Bacterial Cell-surface Displaying Of Glutamate Dehydrogenase And Its Application In L-glutamate Assay

In order to make up for deficiencies including high cost, low specificity and poor stability in traditional glutamate detection, this study developed a new method using microbial surface display technology for whole-cell catalyst to detect glutamate content. Thus, a simple operation, cost-effective method for the sensitive and selective detection of L-glutamate was established. At the same time, this study provided a good theoretical basis to achieve practical application of surface display technology by constructing different surface display systems which explored the effects of his-tag, different expression vectors and expression strains on expression of protein. Proper anchor protein is an important factor to construct microbial surface display system successfully. In this study, the wild type glutamate dehydrogenase encoding gene firstly used come from the genome of Bacillus subtilis originally, and n-terminal domains of ice nucleation protein from Pseudomona borealis were used as anchor protein. The results proved that the fusion protein expressed in inclusion bodies in E.coli cells, which indicating glutamate dehydrogenase was not displayed on the surface of E.coli successfully. Then gldh come from the genome of Thermococcus waiotapuensis originally and n-terminal domains of ice nucleation protein from P.borealis were used to construct microbial surface display system. The results showed that the fusion protein expressed on the surface of E.coli successfully, indicating n-terminal of INP from P.borealis had excellent anchor properties for glutamate dehydrogenase from T.waiotapuensis. It was the first time displaying glutamate dehydrogenase on bacterial surface which provided basic theoretical research and biomaterials in practical applications for the use of microbial surface display systems in biosensor, food testing, pharmaceutical industry etc. All results of this study are showed as follows:1. The construction of glutamate dehydrogenase surface display system using gene from B.subtilis.Constructing glutamate dehydrogenase surface display vectors pET-Inp-gldh, pET-Inp-gldhT and pACY--Inp-gldh using gldh from B.subtilis as target gene and n-terminal domains of ice nucleation protein from P.borealis as anchoring motif. Then they were transformed into E.coli expression strains. The fusion protein were found expressed in the form of inclusion body after SDS-PAGE analysis and activity detection, which suggested gldh gene from B.subtilis incapable of achieving expression in soluble form on the surface of E.coli cells.This part of the study also showed that gldh gene from B.subtilis was expressed in the form of inclusion body regardless of the presence or absence of his-tag and whether using expression vector pET28a (+) or pACYCDuet-1. gldh gene was expressed in the form of inclusion body after plasmid pET-Inp-gldh or pACY-Inp-gldh was transformed into E.coli BL21 (DE3), E.coli transB (DE3), E.coli transetta (DE3) and E.coli BL21 (DE3) plys, which showed the same effects of the above strains on the expression of gldh gene from B.subtilis.2. The construction of glutamate dehydrogenase surface display system using gene from T.waiotapuensis.Constructing glutamate dehydrogenase surface display vector pTInaPb-N-Gldh using gldh from T.waiotapuensis with n-terminal domains of ice nucleation protein as anchoring motif. Then it was transformed into E.coli BL21 (DE3). Localization of glutamate dehydrogenase on the bacterial cell surface was confirmed by SDS-PAGE and enzyme activity assays.The whole cell activity of glutamate dehydrogenase was 3.12 U/OD600, and 90% of the activity was detected in the outer membrane fraction.The optimal temperature and pH for enzyme activity of the bacteria cell-surface displayed glutamate dehydrogenase (bacteria-Gldh) was about 70℃ and 9.0, respectively. Additionally, the fusion protein retained almost 100% of enzyme activity at 4℃ after one month incubation. Transition metal ions could inhibit the enzyme activity to different extent, while common anions had little adverse effect. Importantly, the displayed glutamate dehydrogenase was specific to L-glutamate compared with other glutamate dehydrogenases reported so far. The bacteria-Gldh enabled to catalyze the oxidization of L-glutamate with NADP+ as cofactor, and the resultant NADPH could be detected spectrometrically at 340 nm. The bacteria-Gldh based assay approach showed a wide linear range (10～400 μmol/L) and a low detection limit of 6 μmol/L L-glutamate. Futher, the precise measurement of glutamate in real samples using bacteria-Gldh was accomplished. All results showed that it was a simple, rapid, and cost-effective method for the sensitive and selective detection of L-glutamate.