Gene Cloning,expression And Characterization Of Xylanases Xyn11-1 And Xyn22-1 From Saline-alkaline Soil

Xylanases are critical hemicellulose degrading enzyme,it could degrade xylan into oligosaccharides,which is further synergistically degraded into xylose by glucosidase and other side-chain hydrolases.Xylanase are widely used in medicine,paper-making,animal forage,Marine functional foods and textile industry.Halophilic,basophilic,salt tolerant and alkaline toleran xylanases are suitable for alkaline paper-making production,sewage treatment and marine food production,which could improve production efficiency and reduce production cost.In this study,eleven GH10 and twelve GH11 partial xylanase sequences were obtained from the metagenomic DNA of saline-alkaline soil by GH10 and GH11 xylanase degenerate primers and Touchdown-PCR method.The complete gene sequence of the xylanase xyn11-1 and xyn22-1 was obtained using TAIL-PCR method.xyn11-1 is composed of 699 nucleotides,which encodes 232 amino acids and a termination codon.Xyn11-1 is a novel GH11 xylanase sharing the identity of 79%with the reported GH11 xylanase from Cyphellophora europaea CBS 101466.In order to detect its biological activity,the recombinant plasmid pPIC9K-11-1 was constructed,and expressed in P.pastoris GS115.The specific activity was 123.59 U/mg,Km and Vmax were 3.66 mg · mL-1 and 101.01 ?mol·min-1·mg-1,respectively.The optimal pH of Xyn11-1 is 6.0,and it remains 60.37%relative activity at pH8.0-10.0(alkalophilicity),the residue activity could reach 60.38%after incubation at pH6.0-10.0 for 1 h(alkaline tolerance).The optimal temperature is 50?,the residue activity could reach 82.34%after incubation at 30? for 1 h.Xyn11-1 could retain 77.35%relative activity at 0-4.5 M NaCl(halophilism).xyn22-1 is composed of 690 nucleotides,which encodes 229 amino acids and a termination codon.Xyn22-1 shared the highest identity(98%)with the reported GH11 xylanase from Nocardiopsis valliformis(without protein characterization).the recombinant plasmid Xyn22-1-pET28a(+)was constructed,and expressed in Escherichia coli BL21(DE3)by IPTG.The specific activity was 176.3 U/mg,Km and Vmax were 1.907 mg·mL-1 and 185.19 ?mol · min-1·mg-1,respectively.The optimal pH is 7.0,and it remains 84.28%relative activity at pH6.0-8.0,the residue activity was 60.68%after incubation at pH5.0-11.0 for 1 h(alkaline tolerance).The optimal temperature is 60?,the residue activity could reach 79.65%after incubation at 40? for 1 h.Xyn22-1 could retain 80.04%relative activity at 0-4.5 M NaCl,and retained 54.94-70.34%relative activity at 4.5-5.74 M NaCl(halophilism).Xyn22-1 remained 88.70%residue activity after incubation at 5.0 M NaCl for 10 min,remained 62.8%residue activity after incubation at 5.0 M NaCl for 1 h(salt tolerant).In this study,two xylanases named Xyn11-1 and Xyn22-1 were obtained from the saline-alkaline soil.Xyn11-1 is alkaline-tolerant enzyme,alkalophilicity and halophilism.Xyn22-1 is alkaline-tolerant and salt-tolerant enzyme,halophilism.They has great potential for basic research and industrial applications.