| (-)γ-lactam is the key intermediate raw materials for the synthesis (-) abacavir, which serves as one powerful antiviral agents with enormous value. Compared with the chemical methods, better application prospects were displayed on the spilt (±)γ-lactam by the microbial enzymatic.The y-lactamase from Thermoanaerobacter tengcongensis was expressed in Escherichia coli fused with poly-histidine-tags at both N-terminus and C-terminus. The fusion enzyme could be one-step purified by Ni-chelating agarose and immobilized on the same matrix. The soluble enzyme showed optimum temperature at 80℃and optimum pH at 7.0 whereas immobilized enzyme showed optimum temperature at 90℃and optimum pH at 9.5 respectively. The immobilized y-lactamase showed much higher thermal and pH stability than the soluble enzyme. The immobilized y-lactamase could be applied for 27 batches of 5 h on-column transformation with almost same efficiency at 60℃within 27 days.Lactose was used as an inducer instead of the expensive nonmetabolizable analog of lactose, IPTG. IPTG is satisfactory for small-scale expressions, but not suitable for large-scale fermentations due to its high cost. It was found that lactose could induce foreign protein expression and enhance cell growth during the induction period. The present study, E. coli BL21 (ED3) strain host bacteria,the lactose inducible cloning research in the PET-30 vector in the r-lactam hydrolase gene, induced the greatest expression of lactose can reach 62%. |
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