Cloning,Expression And Purification Of Recombinant Penicillinase TEM-1 And Its Characteristics Investigation

Penicillinases TEM-1 is one of the β-lactamase which have characteristics of hydrolyzed P-lactan ring of penicillins. They have widely used in industry and medicine. The production strain of penicillinase has some shortcomings, such as expensive culture medium, weak ability to produce penicillinase and unstable activity of the enzyme in production process. In this paper molecular clone of the gene encoding penicillinase, high-level expression in E.coli and application of purified penicillinase were studied. Besides, characteristics of penicillinase were also investigated.Objective:Research the plasmid pET22b-penp expression and purification methods in E.coli BL21, and obtain penicillinase TEM-1 with high purity and high activity. Investigate the characteristics of penicillinase TEM-1.Methods:The plasmid pET22b-prop was transformed into E.coli BL21 to construct the recombinant expressing strain. After over-expression via induction of IPTG and breaking cells via ultrasonication, the resulting protein was refolded and purified using DEAE-Sepharose Column. Determine the optimum reaction pH and temperature of penicillinase TEM-1. Investigate its stability in different pH, temperature and accessories. And used microbiological assay to determination its effectiveness.Results:By optimizing expression, we can get 800g cell every 20L fermentation liquid, and expression levels can reach more than 25%. The recombinant TEM-1 was purified by DEAE-sepharose column. The purity of objective protein is greater than 95% and specific activity is greater than 30 million U/mg. The optimum reaction pH of objective protein is 7.0, the optimum reaction temperature is 37 ℃; Objective protein will be stable when the pH is 7.0, temperature is 4℃, and with 10% glycerol or 10% alanine in the system. Penicillinase TEM-1 has a good decomposition to the antibiotics of penicillins and the first generation cephalosporins.Conclusions:1.Constructed engineered bacteria which has efficiently expressing of penicillinase TEM-1.2. Expression and purification method of TEM-1 were established, and the pure enzyme with high concentration and activity was obtained.3. Through the study, get a better understanding to enzymatic properties of TEM-1. Determine the efficacy and action of TEM-1.