| Lipase is a diverse and ubiquitous family of enzymes catalyzing both the hydrolysis and synthesis of various ester compounds. Since its exquisite stability, chemoselectivity, regioselectivity and stereoselectivity, lipase has become one of the most commonly used enzymes in many industrial processes. Lipases are widely distributed throughout animals, plants and microorganisms, and the major lipases used as catalysts are isolated from bacteria.In this thesis, a lipase gene lipK107 was cloned from a screened strain Proteus sp. K107 and heterologously expressed in Escherichia coli BL21 (DE3). Its enzymatic properties and applications in biodiesel production and resolution of a-phenylethanol were investigated. The structure-function relationship of lipase LipK107 was also studied.Section one:Isolated a lipase-producing bacterium, identified its species, cloned the lipase gene and analyzed the sequence.A lipase-producing bacterium K107 was isolated from soil samples of China and identified to be a strain of Proteus sp.. With genome-walking method, the open reading frame of lipase gene lipK107 was cloned. It consisted of 864 nucleotides, encoding a protein of 287 amino acids. Alignment of sequences through BLAST showed that the amino acid sequence of LipK107 shared 79%identity with that from Proteus vulgirus K80, but less than 50% identities with other lipases.Section two:Expressed the lipase gene lipK107 in E. coli BL21 (DE3), purified the LipK107 and investigated its enzymatic properties.The gene lipK107 was cloned into plasmid pET-28a, then expressed in E. coli BL21. After optimization, the recombinant lipase LipK107 could be produced in E. coli mainly as a soluble form.The lipase activity toward p-nitrophenol-laurate was examined using purified LipK107. The LipK107 had an optimal temperature of 35℃, and an optimal pH of 9.0 with good stability. The specific activity of purified LipK107 was 1200 U mg-1 of protein at the optimal condition. The activity of LipK107 was improved when Ca2+was present in the hydrolytic reaction with a final concentration of 5 mM. LipK107 also had an outstanding characteristic of high tolerance to high-polarity solvents, such as methanol. Low-polarity solvents like n-hexane and isooctane had little effect on the lipase activity.Section three:Applied the recombinant E. coli in biodiesel production as whole-cell biocatalyst, explored its characteristics.The recombinant E. coli expressing lipK107 was applied in biodiesel production in the form of whole-cell biocatalyst. It was the first time to use E. coli whole-cell biocatalyst expressing lipase in biodiesel production, and the advantages could be summarized as following:(A) Activity of the biocatalyst increased significantly when cells were permeabilized with cetyl-trimethylammoniumbromide (CTAB); (B) The biodiesel reached a yield of nearly 100%after 12 h reaction at the optimal temperature of 15℃, which was the lowest temperature among all the known catalysts in biodiesel production. The excellent productivity of LipK107 at comparatively low temperature would provide substantial savings in energy consumption as well as mild and clean procedure in conventional industrial process; (C) The E. coli whole-cell biocatalyst showed high activity with wide range of water (30-100%by weight of the substrate), which would make it to be a potential effective enzyme for eliminating substrate availability limitations; (D) The transesterification was carried out efficiently in a mixture containing 5 molar equivalents of methanol to the oil, suggested that LipK107 had high tolerance to methanol; (E) This biocatalyst showed high activity towards many vegetable oils; (F) This biocatalyst displayed exciting stability after repeated usage of 5 cycles. Thus, this whole-cell catalyst with excellent characteristics might become a commercially viable biocatalyst in biodiesel production.Section four:Applied the purified lipase LipK107 into the resolution of a-phenylethanol, optimized the reaction condition, examined the enantioselectivity of LipK107.The capacity of lipase LipK107 catalyzing the kinetic resolution of racemates was investigated, and the transesterification resolution of racemic a-phenylethanol was used as model reaction. The optimal temperature of 20℃further determined that LipK107 was a psychrotrophic lipase in organic reactions. The conversion and eep were greatly dependent on the water concentration, and the LipK107 exhibited high activity when a large amount of water was added into the reaction. Besides, the chain length of acyl donor also had a significant effect on the reaction, and the highest conversion was achieved when methyl palmitate was used. After optimization, the conversion and eep attained to 30.3%and 95.9% respectively, suggested that lipase LipK107 had good enantioselectivity.Section five:Investigated the structure-function relationship by combining the method of molecular modeling with site-directed mutagenesis.Based on the analysis of the model structure of LipK107, a "lid" was found to cover the active site, and different mutations were introduced into the "lid". Results showed that the activities of hydrolysis and synthesis increased 13.3%and 51.1%respectively when the hydrophobicity of "lid" was enhanced. While, the activity of LipK107 decreased more than 70%when the hydrophobicity or electrostatic of "lid" was reduced. Modification of the "lid" domain also affected the substrate preference of LipK107. These results were consistent with the prediction. Therefore, the "lid" had a dramatically effect on lipase activity, and the computer simulations could be used as an efficient method for identifying substitution site, which might open site-directed mutagenesis opportunities to develop highly performing biocatalysts for industrial needs.Three mutants were constructed to examine the effect of native catalytic triad on lipase activity. Results of both hydrolysis and synthesis revealed that the native catalytic triad was crucial for LipK107, and the modification of catalytic triad leaded to great loss of activity.
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