Optimization Of Expression And Modification Of The Catalytic Loop Domain Of Thermomicrobium Roseum Sarcosine Oxidase

Sarcosine oxidase is an important enzyme in the pharmaceutical and food industries,capable of targeting N-methylglycine for demethylation to produce glycine,formaldehyde and hydrogen peroxide,completing reactions that are difficult to perform by chemical demethylation.In this study,we propose to prepare a sarcosine oxidase(Tr SOX)from Thermomicrobium roseum DSM 5159,using the Bacillus subtilis WB600 expression system as a model.In our previous work,Tr SOX has shown excellent thermal stability,resistance to acids,bases and organic solvents,efficient demethylation and a broad substrate profile,but its soluble expression level is still a challenge at this stage.In order to solve this problem and further explore the substrate specificity and chiral selectivity of the enzyme and improve its catalytic performance,the following studies were conducted:(1)Optimization of Tr SOX soluble expression level by promoter reconstruction of p MA5 expression vector.The P_{Hpa II} promoter-35 to the start codon region in the p MA5 vector was replaced with a constitutive promoter(P_arpe,P_ylbp,P₄₃,P_shuttle-09)and an inducible promoter(P_xyl,P_spac,P_grac,P_grac100),and successfully expressed and purified,and finally screened for xylose-induced P_xyl promoter recombination Tr SOX protein yield(U·g^-1 wet cell)was the highest,nearly 20 times higher than that of the natural expression system.(2)Orthogonal experiment L₉(3⁴)was performed to optimize the induction conditions of recombinant B.subtilis WB600/p MA5-P_xyl-trsox and the heat treatment of the crude enzyme solution to complete the final protein folding.The results of the orthogonal experiments were analyzed by SPSS software and the optimal expression conditions were:OD600 nm 2.0,xylose concentration 1.6%(w/v),induction temperature 37°C and induction time 2 h.The crude enzyme solution was heated at different temperatures and times to obtain the optimal conditions of 60 min at 80°C and the protein yield was purified and measured(?g The enzyme was purified and the protein yield(?g of pure enzyme/g of wet bacterial body)was 8times higher than that of the natural plasmid p MA5-P_{Hpa II}-trsox;the catalytic activity(k_cat/K_m)of the N-methyl substrate was increased by thousands of times,indicating that the heat treatment could mimic the natural growth environment of the enzyme and thus assist in the final folding of the protein and improve the catalytic efficiency of the enzyme.The expression level was further increased by about 2.5-fold compared to p MA5-P_xyl-trsox,and the catalytic activity of the enzyme was also significantly increased,indicating that the establishment of the hydrophilic cap may have altered the enzyme surface hydrophobicity,allowing easier access of the N-methylamino acid substrate to the substrate pocket.(3)Modification of the catalytic Loop domain refined the catalytic performance of Tr SOX.Based on homologous sequence comparison,the enzymatic properties of F58Y,F58L,F58G,F58M,S60A,S60G,S60H,P61A,P61E,P61R,F58G/S320R and S60G/S320R mutants were determined by bioinformatics methods such as ancestral sequence reconstruction and were obtained by targeted mutagenesis of Tr SOX.F58M showed no catalytic activity against any substrate;F58G,F58G/S320R and S60H showed a 10-20 fold increase in the catalytic activity against amino acid substrates with hydrophobic side chains;S60G showed a 45-fold higher relative activity(k_cat/K_m)against N-methyl-L-aspartate than the nature enzyme.In addition,only the F58G,F58G/320R,S60H and P61E mutants showed catalytic activity towards pyrrolidine substrates,while the rest of the mutant strains showed no activity.