Cloning,Expression,and Characterization Of Novel Xylanase Genes From Fungal Strains Of Mangrove

Endo-1,4-?-D-xylanase can randomly cleaves xylan backbone and it is the most crucial one among the xylan degrading enzymes.It also has important applications in many industries,such as baking and animal feed.Mangrove is a special category environment for the transition from land to the ocean and contains the rich microbial resources which are vastly unexplored.In this study,xylanase-producing fungal strains were screened from mangrove of Guanxi.And 9 xylanase gene fragments were cloned,3 full-length xylanase genes were obtained by using thermal asymmetric interlaced-PCR procedure.These 3 genes were expressed successfully in Pichia pastoris X33.The recombinant proteins were purified and characterized in detail.The results are as follows:1.Using xylan as the only carbon source of primary screening medium,6 fungal strains which showed different morphology and transparent zones were screened from mangrove of Guanxi.Next,the sequences of 3 GH10 xylanase gene fragments and 6 GH11 xylanase gene fragments were amplified from these strain.These genes showed the highest identities from 78%to 94%with known xylanases.2.Four full-length xylanase genes(a genes were confirmed to be GH10 and other 2 gene were confirmed to be GH11)were obtained by using TAIL-PCR.Then the cDNA sequences of xylanases were cloned and the basic bioinformatics were analyzed:xynMF13-GH10,xynMF13-GH11 and xynMF10-GH11 were 1332bp,696bp and 663bp,encoding the polypeptide of 444,232 and 221 amino acid residues respectively.These three xylanases had similarities of 67%,91%and 83%with known endoxylanases respectively.3.The main enzymatic properties of the purified recombinant xylanase rXynMF13-GH10 are as follows.The specific activity of purifird rXynMF13-GH10 was 1697.03 ± 3.55 U/mg.The optimum pH and temperature of purifird rXynMF13-GH10 were 5.5 and 55 ° C,respectively.And it was resistant to high concentrations of NaCl in the reaction system.The Km and Vmax value were 7.85 ± 0.05 mg/mL and 4166.67 ± 1.54?mol/(mg·min)respectively.The enzyme degraded xylan,producing xylobiose and xylotriose as the predominant products.4.The main enzymatic properties of the purified recombinant xylanase rXynMF10-GH11 are as follows.The specific activity of purifird rXynMF1O-GH11 was 4376.97 ± 5.17 U/mg.The optimum pH and temperature of purif-ird rXynMF10-GH11 were 6.0 and 50 ?,respectively.Moreover,it was resistant to high concentrations of NaCl in the reaction system and stable in NaCl solution of 4 mol/L.The Km and Vmax value were 5.38 ± 0.22 mg/mL and 9615.38 ± 0.09 pmol/(mg·min)respectively.The enzyme degraded xylan,producing a series of xylosaccharides such as xylobiose and so on.5.The main enzymatic properties of the purified recombinant xylanase rXynMF]3-GH11 are as follows.The specific activity of purifird rXynMF13-GHl 1 was 1322.82 ± 4.86 U/mg.The optimum pH and temperature of purifird rXynMF13-GH 10 were 5.0 and 45 ° C,respectively.And it was resistant to high concentrations of NaCl in the reaction system and stable in NaCl solution of 4 mol/L.The Km and Vmax value were 3.16 ± 0.33 mg/mL and 2688,17 ± 1.98 ?mol/(mg·min)respectively.The enzyme degraded xylan,producing a series of xylosaccharides with similar yield.In this study,three new xylanase genes were cloned from the fungi of mangrove and they all successfully expressed in Pichia pastoris.These three weakly acid and mesophilic xylanases have high specific activity,which can lay the foundation for the further study of xylanase gene from microbial origin of the special environment and may find diverse applications in industrial fileds.