| Lipase includes an important group of biotechnologically valuable enzymes which are abundantly distributed in nature. Besides of catalyzing hydrolysis of fats, they also catalyze organic synthesis, transesterification reactions, aminolysis reactions, alcoholysis reactions and acidolysis reactions. During the catalytic process, lipases exhibit wide substrate specificity, chemo-, exquisite regio- and enantio- specificity etc. These properties make the lipases be suitable for applications in various industries such as food, dairy, pharmaceuticals, detergents, textile, biodiesel, cosmetic industries and in synthesis of fine chemicals, agrochemicals, and new polymeric materials. As a result, lipases have become a kind of most valuable biocatalyst. Various lipases with different molecular stability, substrate selectivity and catalytic activity should be prepared for different practical applications and industrial processes. Therefore, to isolate lipase producing strains from disparate sources and exploit their lipases becomes one hot issue.In this paper, symbiotic and epiphytic microbes were isolated from Canarium album (Chinese olive), a plant rich in organic substances and oil, to screen novel lipase producing strains based on the ecological conception. 118 lipase producing strains were isolated from Canarium album, included 75 bacteria, 25 yeasts and 18 fungal strains. Among them, 18 strains (9 bacteria, 4 yeasts and 5 fungi) can secrete lipases with strong organic solvent tolerant stability and esterification synthesis ability. Crude lipases from two of the bacteria strains(B18 and B22), performed higher hydrotic avtivity and organic solvernt tolerant ability than others. According to their 16S rDNA sequences, strains B18 and B22 belong to Enterobacter genus and Citrobacter genus respectively. Both of the genus are common endophytes. And those two strains named as Entrobacteria sp. FN301 for B18 and Citrobacter sp.FN302 for B22, respectively.Lipase from Citrobacter sp.FN302 (CFL302) was purified via ultrafiltration, DEAE Sepharose Fast Flow ion exchange chromatography and Sephacryl S-100 gel chromatography for lipase properities investigation. The purified lipase (CFL302) exhibited a single band on SDS-PAGE by 40 kDa molecular mass. The results show that CFL302 is a thermostable, organic solvents tolerant lipase. And it displays a good catalytic activity between 40℃～60℃and can be stable at pH 4.0～9.0. The optimum temperature was at 45℃and optimum pH about 7.0. Its enzyme activity was improved when presenting in some organic solvents, such as acetone, heptane, hexane, isooctane, benzene and xylene compared to water phase. 10 mmol/L of Ca2+and Mg2+enhanced the enzyme activity of CFL302, as well as Tween20, Tween80 and Urea. CFL302 prefers to hydrolysis the ester with a carbon chain no more than 12 C. The protein N-terminal sequences analysis of CFL302 was N-I-S-C-C-C-R-E-K-P-G-I. According to the NCBI database, no homologous lipase sequence was found by far. It may be a novel lipase with specific structure and propertie.In order to obtain larger amounts of pure enzyme, medium optimization is an effective way. By single-factor experiments, Plackett-Burman and Response Surface Methodology (RSM), the optimum medium for Citrobacter sp.FN302 to produce lipase was achieved as (w/v):maltose 0.93%, yeast extract 1.8%, olive oil emulsion 2.9%, MgSO4·7H2O 0.12%, K2HPO4 0.12%, initial pH 7.5. And the optimum fermentation factors for lipase-producing were about 24h at 30℃and 250rpm with 5% inoculation. The lipase activity of the broth was 6.2U/ml and increased 4.13 fold against at that of the inital.For cloning the lipase gene, DNA library of Citrobacter sp.FN302 were constracted. Genomic DNA from Citrobacter sp.FN302 was digester partially with Sau3AI, and the fragment from 1～5Kb were recovered. Plasmid pUC18 was cleaved with BamHI. And then the genomic DNA were mixed and ligated with the plasmid. Then the recombinant plasmids were transformed into E.coli DH5a competent cells. |
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