Stearothermophilus Bacillus Aminoacylase Gene Cloning, Expression And Cell Immobilization Of E. Coli

Methionine is a sulfur-bearing essential amino acid,which is widely used in forage,pharmaceuticals,health and food industry.Methionine is a high value-added product and one of the imported-dependent amino acids in China.So recent years, looking for a producing method and process of methionine with lower cost and higher output has become a research hot spot in domestic amino acid industry areas.In this study,Bacillus Stearothermophilus was used as an original strain,the amaA gene encoding aminoacylase was cloned and expressed in E.coli.Using N-acetyl-DL-methionine as a substrate,the recombinant E.coli cell immobilization was investigated to explore the application prospect on L-methionine industry.The genome DNA from Bacillus Stearothermophilus was used as a template for PCR.The PCR product was cloned into pMD-18T vector for sequence analysis which indicated the same identity of amaA(Y08753) on Genebank.The plasmid pET-28a(+) and pMD-18T with amaA gene was digested with NdeⅠand EcoRⅠand ligated by T4 DNA ligase to construct amaA-pET28a(+).The recombinant plasmid was transformed into E.coli BL22(DE3) to express the amaA gene.Both SDS-PAGE and aminoacylase assay showed that the cloned strain was effective expressed.The expression condition study included concentration of IPTG,inducing period and induced time etc. The optimized condition was:37℃200rpm shaking;added 0.4mmol/L IPTG when OD₆₀₀ reached 0.8～1.0,culturing 6 hours with IPTG inducement.The enzyme activity was 1,043U/g cells under the optimized condition.Cell immobilization research showed that the optimized condition was 30%of E.coli cell were embed by 3%carrageenan,treated for 20 minutes with 2.25% polyethylene polyamines to permeate and crosslink,then hardened these cells for 20 minutes with 0.15%glutaraldehyde.After immobilization,the cell preserved 83%of enzyme activity.Using N-acetyl-DL-methionine as a substrate,the properties of immobilized bacilli were studied.The results revealed a linear relationship between reaction velocity and substrate concentration when N-acetyl-DL-methionine was under 0.08mol/L. The K_m of free cell was 4.5mmol/L while immobilized cell was 14.7mmol/L at 55℃. Enzyme properties showed that the optimum temperature and pH are 65℃and 7.0 respectively.Compare to free cell,the immobilized cell had a higher thermal and pH stability.After reacting continuously for 10 times,the enzyme activity lost just only 20%.When placed at 4℃for 23 days,73.6%of enzyme activity was preserved compared to original immobilized cell.These results indicated that there is an excellent industrial application prospect of immobilized recombinant E.coli with amaA gene.