Expression Of EG Gene In E. Coli And Expression Of BG Gene In Lactic Acid Bacteria

?Objective?In order to enhance the enzymatic hydrolysis efficiency of cellulose enzyme and improve the utilization rate of cellulose.In this study,the EG gene was successfully cloned and expressed with the carrier of E.coli plasmid basised on previous work.In addition,the bglA and bglB genes were co-expressed with the lactobacillus plasmid.This experiment not only helps understanding the working mechanism of cellulase enzyme,improving the utilization rate of cellulose,but also provides new prospects in the study of new animal feed,which plays an key role in the laboratory.?Methods?(1)Primers were designed based on the EG gene sequence of GenBank b.polymyxa.The EG gene was amplified from the genome of Bacillus polymyxa as a template,and then the obtained target fragment was linked with pBluescript II KS(+).The ligation product was transformed into E.coli DH5?,and the EG gene with the correct sequencing result was digested and then ligated into p ET-28 a plasmid and transferred into E.coli BL21.Finally,double enzyme digestion was performed to verify that the appropriate engineered strain was induced by IPTG.The protein was indued and the protein activity was detected by DNS after purification of the protein by nickel column.(2)Design the upstream primers of the signal peptide according to the L.lactis MG1363 usp45 gene sequence of GenBank,and design the downstream primers of A and B according to the bglA and bglB sequences of the BG gene of GenBank b.polymyxa.Then the upstream of the signal peptide was used as the upstream primer,and downstream of A and B are the downstream primers respectively.The previously secreted expression vectors pNZ-X::bgl A and pNZ-X::bglB were used as templates for PCR.The target fragments RA and RB were amplified and the target fragment was ligated and cloned into the pBluescript II KS(+)vector.The cloned product was transformed into E.coli DH5? and verified by sequencing.After double digestion,the target fragment RARB was obtained.The target fragment was ligated with the p NZ8048 vector to obtain the recombinant plasmid p NZ-X::bglA::X::bgl B which was electrotransformed into the L.lactis NZ9000 competent cells to obtain the recombinant LAB strain.After the expression was induced by nisin,Congo red staining was used.And protein activity was detected by DNS method.?Results?(1)Expression of EG gene in E.coli: A successful cloning and expression vector was constructed,and the recombinant protein was successfully expressed and purified.The size of the protein was about 50 ku.And its activity,by the way of DNS,was determined by DNS method.(2)Co-secretory expression of bglA and bglB in lactic acid bacteria: A co-secretory expression vector was successfully applied and the recombinant protein was expressed and purified.The results showed that the recombinant protein was about 100 ku in size,and the enzyme activity was detected in the supernatant.The recombinant protein can be secreted outside the cell.The results of Congo red staining and DNS detection showed that the co-expressed enzyme activity was higher than that of the two expressed single subunits,and co-expression was beneficial to increase the degradation efficiency of cellulase.? Conclusion ? (1)The cellulose endonuclease EG is one of the important components of cellulase,which mainly acts on the amorphous region of cellulose,and cuts the long chain of cellulose into short chains to produce a large number of small molecules.In this experiment,the E.coli plasmid was used as a vector to successfully clone and express EG gene,which played a certain role in the further research.(2)Both Bgl A BglB in the cellulase system act mainly on cellobiose short-chain cellooligosaccharides,and they are finally hydrolyzed to glucose.In this experiment,the recombinant plasmid pNZ-X::bgl A::X::bglB was successfully constructed and electrotransformed into L.lactis NZ9000 competent cells.Finally,the target protein was successfully expressed.This was not only provided for the study of novel animal feed additives,the also provided conditions for the follow-up work.