The Nitro Reduction Pseudomonas Glutaminase Gene Cloning, Expression And Its Application In Enzymatic Synthesis Of L-theanine

L-Theanine is a unique amino acid and the major umami component of the tea. Many recent researches have revealed that theanine possesed lots of physiological functions such as neuroprotection, blood-pressure reduction, antitumor activity, relaxation and so on. Therefore, more and more attention has been paid to the production and application of theanine, making it more and more popular. The glutaminase (GLS, EC3.5.1.2) catalyzes the hydrolysis of L-glutamine to L-glutamic and ammonia. There are experiments demonstrating that some GLSs can form theanine from L-glutamine and ethylamine as y-Glutamyl transpeptidase (y-GGT, EC2.3.2.2) under the alkaline condition. The existing problems of previous theanine-forming methods are mainly as follows:low yield or too many by-products. The study on obtaining theanine by glutaminase will improve those problems. The major experiment results of this dissertation are as follows:1、In this study, we cloned nucleotide sequences of glutaminase from Pseudomonas nitroreducens for the first time. The length of the gene is909bp, which encodes a protein of302amino acids. According to amino acid sequences alignment, pnGLS shares94%and81%identity with GLS sequences reported from P. putida F1and P. aeruginosa UCBPP-PA14. Meanwhile, pnGLS also contains several key catalyeic residues of the large superfamily of β-lactamases and penicillinbinding proteins, which further proves the glutaminase gene is from P. nitroreducens.2、The present experiment designed specific primers with restriction enzyme sites to amplify the gene. Then, the recombinant plasmid pET.43.1a-pnGLS is constructed and transformed to E. coli BL21.3、The expression conditions of recombinant bacteria has been optimized. When the recombinant bacteria concentration rearches0.6(OD600), the bacteria mixtures are induced by lactose at24℃and180rpm for10hours. The recombinant protein exhibits a high expression up to70%of total cellular proteion. According to the analysis of SDS-PAGE, the protein has a good solubility.4、The reaction conditions for synthesizing theanine were also investigated. Incubation at37℃for5h in5mL reaction system (0.3M glutamine,1.5M ethylamine,1.5transfer crude extract, pH10.0), theanine formation reaches the highest point, and the conversion rate of glutamine as to theanine is about40%. It provides the basis for further construction of glutaminase engineering bacteria to synthesize theanine, and also provides a good prospect for theanine formation.