| Lipase (Lipase, EC3.1.1.3) is an exploded triacylglycerols hydrolytic enzymes, substrates that can notonly triacylglycerol hydrolysis of glycerol and fatty acids, but also the reverse reaction catalyzed hydrolysisreaction. It is not only in heterogeneous systems-play a role (oil-water interface), a lipase and some inthe organic phase can also act as a catalyst. In recent years, with the non-aqueous enzymology deepeninglipase applications beyond the oil-water hydrolysis reaction on the interface range, is widely used in estersynthesis, separation, chemical synthesis intermediate chiral compounds the selective protection, syntheticpolymers, peptide synthesis, etc., in the pharmaceutical, bio-chemical, food and other fields have broadapplication prospects. Microbial lipase lipase wider than the flora and fauna of the reaction pH, suitabletemperature range and good substrate specificity, ease of access to high-purity preparations from industrialproduction, the main source of the current industrial lipase, and different strains derived lipase enzymaticproperties and mechanism of action is different, while different strains and culture conditions necessarynutrients are different, explore the sensitivity of strains of different solvents for research catalysis in organicsolvents laid foundation.In this paper, Xinjiang wine grape endophytes, screening can be produced from organic solvent-resistant strains of lipase, determine its taxonomic status; optimize production target bacteria resistant toorganic solvents lipase fermentation conditions, the ability to improve enzyme production strains, and itscrude enzymatic properties of the enzyme solution was studied. The main results are as follows:1. Resistant to organic solvents Lipase Producing Bacteria and identification: from a grape isolated73endophytes, using rhodamine B screening plate method to get eight lipase producing endophytes, andthen to1%n-heptane alkyl,1%toluene pressure screening by liquid fermentation and genetic stability,resistance to organic solvents to determine the production of lipase enzyme activity within the highestendophyte CJ.26SrDNA-ITS morphological and molecular biology techniques to determine the strain ofAspergillus niger (Aspergillus niger), constructed phylogenetic trees.2. Resistant to organic solvents production optimization CJ lipase fermentation conditions: Theimpact of target strains resistant to organic solvents lipase production medium and fermentation conditionswere studied to obtain the optimum fermentation conditions were: lactose4g, peptone6g, cereal cookingoil5ml, MnCl20.1g, pH7,25%of medium volume, inoculum3%, temperature30℃,150r/min speedtrain72h, this time resistance to organic solvents lipase yield up8.75U/mL.3. CJ production of Crude enzymatic properties of organic solvent-resistant lipase: CJ producedcrude enzyme lipase optimum reaction temperature is40℃, optimum pH7.0; insulation1h retained54.55%of enzyme activity at50℃, has good thermal stability; relatively stable in the pH range of3.0to7.0, there is a certain acid resistance. Metal ions Mn2+on the enzyme activity can promote, Zn2+, Fe2+, Cu2+inhibited the enzyme activity, K+, Ca2+, Na+, Mg2+has little effect on the enzyme activity. Whenthe enzyme blend with sunflower oil and grain as substrate, enzyme activity was228%and180%,significantly higher than the olive oil and the hood. In an organic solvent, and the enzyme activity of thecrude enzyme solution with increasing concentration of the organic solvent is decreased, a longer half-lifeof10%toluene and acetone, respectively, and62.4h60.8h,30%,50%di a longer half-life are toluene,the organic solvent concentration of50%half-life is very low, especially in the half-life of50%ethanol isless than1h. Michaelis constant Km and Vmax are0.032mM,0.066mM/min, we can see the surface of thefat enzyme and substrate affinity bigger. Enzymatic properties suggest, CJ lipase produced a strong organicsolvent tolerance. |
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