Improvement In The Temperature Characteristics Of Xylanase And Its Application In The Extraction Of Total Saponins

Xylanases（EC 3.2.1.8）,generally abbreviated from endo-β-1,4-D-xylanases,can hydrolyze the internalβ-1,4-D-xylosidic linkages of xylans.They have been widely applied in textile,food,paper pulp and fodder industries,as well as in the production of xylooligosacharides and the fuel-grade ethanol.Because xylanases often been applied under high temperature,xylanases with high thermostabilities will have more application potential and development foreground.A gene（Aoxyn11A）encoding a glycoside hydrolase（GH）11family xylanase was cloned from Aspergillus oryzae genome and expressed in Pichia pastoris GS115.Ao Xyn11A was one kind of catalysts displaying high specific activity and enzymatic properties,but low temperature characteristics.In this study,Ao Xyn11A was taken as research objects.To obtain variants with improved temperature characteristics,the gene Aoxyn11A was modified by iterative mutagenesis.Then,variant with high temperature characteristics was applied in the extraction procession of total saponins from Cortex Albizzuae.The optimum extraction condition was established and the yield of total saponins was improved.Based on the homology modeling of Ao Xyn11A and molecular dynamics（MD）simulation for its three-dimensional（3-D）structure,as well as the multiple alignment of the primary structures of Ao Xyn11A and seven thermophilic xylanases,the two nonconservative amino acid residues of Gly²¹ and Ile¹²⁷ that possessing higher B-factor values were modified by iterative-saturation mutangenesis.Using the recombinant plasmid p ET-28a-Aoxyn11A as a template,site-saturation mutagenesis of Gly²¹ in the xylanase-encoding gene Aoxyn11A was performed using the whole-plasmid PCR technique.And then,the mutational transformant library was constructed by transforming p ET-28a-Aoxyn11A^G21X into E.coli BL21.In reference to the thermostability of enzymes,optimal mutational transformant(E.coli/Aoxyn11A^G21I)was screened from library.DNA sequencing results showed that E.coli/Aoxyn11A^G21I expressed a mutant enzyme Ao Xyn11A^G21I with the amino acid residue of Gly²¹ changed by Ile.Then,site-saturation mutagenesis of Ile¹²⁷ in the xylanase-encoding gene Aoxyn11A^G21I was performed.The mutational transformant library was constructed and screened.The results indicated that the temperature characteristics of all variants(Ao Xyn11A^G21I-I127X)were lower than those of Ao Xyn11A and Ao Xyn11A^G21I.Based on the multiple alignment of the primary structures of Ao Xyn11A and seven thermophilic xylanases,the homology modeling of Ao Xyn11A and molecular dynamics（MD）simulation on its 3-D structure,the mutant enzymes Ao Xyn11A^Y13F and Ao Xyn11A^G21I-Y13F were designed.Mutant xylanase-encoding genes Aoxyn11A^Y13F and Aoxyn11A^G21I-Y13F were constructed by mutating a Tyr¹³-encoding codon TAC in Aoxyn11A and Aoxyn11A^G21I into a Phe-encoding TTC by the whole-plamid PCR technique.Aoxyn11A^G21I,Aoxyn11A^Y13F and Aoxyn11A^G21I-Y13F were expressed in P.pastoris GS115,respectively,and enzymatic properties of expressed recombinant products,Ao Xyn11A^G21I,Ao Xyn11A^Y13F and Ao Xyn11A^G21I-Y13F,were analyzed.The temperature optimum(T_opt)of Ao Xyn11A^G21I-Y13F was 60°C,which was 5°C higher than that of Ao Xyn11A^G21I or Ao Xyn11A^Y13F and 10°C higher than that of Ao Xyn11A.Additionally,the half-life of Ao Xyn11A^G21I-Y13F at 50°C(t⁵⁰_1/2)was about 240 min,being 40-,3.4-and 2.5-fold longer than those of Ao Xyn11A,Ao Xyn11A^G21I and Ao Xyn11A^Y13F.The melting temperature（T_m）values of Ao Xyn11A,Ao Xyn11A^G21I,Ao Xyn11A^Y13F and Ao Xyn11A^G21I-Y13F were 52.3,56.5,58.6and 61.3°C,respectively.The above findings indicated that the iterative mutagenesis of both Gly²¹Ile and Tyr¹³Phe improved the temperature characteristics of Ao Xyn11A in a synergistic mode.Besides those,the catalytic efficiency(k_cat/K_m)of Ao Xyn11A^G21I-Y13F was 473.1 m L·mg^-1·s^-1,which was 1.65-fold higher than that of Ao Xyn11A.Ao Xyn11A^G21I-Y13F was applied in the extraction procession of total saponins from Cortex Albizzuae.In reference to the yield of total saponins,the optimun extraction condition was established,as follows:enzymolysis temperature of 45°C,xylanse dosage of 150 U·g^-1,enzymolysis time of 8 h and solid-to-liquid ratio of 1:25（g:m L）.Compared to xylanase enzymolysis method and ethanol reflux method,the yield of total saponins in xylanase-assisted ethanol reflux method was increased by17.55%and 14.66%,respectively.