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

作　　者：黄燕[1] 宿玲恰[1] 吴敬[1] 

机构地区：[1]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

出　　处：《生物技术通报》2016年第6期199-204,共6页Biotechnology Bulletin

基　　金：国家杰出青年基金项目(31425020);111计划(111-2-06)

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)/p ET-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/m L,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p H5.0,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。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.

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 

分 类 号：TQ920.6[轻工技术与工程—发酵工程]