Directed Evolution Of Transglutaminase And The Research On Relationship Between Structure And Function Of Its Mutants

Transglutaminase(EC2.3.2.13,Transglutaminase,TGase)is a transferase that catalyzes the acyl transfer reaction.It can achieve crosslinking within and between protein molecules.At present,Streptomyces mobaraensis transglutaminase(MTG)has been extensively used in food,medicine and textile fields.Because the processing is usually carried out at high temperature.Therefore,screening MTG mutants with high specific enzyme activity and good thermal stability is great significance for improving MTG catalytic efficiency.In this study,the zymogen gene promtg of MTG was amplified using the Streptomyces mobaraensis genome as the template and constructed into the secretory expression vector pXMJ19L.The constructed plasmid with the gene of interest would be transferred to E.coli DH5? for amplification,and finally transferred into Corynebacterium glutamicum ATCC13032 to obtain the recombinant strain ATCC13032/pXMJ19L-promtg to achieve PROMTG secretory expression.Furthermore,random mutation of promtg by error-prone PCR technology,combined with the above-mentioned expression system of Corynebacterium glutamicum to construct a mutation library.Finally,MTG mutant E164L with high specific activity and high thermal stability was obtained through high-throughput screening.The activity of wild-type MTG(WT)and mutant E164L were measured,and the specific activity were improved at 20?-50?.The specific activity of E164L(54.8 U/mg)is 1.95 times that of WT(26.8 U/mg)at 50?.In addition,through the analysis of enzymatic properties,the results showed that the optimum temperature of both WT and E164L were 50? and the optimum pH were 6.Meantime,the thermal stability of WT and E164L at 50? has been measured,compared with WT,the half-life of E164L was increased by 1.66 times.The conformation of E164L and WT was studied by using molecular dynamics simulation.The molecular dynamics(MD)simulation results indicated that the mutation Glu164Leu resulted in the weaker interactions of Aspl59-Glu164 and Gly228-Leu231,leading to the enhanced instability of Ile240-Asn253 linked to Gly228-Leu231 by eight residues.It further caused the reduced interactions between loop region 1(Ile240-Asn253)and loop region 2(His277-Met288),facilitating the access of the substrate molecule to the active site.This study improved the understanding of structure-activity relationship for MTG adapted to high temperature conditions.It also provides theoretical foundation and preliminary information for engineering MTG with enhanced characteristics to meet the industrial requirements.