| Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) catalyze the hydrolysis and the synthesis of esters formed from glycerol and long-chain fatty acids. Lipases are widely used in the speciality organic syntheses, including food processing, production of cosmetics, processing of fats and oils, pharmaceuticals, paper manufacture, detergents formulations, the synthesis of fine chemicals and so on. In this thesis, lipase-producing marine microorganisms were isolated. The objective strain was treated by mutations, and the enzyme production of the mutated strain as well as the properties of the crude enzyme were studied. The research results were as follows:A lipase-producing marine microorganism was isolated from five different samples, such as the intestinal contents of eeland soil samples. The lipase produced by strain ZF-16 had the highest activity to catalyze glycerolysis of oil, and the enzyme production of strain ZF-16 was 44.00 U/mL (p-NPP mcolorimetric methods) or 6.34 U/mL (titrimetric methods). Strain ZF-16 was identified as Burkholderia cepacia according to its morphology characteristics and the sequence of 16S rDNA, and it was named B. cepacia ZF-16. The enzyme production of the mutated strain obtained by UV-microwave-DES combined mutation was 12.71 U/mL (titrimetric methods). Compared with the original strain, the enzymatic activity of the mutated strain increased 100.5%, and the genetic stability was good.The optimal liquid culture medium and fermentation conditions for B.cepacia ZF-16-113 lipases production were investigated and the results were as follows:0.5% corn starch,3.0% peptone,10.0%(v/v) olive oil,0.05% MgSO4·7H2O,0.1% K2HPO4, medium initial pH was 6.0～7.0; incubation temperature was 30℃, liquid volume was 50 mL/250mL, inoculums size was 2.0%, inoculum age was 18 h, fermentation time was 68 h. Under those conditions, the enzymatic activity of B. cepacia ZF-16-113 were as high as 72.59 U/mL, and it increased 4.71-fold.After extracting by 2.0%(v/v) chloroform to remove grease, salting in by 10% ammonium sulfate and salting out by 60% ammonium sulfate, the B.cepacia ZF-16-113 lipases were obtained and the enzymatic properties were studied. The optimal pH and temperature of the lipase were pH 9.0 and 75℃, respectively. The lipase could maintain 80% activity in the pH range of 5.0～10.0 after incubation at 25℃ for 1 h. The lipase could maintain 80% activity below 40℃, but it inactivated rapidly above 40℃, the activity was only 6.8% at 75℃ for 1 h. The activity of lipase was activated by 1 mM Na+ and K+ ions, and intensely activated by Ca2+, but it was strongly inhibited by Zn2+. Besides, the activity of lipase was inhibited by SDS (ionic surfactant). Tween-20, Tween-80 and TritonX-100 (nonionic surfactant) could definitely activate the enzyme. The stability of the lipase was relatively well at hydrogen peroxide (oxidizing agent), and poor at sodium hypochlorite (oxidizing agent), and rather well at organic solvent with weak polarity, such as diethyl ether, carbon tetrachloride, toluene, benzene, cyclohexane, n-hexane and n-heptane. The lipase could degrade substrates with different chain length from C4 to C18, especially tricaprylin (C8). The B.cepacia ZF-16-113 lipases show normal Michaelis-Menten kinetics using p-NPP as substrate, and the Km and Vmax value were 7.24 mmol/L and 114.94 U·mL-1·min-1, respectively. |
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