| Lipases (EC.3.1.1.3, triacylylgcerol acylhydrolases) are a group of enzymes having the ability to hydrolyze triacylglycerols at an oil water interface. They widely distributed among microorganisms, plants and animals. They can catalyze the reactions of ester hydrolysis, ester synthesis and transesterification, which are widely used in the processing of fats and oils, detergents and degreasing formulations, food processing, the synthesis of fine chemicals and pharmaceuticals, paper manufacture, and production of cosmetics. To explore their biotechnological potentials, microbial lipases have attracted much attention. Bacillus subtilis, a family of gram-positive bacterium, that is studied extensively and used widely, has great potential in many fields. At present, B. subtilis genome has been sequenced and 53% of gene sequences have been reported. Its classification as a Generally Regarded As Safe (GRAS) organism by the Food and Drug Administration of USA (FDA) has made it an attractive expression host to produce proteins of commercial interest. Recently lipases from B. subtilis have attracted more focus since it shows the potentials used in food and chemistry industries. However the expression of lipase in wild type B. subtilis is low efficient, and this limited its utilization widely. In order to increase the lipase yield and characterize the lipase of B. subtilis, DNA recombinant technology was used to increase the production of enzyme. It is shown that lipases from B. subtilis had much higher activity in B. subtilis system than in others, because lipase is prone to be folded correctly. Up to now, there is no suitable plasmid to express lipase in B. subtilis, so it is necessary to design and construct a new plasmid for overexpression of lipase. The vegetative sigma factor, s43, can recognize a strong promoter regulating the expression of lipase in B. subtilis, especially –35 and –10 region of lipase gene expression. According to these informations, we designed two DNA fragments including five restriction-enzyme sites, a promoter sequence recognized by s43, a ribosome binding site (rbs) and a terminator suitable to B. subtilis. The DNA fragments were incorporated into pBD64 and a new plasmid pBSR2 was constructed. The lipase gene from strain IFFI10210 was cloned and overexpressed in B. subtilis A.S.1.1655. The purified recombinant lipase was characterized in the aspects of optimum reaction condition, substrate specificity, and effects of metal ions and sodium taurocholate. A novel plasmid, pBSR2, was constructed by incorporating a strong lipase promoter and a terminator into the original pBD64. A mature lipase gene from Bacillus subtilis strain IFFI10210, an existing strain for lipase expression, wascloned into the plasmid pBSR2 and transformed into Bacillus subtilis A.S.1.1655. Thus an overexpression strain, BSL2, was obtained. The yield of lipase is about 0.3U per ml of culture and 100 folds higher than that in Bacillus subtilis strain IFFI10210. The recombinant lipase was purified in a three-step procedure involving ammonium sulfate fractionation, ion exchange and gel filtration chromatography. Characterizations of the purified enzyme revealed a molecular mass of 24kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, maximum activity at 43oC and pH8.5 for hydrolysis of p-nitrophenyl caprylate. The values of Km and Vm were found to be 0.37mM and 303μmol mg-1min-1, respectively. The substrate specificity study showed that p-nitrophenyl caprylate is a preference of the enzyme. The metal ions Ca2+, K+ and Mg2+ can activate the lipase,whereas Fe2+, Cu2+ and Co2+ inhibited it. The activity of the lipase can be increased about 48% by sodium taurocholate at the concentration of 7mM and inhibited at concentrations over 10mM. Through two cycles of error prone PCR and one cycles of DNA shuffling, coupled with a sensitive screening method, a mutant BSL was obtained. Its catalytic activity was 4.5-fold higher than that of wild BSL. DNA sequence revealed that three bases were changed, but two of them result in substitutions of anmio acids, A349T (Lys74Asn), G493C(Val106Leu), and the other one of them, C415G, is synoymcodon mutation.. Protein analysis illustrated the stability and activity improvement of mutant lipase. Optically active alcohol, such as chiral 2-octanol, is a very useful intermediate. It was widely used in the production of pesticide and liquid crystals. In this thesis, the resolution of rematic 2-octanol catalyzed by lipase was...
|
PDF Full Text Request