Screening Of Thermostable Acid Lipase Strain And Its Lipase Characterizations

A thermostable acidic lipase producing strain B-36was isolated from soil rich in oil esters. Research was conducted to have the strain identified and selected through mutagenesis, and optimize its fermentation conditions. The lipase produced by strain B-36was purified and the enzymatic properties were studied.(1)Isolation and screening of strains producing thermostable acidic lipase:50soil samples from the western suburb of Zhengzhou City were collected.48lipase-producing strains were isolated by using bromocresol purple screening plate.16strains with enzyme activity between9.0-9.6U/mL were identified at acidic tributyrin plate, and we obtain B-36, a strain preferable to produce enzyme.(2)Classification and identification of strain B-36:According to morphological, physiological and biochemical characteristics and16S rDNA gene sequence analysis, the strain was identified the bacterium Klebsiella sp., eventually named by Klebsiella sp.B-36.(3) Mutation breeding:To improve the lipase-producing activity, Klebsiella sp.B-36was mutated with UV, microwave and DES. The mutant DES-5with better stability and higher enzyme activity was obtained, and the enzyme activity was77.5%higher than the original strain.(4)The study of fermentation conditions:The fermentation medium was optimized by using single factor and orthogonal experimental design L9(33). Study showed that the optimal combination of fermentation medium (%) was:yeast extract0.8, peptone3and olive oil0.8. The fermentation conditions were optimized by conducting the single factor experiment and response surface experimental design. The optimal fermentation conditions were:pH4.85, temperature46.5℃, the liquid volume in60mL/250mL, inoculum size2.12%, and the enzyme activity was increased from17.23U/mL to21.79U/mL.(5)Separation and purification of lipase from Klebsiella sp.B-36:The fermentation broths were centrifuged and discarded the pellet. Supernatants were purified by using70%ammonium sulfate precipitation, dialyzed and Sephadex G-75chromatography, etc. The protein purity was determined via SDS-PAGE gel electrophoresis, and the electrophoretic pattern was a single band with a molecular weight of27.19KDa, indicating that a relatively pure protein sample was obtained. After the above purification process, the specific activity of purified lipase reached367.42U/mg, and the purification fold and the recovery rate were10.29times and26.45%, respectively.Enzymatic properties of the purified lipase showed that:The optimum temperature and pH for the purified lipase was60℃and4.0, respectively, and the enzyme had good thermal stability in the range of60℃, while the enzyme has a better stability under acidic conditions. The metal ions of Mg2+, Ca2+, and Na+significantly enhanced the lipase activity, whereas Fe3+Cu2+and Zn2+reduced the lipase activity, in general determining that the enzyme is a metal ion dependent enzyme.(6)Immobilized lipase Klebsiella sp.B-36was used to catalyze transesterification reaction in order to produce biodiesel. Studies have showed that lipase of Klebsiella sp.B-36can be used in catalytic synthesis of biodiesel. The effects of the immobilize lipase dosage, temperature, reaction time, methanol to oil molar ratio were determined. The results showed that immobilize lipase dosage3g, temperature40℃, reaction time24h, methanol to oil molar ratio9:1(Every8h plus1/3of the total amount of methanol), and the conversion rate could reach31.65%.In general, this article reported that Klebsiella sp. can produce thermostable acidic lipase, and preliminary study was conducted on artificial mutation, fermentation conditions, enzymatic properties and enzymatic synthesis of biodiesel. Results indicate that Klebsiella sp. has great potential in development and application, and it can be used as the starting strain of microbial lipase. The study established a theoretical foundation for strain improvement and construction of genetically engineered bacteria producing efficient lipases.