Gene Cloning, Heterologous Overexpression And Enzymatic Characterization Of Two Novel Xylanases From Penicillium Sp. F63 CGMCC1669

Xylan is heterogeneous polysaccharide consisting of a polymer ofβ-1,4-linked xylose units with various substituted side chains.As the major component of plant cell wall,xylan, after cellulose,is the second most abundant renewable natural resources.The complete degradation of xylan is accomplished by the synergistic action of various enzymes,of which the most important are endo-β-1,4-xylanases(EC3.2.1.8).Xylanases have broad potential applications in the fields of paper and'feed industry,food processing,textile,energy science and environmental protection.A variety of microorganisms,bacteria and filamentous fungi,have been reported to produce xylanases.Filamentous fungi are the ideal source of xylanases due to their secretion expression,high xylanase productivity and easy cultivation.To date,several xylanase genes from Penicillium spp.have been cloned and expressed.However,the use of fungal xylanases is still limited by high production cost.Therefore,highly desirable xylanases with excellent properties and high yield are still in significant need.In the present paper,we described the cloning of two xylanase genes(xyn11F63 and xyn10F63) from Penicillium sp.F63 CGMCC 1669, overexpression of xyn11F63 in Pichia.pastoris and characterization of the recombinant xylanase.In the present study,we confirmed the xylanase-producing ability of the native strain of Penicillium sp.F63 through shake flask fermentation using Mandels-Weber's medium with birchwood xylan as the induction substrate.Two xylanase-encoding genes,xyn11F63 and xyn10F63,were cloned from Penicillum sp.F63 CGMCC1669 using degenerated PCR,thermal asymmetric interlaced(TAIL)-PCR and reverse-transcription(RT)-PCR techniques.As for the gene xyn11F63,the full-length chromosomal DNA consists of 724 bp including a 73-bp intron, and encodes a 217-amino acid family 11 xylanase.Moreover,the chromosomal DNA of xyn10F63 have a size of 1,338 bp,interrupted by 4 introns with lengthes of 64,52,52,72 bp, respectively,in which an open reading frame of 1098 bp,encoding a family 10 xylanase,was identified.The deduced amino acid sequence of xyn11F63 shows the highest identity of 70%to the xylanse from Penicillium sp.strain 40,while that of xyn10F63 is 81%compared to the xylanase from Penicillium chrysogenum.The results indicate that the two xylanase genes are novel ones.After the constructed recombinant plasmid pPIC9-xyn11F63 transformation into P. pastoris GS115,The gene xyn11F63 was functionally expressed in the recombinant P.pastoris, with a xylanse activity of 516 U/ml,which is the highest level reported to date,after the methanol induction for 72 h.After purification to electrophoretic homogeneity,the recombinant xylanase showed maximal activity at pH 4.5 and 40℃,was stable at acidic buffers of pH 4.5～9.0,and was resistant to proteases(proteinase K,trypsin,subtilisin A andα-chymotrypsin).The specific activity,K_m,and V_max for oat spelt xylan substrate was 7,988 U mg^-1,22.2 mg ml^-1,and 15,105.7μmol min^-1 mg^-1,respectively.Moreover,the recombinant xylanase rXYN11F63 has excellent substrate specificity,and no cellulose activity was detected.These properties make XYN11F63 a potential economical candidate for use in paper,feed and food industrial applications.