Cloning and Expression of Glutaminase New Gene in the Sauce Billet Metagenomic

摘要

Aim to study the Glutaminase gene structure and function the prokaryotic expression vector pEAZY El-glsA927 is construnted from the new genes glutaminase named glsA927 which is cloned in the traditional sauce billet microbial metagenomic and connected to pEAZY El carrier. After the recombinant plasmid is passed into E. coli BL21 and induced by gene expression and after purified by the Ni column, express product detect the enzyme activity by means of bacteria glutaminase activity quantitative detection kit. Sequence analysis results show that the gene length of glsA927 is 927 bp, which has the highest sequence homology (AF057158.1), 94.82%, reported on GENBANK, with 16 different amino acids. Protein expression analysis results show that when the concentration of IPTG tendency is 0.2 mmol/L, protein expression of induced 4 h has the highest amount. The Ni ion affinity chromatography purification of recombinant glutaminase ihs-glsA927, preliminarily determine glutaminase activity to a maximum of 468.7 U mu/g, SDS-PAG and Western results show that recombinant glutaminase His-glsA927 which is purified by the Ni ion affinity chromatography could be preliminarily determined that the glutaminase activity can reach as much as 468.7 U/μg and not easy to be affected by the concentration of NaCl.