Analysis And Expression Of Xylanase Genes From Two Microbial Sources

BackgroundXylan is a polypentacarbonic sugar with very complex structure.Most of the xylans aer not utilized.Xylan needs to be degraded into Xylooligosaccharides by xylanase before it can be used effectively.Therefore,the construction of xylanase producing microorganism engineering bacteria can enrich the source of xylanase and make it more widely used in many fields such as industry,agriculture,food and feed processing.Objectives1.To supplement the types of xylanase at present,new xylanases were isolated from Aspergillus niger and Alteromonas macleodii.2.To broaden the way of xylanase research,recombinant engineering strains producing xylanase were constructed.3.To lay the foundation for further molecular modification of the xylanases,the fermentation conditions of the recombinant engineering strains were optimized and their enzymatic properties were determined.Methods1.The xylanase gene obtained from Aspergillus niger was transformed into Bacillus subtilis WB600 by electroporation,and the fermentation optimization and enzymatic properties were determined at the laboratory levels.2.To determine the xylanase gene,A.macleodii from marine microorganisms was screened and sequenced.3.The xylanase genes were analyzed by bioinformatics,and in order to express xyn ZT-1 gene at higher levels,the gene fragments were codon optimized and then synthesised.4.To obtain high copy transformants,the recombinant xylanase genes were transformed into E.coli BL21(DE3)and P.pastoris GS115,respectively.5.The recombinant xylanases were expressed in shake flask and their enzymatic properties were determined.Results1.Three xylanase genes from A.niger were successfully expressed in B.subtilis,and the enzyme activities of the recombinant strains.Under the conditions of inoculation amount of 1.0%,inoculum age of 14 h,loaded liquid of 50 m L and temperature 34?,the enzyme activity of the recombinant WB600/p WB980/xyn ZF-2 was 0.168 U/m L.Under the conditions of inoculation amount of 1.5%,inoculum age of 14 h,loaded liquid of 30 m L and temperature 37?,the enzyme activity of the recombinant WB600/p WB980/xyn ZF-318was 0.359 U/m L.Under the conditions of inoculation amount of 1.5%,inoculum age of 12h,loaded liquid of 30 m L and temperature 39?,the enzyme activity of the recombinant WB600/p WB980/xyn ZL was 0.179 U/m L.2.Three xylanase genes were obtained by sequencing the whole genome of A.macleodii and submitted to Gen Bank with the login numbers of MT814835(xyn ZT-1),MT814836(xyn ZT-2),and MT814837(xyn ZT-3).3.The results showed that the full length of Xyn ZT-1,Xyn ZT-2 and Xyn ZT-3 were 831bp,1026 bp and 2967 bp,respectively.The number of amino acids encoded by mature peptides were 247-aa,342-aa and 989-aa,respectively.They belong to GHF11,GHF43 and GHF10,respectively.4.The engineering strains of xylanases,named BL21/xyn ZT-1,BL21/xyn ZT-2,GS115/xyn ZT-3,were successfully constructed,and their fermentation conditions were optimized.The recombiant enzyme activities were 0.198 U/m L,0.187 U/m L,and 1.430U/m L,respectively.5.The recombinant xylanase was purified by Ni~+chromatography and treated by dialysis bag,and its SDS-PAGE electrophoresis and enzymatic properties were determined.The optimum reaction temperature of the three recombinant xylanases was 45?.When the temperature was higher than 50?,the enzyme activity of the recombinant Xyn ZT-1 and Xyn ZT-3 decreased rapidly and even lost.The enzyme stability of Xyn ZT-2 was better.The optimum p H of the recombinant enzyme Xyn ZT-1 was 5.0,while the optimum p H of the recombinant Xyn ZT-2 and Xyn ZT-3 was 6.0.The p H stability of Xyn ZT-1 was better than that of Xyn ZT-2 and Xyn ZT-3.Conclusions1.Three xylanase genes were obtained from A.niger and heterologously expressed in B.subtilis,and their expression conditions were optimized.2.Three xylanase genes xyn ZT-1,xyn ZT-2 and xyn ZT-3 were obtained from A.macleodii,which enriched the new xylanase enzymes.3.The xylanases were heterologously expressed in E.coli BL21(DE3)and P.pastoris GS115,respectively,and the purified recombinant xylanases were obtained,and the enzymatic properties were determined.