TW200951220A

TW200951220A - Method of bioconversion of glutamate by glutamate decarboxylase from molecular gene cloning organisms for enrichment of GABA (γ-aminobutyric acid)

Info

Publication number: TW200951220A
Application number: TW97120392A
Authority: TW
Inventors: Hung-Chien Chien; Allen Chien
Original assignee: Hung-Chien Chien
Priority date: 2008-06-02
Filing date: 2008-06-02
Publication date: 2009-12-16
Also published as: TWI454576B

Abstract

A kind of substance containing glutamate can be offered a kind of enzyme to produce GABA (γ -aminobutyric acid). It is including a recombinant technology to bioproduce the glutamate decarboxylase (GAD3). A kind of substance containing glutamate was decarboxylated to GABA by glutamate decarboxylase.

Description

200951220 九、發明說明：【發明所屬之技術領域】利用含有糙胺酸（glutamate)的物質，以麵胺酸脫羧基酶 (glutamate decarboxylase; GAD3 )生合成 GABA(y-胺基丁酸) 的一種方法，包括：利用轉殖重組基因方法來生產麩胺酸脫羧基酶；該麩胺酸脫羧基酶可將物質中之麩胺酸 (glutamate)，經去叛基後產生GABA。【先前技術】 ❹ 文獻所記載的生產GABA(Y-胺基丁酸)如United States200951220 IX. Description of the invention: [Technical field of the invention] A method for synthesizing GABA (y-aminobutyric acid) by using glutamate decarboxylase (GAD3) using a substance containing glutamate The method comprises the steps of: producing a glutamic acid decarboxylase by using a recombinant recombinant gene method; and the glutamic acid decarboxylase can produce GABA after decarboration. [Prior Art] ❹ The production of GABA (Y-aminobutyric acid) as reported in the literature, such as United States

Patent 20020106424 ( γ -Aminobutyric Acid -containing natural food material and methold for manufacturing the same)及 J.S.Patent 20020106424 ( γ -Aminobutyric Acid - containing natural food material and methold for manufacturing the same) and J.S.

Tsai, Y.S. Lin, B.S. Pan and T.J. Chen, Biochemistry Volume 41. *Tsai, Y.S. Lin, B.S. Pan and T.J. Chen, Biochemistry Volume 41. *

Issue 6, June 2006, Pages 1282-1288. (Antihypertensive peptides and γ-aminobutyric acid from prozyme 6 facilitated lactic acid bacteria fermentation of soymilk)是應用自然界之微生物本身就有麵胺酸脫敌基酶glutamate decarboxylase ( GAD )之天然酵素將大自然食物中之麵胺酸（glutamate)，分解轉化為γ -aminobutyricacid，增加食品之營養價值。本發明係利用轉殖重組基因方法來生產新的麵胺酸脫敌基酶glutamate decarboxylase (GAD3)，不僅用於食品也可用於工業上大量生產 GABA 〇麵胺酸脫竣基酶 glutamate decarboxylase ( GAD3 )能將 200951220 glutamate上的羧基去除’形成GABA(广胺基丁酸）。它的受質以及產物皆是人體的神經傳導物質。人體若缺乏了 GAD貝,】無法正常產生GABA來抑制神經分泌g 1 utamate產生的衝動，病變形成過度緊張、癲癇症、躁鬱症、狂躁症、失眠、末端顫抖或是帕金森氏症甚至是老年癡呆症。【發明内容】本研九成功將根據NCBI上為⑽·㈣/沿orjFzoe新妨 ❹ glutamate decarboxylase GAD3的基因序列設計出的引子 (primer) ’ 經由 genetic engineering 選殖 6>/7么從教的 glutamate decarboxylase GAD3 基因（大小 1，557 bp) ’在具有強勢的起動子(pr〇m〇ter)的表現載體上，大量轉錄目標基因，緊接轉譯出的蛋白和網路來源資料庫相似性高達 97%的基因序列，並成功選殖入pQE3〇表現載體轉殖進入五 coZ/NovaBlue。經過使用IPTG的誘導— promoter後，這些 ❹ transformants將這段基因完整的轉譯出來，並帶有6個HisTag 以利使用Co -NTA affinity column來將酵素純化，最後再自行改良 Jacobs, W· A.所提出的〇-phathaldehyde-sulf ite derivation活性測定法，成為快速的酵素活性定量測定法，發現的 glutamate decarboxylase GAD.3 為一種嶄新且優良有效的GABA生產酵素。目前正使用 glutamate decarboxylase GAD3 開發工業化生產 GABA 產程。本發明主要分析出glutamate decarboxylase GAD3的酵素 6 200951220 特性’其中酸鹼環境、離子需求、受質抑制、產物抑制甚至探討到glutamate decarboxylase GAD3對於其他胺基酸受質的催化性、對於味精催化性，以利日後學術界酵素結構分析以及活性區分析有所幫助，甚至推動GABA在未來生機發酵大置瓜ie的可3b性或疋早純的GAD次級產物製造，希望能為精神患者有所幫助。【實施方式】 ❹ Glutamate decarboxylase GAD3 可以將 glutamate 上的鲮基去除，形成GABA。它的受質以及產物皆是人體的神經傳導物質glutamate ’以及神經傳導抑制劑GABA (γ_胺基丁酸， γ-aminobutyric acid)(圖 1)。本研究抽取chromosome DNA，以洋菜膝體電泳做檢測。此次所抽Chr〇m〇S〇me DNA其〇1>260 = 1. 855 ’ 0D28。= 0. 88卜 OD32〇=〇. 〇41，〇D26〇 / 〇D28。的比值為 © 2. 106 ’大於數值1. 8很多，代表DNA濃度很高，含有少許的 RNA(或沒含RNA );而OD32〇 / 0]½的比值為〇. 〇22，代表所 S的_類的量很少或沒有，DNA很純。核酸對波長260 nm之 0. D·值為1時，雙股DNA的濃度為5〇 / ml，故本研究所抽取的 chromosome DM 濃度為 6, 183 / ml ’而DNA長度遠遠大於3. 〇 Kb。利用也遍1限制柄酵素處理後的s 染色體DNA為模板，加入前端帶有万棚{ji限制酶酵素切位與後 200951220 端帶有兌/1限制酶酵素切位的引子，混合置入PCR(聚合酶連鎖反應）反應中，夾取出glutamate decarb〇Xyiase GAD3 基因（圖3)，確定NCBI公佈片段存在，且作為接下來轉殖入厶co/乂 NovaB 1 ue大腸菌品系所用。依照glutamate decarboxylase GAD3基因序列，在基因 ORF的起始與終止之位置分別設計一條含有不同限制酶切位的引子。利用聚合酶連鎖反應，將glutamate decarboxylase GAD3基因由基因體上大量複製出來，經限制梅剪切後，選殖入PQE30表現載體(Qiagen c〇)。再轉形入五.cc>/z.Issue 6, June 2006, Pages 1282-1288. (Antihypertetic peptides and γ-aminobutyric acid from prozyme 6 facilitated lactic acid bacteria fermentation of soymilk) is the application of natural microbes themselves to the glutamate decarboxylase (GAD) The natural enzyme decomposes glutamate in natural foods into γ-aminobutyric acid, increasing the nutritional value of food. The invention utilizes the method of transgenic recombinant gene to produce a new glutamate decarboxylase (GAD3), which can be used not only in food but also in industrial production of GABA glutamate decarboxylase (GAD3). ) The carboxyl group on 200951220 glutamate can be removed to form GABA (polyaminobutyric acid). Its receptors and products are the nerve conduction substances of the human body. If the human body lacks GAD shell, it cannot produce GABA normally to inhibit the impulse of neuronal secretion of g 1 utamate, excessive tension, epilepsy, bipolar disorder, mania, insomnia, terminal tremor or Parkinson's disease or even old age. Dementia. [Summary of the Invention] This research will succeed in designing a primer based on the gene sequence of glutamate decarboxylase GAD3 on the NCBI (10)/(iv)/orjFzoe. The genetic glutamate is taught by genetic engineering 6/gt; Decarboxylase GAD3 gene (1,557 bp in size) 'On the expression vector with a strong promoter (pr〇m〇ter), a large number of target genes are transcribed, and the similarity between the translated protein and the network source database is as high as 97 % of the gene sequence was successfully cloned into the pQE3〇 expression vector for propagation into five coZ/NovaBlue. After using IPTG induction-initiator, these ❹ transformants completely translated the gene and carried six HisTags to purify the enzyme using the Co-NTA affinity column, and finally improved Jacobs, W·A. The proposed 〇-phathaldehyde-sulfite derivation activity assay became a rapid quantitative assay for enzyme activity, and the glutamate decarboxylase GAD.3 was found to be a new and excellent GABA producing enzyme. The industrial production of GABA is currently being developed using glutamate decarboxylase GAD3. The invention mainly analyzes the enzyme 6 of the glutamate decarboxylase GAD3 200951220 characteristic 'in which the acid-base environment, ion demand, substrate inhibition, product inhibition or even the glutamate decarboxylase GAD3 catalytic effect on other amino acid acceptors, for MSG catalytic properties, Eli's future academic enzyme structure analysis and active area analysis have helped, and even promoted the production of GAB secondary products of GABA in the future, which is expected to be helpful for mental patients. . [Examples] ❹ Glutamate decarboxylase GAD3 can remove sulfhydryl groups on glutamate to form GABA. Its receptors and products are the human neurotransmitter glutamate ‘and the neurotransmitter GABA (γ-aminobutyric acid) (Fig. 1). In this study, chromosome DNA was extracted and tested by electrophoresis of Chinese cabbage. The CHr〇m〇S〇me DNA extracted this time was 〇1>260 = 1. 855 ′ 0D28. = 0. 88卜 OD32〇=〇. 〇41, 〇D26〇 / 〇D28. The ratio is © 2. 106 'greater than the value 1. 8 is a lot, representing a high DNA concentration, containing a little RNA (or no RNA); and the ratio of OD32〇 / 0]1⁄2 is 〇. 〇22, representing the S The amount of _ class is small or absent, and the DNA is pure. The nucleic acid has a wavelength of 260 nm. When the D· value is 1, the concentration of the double-stranded DNA is 5〇/ml, so the concentration of the chromosome DM extracted in this study is 6,183 / ml ' and the DNA length is much larger than 3. 〇Kb. The s chromosomal DNA after the restriction of the enzyme treatment was used as a template, and the primer with the cut-off of the ji shackle and the cut-off of the restriction enzyme enzymatic enzyme at the end of 200951220 was added to the PCR. In the (polymerase chain reaction) reaction, the glutamate decarb〇Xyiase GAD3 gene was extracted (Fig. 3), and the NCBI published fragment was confirmed and used as the next transgenic 厶co/乂NovaB 1 ue coliform strain. According to the glutamate decarboxylase GAD3 gene sequence, a primer containing different restriction enzyme sites was designed at the start and end of the gene ORF. Using the polymerase chain reaction, the glutamate decarboxylase GAD3 gene was largely reproduced from the genome, and after restriction of the plum, it was selected into the PQE30 expression vector (Qiagen c〇). Then turn into five.cc>/z.

NovaBlue ’以表現出N端帶有六個Histidine的glutamate decarboxylase GAD3(圖 2)。利用 Viogene Gel-MTM Gel extraction system 套組 (Viogene，California，U.S. A.)進行 DNA 片段的回收及純化PCR後的產物，以及pQE 30大腸菌選殖載體，經過兩限制酶(及洲1、&/1)處理後’ligati〇n前的準備確定（圖4)。經過 Transformation 進入 NovaBlue 帶有 glutamate decarboxylase GAD3基因之PQE30載體（圖5)，從篩選出的 transformants中純化出來再以兩限制酶(万棚hi、兔/ j)處理後’確定選殖成功，以供定序進一步確定（圖6)。以上 ’ 所選殖到的 giutamate decarboxylase GAD3 基因’其〇RF ( Open Reading Frame ) 200951220 全長有1，557 bp。將定序完成後轉譯出的胺基酸序列利用 ExPASy網站上的軟體，計算結果顯示，glutamate decarboxylase GAD3分子量為56 kDa來自第8號 transformant定序出1，557 bp與NCBI提供之原序列相似度尚達97%，且不帶有***子intr〇n的一段基因（圖11)。在純化與回收glutamate decarboxylase GAD3前，確定各成功的transformants具有蛋白表現，利用sonicatiori超因波震盈處理破函後’將其蛋白與未選殖入glutamate decarboxylase GAD3基因的空pQE 30做對照，已確定其中的差異性’最後仍選出第8號transformants作為往後實驗用。接著在 glutamate decarboxylase GAD3基因的N端額外加入6個histidine的DNA序列，利用 TALON metal affinity resins ( CLONTECH Laboratories， Inc.)純化 glutamate decarboxylase GAD3，將回收的蛋白液經 SDS-PAGE 分析結果得知，glutamate decarboxylase GAD3蛋白質大小約56 kDa (圖7)。使用Bio-rad assay债測 0D595的結果，將吸光值帶入BSA蛋白濃度標準曲線換算，可以得到2. 21 /zg/# 1的純酵素。此酵素準備活性測定。製作實驗組1倍酵素、2倍酵素以及對照組熱殺死酵素、無加入酵素、無反應受質等五組做下列比較實驗，掃描330 nm 至780 nm光波，繪出曲線後比較各差別，結果發現，具有活 200951220 性酵素在藍綠光區〔630 nm〕具有顯著吸光，表示giutamate decarboxylase GAD3 催化 glutamate 後將其 carb〇Xyi-Gr〇up 釋出’使其溴甲酴綠原先酸性褐黃色偵測出鹼性的藍綠色，證明此酵素具有催化活性。 (〇-pha tha 1 d i a 1 dehyde/Su 1 f i t e)反應線性回歸標準曲線一配置20序列稀釋不同濃度GABA與0ΡΑ反應，製作測定330 nm 光波’其GABA與0PA的異構物吸收波。繪成反應初速度精確度高達R2=0. 9997的線性函數，且涵蓋超廣範圍，回歸了〇 “Μ 至2. 5 mM ’以利往後實驗所用。在16種共輕酸緩衝溶液65 mM (Citrate pH 3、Citrate pH 4、Acetate pH 4、Acetate pH 5、Acetate pH 5. 5、Acetate pH 6、Phophate pH 6、Phophate pH 6. 6、Phophate pH 7、 Phophate pH 8、Borate pH 8、Borate pH 8. 5、Borate pH 9、 Clark & Lubs pH 9、Clark & Lubs pH 9. 5、Clark & Lubs pH 10)進行 glutamate decarboxylase GAD3 催化 L-glutamate 成為GABA，用330 nm吸收光偵測其GABA與0ΡΑ的異構物吸收波。得到比較催化效率與各pH曲線，發現最適酸鹼環境65 mM phosphate buffer pH 6. 6 最好。最適緩衝液 65 mM phosphate buffer pH 6. 6 中， glutamate decarboxylase GAD3反應於8種不同溫度底下（30 200951220 °C, 31.3 °C, 33.8 °C, 37.4 °C, 42.2 °C, 45.9 °C, 48 4 °C，50 °C)，用330 nm吸收光偵測其GABA與OPA的異構物吸收波。得到催化效率與溫度關係，發現37°C為最適 glutamate decarboxylase GAD3 催化溫度。本反應於7種不同金屬鹽類環境50 (C0CI2，CaCL·NovaBlue's showed glutamate decarboxylase GAD3 with six Histidines at the N-terminus (Fig. 2). DNA fragment recovery and purification of the PCR product using the Viogene Gel-MTM Gel extraction system kit (Viogene, California, USA), and the pQE 30 coliform selection vector, after two restriction enzymes (and continent 1, & / 1 After the processing, the preparation before the 'ligati〇n is determined (Fig. 4). After Transformation, enter the NovaQ PQE30 vector carrying the glutamate decarboxylase GAD3 gene (Fig. 5), and purify it from the selected transformants and then treat it with two restriction enzymes (Wanpeng hi, rabbit/j) to determine the success of the selection. The sequencing is further determined (Figure 6). The above-mentioned giutamate decarboxylase GAD3 gene was selected as its RF (Open Reading Frame) 200951220 with a total length of 1,557 bp. The amino acid sequence translated after the sequencing was completed using the software on the ExPASy website, and the results showed that the molecular weight of glutamate decarboxylase GAD3 was 56 kDa from the No. 8 transformant sequenced 1,557 bp and the original sequence similarity provided by NCBI. It is 97%, and does not have a gene inserted into the intr〇n (Fig. 11). Before purifying and recovering glutamate decarboxylase GAD3, it was determined that each successful transformant had protein expression, and after using the sonicatiori super-wave shock treatment to break the letter, 'the protein was compared with the empty pQE 30 which was not selected into the glutamate decarboxylase GAD3 gene. Determine the difference in it's final selection of the number 8 transformants for later experiments. Then, 6 DNA sequences of histidine were additionally added to the N-terminus of the glutamate decarboxylase GAD3 gene, and glutamate decarboxylase GAD3 was purified by TALON metal affinity resins (CLONTECH Laboratories, Inc.). The recovered protein solution was analyzed by SDS-PAGE, and glutamate was obtained. The decarboxylase GAD3 protein is approximately 56 kDa in size (Figure 7). Using the results of the Bio-rad assay debt measurement 0D595, the absorbance value was taken into the standard curve of the BSA protein concentration to obtain a pure enzyme of 2.21 /zg/#1. This enzyme is prepared for activity assay. The experimental group 1 times of enzyme, 2 times of enzyme and the control group of heat-killing enzyme, no enzyme, no reaction, and other five groups were used to do the following comparison experiments. The light waves from 330 nm to 780 nm were scanned, and the curves were compared to compare the differences. It was found that the active 200951220 sex enzyme had significant absorbance in the blue-green region [630 nm], indicating that the giutamate decarboxylase GAD3 catalyzes the glutamate and releases its carb〇Xyi-Gr〇up' to make its bromoformine green acid yellow brown. An alkaline blue-green color was detected, demonstrating that the enzyme is catalytically active. (〇-pha tha 1 d i a 1 dehyde/Su 1 f i t e) Reaction Linear Regression Standard Curve 1 Configure a 20-sequence dilution of different concentrations of GABA and 0ΡΑ to prepare an absorbance wave for the determination of the 330 nm light wave's GABA and 0PA. The linear function of the initial velocity of the reaction is as high as R2 = 0.999, and covers a wide range, and the regression is 〇 "Μ to 2.5 mM ' for later experiments. In 16 kinds of light acid buffer solution 65 mM (Citrate pH 3, Citrate pH 4, Acetate pH 4, Acetate pH 5, Acetate pH 5. 5, Acetate pH 6, Phophate pH 6, Phophate pH 6.6, Phophate pH 7, Phophate pH 8, Borate pH 8, Borate pH 8. 5, Borate pH 9, Clark & Lubs pH 9, Clark & Lubs pH 9. 5, Clark & Lubs pH 10) glutamate decarboxylase GAD3 catalyzes L-glutamate to GABA, absorbs light at 330 nm The absorption wave of GABA and 0ΡΑ isomers was measured. The catalytic efficiency and the pH curve were obtained. It was found that the optimum acid-base environment 65 mM phosphate buffer pH 6. 6 was the best. The optimum buffer was 65 mM phosphate buffer pH 6. 6 Glutamate decarboxylase GAD3 reacts under 8 different temperatures (30 200951220 °C, 31.3 °C, 33.8 °C, 37.4 °C, 42.2 °C, 45.9 °C, 48 4 °C, 50 °C), absorbs at 330 nm Light is detected by the absorption of isomers of GABA and OPA. Catalytic efficiency and temperature are obtained. Relationship, found that 37 ° C is the optimum glutamate decarboxylase GAD3 catalytic temperature. The reaction in 7 different metal salt environments 50 (C0CI2, CaCL·

NiCh，MgS〇4，C11SO4，ZnS〇4，FeS〇4)的 glutamate decarboxylase GAD3反應10分鐘在最適反應溫度37，用 © 330 nm吸收光偵測其GABA與OPA的異構物吸收波。設定無離子加入的對照組為100%，得到催化效率與不同金屬離子環境的百分比 Bar Chart 〇發現 glutamate decarboxylase GAD3 酵素不受金屬離子影響其活性。接者’分別在最適緩衝溶液(在此為65 mM phosphate buffer pH 6. 6)下溶入 5 mM 的 9 種不同受質（L-tyrosine, D-tyrosine, phenylalanine, tryptophan, lysine, ❹ para-hydroxybenzoate, leucine, glycine, raonosodium L-glutamate ) ’ 將此溶劑各與 glutamate decarboxylase GAD3反應10分鐘在最適反應溫度37 °C。OPA異構反應後，分光光度計下掃瞄200 nm至600 nm。劃出曲線觀察與未加入酵素催化的溶劑對照組作比較，發現以不同光波掃瞄來判定 glutamate decarboxylase GAD3具有催化該受質活性。比較各受質的實驗組與對照組之曲線異同後，發現5種受質 11 200951220 L-tyrosine , D-tyrosine , L-tryptophan, para-hydroxyl benzoate 和 mono-sodium L-glutamate 可被 glutamate decarboxylase GAD3 催化。不過 phenylalanine, lysine, leucine,和glycine四種受質無法被glutamate decarboxylase GAD3催化。圖中210 nm突幅peak不能判定為具有活性，因0ΡΑ已將其溶劑内容物異構苯環化，具有300 nm以上吸光，該210 nm突幅的peak以證實為保存glutamate ❹ decarboxylase GAD3溶劑中imidazole的吸光曲線，故排除之。本實驗調製4種不同廠牌【大王、味王、味全、味正(味丹）】的monosodium L-glutamate(味精），分別溶入最適緩衝溶液 65 mM phosphate buffer pH 6. 6，與 glutamate decarboxylase GAD3在最適反應溫度37 °C下反應10分鐘。在分光光度計下偵測330 nm光波，其GABA與0ΡΑ的異構物吸 ® 收波。此法晝出百分比Bar Chart後用以比較單純催化 L-glutamate對照組設定為100%，發現催化活性值最顯著的為味全味精（圖8)。不同濃度味全 MSG 對 glutamate decarboxylase GAD3 的受質抑制測定，配製不同濃度的味全MSG(37raM、10 mM、5 mM、 2. 5 mM、1. 25 mM)，分別溶於最適緩衝溶液65 mM phosphate buffer pH 6. 6，與 glutamate decarboxylase GAD3 反應 10 12 200951220 分鐘在最適反應溫度37 °C。在分光光度計下偵測330 nm光波’其GABA與0ΡΑ的異構物吸收波。收集各實驗組數據，繪成百分比曲線觀察MSG濃度上升時對酵素催化的活性影響。得到結果5 mM受質濃度為最佳（此部份設定為1〇〇%)，且符合以往實驗經驗。一旦高於5 mM便會出現受質抑制此酵素反應的結果，在14 mM濃度下明顯的發現glutamate decarboxylase GAD3 活性驟降高達 50%(圖 9)。接著’產物抑制作用測定中’配製不同濃度的gaba(ioThe glutamate decarboxylase GAD3 reaction of NiCh, MgS〇4, C11SO4, ZnS〇4, FeS〇4) was carried out for 10 minutes at an optimum reaction temperature of 37, and the absorption wave of the isomers of GABA and OPA was detected by absorption light at 330 nm. The control group with no ion addition was set to 100%, and the percentage of catalytic efficiency and different metal ion environments was obtained. Bar Chart 〇 It was found that the glutamate decarboxylase GAD3 enzyme was not affected by metal ions. The recipients were dissolved in 5 mM of 9 different receptors (L-tyrosine, D-tyrosine, phenylalanine, tryptophan, lysine, ❹ para- in the optimal buffer solution (here 65 mM phosphate buffer pH 6.6). Hydroxybenzoate, leucine, glycine, raonosodium L-glutamate ) ' The solvent was reacted with glutamate decarboxylase GAD3 for 10 minutes at an optimum reaction temperature of 37 °C. After the OPA isomerization reaction, the spectrum is scanned from 200 nm to 600 nm under a spectrophotometer. The curve observation was compared with the solvent-free control group without enzyme addition. It was found that glutamate decarboxylase GAD3 was catalyzed by different light scanning. Comparing the curves of the experimental group and the control group, it was found that 5 kinds of receptors 11 200951220 L-tyrosine, D-tyrosine, L-tryptophan, para-hydroxyl benzoate and mono-sodium L-glutamate can be glutamate decarboxylase GAD3 catalytic. However, the four substrates of phenylalanine, lysine, leucine, and glycine were not catalyzed by glutamate decarboxylase GAD3. In the figure, the 210 nm swell peak cannot be judged to be active. Since the oxime has been cyclized in the solvent content, it has a absorbance of more than 300 nm. The peak of the 210 nm swell is confirmed to be the solvent for preserving glutamate ❹ decarboxylase GAD3. The absorption curve of imidazole is excluded. This experiment modulates the monosodium L-glutamate (monosodium glutamate) of 4 different brands [Dawang, Weiwang, Weiquan, Weizheng (Wei Dan)], respectively dissolved into the optimal buffer solution 65 mM phosphate buffer pH 6. 6, and glutamate Decarboxylase GAD3 was reacted for 10 minutes at an optimum reaction temperature of 37 °C. The 330 nm light wave is detected by a spectrophotometer, and its GABA and 0ΡΑ isomers are absorbed. This method used a percentage of Bar Chart to compare the purely catalyzed L-glutamate control group to 100%, and found that the most significant catalytic activity value was the whole MSG (Fig. 8). Different concentrations of MSG were used to determine the inhibition of glutamate decarboxylase GAD3. Different concentrations of MSG (37raM, 10 mM, 5 mM, 2. 5 mM, 1.25 mM) were prepared and dissolved in the optimal buffer solution 65 mM. Phosphate buffer pH 6. 6, reacted with glutamate decarboxylase GAD3 10 12 200951220 min at an optimum reaction temperature of 37 °C. Under the spectrophotometer, the 330 nm light wave is detected as its GABA and 0 异构 isomer absorption wave. The data of each experimental group were collected, and a percentage curve was plotted to observe the effect of the enzyme concentration on the activity of the enzyme when the MSG concentration was increased. The result is that the 5 mM substrate concentration is optimal (this part is set to 1%) and is consistent with previous experimental experience. Once above 5 mM, the result of inhibition of this enzyme reaction was observed, and a significant drop in glutamate decarboxylase GAD3 activity was found to be as high as 50% at 14 mM (Fig. 9). Then in the 'product inhibition assay', different concentrations of gaba (io) were prepared.

mM、5 mM、2. 5 mM、1. 25 mM、OmM 對照組），並溶於 50 mM 的Tris buffer pH 8. 5中，與酵素在25°C作處理。處理完成的酵素’使用 65 mM phosphate buffer pH 6. 6 清洗 Molecular Cut-off l〇kDa Column (Milli-Q ) 3 次，使glutamate decarboxylase GAD3 中 50 mM Tris buffer pH 8. 5 換成 65 mMmM, 5 mM, 2.5 mM, 1.25 mM, OmM control), and dissolved in 50 mM Tris buffer pH 8.5, treated with enzyme at 25 °C. The treated enzyme was washed 3 times with 65 mM phosphate buffer pH 6. 6 Molecular Cut-off l〇kDa Column (Milli-Q), and 50 mM Tris buffer pH 8.5 in glutamate decarboxylase GAD3 was changed to 65 mM

Phosphate buffer pH 6. 6(此過程主要用意為去除殘餘 GABA)。將最後〉辰成 200 y 1 溶於 65 mM phosphate buffer pH 6. 6的酵素分別回收於Eppendorf中。Phosphate buffer pH 6. 6 (this process is primarily intended to remove residual GABA). The enzymes dissolved in 65 mM phosphate buffer pH 6. 6 were separately recovered in Eppendorf.

配置 5 mM L-glutamate 溶於 65 mM phosphate buffer pH 6. 6中，與處理過不同濃度的GABA的glutamate decarboxylase GAD3酵素進行催化1〇分鐘在最適反應溫度下 37 C °在分光光度計下偵測330 nm光波，其GABA與0PA的異構物吸收波（圖10)。整理實驗數據繪成百分比曲線，來探 13 200951220 討其酵素產物濃度提高時，對glutamate decarboxyl&se 的活性影響。將0 mM對照組設定為100%，可以觀察出卷酵素催化環境下，經5 mM GABA處理後酵素，發現活性高達坨以、可見得產物 GABA 對 glutamate decarboxylase GAD3 具有、酵素催化活性（圖10)。【圖式簡單說明】〇【主要元件符號說明】5 mM L-glutamate was dissolved in 65 mM phosphate buffer pH 6. 6 and catalyzed with glutamate decarboxylase GAD3 enzyme treated with different concentrations of GABA for 1 〇 at 37 ° C at the optimum reaction temperature under spectrophotometer The 330 nm light wave absorbs the wave of the GABA and 0PA isomers (Figure 10). The experimental data was compiled into a percentage curve to investigate the effect of glutamate decarboxyl&se on the increase in the concentration of the enzyme product. By setting the 0 mM control group to 100%, it was observed that the enzyme was treated with 5 mM GABA in a catalytic enzyme environment, and it was found that the activity of GABA was found to be catalyzed by glutamate decarboxylase GAD3 (Fig. 10). . [Simple description of the diagram] 〇 [Main component symbol description]

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Claims

200951220 、十、申請專利範圍： 1. 一種製造GABA ( r_胺基丁酸）的方法，包括； (a) 利用一轉殖重組基因方式來生產一麩胺酸脫羧基酶；以及 (b) 將該越胺酸脫叛基酶加入一麵胺酸以產生該gaba。 2. 如申請專利範圍第1項之方法，步驟(a)包含： (al)操取自一來之一韵基因； ❹ （a2)將該职说基因接上一特定質體； (a3)將該特定質體轉殖入一痔定穿主；以及 (a4)利用該特定瘃主生產該麩胺酸脫羧基酶。 • 3.如申請專利範圍第2項之方法，其中（a2)該特定質體係— • pQE30 質體。 4.如申請專利範圍第2項之方法，其中（a3)該#定瘃主係一大腸桿菌。 ❿ 5.如申凊專利範圍第2項之方法，更包含： (a5)以affinity column純化該麵胺酸脫羧基酶。 6. 如申請專利範圍第2項之方法，其中（al)該微^基因之序列，是不帶有***子intron的基因，其長度為1557^。 7. 如申明專利範圍第2項之方法’其中（a4)生產該麵胺酸脫幾基酶’其分子量為56 kDa。 8. 如申睛專利範圍第1項之方法，於步驟(b)包含： (bl)邊麵胺酸脫羧基酶將該楚胺酸去竣基，產生該Gaba ; 15 200951220 ⑽該GABA不會抑制該麵胺酸脫叛基酶的催化活性； (Μ)該紐酸最適濃度為5mM，但在高濃度下將使該麵胺酸脫羧基酶活性下降； (b4) 4 反應最適條件為 rc 及 pjj 6. 6 phosphate buf fer (65 mM);以及 (b5)該麩胺酸脫鲮基酶的比活性是15,〇〇〇 u/mg。 9.如申請專利範圍第8項之方法，其中（bl)該麩胺酸脫羧基酶可將其他氨基酸去除叛基，其中包含：L-tyrosine, D-tyrosine, L-tryptophan, para-hydroxylbenzoate, 和 mono-sodium L-glutamate ° 10.如申請專利範圍第8項之方法，其中（bl)該麩胺酸脫羧基酶的活性不受金屬鹽類如C0CI2，CaCh, NiCh，MgS〇4，CuS〇4, ZnS〇4，FeS〇4 的影響。200951220, X. Patent application scope: 1. A method for producing GABA (r-aminobutyric acid), comprising: (a) producing a glutamic acid decarboxylase by using a transgenic recombinant gene; and (b) The valine acid detrest enzyme is added to the monoamine to produce the gaba. 2. For the method of claim 1 of the patent scope, step (a) comprises: (al) taking one of the rhyme genes; ❹ (a2) attaching the job gene to a specific plastid; (a3) The specific plastid is transferred into a quinone; and (a4) the glutamic acid decarboxylase is produced using the particular steroid. • 3. For the method of claim 2, (a2) the specific quality system – • pQE30 plastid. 4. The method of claim 2, wherein (a3) the #定瘃主系-E. coli. ❿ 5. The method of claim 2, further comprising: (a5) purifying the face acid decarboxylase with an affinity column. 6. The method of claim 2, wherein (al) the sequence of the microgene is a gene without an insert intron having a length of 1557^. 7. The method of claim 2, wherein (a4) producing the face acid deaminase has a molecular weight of 56 kDa. 8. The method of claim 1, wherein in step (b): (bl) a facial acid decarboxylase deactivating the sulphate to produce the Gaba; 15 200951220 (10) the GABA does not Inhibition of the catalytic activity of the face acid detoxification enzyme; (Μ) The optimum concentration of the acid is 5 mM, but at a high concentration will reduce the activity of the face acid decarboxylase; (b4) 4 The optimal reaction condition is rc And pjj 6. 6 phosphate buf fer (65 mM); and (b5) the specific activity of the glutamate deaminase is 15, 〇〇〇u/mg. 9. The method of claim 8, wherein the (bl) glutamic acid decarboxylase degrades other amino acids, including: L-tyrosine, D-tyrosine, L-tryptophan, para-hydroxylbenzoate, And mono-sodium L-glutamate ° 10. The method of claim 8, wherein (bl) the activity of the glutamic acid decarboxylase is not affected by metal salts such as COCI2, CaCh, NiCh, MgS〇4, CuS 〇4, ZnS〇4, the effect of FeS〇4.