重组谷氨酸脱羧酶制备γ-氨基丁酸的工艺条件优化

doi:10.13560/j.cnki.biotech.bull.1985.2016.06.029

生物技术通报 ›› 2016, Vol. 32 ›› Issue (6): 199-204.doi: 10.13560/j.cnki.biotech.bull.1985.2016.06.029

重组谷氨酸脱羧酶制备γ-氨基丁酸的工艺条件优化

黄燕, 宿玲恰, 吴敬

江南大学食品科学与技术国家重点实验室生物工程学院工业生物技术教育部重点实验室，无锡 214122

收稿日期:2015-09-17 出版日期:2016-06-27 发布日期:2016-06-28
作者简介:黄燕，女，硕士研究生，研究方向：重组酶的制备和应用；E-mail：18206180508@163.com
基金资助:
国家杰出青年基金项目(31425020)，111计划(111-2-06)

Optimization of γ-aminobutyric Preparation by Recombinant Glutamate Decarboxylase

HUANG Yan, SU Ling-qia, WU Jing

State Key Laboratory of Food Science and Technology，School of Biotechnology and Key Laboratory of Industrial Biotechnology of Ministry of Education，Jiangnan University，Wuxi 214122

Received:2015-09-17 Published:2016-06-27 Online:2016-06-28

摘要/Abstract

摘要： 谷氨酸脱羧酶，一种磷酸吡哆醛(PLP)依赖性酶，能专一、不可逆地催化L-谷氨酸脱羧得到γ-氨基丁酸(GABA)。构建了产Lactobacillus brevis WJH3 谷氨酸脱羧酶重组大肠杆菌E.coli BL21(DE3)/pET-24a-gad，以此作为菌种进行摇瓶发酵诱导培养，发酵过程中一次性添加0.05 mmol/L PLP培养24 h，破壁上清酶活达81.7 U/mL，是不添加PLP对照酶活的1.8倍。对酶转化L-谷氨酸钠生成GABA反应条件进行了优化，结果表明，在转化体系不添加PLP的情况下，底物谷氨酸钠浓度为250 g/L，反应初始pH5.0，温度37℃，加酶量60 U/g 底物，转速200 r/min，在此条件下反应18 h，GABA转化率达到100%，为γ-氨基丁酸的工业化生产奠定基础。

关键词: 谷氨酸脱羧酶, 酶转化, 磷酸吡哆醛, 谷氨酸钠, γ-氨基丁酸

Abstract: Glutamate decarboxylase(GAD)，a pyridoxal 5'-phosphate(PLP)-dependent enzyme，irreversibly catalyzes the decarboxylation of L-glutamate to be the valuable food additive γ-aminobutyric acid(GABA). In this study，a recombinant Escherichia coli BL21(DE3)/pET-24a-gad producing Lactobacillus brevis WJH3 GAD was constructed as strain in the flask culturing of fermentation and induction. The activity of GAD produced in the supernatant of culturing for 24 h medium supplemented one-time with 0.05 mmol/L PLP was 81.7 U/mL，and this was 1.8-fold of that without PLP supplementation. Furthermore，the condition for GABA preparation by enzymatic conversion was optimized；under the condition of 250 g/L monosodium glutamate(MSG)，pH5.0，37℃，60 U GAD per gram substrate incubated for 18 hours，and rotation rate 200 r/min，100% of the MSG was transformed into GABA. These results establish the utility of PLP supplementation and lay the foundation for large-scale enzymatic production of GABA.

Key words: glutamate decarboxylase, enzymatic conversion, pyridoxal 5&apos, -phosphate, monosodium glutamate, γ-aminobutyric

黄燕, 宿玲恰, 吴敬. 重组谷氨酸脱羧酶制备γ-氨基丁酸的工艺条件优化[J]. 生物技术通报, 2016, 32(6): 199-204.

HUANG Yan, SU Ling-qia, WU Jing. Optimization of γ-aminobutyric Preparation by Recombinant Glutamate Decarboxylase[J]. Biotechnology Bulletin, 2016, 32(6): 199-204.

参考文献

[1]Manyam BV, Katz L, Hare TA, et al. Isoniazid-induced elevation of CSF GABA levels and effects on chorea in huntington’s disease[J]. Annals of Neurology, 1981, 10(1)：35-37.
[2]Wong T, Guin C, Bottiglieri T, et al. Gaba, γ-hydroxybutyric acid, and neurological disease[J]. Annals of Neurology, 2003, 54(S6)：S3-S12.
[3]Inoue K, Shirai T, Ochiai H, et al. Blood-pressure-lowering effect of a novel fermented milk containing γ-aminobutyric acid(GABA)in mild hypertensives[J]. European Journal of Clinical Nutrition, 2003, 57(3)：490-495.
[4]杨胜远, 陆兆新, 吕凤霞, 等. γ -氨基丁酸的生理功能和研究开发进展[J]. 食品科学, 2005, 26(9)：546-551.
[5]Meldrum BS. Epilepsy and y-aminobutyric acid-mediated inhibition[J]. Int Rev, Neurobiol, 1975, 17(1).
[6]Lammens TM, Franssen MCR, Scott EL, et al. Synthesis of biobased N-methylpyrrolidone by one-pot cyclization and methylation of γ-aminobutyric acid[J]. Green Chemistry, 2010, 12(8)：1430-1436.
[7]Saskiawan I. Biosynthesis of polyamide 4, a biobased and biodegradable polymer[J]. Microbiology Indonesia, 2008, 2(3)：5.
[8]Kawasaki N, Nakayama A, Yamano N, et al. Synthesis, thermal and mechanical properties and biodegradation of branched polyamide 4[J]. Polymer, 2005, 46(23)：9987-9993.
[9]Li H, Qiu T, Huang G, et al. Production of gamma-aminobutyric acid by Lactobacillus brevis NCL912 using fed-batch fermentation[J]. Microb Cell Fact, 2010, 9(9)：85.
[10]Plokhov AY, Gusyatiner MM, Yampolskaya TA, et al. Preparation of γ-aminobutyric acid using E. coli cells with high activity of glutamate decarboxylase[J]. Applied Biochemistry and Biotechnology, 2000, 88(1-3)：257-265.
[11] 范恩宇. 短乳杆菌谷氨酸脱羧酶基因的克隆与表达[D]. 杭州：浙江大学, 2010.
[12] Fan E, Huang J, Hu S, et al. Cloning, sequencing and expression of a glutamate decarboxylase gene from the GABA-producing strain Lactobacillus brevis CGMCC 1306[J]. Annals of Microbiology, 2012, 62(2)：689-698.
[13]朱孔亮. 泡菜用乳酸菌的筛选, 高密度培养及菌剂配方的研究[D]. 无锡：江南大学, 2014.
[14]Chen X, Su L, Wu D, et al. Application of recombinant Bacillus subtilis γ-glutamyltranspeptidase to the production of l-theanine[J]. Process Biochemistry, 2014, 49(9)：1429-1439.
[15] Gut H, Pennacchietti E, John RA, et al. Escherichia coli acid resis-tance：pH-sensing, activation by chloride and autoinhibition in GadB[J]. The EMBO Journal, 2006, 25(11)：2643-2651.
[16]吴晓燕, 钱绍松, 李加友, 等. 酶法制备 D-谷氨酸和 γ-氨基丁酸的工艺研究[J]. 化工进展, 2005, 24(8)：889-892.
[17]田灵芝, 徐美娟, 饶志明. 一株重组大肠杆菌/pET-28a-lpgad 的构建及其高效生产 γ-氨基丁酸转化条件的优化[J]. 生物工程学报, 2012, 28(1)：65-75.
[18]Binh TTT, Ju WT, Jung WJ, et al. Optimization of γ-amino butyric acid production in a newly isolated Lactobacillus brevis[J]. Biotechnology Letters, 2014, 36(1)：93-98.
[19]Shi X, Zheng B, Chang C, et al. Enzymatic Bioconversion for γ-Aminobutyric Acid by Lactobacillus brevis CGMCC No. 3414 Resting Cells[M]//Springer Berlin Heidelberg：Advances in Applied Biotechnology, 2015：609-617.
[20] 夏江, 梅乐和, 黄俊, 等. 产 γ-氨基丁酸的乳酸菌株筛选及诱变[J]. 核农学报, 2006, 20(5)：379-382.
[21] Li H, Qiu T, Huang G, et al. Production of gamma-aminobutyric acid by Lactobacillus brevis NCL912 using fed-batch fermentation[J]. Microb Cell Fact, 2010, 9(9)：85.
[22] 范恩宇. 短乳杆菌谷氨酸脱羧酶基因的克隆与表达[D]. 杭州：浙江大学, 2010.

重组谷氨酸脱羧酶制备γ-氨基丁酸的工艺条件优化

Optimization of γ-aminobutyric Preparation by Recombinant Glutamate Decarboxylase

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