Genome Sequencing, Amidase Genes Cloning And Heterologous Expression Of Paracoccus Marcusii W10173

Amidase can hydrolysis amide to corresponding carboxylic acids and ammonia. It has an extremely important role in the pharmaceutical, foods, textile and leather processing.L-asparaginase is widely used in the treatment of childhood acute lymphoblastic leukemia(ALL) clinically. L-glutaminase is a key and rate-limiting enzyme in glutamine decomposition, which has an important relationship with tumor growth, angiogenesis and tumor immunity. Steroselective piperazine-2-carboxamidase can hydrolysis piperazine-2-carboxamide to produce the chiral drug intermediates of piperazine-2-carboxylic acid, their different individual enantiomers(R and S type) have a plurality of different derivatives of pharmacological active. In previous work, the(S)-amidase produced by Paracoccus marcusii W10173, which can selective transform the S-enantiomer of(R,S)-piperazine-2-carboxamide to yield S-piperazine-2-carboxylic acid was screened by our lab. In this study, we obtained the whole-genome sequencing of Paracoccus marcusii W10173 by whole-genome sequencing technology. 5 amidase encoding genes were found though genome annotation, and they were cloned and heterologous expressed in order to explore amidase resource of the strain further and provide the basis for the production and application of amidases.The 5 amidase encoding genes we get by analyzing the whole-genome sequence,including L-glutaminase(PM1509), L-asparaginase(PM1383, PM3012) and the potential piperazine-2-carboxamidease(PM2486, PM3475). PM1509, PM1383, PM3012 were cloned and constructed prokaryotic expression vector successfully. The induction condition was determined by adding 0.8 mmol/L IPTG when the bacterial density OD600 was about 0.6, and then the recombinant bacteria was induced for 5 h at 18 ℃. The recombinant protein accounted for 30.5 %, 23.8 %, 20.3 %, respectively, of the total protein. The recombinant proteins were accumulated in the form of inclusion body, when purified using affinity chromatography with Ni-colum after sonication treatment, the yields of purified proteins were2.53 mg/mL, 2.13 mg/mL, 1.62 mg/mL respectively. After renaturation, the expression quantity of purified proteins were 0.41 mg/mL, 0.34 mg/mL, 0.31 mg/mL, respectively. And the recovery rates were 16.2 %, 15.9 %, 19.1 %, respectively. L-asparagine and L-glutamine waere used as substrate respectively to determine refolded proteins for enzyme activity. The result shows that the refolded proteins can hydrolysis L-asparagine, L-glutamine to produce L-aspartate, L-glutamate, respectively.We predicted the secondary structure composition of PM2486 and PM3475 by SOMPAsoftware and Predict Protein database, it showed that there are a little β- fold and lots of random coils, in addition, PM2486 and PM3475 also contain Cys that can form disulfide bonds by Predict Protein database predicted. These factors result in the possibility of a large inactive protein after refolding. Therefore, we select Pichia pastoris GS115 as host to carry out eukaryotic expression. Majority transformants are His+, so methanol was used to induce protein expression. Transformants induced and the target protein is in supernatant and intracellular by SDS-PAGE, piperazine-2-carboxamide as a substrate was subjected to measure activity in order to determine the enzyme activity. The results showed that the recombinant protein of PM2486 has enzyme activity, but PM3475 not. The inducing conditions for transformant PM2486-5 that contains high protein content in culture supernatant for preliminary screening was optimized further. The daily sample testing showed that the longer induction time prolonging, the more amount of protein expression accumulated,and the recombinant protein concentration is 0.519 mg/mL in culture supernatant after 6 d expressed by Bradford assay.