| Lipase have catalytic effect on some ester with big or small molecular weight, the polyol ester, amide compounds and some polybasic acid ester etc substrates. Current studies of lipase, mainly concentrated in the development of green energy and chiral resolution of drug intermediates, etc. Because of lipase from aspergillus oryzae showed high stereo selectivity to some material with chiral properties, therefore it has the bigger potential applications in production. In this paper, studied the separation and purification of intracellular enzyme, enzyme properties, the immobilization of lipase, resolution of ethyl-2-(4-hydroxyphenoxy) propionate.First, break the cell wall, and then the optimization the extraction conditions of lipase. Extraction of lipase with Tris-HCl (pH8.5, 0.05mol/L) in 1～1.5h. In this condition, the extraction yield of lipase is highest.Second, the lipase was purified by ammonium sulfate precipitation, hydrophobic interaction chromatography. One lipase was obtained and had the molecular weight of 25.8 kDa determined by SDS-PAGE. The enzyme activity recovery of lipase was 11.4%. The purification fold of Lipase was 34.Third, we studied the characterization of the purified lipase.(1)The optimum reaction temperature of lipase is 35℃, under 40℃ to maintain a relatively stable state, when the temperature arrived the degree of more than 40℃, enzyme become instability. The optimum pH of the enzyme reaction is 7.5, different buffer system’s influence on the enzyme activity is also different. When the pH is controlled in 7.0, it maintains relative stability of the enzyme activity. When the pH enhanced or reduced, enzyme activity loss is obvious.(2) The effect of metal ions on enzyme activity is mainly embodied as follows, the inhibitory effect of Zn2+, Cu2+, Na+ on lipase is small. K+, Mg2+, Al3+ have certain inhibitory effect on lipase, and the inhibitory effect of Fe2+ is very obvious, Ca2+, Mn2+ have promoting effect on lipase.(3)In the Surfactant, TrionX-100, twain-80, twain-20 has certain promoting effect on enzyme activity. SDS inhibits the enzyme activity. The inhibitors of EDTA activated the enzyme.(4) In the influence of organic solvents on enzyme activity, n-hexane, n-heptane and the normal octane play a role in promoting the enzyme activity of lipase. And the dimethyl sulfoxide, methanol, ethanol, acetone and isopropanol for inhibited the enzyme activity of lipase.(5) In the substrate specificity of enzymes, found that the lipase has high catalytic activity on p-nitrophenyl esters with short chains carboxylic group (C2).And the olive oil and triolein have the same effect.(6) In the Zymography experiments, we found a transparent hydrolysis on the substrate color plate. This indirect the purification of the enzyme lipase is we wanted.(7) The Km and Vmax of lipase for pNPA were 18.84 mmol/L and 7.3 mmol/L·min.(8) With different adsorption method and embedding method for immobilizing the lipase, we found that with ion exchange adsorption immobilized lipase, get the highest recovery rate of enzyme activity. And when adsorption temperature is 35℃, the adsorption time is 12 hours, enzyme liquid concentration containing 1.5 mL liquid enzyme in 5 mL, the effect is best.(9)We have researched the condition in resolution of ethyl-2-(4-hydroxyphenoxy) propionate. The reaction medium is phosphate buffer at pH8.00.2 mol/L, added enzyme content is 2%, the amount of substrate was 1%, the reaction time is 2.5 h. |
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