Expression, Purification And Properties Of An (+) γ-Lactamase

γ-Lactamase is one kind of novel amidase.It can hydrolyze the amide bonds of the amides.(+)γ-lactamses can be applied in the kinetic resolution of racemic y-lactam, which can produce(-)γ-lactam efficiently. The(-)γ-lactam enantiomer is an important intermediate material for the synthesis of (-)abacavir, which serves as one powerful antiviral agents with great enormous value. Advantages such as mild reaction conditions, stereospecificity, high specificity toward the substrate, low energetic consumption, biocatalyst reuse capability, and high-quality bioconversion make biocatalysis an alternative method to chemical synthesis of certain products.Compared with the chemical methods, better application prospects were displayed on the spiltγ-lactam by the microbial enzymatic.Theγ-lactamase gene from Microbacterium hydrocarbonoxydans was cloned by PCR using primers based on the N-terminal and C-terminal amino-acid sequences of native protein.The gene could be highly expressed in Escherichia coli fused with poly-histidine-tags at both N-terminus and C-terminus.The recombinant enzyme could be one-step purified by Ni-chelating agarose. Under optimum conditions,the yield of (-)γ-lactam was 45% and e.e. value was 99%.It has a molecular weight of 18KDa. Mgnco had two distinct domains:the ferredoxin domain fuses withγ-lactamase domain.Mgnco showed optimum temperature at 30℃and optimum pH at 6.5,and it denaturalizated when the temperature was risen to 50℃in 10 min. Mcihaelis constant of Mgnco was 4.41mmol/L and Vmax was 0.137mol·L-1·min. Fe2+(2mmol·L-1) and DTT (<10mmol·L-1)enhanced the enzyme activity.Mgnco was strongly inhibited by PMSF. NAD, NADH, NADPH,FAD and FMN did not take effect on activity of Mgnco.