基于基因组挖掘技术的新型谷氨酸脱羧酶基因挖掘及表达鉴定

Gene Mining,Expression and Identification of Novel Glutamate Decarboxylases Based on Genome Mining

采用基因组挖掘技术,以来源于Lactobacillus brevis活性较高的谷氨酸脱羧酶(glutamate decarboxylase,GAD)LbGAD为探针,从乳酸菌(Lactococcuslactis、Lactobacillussenmaizukei)和肠球菌(Enterococcus sulfureus)的基因组中挖掘到了3个假定的GAD基因(LlGAD、LsGAD和EsGAD)。借助pET28a质粒分别实现了这4个基因在大肠杆菌BL21中的表达,其中LsGAD和LlGAD的表达产物可溶性较好,相应发酵液中GAD活力分别为34.17、38.91 U/mL。Ls GAD的比活力、温度特性、pH值特性和K_(cat)/K_(m)值也明显优于其他几个酶。此外,初步研究了全细胞催化L-谷氨酸制备γ-氨基丁酸(γ-aminobutyric acid,GABA)的工艺,6 g/L的L-谷氨酸经过24 h转化后,GABA得率最高可达58%。本研究实现了GAD从基因组数据到真实酶的跨越,获得了1个性能优良的GAD,并初步实现了GABA的生物合成,为实现GABA低成本、规模化的生物合成提供了科学依据。

In this study,three hypothesized glutamate decarboxylases(GAD)genes from Lactococcus lactis,Lactobacillus senmaizukei and Enterococcus sulfureus,respectively named as LlGAD,LsGAD and EsGAD,were excavated using genome mining technology with the high-activity GAD gene(LbGAD)from Lactobacillus brevis as the probe.By means of pET28a plasmid,these four genes were expressed in E.coli BL21.The expressed products of LsGAD and LlGAD had a good solubility,exhibiting GAD activity of 34.17 and 38.91 U/mL,respectively in the fermentation broths.The specific activity,temperature characteristics,pH characteristics and K_(cat)/K_(m)value of LsGAD were significantly superior to those of the other GAD enzymes.In addition,the biosynthesis ofγ-aminobutyric acid(GABA)from L-glutamic acid by whole-cell catalysis was studied.When L-glutamic acid at 6 g/L was transformed for 24 h,the maximum yield of GABA of 58%was obtained.In conclusion,this study has realized a leap from genomic data to real enzymes by obtaining GAD with excellent performance as well as the biosynthesis of GABA,which will lay a solid foundation for the low-cost and large-scale biosynthesis of GABA.