胺酸合成酶複合體。此酵素複合 φ 體位於細胞膜上，在三磷酸腺苷（ATP)及麩胺酸之存在下， 可合成出PGA。依照PGA生產時是否需要麩胺酸之存在， 可將生產PGA之芽孢桿菌屬菌種分為兩群，一群為需要麩 胺酸存在才會生產的菌種如 5· ATCC9945A，另一群則為不需麩胺酸即可生產的菌種如 B. licheniformis A35。 在產業應用方面，由於聚麩胺酸及其衍生物具有無 毒、水溶性、可食性、生物可分解性、多陰離子（polyanonic) 200932911 及保濕性等性質，可加以運用於食品、畜產、化妝品、醫 學及環境保護等領域，目前研究人員亦致力篩選高聚麩胺 酸產量之菌株、探討最適發酵及純化條件及開發聚麩胺酸 及其衍生物之新用途。 【發明内容】 因此本發明就是在提供一種聚麩胺酸生產菌株及利 用聚麩胺酸生產菌株生產聚麩胺酸的方法。 ^ 聚麵胺酸生產菌株（枯草桿菌PGA1 ; PGA1)係衍生自枯草桿菌168衍生株DB430,具 有一持續型強表現元件SECS-3嵌入以以//以168衍生株 DB430染色體之聚麩胺酸合成相關基因少wsC及之 上游，具有產生T-聚麩胺酸之能力。枯草桿菌PGA1寄存 於食品工業發展研究所（FIRDI)，編號BCRC910381。 利用枯草桿菌PGA1生產聚麩胺酸的方法包含以一特 定培養液培養枯草桿菌PGA1，並將分泌至培養液中的τ-Ο 聚麩胺酸以酒精沈澱分離得到。 根據上述可知，本發明之枯草桿菌PGA1利用嵌入持 續型強表現元件SECS-3於聚麩胺酸合成相關基因ywiC：及 之上游，配合本發明之提出之培養液配方，可提升 菌體密度及聚麩胺酸產量，在環境中沒有麩胺酸存在下 即可持續大量表現r -聚麩胺酸，具有生產快速、簡便等產 業利用價值。 6 200932911 【實施方式】 由於枯草桿菌凡168之基因體序列已解序完 成，發現5· 168基因體中帶有與聚麵胺酸合成相 關之基因ywiC及然而凡168卻無法生產 PGA，推測5.168無法生產PGA之原因乃是由於 無法有效表現聚麩胺酸合成酶複合體所致。 本發明實施例之策略為將凡㈣以出168衍生株DB430 (He, X.S., Shyu, Y.T., Nathoo, S., Wong, S.L., and Doi, R.H. © 1991. Construction and use of a Bacillus subilis mutant deficient in multiple protease genes for the expression of eukaryotic genes. Ann. N. Y. Acad. Sci. 646: 69-77.)染色 體中之；;wsC上游DNA序列置換為人工合成之持續型強 表現元件SECS-3，SECS-3之序列如SEQ ID ΝΟ:1所示。 SECS-3 由 UPve#(TCTTAACGTTGATATAATTTAAATT) + 人工合成強啟動子（TTGACA-17nt-TATAAT)+SDl序列 (AGTGAGGTG)今Opt SD2 序列(AAAGGAGG)所組成，藉 Ο 由SECS-3可使枯草桿菌DB430大量表現聚麩胺酸合成相 關酵素，並成為PGA生產株。 依照本發明之實施例，聚麩胺酸生產菌株（枯草桿菌 PGA1 ; PGA1)之選殖係先構築一帶有部分 ywiCDNA片段之嵌入型載體，再利用一勝任細胞轉形法 將此載體導入枯草桿菌DB430中。 請參照第1圖，為本發明之帶有部分少DNA片段 之嵌入型載體P177-PGAI的基因排列示意圖。此DNA片段 7 200932911 中含有持續型強表現元件SECS-3、部分聚麩胺酸合成酵素 複合體少^^匚基因片段、terminator(終止子）及T1 terminator ° 依照本發明之實施例，載體P177-PGAI之構築係利用 枯草桿菌之基因體去氧核糖核酸為模板，以專一性引子組 合 PpgaNdelF (SEQ ID NO : 2)及 PpgaSallR (SEQ ID NO : 3)進行部分聚麩胺酸合成酵素複合體DNA片段之擴 增。增幅之聚合酶連鎖反應產物以限制酶iW/el及Sfl/I剪 © 切後，利用T4 DNA ligase(接合酶）將DNA片段接入以相 同限制酶剪切之帶有SECS-3的質體pSECS-3GFPT中，使 含部分聚麩胺酸合成酵素複合體ywsC DNA的片段位於 SECS-3之下游，再將黏合產物電轉形入大腸桿菌JM109 中，隨機挑選轉形株抽取其質體，進行限制酶剪切及DNA 定序確認。 將DNA序列正確無誤之質體命名為pSECS-3PGAI。 PSECS-3PGAI以限制酶5g/II及剪切後，回收分子 © 量約0.4 Kb之DNA片段，此DNA片段中含有持續型強表 現元件SECS-3、與宿主染色體同源的部分聚麩胺酸合成酵 素複合體少wiC基因片段、λί0終止子及T1終止子》 利用T4 DNA ligase將此DNA片段接入以相同限制酶剪 切之質體pl77中，質體pl77搭配枯草桿菌宿主為一高效 率的質體-宿主轉移系統，質體pl77帶有抗抗生素基因作 為選擇性標總（selective marker)，且質艘pi 77在枯草桿菌 宿主中不能複製。將黏合產物電轉形入大腸桿菌JM109 8 200932911 中。隨機挑選轉形株抽取其質體，進行限制酶剪切及DNA 定序確認。將DNA序列正確無誤之質體命名為 P177-PGAI。 質體P177-PGAI上所帶之持續型強表現元件 SECS-3、部分聚麵胺酸合成酵素複合體基因、λί0 終止子及Τ1終止子基因片段可利用勝任細胞轉形過 程中DNA片段化之特點，使與宿主染色體同源的部分 基因片段與宿主染色體上的基因進行單一交叉 © (single-cross over)，於宿主染色體上的少基因上發生基 因重組，而***染色體。 枯草桿菌勝任細胞之製備步锁如下：挑取 JM109(pl77-PGAI)單—菌落接種於 lb (Luria-Bertani)培 養基中，於37°C、150rpm之條件下進行振盪培養。經隔 夜培養後，取適量菌液接種於新鮮SpC培養基（20ml T base, 0.2 ml 50%(W/V) glucose, 0.3 ml 1.2% (W/V) MgS〇4.7H2〇, 0.4 ml 10%(W/V) Bacto yeast extract, 0.5 ml 〇 1% (W/V) casamino acids ； T base ： 0.2 % (NH4)2S04 ' 1.83 % ΚΗ2Ρ〇4·3Η20'0.6% KH2PO4 ' 0.6% Trisodium citrate 2.0%。）中，將起始菌液濃度調整至A600=0.1。之後於37 °C、180 rpm之條件下進行振盪培養，當生長期由對數期 進入平穩期時，取培養液接種於新鮮配製的SPII培養基 (SPII: 200 ml T base, 2 ml 50%(W/V) glucose, 14 ml 1.2% (W/V) MgS04.7H20, 2 ml 1% (W/V) casamino acids, 2 ml 10% (W/V) bacto yeast extract, 1 ml 0.1 M CaC12 ； T base ： 9 200932911 0.2 % (NH4)2S04、1.83 % ΚΗ2Ρ〇4_3Η20、0.6% ΚΗ2Ρ〇4、 0.6% Trisodium citrate 2.0%。）中，加入之培養液體積為 SPII培養基體積之1/10,於37°C，120rpm下進行振盪培 養，培養至菌液濃度約為A_=0.3〜0.4時，離心（6000 X g， 10分鐘）收集菌體並保留上清液，上清液中含有勝任及產 孢因子（competence and sporulation factor)，可誘發勝任狀 態之發生。將菌體懸浮於適量上清液及甘油中’均勻混合 後，進行分裝（0.5 mL/管），利用液態氮急速凍結後，貯存 φ 於-70°C備用。 進行DNA轉形試驗時，自-8(TC冰箱中取出勝任細 胞，利用37 °C水浴進行快速解凍，並加入等倍體積之 SPIIE，再與適量質體pl77-PGAI混合後，於37°C下進行 振瘼培養（180 rpm)。培養30分鐘後，加入SB培養基（3.5 % tryptone、2.0 % yeast extract、0.5 % NaCl，pH 7.5 ° ) ’ 於37°C、180 rpm之條件下，繼續振盪培養〆小時。之後’ 離心（10000 X g，10分鐘）收集菌體，將其塗:抹於含適量抗 Q . 生素之培養基。於37°C下靜置培養16至24小時即可觀 察结果。 請參照第2圖，為質鱧pl77-PGAI與宿主染色體發生 單〆交叉的基因排列示意圖。當P177-PGAI在宿主染色體 上成功發生單一交又（single-cross over)而造成基因重組’ 質艘P177-PGAI的抗抗生素之基因（Cmr)將會嵌入染色體 中，並賦予轉形株抵抗氯黴素之能力，可藉由含氯黴素的 培養基篩選出轉形株。此外轉形株之持續梨強表現元件 200932911 SECS-3會座落於聚麩胺酸合成相關基因ywsC及： 之上游，促進聚麵胺酸合成相關基因ywsC及基因 之表現，使轉形株菌株具有生產7-聚麩胺酸之能力，且轉 形株菌落會有黏液狀（mucoid)之外觀。隨機挑選多株具有 黏液性狀之轉形株，抽取菌體之染色體DNA，利用PCR 方式確認這些菌株的確為質體P177-PGAI嵌入成功之聚麩 胺酸生產株。聚麩胺酸生產株命名為枯草桿菌PGA1 (5acz7/ws PGAI )，並寄存於食品工業發展研究所 ❿ （FIRDI)，編號 BCRC910381。 進一步將枯草桿菌PGA1培養於LB液體培養基中， 於37°C培養12及24小時後，收集上清液，利用γ-聚麩 胺酸可被酒精沉澱之特性分離培養液中Τ·聚麩胺酸。將培 養液離心（21910 X g，15分鐘）後，取出適量上清液，放置 於微量離心管中並加入4倍體積之冰冷乙醇，均勻混合 後，可見棉絮狀之物質產生，此即為粗聚麩胺酸。於4°C 下放置隔夜，經離心（21910 X g，15分鐘）並倒除上清液 © 後，將沉澱物放置於室溫下乾燥得到γ-聚麩胺酸。依照本 發明之另一實施例，可利用類似之步驟進行γ-聚麩胺酸之 大量分離，差別在於將培養液離心之前先以酸（例如鹽酸） 滴定至約pH 3後，再以5000 X g、30分鐘之條件進行離 心，之後以類似前述之步驟得到γ-聚麩胺酸。 利用蛋白質電泳觀察枯草桿菌PGA1生產γ-聚麩胺酸 之情形。結果顯示，轉形株均於培養24小時後之γ-聚麩 胺酸產量較高。 200932911 依照本發明之另一實施例，將枯草桿菌PGA1培養於 一 Medium A (3.3 % tryptone、2.0% yeast extract、0.74% NaCl、0.8% Na2HP04，0.4 % KH2P〇4、2.0% casamino acids、1.0% glucose 及 0.06 mM M11CI2，pH 7.5)中，於 37°C培養48小時後，可提升菌體之密度及γ-聚麩胺酸。請 參照表一，為不同培養基對本發明之枯草桿菌PGA1的生 長及聚麩胺酸產量的影響。所有的菌株皆於37°C下培養 48小時。 ❹ 表一、不同培養基對本發明之聚麩胺酸生產株之生長及 γ-PGA產量的影響 聚麩胺酸生產 株 培養基 菌量（A600) γ-PGA產量 (g/L) A LB 1.68 4.75 Medium A 9.13 55.25 B LB 1.40 4.75 Medium A 9.51 53 由表一可看出將本發明之枯草桿菌PGA1接種於 Medium A。中，可提升菌體之密度及y -聚麩胺酸之產量。 菌體培養48小時後之τ -聚麩胺酸產量可達50 g/L以上。 由這些結果顯示，本發明利用嵌入性策略將聚麩胺酸合成 酶複合體結構基因上游之DNA序列置換為持續型強表現 元件可使枯草桿菌轉形株（枯草桿菌PGA1)生產τ -聚麩 胺酸，配合MediumA可進一步提升r-聚麩胺酸之產量。 雖然本發明已以實施例揭露如上，然其並非用以限定 本發明，任何熟習此技藝者，在不脫離本發明之精神和範 12 200932911 圍内，當可作各種之更動與潤飾，因此本發明之保護範圍 當視後附之中請專利範圍所界定者為準。 【圖式簡單說明】 為讓本發明之上述和其他目的、特徵、優點與實施例 能更明顯易懂，所附圖式之詳細說明如下： 第1圖為本發明之帶有部分少wsCDNA片段之敌入型 ® 載體P177-PGAI的基因排列示意圖。 第2圖為質體P177-PGAI與宿主染色體發生單一交又 後的基因排列示意圖。 【主要元件符號說明】 無 〇 13200932911 IX. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a polyglutamic acid producing strain and a method for producing polyglutamic acid using a polyglutamic acid producing strain, and in particular to a kind of polyglycolic acid Production method of glutamic acid Bacillus subtilis strain and γ-poly glutamic acid [Prior Art] ® Poly-Y-glutamic acid (PGA) is an unusual anionic polypeptide that consists of D-type and L-type glutamic acid or all-type L-type glutamine. The acid is formed by polymerization of hydrazine:-amine group and γ-hydroxy group. This substance was first discovered in the capsule of 5. The successive studies found that the specific species of the genus Bacillus such as 5. var. natto anti-B. licheniformis will also bombard this substance. The synthesis of polyglutamic acid is related to the PGA operon, which exhibits a polyglutamic acid synthase complex. This enzyme complex φ body is located on the cell membrane, and PGA can be synthesized in the presence of adenosine triphosphate (ATP) and glutamic acid. According to whether the presence of glutamic acid is required in the production of PGA, the Bacillus species producing PGA can be divided into two groups, one group is a strain that requires glutamic acid to produce, such as 5·ATCC9945A, and the other group is not. A strain that can be produced by glutamic acid such as B. licheniformis A35. In industrial applications, polyglutamic acid and its derivatives can be used in food, livestock, cosmetics, etc. because of their non-toxic, water-soluble, edible, biodegradable, polyanonic 200932911 and moisturizing properties. In the fields of medicine and environmental protection, researchers are also working to screen strains of high-glutamic acid production, explore optimal fermentation and purification conditions, and develop new uses of polyglutamic acid and its derivatives. SUMMARY OF THE INVENTION Accordingly, the present invention is directed to a method for producing a polyglutamic acid producing strain and a polyglutamic acid producing strain for producing polyglutamic acid. ^ Polyglycine production strain (Bacillus subtilis PGA1; PGA1) is derived from Bacillus subtilis 168 derivative DB430, with a continuous strong performance element SECS-3 embedded to / / 168 derivative DB430 chromosome polyglutamic acid The synthesis-related gene has less wsC and upstream, and has the ability to produce T-poly glutamic acid. Bacillus subtilis PGA1 is deposited with the Food Industry Development Institute (FIRDI) under the number BCRC910381. The method for producing polyglutamic acid using Bacillus subtilis PGA1 comprises culturing Bacillus subtilis PGA1 in a specific culture solution, and isolating the τ-Ο polyglutamic acid secreted into the culture solution by alcohol precipitation. According to the above, the Bacillus subtilis PGA1 of the present invention utilizes the embedded strong expression element SECS-3 in the upstream of the polyglutamate synthesis-related gene ywiC: and the formulation of the culture solution proposed by the present invention can increase the cell density and The yield of polyglutamic acid can be a large amount of r-polyglutamic acid in the presence of glutamic acid in the environment, which has the industrial utilization value of rapid and simple production. 6 200932911 [Embodiment] Since the genome sequence of B. subtilis 168 has been sequenced, it is found that the gene ywiC associated with polyaminic acid synthesis in the 5·168 gene body and the PGA cannot be produced in the 168 gene, speculating 5.168 The reason for the inability to produce PGA is due to the inability to effectively express the polyglutamate synthase complex. The strategy of the embodiment of the present invention is to use 168 derivatives DB430 (He, XS, Shyu, YT, Nathoo, S., Wong, SL, and Doi, RH © 1991. Construction and use of a Bacillus subilis mutant deficient In multiple protease genes for the expression of eukaryotic genes. Ann. NY Acad. Sci. 646: 69-77.) in the chromosome;; wsC upstream DNA sequence replaced by synthetic continuous strong performance element SECS-3, SECS- The sequence of 3 is shown as SEQ ID ΝΟ:1. SECS-3 consists of UPve#(TCTTAACGTTGATATAATTTAAATT) + synthetic strong promoter (TTGACA-17nt-TATAAT) + SDl sequence (AGTGAGGTG), which is composed of the current Opt SD2 sequence (AAAGGAGG), which can be used to make a large number of Bacillus subtilis DB430 by SECS-3. It is a polyglycine synthesis-related enzyme and becomes a PGA production strain. According to an embodiment of the present invention, a polyglutamic acid producing strain (Bacillus subtilis PGA1; PGA1) is firstly constructed with an embedded vector having a partial ywiC DNA fragment, and then introduced into the Bacillus subtilis by a competent cell transformation method. DB430. Referring to Fig. 1, there is shown a schematic diagram of the gene arrangement of the embedded vector P177-PGAI with a partial DNA fragment of the present invention. This DNA fragment 7 200932911 contains a sustained strong expression element SECS-3, a partial polyglutamic acid synthase complex, a small gene fragment, a terminator, and a T1 terminator °. According to an embodiment of the present invention, the vector P177 - PGAI is a partially glutamate synthetase complex using Pseudomonas aeruginosa DNA as a template and a specific primer combination PpgaNdelF (SEQ ID NO: 2) and PpgaSallR (SEQ ID NO: 3) Amplification of DNA fragments. The amplified polymerase chain reaction product was digested with restriction enzymes iW/el and Sfl/I, and the DNA fragment was ligated with the same restriction enzyme with the SECS-3 cleavage using T4 DNA ligase (ligase). In pSECS-3GFPT, the fragment containing part of the polyglutamate synthetase complex ywsC DNA was located downstream of SECS-3, and then the adhesive product was electrotransformed into Escherichia coli JM109, and the transformed strain was randomly selected to extract the plastid. Restriction enzyme cleavage and DNA sequencing confirmation. The plastid with the correct DNA sequence was named pSECS-3PGAI. PSECS-3PGAI recovers a DNA fragment of about 0.4 Kb with restriction enzyme 5g/II and after cleavage. This DNA fragment contains a persistent strong expression element SECS-3, a partial polyglutamic acid homologous to the host chromosome. Synthetic enzyme complex with less wiC gene fragment, λί0 terminator and T1 terminator. This DNA fragment was ligated into the plastid pl77 with the same restriction enzyme by T4 DNA ligase, and the plastid pl77 was matched with the Bacillus subtilis host for high efficiency. The plastid-host transfer system, the plastid pl77 carries an anti-antibiotic gene as a selective marker, and the pi 77 is not replicable in the B. subtilis host. The adhesive product was electrotransformed into E. coli JM109 8 200932911. Randomly selected transgenic plants were used to extract their plastids for restriction enzyme cleavage and DNA sequencing confirmation. The plastid with the correct DNA sequence was named P177-PGAI. The persistent strong expression element SECS-3, part of the polyaminan synthase complex gene, λί0 terminator and Τ1 terminator gene fragment on the plastid P177-PGAI can be used for DNA fragmentation during competent cell transformation. It is characterized in that a partial gene fragment homologous to the host chromosome is single-crossed with a gene on the host chromosome, and a gene is recombined on a small gene on the host chromosome, and is inserted into the chromosome. The preparation step of Bacillus subtilis competent cells was as follows: JM109 (pl77-PGAI) single colonies were picked and inoculated in lb (Luria-Bertani) medium, and shake culture was carried out at 37 ° C and 150 rpm. After overnight incubation, an appropriate amount of bacterial solution was inoculated into fresh SpC medium (20 ml T base, 0.2 ml 50% (W/V) glucose, 0.3 ml 1.2% (W/V) MgS〇4.7H2〇, 0.4 ml 10% ( W/V) Bacto yeast extract, 0.5 ml 〇1% (W/V) casamino acids ; T base : 0.2 % (NH4)2S04 ' 1.83 % ΚΗ2Ρ〇4·3Η20'0.6% KH2PO4 ' 0.6% Trisodium citrate 2.0%. In the case, the initial bacterial solution concentration was adjusted to A600 = 0.1. Then, shake culture was carried out at 37 ° C and 180 rpm. When the growth phase entered the stationary phase from the log phase, the culture solution was inoculated into freshly prepared SPII medium (SPII: 200 ml T base, 2 ml 50% (W). /V) glucose, 14 ml 1.2% (W/V) MgS04.7H20, 2 ml 1% (W/V) casamino acids, 2 ml 10% (W/V) bacto yeast extract, 1 ml 0.1 M CaC12 ; T Base : 9 200932911 0.2 % (NH4)2S04, 1.83 % ΚΗ2Ρ〇4_3Η20, 0.6% ΚΗ2Ρ〇4, 0.6% Trisodium citrate 2.0%.), the volume of the culture medium added is 1/10 of the volume of the SPII medium at 37°. C, shaking culture at 120 rpm, culture until the concentration of the bacterial solution is about A_=0.3~0.4, collect the cells by centrifugation (6000 X g, 10 minutes) and retain the supernatant. The supernatant contains the competent and sporulation factors. (competence and sporulation factor), can induce the occurrence of competent state. The cells were suspended in an appropriate amount of supernatant and glycerin, and then uniformly mixed, and then dispensed (0.5 mL/tube), and rapidly frozen with liquid nitrogen, and stored at φ at -70 °C for use. In the DNA transformation test, the competent cells were taken out from the -8 (TC refrigerator), rapidly thawed in a 37 °C water bath, and an equal volume of SPIIE was added, and then mixed with an appropriate amount of plastid pl77-PGAI at 37 ° C. The vibrating culture (180 rpm) was carried out. After culturing for 30 minutes, SB medium (3.5% tryptone, 2.0% yeast extract, 0.5% NaCl, pH 7.5 °) was added to continue to oscillate at 37 ° C and 180 rpm. After culturing for a few hours, the cells were collected by centrifugation (10000 X g, 10 minutes), and applied to a medium containing an appropriate amount of anti-Q. sin. The cells were allowed to stand at 37 ° C for 16 to 24 hours to observe. Results. Please refer to Figure 2 for a schematic diagram of the gene arrangement of the 鳢 pl77-PGAI and the host chromosome. When P177-PGAI successfully single-cross over the host chromosome, the gene recombination The anti-antibiotic gene (Cmr) of P177-PGAI will be inserted into the chromosome and confer the ability of the transgenic strain to resist chloramphenicol. The transformed strain can be screened by the chloramphenicol-containing medium. Continuous pear strong performance component 200932911 SECS-3 It is located upstream of the polyglutamate synthesis-related gene ywsC and: to promote the expression of the ywsC and genes of the polyaminic acid synthesis gene, so that the transgenic strain has the ability to produce 7-poly glutamic acid, and the transformation The colonies of the strains will have the appearance of mucoid. A variety of transgenic plants with mucous traits were randomly selected, and the chromosomal DNA of the cells was extracted. It was confirmed by PCR that these strains were indeed plastid P177-PGAI embedded in polyglutamate. Acid production strain. The polyglutamic acid production strain is named Bacillus subtilis PGA1 (5acz7/ws PGAI) and deposited in the Food Industry Development Research Institute (FIRDI), No. BCRC910381. Further, Bacillus subtilis PGA1 is cultured in LB liquid medium. After incubation at 37 ° C for 12 and 24 hours, the supernatant was collected, and the γ-poly glutamic acid was separated from the culture solution by the characteristic of γ-poly glutamic acid precipitated by alcohol. The culture solution was centrifuged (21910 X g, 15 After a minute, take out the appropriate amount of supernatant, place it in a microcentrifuge tube and add 4 volumes of ice-cold ethanol. After mixing evenly, the cotton-like substance is produced, which is the crude polyglutamic acid. At 4 ° C Placement After centrifugation (21910 X g, 15 minutes) and removing the supernatant ©, the precipitate is dried at room temperature to obtain γ-poly glutamic acid. According to another embodiment of the present invention, a similar The step is to carry out a large amount of separation of γ-polyglutamic acid, the difference is that the culture solution is titrated to about pH 3 with an acid (for example, hydrochloric acid) before centrifugation, and then centrifuged at 5000 X g for 30 minutes, and then similar to the foregoing. The step of obtaining γ-poly glutamic acid. The production of γ-poly glutamic acid by Bacillus subtilis PGA1 was observed by protein electrophoresis. The results showed that the yield of γ-poly glutamic acid was higher in the transgenic plants after 24 hours of culture. 200932911 According to another embodiment of the present invention, Bacillus subtilis PGA1 is cultured in a Medium A (3.3% tryptone, 2.0% yeast extract, 0.74% NaCl, 0.8% Na2HP04, 0.4% KH2P〇4, 2.0% casamino acids, 1.0%). Glucose and 0.06 mM M11CI2, pH 7.5), after incubation at 37 ° C for 48 hours, can increase the density of the cells and γ-poly glutamic acid. Please refer to Table 1 for the effect of different media on the growth of the Bacillus subtilis PGA1 of the present invention and the yield of polyglutamic acid. All strains were cultured for 48 hours at 37 °C. ❹ Table 1. Effect of different media on the growth and γ-PGA yield of the polyglutamic acid producing strain of the present invention. Polyglutamate producing strain medium amount (A600) γ-PGA yield (g/L) A LB 1.68 4.75 Medium A 9.13 55.25 B LB 1.40 4.75 Medium A 9.51 53 It can be seen from Table 1 that the Bacillus subtilis PGA1 of the present invention was inoculated to Medium A. The density of the cells and the yield of y-poly glutamic acid can be increased. After 48 hours of cell culture, the yield of τ-poly glutamic acid can reach 50 g/L or more. These results show that the present invention utilizes an intercalation strategy to replace the DNA sequence upstream of the polyglutamate synthase complex gene into a sustained strong expression element to enable the production of τ-polygluten by Bacillus subtilis transformant (Bacillus subtilis PGA1). Amino acid, combined with MediumA, can further increase the yield of r-poly glutamic acid. Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention, and the present invention can be variously modified and retouched without departing from the spirit of the present invention and the scope of the present invention. The scope of protection shall be subject to the definition of patent scope in the attached annex. BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; Schematic diagram of the gene arrangement of the enemy type vector P177-PGAI. Figure 2 is a schematic diagram showing the arrangement of genes after plastid P177-PGAI and host chromosomes are single-crossed. [Main component symbol description] None 〇 13