Identification And Fermentation Optimization Of A Lipase Producing Strain F. Oxysporum_SHM

Lipase is an enzyme which can catalyzes the hydrolysis of fat into glycerol and fatty acids. Animals, plants and micro-organisms all can secrete lipase. Among them, microbial lipases which are widely used in the production of industrial, decontamination and food industry and other areas of fat. Domestic and international studies involving lipase enzyme catalyzed structural properties of fat, high-yield strain breeding and fermentation conditions and other aspects of control. This study is identified by screening to obtain a proprietary lipase producing strain F. Oxysporum_SHM and explore its fermentation conditions and crude enzyme lipase enzyme properties.First use olive oil as the sole carbon source to screen micro-organisms from the oil laboratory air, stove of residents’ home and the soil near the canteen. After primary and secondary screening of enzyme activity to obtain a relatively high strain Y18. By morphological and molecular characterization to determine their belonging Monilianceae, Fusarium LK., Fusarium Oxysporum, so preliminary named F. Oxysporum_SHM.Then the fermentation conditions were optimized. After election of the suitable basal medium, we conduct the single factor optimization with carbon source, nitrogen source, KH2PO4, Mg SO4, p H and medium volume. And use response surface design(CCD) to determine the optimum conditions of fermentation, That is: 1% of linseed oil, 0.5 g/L Mg SO4 ? 7H2 O, 2.0 g/L K2HPO4 ? 3H2 O, 8.0 g/L sucrose, 30.0 g/L tryptone, p H8.0, liquid volume 30.0 m L/250 m L. Under these conditions, forecast fermentation lipase activity was 5566.85 U/m L, the actual measured fermentation enzyme activity of 5216.23 U/m L, close to model predictions.Finally, the enzymatic properties of F. Oxysporum_SHM crude lipase was discussed. By ammonium sulfate precipitation, dialysis desalination, and Mono Q anion exchange chromatography to obtain a purer F. Oxysporum_SHM lipase, compared with the fermentation broth the specific lipase activity promoted by 27.10 folds. After dialysis desalting crude freeze dried lipase properties analysis, it shows that the enzyme optimum temperature is 40 ℃, and stable temperature range is below 45 ℃; The enzyme optimum p H is 9.0, stable p H is in the range of p H 3.0 to 10.0; Ca2+, Fe2+, Mn2+ can significantly enhanced the lipase activity, which Fe2+ significantly effects on the enzyme activity by increased 181%, Mg2+, Cu2+ on the enzyme activity inhibition were retained 83.82% and 76.98% of the residual enzyme activity; the determination of the lipase hydrolyzed p-NPP Km of 270.36 mg/L, Vmax of 25.91 nmol/min ? mg by Lineweaver-Burk plot of purified lipase; it shows that the F. Oxysporum_SHM lipase has higher lipase activity than the two commodities lipase which the lipase activity is only 6.2% and 13.6% by comparison of two commodities lipase activity.