Research On The Microbial Screening Of Esterase For Hydrolyzing Lactic Esters And Characteristics Of Enzymatic Reaction

In this paper, a strain was selected with high esterase activity. Its characteristics of production and enzymology and separation of L-lactic acid were investigated.Through primary screening and rescreening, a fungus, F-16, with high esterase activity was selected. Its product was relatively pure L-lactic acid. According to the Fungus Taxonomy, it belongs to Hyphomycetales, Hyphomycetes, Deuteromycotina, Eumycota, Fungi.The growth of F-16 and the production of enzyme were affected by microbial medium, including C source, N source, mineral, initial pH of medium, rotating rate, culture time and culture temperature. The results showed that the optimal N sources were peptone, corn steep liquor and yeast extract; the optimal C sources were sucrose, glucose and maltose; the optimal minerals were MgSO4 7H2O, KHPCU and CuSO4 5H2O. And the optimal culture conditions were temperature 30, rotating rate 200 r/min and culture time 72 hours.On the base of the single factor, three C sources, three N sources and mineral were selected for the Homogeneous Test. The results of the coefficients showed that the influence of peptone was the most important; then corn steep liquor, lactose and CuSO4 5H2O; the effects of others were small. The fermentation with 10L mechanically stirred fermentor was 20 hours shorter than that in the conical flasks with the highest esterase activity 14.6 U/ml, which was 30% than that in the conical flasks.The optimal reaction conditions of esterase were temperature 35 and pH 9.0. The esterase was stable under 40 between 5.0-9.0. Tests were carried out to examine the substrate characteristics of the esterase and the results showed that the esterase could hydrolyze not only lactic acetate, triacetin, and so on, with low molecular weight, but also the esters with high molecular weight. The study on the kinetics of the enzyme showed that the enzymatic reaction with lactic acetate as the substrate accorded with the Michaelis-Menten equation. It Michaelis constant Kmwas 5.77, and the maximum velocity was 0.45 u mol/L min.In order to separate the L-lactic acid and lactic acetate, the adsorption chromatography was utilized besides the extraction with ethyl ether. The operation conditions for resin A was initial 15% lactic acid, pH 6.5, temperature 30, flow velocity 2.28 ml/min taking the condition of optimal enzymatic reaction, the amount of adsorption and natural hydrolyzation of lactic acetate into account. The column was eluated with methanol and 60 water and methanol was better than 60 water.