Mutation Breeding Of Lipase Production Strain Aspergill Us Oryzae WZ007and Purification Of Lipase From The Mutation Strain

Lipases (triacylglycerol hydrolases) catalyze the hydrolysis, esterification, transesterification, widely used in detergents, food, medicine, leather, textile and other industries. In recent years, most of the research works in this field focused on energy development and resolution of chiral compounds. Lipase from Aspergillus oryzae has an important industrial application prospects due to its excellent catalytic activity and optical resolution for chiral compounds. In this paper, mutation breeding of A. oryzae WZ007, fermentation conditions, purification and enzymatic properties of lipases from the mutation strain Cs007were studied.First,137Cs y ray was used to induce mutations with enhanced yield of lipase in A. oryzae WZ007. A mutant strain Cs007with about110%increase in lipase yield compared with the wild strain was obtained Second, different carbon sources and nitrogen source affecting cell growth and lipase yield of mutain strain Cs007were investigated by single-factor experiments. The optimal carbon and nitrogen source for cell growth and lipase production were olive and peptone, respectively. The maximum extracellular and mycelium bound enzyme activities were obtained when the concentrations of olive and peptone were1%(V/V) and2.2%(W/V), respectively. The extracellular and mycelium bound lipase activities achieved6.06U/mL and365.5U/g dry biomass using pNPA assay. The extracellular and mycelium bound lipase activities achieved28.5U/mL and1150.5U/g dry biomass using olive assay. Next, extracellular lipase production in A. oryzae Cs007were further optimized by response surface analysis methodology (RSM). Consequently, the optimum culture medium were composed of olive oil1%(V/V), peptone1.76%(W/V), KH2PO40.11%(W/V), MgSO40.05%(W/V), NaCl0.05%(W/V) and gum arabic0.37%(W/V). After48h incubation with the medium (initial pH5) under the condition of200rpm and30℃, the maximum lipase activity achieved6.49U/mL, which led to a2.32folds increase as compared to the initial result2.8U/mL.Third, lipase from A. oryzae Cs007was purified homogeneity using ammonium sulfate precipitation, Q Sepharose Fast Flow anion exchange chromatography and Superdex G-75gel filtration. Two lipases (Lip I, and Lip Ⅱ) were obtained and had the same subunit molecular weight of25, 000determined by SDS-PAGE. The enzyme activity recoveries of Lip I and Lip Ⅱ were4.98%and0.94%, respectively. The purification folds of Lip Ⅰ and Lip Ⅱ were31.03and29.82, respectively.Last, enzymatic properties of purified Lip Ⅰ and Lip Ⅱ were investig-ated.(1) The optimum temperatures for Lip Ⅰ and Lip Ⅱ were35℃and40℃, respectively. Two lipases were stable below40℃, and the stability declined rapidly as soon as the temperature increased up to50℃. The optimum pH for Lip Ⅰ and Lip Ⅱ were both7.5. Two lipases were stable in the pH range from5to7.5.(2) Both the enzyme activities of two lipases were inhibited by Cu2+、 Ag+and SDS, and the activity of Lip Ⅰ was stimulated by EDTA.(3) Lip Ⅰ had a poor stability in isopropanol and butanol, and Lip Ⅱ had poor stability in isopropanol, acetone and toluene. The enzyme activities of Lip Ⅰ and Lip Ⅱ were both improved greatly in cyclohexane and heptane.(4) Lip Ⅰ and Lip Ⅱ had high catalytic activity on p-nitrophenyl esters with short chains carboxylic group (C2), as well as olive oil and triolein.(5) The Km and Vmax of Lip Ⅰ for pNPA were0.99μmol/mL and13.51μmol/mL·min. The Km and Vmax of Lip Ⅱ for pNPA were1.1μmol/mL and12.5μmol/mL·min.