| Optically pure Vince lactam is an important precursor for the synthesis of many chemicals and drugs like anti-AIDS drug, Abacavir and anti-viruses drug, Peramivir, which generate enormous profits every year. Among all the synthetic approaches, enantioselective biocatalysis of racemic γ-lactam is an efficient and environmental-friendly way to synthesize optical pure γ-lactam. Previous studies proved that a(+)-γ-lactamase producing strain Delftia sp. CGMCC 5755 was active in hydrolyzing 100 g·L–1 racemic γ-lactam, achieving in conversion rate of 53.7% and 99.9% ee. Our studies are focused on the soluble heterologous expression of(+)-γ-lactamase from Delftia sp. aiming at reducing the fermentation process costs and increasing productivity.Based on the Delftia acidovorans SPH-1 genome sequences, a genome hunting strategy was adopted to identify the(+)-γ-lactamase coding genes. Using “acetamidase/formamidase/arylformamidase” as the keywords, five putative(+)-γ-lactamase genes were selected and heterologously expressed in Escherichia coli. Among them, lm2 and lm3 coding enzymes could hydrolyze(+)-γ-lactam but showed little activity, which could be attributed to the inclusion body formation. The lactamase encoded by lm3 displayed the highest activity is disiganated as De Lm.To promote the soluble expression of(+)-γ-lactamase, various strategies were attempted. After optimizing induction temperature and culturing medium, the solubility of De Lm was not dramatically improved. T5 promoter was adopted to decrease the transcription level but failed in improving the solubility. Recombinant E. coli BL21(DE3)/p ET43a-delm strain was constructed with a solubilization tag. The fusion protein Nus A-De Lm was mostly soluble according to the SDS-PAGE analysis and its enzyme activity increased to 1.02 U·gDCW–1. Molecular chaperones, including Gro ES-Gro EL, Dna K-Dna J-Grp E and Tig were not helpful in improving the(+)-γ-lactamase solubility. Another prokaryotic heterolotgous expression system Bacillus subtilis was then tested for the expression of(+)-γ-lactamase. Recombinant B. subtilis 168/p MA5-delm showed increased enzyme activity by 58% compared with that in recombinant E. coli and no inclusion body formed.The whole cell catalysis conditions of recombinant B. subtilis 168/p MA5-delm in enantioselective resolution of rac-γ-lactam are optimized to be at 30 °C and p H 9.0, respectively. Optimal agitation speed is determined to be 300 r·min–1. No substrate inhibition is observed when whole cells are incubated against as high as 250 g·L–1 γ-lactam. The apparent kinetic parameters of Vmax and Km are calculated to be 0.595 mmol·min–1·gDCW–1 and 378 mmol·L–1(about 41 g·L–1), respectively. The recombinant B. subtilis is further applied in the preparation of(–)-γ-lactam. In a 500 m L reaction, 5 g lyophilized cells are employed in enantioselective hydrolysis of 50 g γ-lactam under optimized conditions.(+)-γ-Lactam is completely hydrolyzed after 22.5 h, resulting in 55.2% conversion and 98.6% ee. After extracted with dichloromethane for three times, the organic phases are combined, dehydrated and evaporated to obtain 14 g(–)-γ-lactam with 56% recovery rate and 99% optical purity. In a word, the recombinant B. subtilis 168/p MA5-delm whole cells are promising biocatalysts and can be applied in the synthesis of optically active γ-lactam. |
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