Characterization Of Maleate Cis-trans Isomerase From Serratia Marcescens And The Fermentation Optimization

Maleate cis-trans isomerase (EC5.2.1.1, MaiA) specifically catalyzes geometricisomerization of fumarate from maleate, while carbon-carbon double bond will not bemigrated. Fumarate, the product of the reaction, has extensive market development prospectsin food, chemical industry, medicine, coating, resin and plasticizer industries. As theproduction of fumarate through biological synthesis is more enviromental friendly thanchemical synthesis, theory study and efficient preparation of the MaiA show socialsignificance and economic advantage.In this research, maiA gene from Serratia marcescens has been cloned in Escherichiacoli. Recombinant maleate cis-trans isomerase has been expressed, purified and characterized.Furthermore, fermentation conditions of the recombination strain has been optimised in shakeflasks to improve the production of the recombination enzyme. The main contents of thisstudy are as follows:(1) The maiA gene from S. marcescens was inserted in pET-24a(+) and the resultingplasmid was transferred to E. coli BL21(DE3) to construct E. coli BL21(DE3)/pET-24a(+)-maiA. According to the SDS-PAGE, recombinant MaiA has been expressedsuccessfully after being induced. Then the purified recombinant maleate cis-trans isomerasewas obtained by affinity chromatography.(2) Enzymatic properties of MaiA show that the optimum temperature was37℃andthe optimum pH was8.4. Under the optimal conditions, the Kmvalue was4.20mmol·L-1, theVmaxvalue was1.27mmol·min-1, the kcatvalue was4.38s-1and the catalytic efficiency(kcat/Km) was1.04L·mmol-1·s-1.(3) We optimized the fermentation condition of MaiA by E. coli BL21(DE3)/pET-24a(+)-maiA in shake flasks. First, the constituents of fermentation medium, includingcarbon source, nitrogen source and metal ions, were optimized through single factor tests.Then we conducted L9(34) orthogonal tests on these constituents. It indicated that the theoptimal carbon source was glycerol (10g·L-1), nitrogen source was yeast extract (12g·L-1)and peptone (12g·L-1), metal ion was Mg2+(2mmol·L-1). According to single factor tests, theoptimal volumn of fermentation medium in250mL flask was30mL and the optimal rotatingspeed was200r·min-1. After optimization, the enzyme activity reached88.2U·mL-1, about21%higher than the initial level.