Gene Discovery,Characterization Of Sucrose Phosphorylase And Its Research In α-arbutin Biosynthesis

Hydroquinone(HQ)is a natural and efficient whitening ingredient,which has been widely added to whitening products,but it has certain cytotoxicity.As a natural derivative of HQ,α-arbutin has the advantages of good stability and few toxic side effects,and has great application value in cosmetics,food,medicine and other fields.At present,enzymatic catalysis is generally recognized as one of the safest and most efficient ways to synthesize α-arbutin.Sucrose phosphorylase(SPase)is one of the most widely used enzymes to synthesize α-arbutin.The principle is to transfer the glucoside from sucrose to HQ molecule to obtain α-arbutin.However,the SPases that have been screened for α-arbutin production have the defects of few species and low catalytic activity.Therefore,the aim of this study is to mine a novel SPase with high catalytic activity from biological databases,characterize it enzymically,apply it to the biosynthesis of α-arbutin,and finally modify it through semi-rational design to improve its catalytic activity.The specific research content is as follows:(1)In this study,five different microbial SPase genes were selected for codon optimization and synthesis by sequence alignment.The synthesized target gene was cloned into the recombinant vector p ET20b(+),and then the recombinant plasmid was transferred into Escherichia coli cells for expression.We successfully solved the problem that the expression of SPase in E.coli was in the form of insoluble and inactive inclusion bodies by introducing the inducible molecular chaperone plasmid p G-Tf2 to express the molecular chaperone protein Gro ES-Gro EL and Tf factor.Based on the preliminary analysis of the catalytic properties of the crude enzyme solution,Pe SP with a high level of a-arbutin synthesis was selected as the starting enzyme for subsequent studies.Pe SP was derived from P.elgii.After optimizing the co-expression conditions,IPTG at a final concentration of 0.05 m M and 10 ng×m L-1 tetracycline were added to the medium at an OD600 of 0.9 as an inducer,and the cells were cultured at 20℃ and 180 rpm for 20 hours.The soluble protein level was the highest,and the enzyme specific activity of Pe SP increased from 77.63 U×mg-1 to 91.64 U×mg-1.Optimization was performed by coexpression conditions.(2)The optimum temperature of the purified recombinant Pe SP was 35℃ and the optimum p H was 7.0.The half-life at 35℃ was 3.6 h.The recombinant Pe SP was stable at p H 6.5-8.5 and sensitive to high concentrations of metal ions.The Km,Vmax and Kcat values of sucrose as the substrate were 14.50 m M,227.27 μmol×mg-1×min-1 and 141.88min-1,respectively.The optimum reaction conditions for the biosynthesis of α-arbutin from recombinant Pe SP were optimized.We found that the maximum yield of a-arbutin was 52.60 mg×m L-1 and the HQ conversion rate was 60.9% at 30 ℃ and p H 7.0 when 200U×m L-1 Pe SP was mixed with sucrose and hydroquinone in the molar ratio of 5:1 for 25 h.(3)To improve the catalytic capacity of Pe SP,a semi-rational modification was carried out.Two mutants(K160E and R348I)with enhanced Pe SP activity were obtained by site-directed saturation mutagenesis of three amino acid residues that most significantly affected the activity of Pe SP.The enzymatic properties of the mutants were analyzed and the conditions for synthesizing arbutin were optimized.The specific activities of mutants K160 E and R348 I were found to be 215.42 U×mg-1 and 202.36 U×mg-1,which were increased by 1.27 and 1.19 times,respectively.The Kcat values of mutants K160 E and R348 I were 204.25 min-1 and 173.92 min-1,respectively.Compared with the wild type Pe SP,the mutant Pe SP was increased by 1.44 and 1.22 times,indicating that the substrate affinity of the mutant Pe SP was increased compared with the wild type,but the thermal stability of the mutant Pe SP was decreased.The highest yield of a-arbutin synthesized by HQ transformed by the mutants was 65.73 mg×m L-1 and 58.73 mg×m L-1,which was increased to 1.25 and 1.12 times of the original,respectively.The HQ conversion rate was 79.01% and 70.60%,which was increased to 1.09 and 1.06 times of the original,respectively.In addition,the key amino acid residues that affect the stability of Pe SP were also analyzed in this study,and amino acid residues that have a greater influence on the thermal stability of Pe SP were found,which provides a reference for optimizing the stability of SPase.