Directed Evolution Of A Lipase From Bacillus Subtilis

Lipases (EC.3.1.1.3, triacylylgcerol acylhydrolases) are a group of enzymeshaving the ability to hydrolyze triacylglycerols at an oil water interface. Theywidely distributed among microorganisms, plants and animals. They can catalyzethe reactions of ester hydrolysis, ester synthesis and transesterification, which arewidely used in the processing of fats and oils, detergents and degreasingformulations, food processing, the synthesis of fine chemicals andpharmaceuticals, paper manufacture, and production of cosmetics. To exploretheir biotechnological potentials, microbial lipases have attracted much attention.Bacillus subtilis, a family of gram-positive bacterium, that is studiedextensively and used widely, has great potential in many fields. At present, B.subtilis genome has been sequenced and 53% of gene sequences have beenreported. Its classification as a Generally Regarded As Safe (GRAS) organism bythe Food and Drug Administration of USA (FDA) has made it an attractiveexpression host to produce proteins of commercial interest. Recently lipases fromB. subtilis have attracted more focus since it shows the potentials used in food andchemistry industries. However the expression of lipase in wild type B. subtilis islow efficient, and this limited its utilization widely. In order to increase the lipaseyield and characterize the lipase of B. subtilis, DNA recombinant technology wasused to increase the production of enzyme. It is shown that lipases from B. subtilishad much higher activity in B. subtilis system than in others, because lipase isprone to be folded correctly. Up to now, there is no suitable plasmid to expresslipase in B. subtilis, so it is necessary to design and construct a new plasmid foroverexpression of lipase.The vegetative sigma factor, s43, can recognize a strong promoter regulatingthe expression of lipase in B. subtilis, especially –35 and –10 region of lipasegene expression. According to these informations, we designed two DNAfragments including five restriction-enzyme sites, a promoter sequence recognizedby s43, a ribosome binding site (rbs) and a terminator suitable to B. subtilis. TheDNA fragments were incorporated into pBD64 and a new plasmid pBSR2 wasconstructed. The lipase gene from strain IFFI10210 was cloned and overexpressedin B. subtilis A.S.1.1655. The purified recombinant lipase was characterized inthe aspects of optimum reaction condition, substrate specificity, and effects ofmetal ions and sodium taurocholate.A novel plasmid, pBSR2, was constructed by incorporating a strong lipasepromoter and a terminator into the original pBD64. A mature lipase gene fromBacillus 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 about0.3U per ml of culture and 100 folds higher than that in Bacillus subtilis strainIFFI10210. The recombinant lipase was purified in a three-step procedure involvingammonium sulfate fractionation, ion exchange and gel filtration chromatography.Characterizations of the purified enzyme revealed a molecular mass of 24kDa insodium dodecyl sulfate-polyacrylamide gel electrophoresis, maximum activity at43oC and pH8.5 for hydrolysis of p-nitrophenyl caprylate. The values of Km andVm were found to be 0.37mM and 303μmol mg-1min-1, respectively. The substratespecificity 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% bysodium taurocholate at the concentration of 7mM and inhibited at concentrationsover 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. Itscatalytic activity was 4.5-fold higher than that of wild BSL. DNA sequencerevealed that three bases were changed, but two of them result in substitutions ofanmio acids, A349T (Lys74Asn), G493C (Val106Leu), and the other one of them,C415G, is synoymcodon mutation. Protein analysis illustrated the stability andactivity improvement of mutant lipase.