| ?Objective? As the research object to the Bacillus subtilis(B.subtilis),with coexpression ?-glucosidase genes bgl A and bgl B,improve the enzyme activity of ?-glucosidase.And ?-glucosidase gene bgl A,bgl B and endo ?-glucanase gene EG from B.polymyxa were linked into the secretion type expression vector of L.lactis p NZ-X(p NZ8048::usp45),obtained the secretion type recombinant strains of lactic acid bacteria(LAB).This study provided a feasible solution for the recombinant expression of cellulase in the food grade strain,which layed a foundation for the research of the degradation of straw.?Methods? ?Downloading Bacillus polymyxa bgl A?bgl B?EG gene from Gen Bank,and designing primers.Total DNA as a template to amplify bgl A?bgl B?EG gene and connect them with p Bluescript II KS(+)and transform E.coli DH5?,after screening the positive clones,the plasmids were extracted and identified by double enzyme digestion and sequencing.Otained bgl A?bgl B?bgl gene from the positive plasmids and connect them with p ET-28 a and transform E.coli C41,the three recombination strains were designated as EA?EB and co-expression EAB.The strains EA and EB were mixed at ratios of 1:1?1:2?2:1 and their total ?-glucosidase activity was compared with those of each single enzyme activity group?the co-expression strain and mixed expression strains.?Downloading L.lactis MG1363 gene from Gen Bank,and designing primers.Total DNA as a template to amplify usp45 gene and connect it with p MD18-T and transform E.coli DH5?.The plasmid was identified by double enzyme digestion and sequencing.Otained usp45 gene from the positive plasmids and connected it with p NZ8048 and transform E.coli DH5?,obtained the secretory type expression vector p NZ-X.?The B.polymexa bgl A?bgl B?EG gene sequencing right from the positive recombinant plasmid were cut,respectively connecting p NZ-X into E.coli DH5?,proved correct after the extraction of recombinant plasmid transfected into L.lactis NZ9000,the nisin induced by enzyme activity determination of secretory expression Bgl A,Bgl B and EG.?Results? ?Expression of recombinant ?-glucosidase in E.coli: the result of SDS-PAGE analyses showed that both Bgl A and Bgl B were 50 ku,and the size of these proteins in the EA and EB mixed cultures was also 50 ku.The size of Bgl in the co-expression strain EAB was 100 ku.The results indicated that a Bgl complex was able to form in cells,but not in vitro.In an enzyme activity assay,the activity of Bgl from the co-expression strain was not significantly different from that of bgl in B.polymyxa,but it was significantly higher than those of Bgl A in EA and Bgl B in EB(P<0.05).The results of congo red staining also showed that the enzyme activity of Bgl was significantly higher than those of Bgl A and Bgl B.?Expression of cellulase gene in LAB: the size of Bgl A?Bgl B?EG were all 50 ku.The enzyme activity showed that the enzyme activity of B.polymyxa was significantly higher than that of the recombinant strains of enzyme by DNS(P<0.05),but has a certain activity.Congo red staining results also showed that the secreted recombinant LAB could produce the enzyme hydrolysis significantly.?Conclusions? ?In this study the ?-glucosidase two subunits of bgl A and bgl B were coexpressed in E.coli C41,than the each single and mixed expression of the enzyme activity increased four times its activity,no significant difference the original strain enzyme,the results of this study on cellulose degradation and gene coexpression to provide experimental materials.?In this study,the cloned ?-glucosidase gene bgl A,bgl B and EG from Bpolymyxa,and respectively connected with a signal peptide secreted,successfully constructed secretory expression vector p NZ-X:: bgl A,p NZ-X:: bgl B and p NZ-X:: EG. |
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