| S.marcescens can secrete a non-specific phospholipase A2,Phospholipase A2mainly by Ca2+distribution in the body,Can be divided into secretory (PLA2-Ⅰ andPLA2-Ⅱ)and cytosolic(PLA2-IIIandPLA2-Ⅳ).This paper successfully cloned phospholipase A2gene (plA2) from the viscous marcescens.connection with pUCm-T cloning vector become pUCm-T-plA2vector. After sequenced confirm the expression vector is right.The combinant expression plasmid pET-15b-plA2, transformed into BL21(DE3), IPTG induced expression, expression conditions were optimized. Phospholipase A2is identified by SDS-PAGE electrophoresis, molecular mass about27KD, mainly in the form of inclusion body. Optimization of expression conditions is to determine the optimal conditions:pH6.5,37℃.Transfer amount is1:60, IPTG final concentration is0.4mM, and inducing6h. Phospholipase A2was purified by Nickel column. The purified samples were measured activity, the specific activity of the enzyme is137U/mg. Lecithin and soybean lecithin were measured as a recombinant phospholipase substrates, Km values were0.059mM and0.124mM, determining optimal recombinant phospholipase substrate is soy lecithin.Different substrate concentration, PH, ionic strength buffer phospholipase activity of the recombinant were analyzed to determine the concentration of substrate7%, PH6.0, ionic buffer30Mm get the maximum activity. At the same time the use of recombinant enzyme degumming soybean oily applications, experiments show that when unglued time3h, enzyme dosage15μL/Kg, when the temperature is40℃unglued best, In this paper developed for large-scale application of enzymatic degumming lay basic, low cost, to overcome the pollution generated by the chemical degumming process is utilized to generate the fatty acid chain phospholipid hydrolysis lysophospholipids, lysophospholipid having good water resistance, hydration can remove it. |
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