Construction And Study Of A Cellulosome Containing Xylanase And Cellulase And Its Degradation Effect On Avicel

The "hard-to-degradate" nature of lignocellulose has made the rich cellulose resources not used effectively in animal husbandry.Cellulose-decomposing bacteria can assemble various cellulase and hemicellulase into cellulosomes by using scaffoldin,thereby achieving the purpose of efficient degradation of cellulose by various enzymes.Artificially designed cellulosomes can provide solutions for efficient degradation of cellulose.The host strains of the artificially designed cellulosomes usually use Escherichia coli,which is easy to achieve over-expression of the protein;Bacillus subtilis is a beneficial bacteria with good protein secretion.It is a new host bacteria for expressing cellulosomes.Therefore,this experiment first designed there genes that constitute the cellulosome,the scaffoldin genes scaf containing cellulose-binding domain and two different adhesion domain genes came from Clostridium thermocellum and Clostridium cellulolyticum;The recombinant xylanase gene xyn10 B was designed using the Thermobifida fusca xylanase gene and the C.thermocellum dockerin gene doc2;Similarly,the recombinant cellulase gene cel48 F was designed using the cellulase gene and dockerin gene doc1 of C.cellulolyticum.Then the expression vectors for E.coli and Bacillus subtilis of Scaf,Xyn10 B,and Cel48 F were constructed.In addition,Scaf,Xyn10 B,and Cel48 F were expressed and purified.Finally,Scaf,Xyn10 B and Cel48 F were assembled into the cellulosome,and the effect of the cellulosome on degradation of cellulose was examined.The results were shown as follows:1?The genes of the cellulosome,namely scaffoldin gene scaf,recombinant xylanase gene xyn10 B and cellulase gene cel48 F met the basic requirements for the design of cellulosome chimera and could be used to construct expression vectors.2?The E.coli BL21(DE3)expression vectors pET-scaf?pET-cel48F?pET-xyn10 B and B.subtilis 1A747 expression vectors pET-scaf?pET-cel48F?pET-xyn10 B of the cellulosome elements Scaf,Cel48 F and Xyn10 B were successfully constructed and could be used to express the target proteins.3 ? The soluble expression of Scaf,Cel48 F and Xyn10 B in E.coli BL21(DE3)was successfully achieved,but the expression in B.subtilis 1A747 was not achieved.4?The cellulosome elements Scaf,Cel48 F and Xyn10 B could be assembled in vitro,and the cellulosome enzyme activity(0.107±0.013 U/mL)was significantly higher than that of the free enzymes mixture(0.084±0.001 U/mL).In conclusion,this study demonstrated that the assembly of free enzymes Xyn10 B and Cel48 F into the cellulosome by scaffoldin Scaf could increase the ability of the two enzymes to degrade cellulose.