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  

Construction of a recombinant Escherichia coli BL21/pET-28a-lpgad and the optimization of transformation conditions for the efficient production of γ-aminobutyric acid

作　　者：田灵芝[1,2] 徐美娟[1,2] 饶志明[1,2] 

机构地区：[1]江南大学工业生物技术教育部重点实验室,江苏无锡214122 [2]江南大学应用微生物与代谢工程研究室,江苏无锡214122

出　　处：《生物工程学报》2012年第1期65-75,共11页Chinese Journal of Biotechnology

基　　金：国家重点基础研究发展计划(973计划)(No.2012CB725202);教育部新世纪优秀人才计划(No.NCET-10-0459);教育部111引智计划(No.111-2-06);国家自然科学基金(No.30970056);国家高技术研究发展计划(863计划)(No.2007AA02Z207);中央高校基本科研业务费专项资金(No.JUSRP31001);江苏省优势学科工程项目资助~~

摘　　要：为实现微生物法高效率生产γ-氨基丁酸(GABA),从一株经多次诱变筛选的具有较高谷氨酸脱羧酶(GAD)活力植物乳杆菌GB 01-21全基因组DNA中PCR扩增获得GAD酶基因lpgad,构建重组质粒pET-28a-lpgad,在大肠杆菌E.coli BL21(DE3)中高效诱导表达。并采用Ni柱亲和层析纯化获得重组GAD,并对其酶学性质进行初步研究,为改良转化工艺提高GABA产量提供可靠理论依据。结果显示,重组大肠杆菌中GAD酶活显著提高,可达8.53 U/mg,是植物乳杆菌GB 01-21中GAD酶活的4.24倍。将该重组菌应用于转化L-谷氨酸生产GABA,5 L发酵罐水平转化24 h产量可达143.5 g/L,摩尔转化率为97.32%,是植物乳杆菌GB 01-21的2.19倍。纯化后酶学性质进行初步研究表明:其最适pH为4.8;最适温度为37℃;Ca2+、Mg2+对其有较强的激活作用,将上述实验结果用于转化条件的优化,最终5 L发酵罐上进行转化实验,批次添加底物L-谷氨酸共600 g,转化24 h,GABA累计浓度可达204.5 g/L,摩尔转化率为97.92%,与最初转化条件相比,GABA浓度提高了42.5%,为其工业化应用打下了良好的基础。In order to enhance γ-aminobutyric acid production from L-glutamate efficiently,we amplified the key enzyme glutamate decarboxylase（GAD） encoding gene lpgad from the strain Lactobacillus plantarum GB 01-21 which was obtained by way of multi-mutagenesis and overexpressed it in E.coli BL21.Then we purified GAD by Ni-NTA affinity chromatography and characterized the enzyme to optimize the conditions of the whole-cell transformation.The results showed that the recombinant E.coli BL21（pET-28a-lpgad） produced 8.53 U/mg GAD,which was increased by 3.24 fold compared with the GAD activity in L.plantarum.The optimum pH and temperature of the enzyme were pH 4.8 and 37 °C,respectively.At the same time,we found that Ca2＋ and Mg2＋ could increase the activity significantly.Based on this,we investigated γ-aminobutyric acid transformation in 5 L fermentor under the optimum transformation conditions.Accordingly,the yield of γ-aminobutyric acid was 204.5 g/L at 24 h when the 600 g L-glutamate was added and the mole conversion rate had reached 97.92%.The production of γ-aminobutyric acid was improved by 42.5% compared with that under the unoptimized transformation conditions.This paved a way for the γ-aminobutyric acid construction of the industrial applications.

关 键 词：Γ-氨基丁酸 谷氨酸脱羧酶 重组大肠杆菌 全细胞转化 优化 

分 类 号：TQ921.4[轻工技术与工程—发酵工程]