Heterologous Expression And Site-directed Mutation Of Alliinase And Its Isozymes In Escherichia Coli

Allicin(diallyl thiosulfinate)is considered to be one of the main biologically active organic sulfur compounds synthesized in Allium plants,which is the most abundant thiosulfinate in fresh garlic with anti-cancer and bacteriostatic effect,and shows great potential in the treatment of many diseases.However,although allicin has a wide market application potential,its high instability has limited its further development.In practical applications,alliin and alliinase are usually made into separate preparations,and then mixed when needed to generate allicin in situ.Therefore,the market potential and application value of allicin are closely related to alliinase.The efficient and convenient production of allinase will provide a good foundation for the in-depth research and market application of garlic and allicin,and provide reference for the further development of efficient allinase compound preparation in the field of medical care.Escherichia coli is one of the preferred producers of recombinant proteins.The purpose of this project is to use E.coli as the expression host to efficiently and quickly produce proteins that can catalyze alliin into allicin.First,allinase from garlic bulbs with high catalytic activity,clear structure and available sequence was selected as the target protein and heterologous expressed by E.coli BL21(DE3),but the results showed that the alliinase from garlic bulb was expressed intracellularly as inclusion bodies.We therefore optimized its expression by several molecular modification methods,however,the experimental results were not satisfactory,suggesting that the alliinase derived from garlic bulbs was difficult to express as soluble proteins with catalytic activity in E.coli.Alternative alliinase isozymes were then selected in this study,two sources of alliinase isozymes,the methionine γ-lyase G107H of Brevibacterium linens and methionine γ-lyase C115H of Clostridium sporogenes,were investigated and we found that the methionine y-lyase C115H from C.sporogenes showed good activity to catalyze the production of pyruvate from alliin and allicin at the same time,which meets our expectation of producing alliinase isozymes efficiently.We investigated the effect of His-tag fusion at the N-terminus or C-terminus of the methionine γ-lyase C115H on the expression of the target protein,and found that when the His-tag fusion was at the C-terminus of the target gene,the expression level of the recombinant protein was higher in E.coli,and the enzyme activity(U/mg)was also higher,reaching 0.60 U/mg.After further purification with Ni-NTA agarose resin,the activity of the recombinant methionine y-lyase C115H reached 2.17 U/mg,which was 3.6 times higher than that before purification.In order to further improve the catalytic activity of methionine γ-lyase Cl 15H derived from C.sporogenes to alliin,a site-directed mutagenesis method was used to modify the target protein.By using the ClustalW function in the MEGA7 software,the methionine y-lyase sequences of 14 sources were compared and analyzed.15 amino acids were selected as mutation sites,and the crude enzyme in the intracellular supernatant of the mutated recombinant strain was determined.The activity of the mutants S59T and L265I was found to be significantly higher,and purification of these two mutant proteins revealed that compared with the control protein(2.17 U/mg),the enzyme activities of the S59T and L265I mutants reached 4.02 U/mg and 5.01 U/mg,respectively,which were 1.85 and 2.31 times for the control protein.Finally,we explored whether the amino acid hydrophobicity of the Leu265 mutation site is the main factor affecting enzyme activity.We mutated this site to five other hydrophobic amino acids and determined the activity in the intracellular supernatant of crude enzyme.The results showed that after mutation to other hydrophobic amino acids,the enzyme activity of the target protein decreased significantly.This indicates that the improvement of enzyme activity by L265I mutant was not due to hydrophilicity,but possibly the interaction between side chain groups and substrates has changed.We also compared the optimal reaction temperature and pH of the L265I mutant protein and the control protein,and the results were similar.This may indicate that mutations at the Leu265 site did not obviously alter the structure of the protein.