Effect Of Surface Modification Of Acyltransferase From Mycobacterium Smegmatis On Enzymatic Constant

Surface modification techniques for enzyme molecules refer to the use of protein engineering techniques to replace certain amino acid residues on the surface of an enzyme protein molecule,or to couple certain chemical groups to specific locations on the surface of an enzyme molecule.Improve the stability or catalytic activity of the enzyme protein in a specific solvent by modifying the surface of the enzyme protein molecule,Regulating the aggregation behavior of enzyme molecules in specific solvents,Improve the immobilization effect of the enzyme molecule,and so on.Surface modification techniques are increasingly used in the modification of enzyme molecules.In this paper,Mycobacterium smegmatis acyl transferase?MsAcTase?with promiscuous activity was studied.First,using the site-directed mutagenesis technique to introduce mutated amino acid residues at different positions on the surface of the MsAcTase molecule.Secondly,the mutated amino acid residue was used to modify the glycosylation of the mutated MsAcTase.In order to achieve this goal,two technical routes of in vivo glycosylation modification and in vitro glycosylation modification would be adopted.That was,the MsAcTase mutant-encoding gene was introduced into Heterologous expression in Pichia pastoris and Escherichia coli,respectively.The mutant gene was expressed in Pichia pastoris,and the glycosylation modification of the recombinant protein was carried out by using the glycosylation modification system of Pichia pastoris.The recombinant protein expressed in E.coli was intended to be modified in vitro by glycosylation.The specific experimental results were as follows:Using the YASARA software,combined with the resolved MsAcTase octamer structure,a suitable mutation site was screened on the surface of the MsAcTase molecule.A mutant gene intended to be glycosylated in vivo,and a mutant having an Asn-Xaa-Ser/Thr sequence is screened and constructed.A mutant gene intended to be glycosylated in vitro was introduced into a Cys mutation at a suitable site on the surface of the MsAcTase molecule.After analysis and screening,three potential candidate mutants of MsAcTase-Q¹⁵⁹N,MsAcTase-I¹⁸⁶N and MsAcTase-D²⁰³S were screened as mutants with glycosylation in vivo.MsAcTase-C⁷S/C⁷⁷S/A¹⁴³C and MsAcTase-C⁷S/C⁷⁷S/S¹⁸⁴C were screened as candidate mutants for in vitro glycosylation modification.The above mutated genes were introduced into Escherichia coli,recombinantly expressed,and after isolation and purification,the basic enzymatic properties were determined,and the mutation effect was evaluated.The results showed that the specific activity of MsAcTase-I¹⁸⁶N was reduced to 74%of wild-type MsAcTase,respectively,while the specific activity of mutant MsAcTase-D²⁰³S and mutant MsAcTase-Q¹⁵⁹N did not change significantly.The k_cat/K_m of MsAcTase,MsAcTase-Q¹⁵⁹N and MsAcTase-D²⁰³S were determined by ethyl acetate as the substrate:219.9 L·mmol^-1·min^-1,147.3 L·mmol^-1·min^-1,respectively.163.0 L·mmol^-1·min^-1.The specific enzyme activities of MsAcTase-C⁷S/C⁷⁷S/A¹⁴³C and MsAcTase-C⁷S/C⁷⁷S/S¹⁸⁴C were decreased compared with wild-type MsAcTase,and the specific activity of mutant MsAcTase-C⁷S/C⁷⁷S/A¹⁴³C was reduced to 70%of wild-type MsAcTase.The specific activity of the enzyme MsAcTase-C⁷S/C⁷⁷S/S¹⁸⁴C was reduced to 55%of the wild-type MsAcTase.The k_cat/K_m measured by ethyl acetate was 219.9 L·mmol^-1·min^-1,144.6 L·mmol^-1·min^-1,and 124.3L·mmol^-1·min^-1,respectively.After significant analysis,there were no significant differences except the mutants MsAcTase-I¹⁸⁶N,MsAcTase-C⁷S/C⁷⁷S/A¹⁴³C and MsAcTase-C⁷S/C⁷⁷S/S¹⁸⁴C.The gene encoding the wild-type perhydrolase MsAcTase and its mutants MsAcTase-Q¹⁵⁹N and MsAcTase-D²⁰³S was introduced into Pichia pastoris for expression.The specific activities of the recombinant proteins determined after separation and purification were:The specific activities of MsAcTase,MsAcTase-Q¹⁵⁹N and MsAcTase-D²⁰³S were 47.6 U/mg,56.4 U/mg and 47.9 U/mg,respectively.Using ethyl acetate as substrate,the k_cat/K_m of MsAcTase,MsAcTase-Q¹⁵⁹N and MsAcTase-D²⁰³S were:47.0 L·mmol^-1·min^-1,65.6 L·mmol^-1·min^-1,61.5 L·mmol^-1·min^-1,respectively.The glycosylation modification of the recombinant MsAcTase mutant was verified by PAS staining and glycosidase digestion.The results showed that none of the two mutants were modified by glycosylation.