Research On Properties Of Serine Hydroxymethyltransferase And Its Molecular Dynamics Simulation

L-serine had been widely used in many fields such as pharmaceutical and food industry. C urrently, the most promising method to produce L-serine is using Serine hydroxylmethyltransferase(SHMT) to catalyze the interconversion between serine and glycine. The method has many advantages, such as mild reaction conditions and single product. The poor thermal stability of most SHMTs restricts their application in the industry. Through the protein modification to improve the thermal stability of the SHMT is one of the research hotspot at present.The SHMT from Escherichia coli can be model enzyme to provide theoretical basises for the study of other enzymes, because it has been most studied. SHMT from E.coli SRZ018 was selected as the research object in this paper. The properties of purified protein was studied. Predicted sites of mutation by means of molecular dynamics simulation was expected to provide a theoretical basis for the site directed mutagenesis of SHMT. The main results are as follows:(1) The prokaryotic expression and protein purification system of SHMT were established. It‘s successed in amplifing glyA which is the encoding gene of SHMT from E.coli SRZ018 and constructing recombinant plasmid pET-28a-glyA. A large number of SHMT was expressed in E.coli BL21. The concentration of imidazole in elution buffer was determined as 150 mmol /L by using N i-NTA resin to purify SHMT. Finally we got 1 mg/mL enzyme protein solution.(2) The reaction conditions were builded and optimized. The concentration of factors affecting the in vitro system such as C TAB, PLP and DL-?-phenylserine, was optimized respectively. SHMT works best when reaction system containing 0.03 g/L C TAB, 50 ?mol/L PLP and 200 mmol/L DL-?-phenylserine.(3) The p H stability and thermal stability of SHMT was studied respectively. The optimum p H of SHMT is 7.5. After treated at p H 6.5-8.0 for 1 h, the enzyme activity remained more than 85% compared with the untreated group, indicated that it has good pH stability. The optimal temperature of SHMT is 40?. After treated at temperature 35-70? for 10 min, SHMT only remained less than 80% of the enzyme activity, indicated that it has poor thermal stability.(4) Mutant site was determined by the molecular dynamics simulation. The RMSF value, which was analyzed by molecular dynamics simulation software Gromacs, indicated the poor stability of G151 in SHMT. The mutation of G151 to A151 may improves the thermal stability of SHMT suggested by molecular dynamics simulation.