Expression Of Candida Antarctica Lipase B In Lactococcus LactisMG1363

In recent years,it has become a resesrch focus in molecular biology of lactic acidbacteria that exploring some genes with practical applications significance by cloning andexpression in lactic acid bacteria and constructing a food-grade selection marker inLactococcus lactis expression system.Objective:The Candida antarctica lipase B(CALB) gene was cloned into a food-grade expressionvector pMG36e-NisI which has a Emrselection marker renamed pMG36e-NisI-CALB andwas expressed in L. lactis MG1363. Then, the usp45,CALB,Spax gene were cloned into aexpression vector pMG36e-nisI renamed pMG36N-UCS which display Candida antarcticaLipase B on the Surface of L.lactis,so,the feasibility of whole-cell biocatalyst wasexplored.The prenisin gene was cloned in T-vector and sequenced for further research.Methods:1)According to the CALB sequence, a pair of primers was designed to amplify theCALB. The CALB gene was cloned into a food-grade expression vector pMG36e-nisI2）PCR and enzyme digestion was applied to screen the recombinant plasmid and thesame methods was used to screen the recombinant L.Lactis after each plasmid wastransformed into the bacteria by electroportion exclusively.Then,in order to construct afood-grade vector pMG36N-CALB, erythromycin resistance gene from the recombinantplasmid pMG36e-NisI-CALB was knocked out by redesigning the pair of specific primers.3) The genome of MG1363and Staphylococcus aureus was extracted to provide thetemplate for PCR the gene of usp45and Spax. And the CALB gene was cloned.Then thePCR production was cloned to a food-grade vector pMG36e-nisI.4) PCR and enzyme digestion was applied to screen the recombinant plasmid and thesame methods was used to screen the recombinant L.Lactis after each plasmid wastransformed into the bacteria by electroportion exclusively.Then,In order to construct afood-grade vector pMG36N-UCS, erythromycin resistance gene from the recombinantplasmid pMG36e-NisI-UCS was knocked out by redesigning the pair of specific primers.5)The lipase activity was determined to study the properties of the CALB. 6)The genome of LN1.28(L.lactis which produces nisin) was extracted to provide thetemplate for PCR the gene of prenisin. And the PCR production was cloned to T vector andsquencedresults1)pMG36e-NisI-CALB was constructed,then pMG36N-CALB was constructed and wasexpressed in L. lactis MG1363.2）pMG36e-NisI-UCS was constructed, then pMG36N-UCS was constructed and wasexpressed in L. lactis MG1363.3）The optimal conditions of enzymatic hydrolysis were as follows: pH value of8.0~8.5and temperature of40~45℃4）The prenisin gene was loned into T-vector.