Cloning, Expression And Directed Mutagenesis Of Xylanase Gene From Aspergillus Usamii

Xylanase(EC 3.2.1.8) can hydrolyzeβ-1,4-glycosidic linkages of the xylan backbone to produce xylooligosaccharides and D-xylose.Hence,it is the crucial enzyme component of xylanolytic enzyme systems.It broadly exists in microorganisms and has wide commercial application in industrial processes,such as feed,paper,foodstuff,medicine and energy industries.Based on the information of N-terminal amino acid sequence of XynⅡfrom Aspergillus usamii E001,the appropriate codon usage of Aspergillus and 3'-terminal poly(A) of eukaryotic mRNA,the XynⅡcDNA was amplified by RACE method,and then the DNA sequence was amplified using specific primers of cDNA.Both sequences of cDNA and DNA were analyzed and were submitted to GenBank(Accn:DQ 114485,DQ 191144).The amino acid sequence had higher similarity with those of G/11 family xylanases reported from other microorganisms.Compared with other Aspergillus sp.,the highest similarity was up to 89% with Aspergillus niger(GenBank Accn:ANU39784).XynⅡcDNA fragment encoding mature peptide was inserted into the plasmid pET-28a(+) and expressed in E.coli BL21-CodonPlus(DE3)-RIL.A maximum activity of 49.6 U mg^-1 was obtained from cellular extract of E.coli BL21-CodonPlus(DE3)-RIL harboring pET-28a-xynlI.Then the mature peptide cDNA was cloned into the Pichia pastoris expression vector pPIC9K,resulting in the recombinant plasmid pPIC9K-xynⅡ.Linearized with SalⅠ,pPIC9K-xyn//was transformed into P.pastoris GS 115 and KM71,respectively. After selection,the recombinant P.pastoris PXGL98(Mut+) and PXKL29(Muts) were obtained.Both of the recombinant strains could secrete functional xylanase,and in shake-flask culture induced with methanol,the maximal enzymatic activities of PXGL98 and PXKL29 were up to 3139.68 U/rag and 3846.83 U/mg,respectively.A homology modeling of XynⅡwas constructed by SWISS-MODEL and BLAST. Mutational analysis of the xynⅡgene products showed that Glu-79 and Glu-170 were the important catalytic amino acid residues in the active site.A conserved amino acid,Asp-37 had been found in the catalytic domain betweenβ-sheet A3 and B3 in the tertiary structure,which influenced the pH properties of enzyme.Then a D37N mutation was introduced in XynⅡby site-directed mutagenesis.The mutant xylanase(XynⅡ_D37N) expressed in P pastoris were purified and its enzymatic properties were determined.The result revealed that the optimal pH of XynⅡD37N was increased from 4.2 to 5.3 and the pH stability of XynⅡ_D37N was changed from pH 3.0-7.5 to pH 3.0-9.0.The mutant xylanase XynⅡ_D37N was a good material for further research in the relationship between structure and function of xylanase.Replacing several serine and threonine residues on the Ser/Thr surface of XynⅡwith arginines effectively increased the thermostability of the enzyme.The substitution of Ser and Thr residues on the Ser/Thr surface of the enzyme with four(ST4) or five arginines(ST5) led to an increase in optimal temperature of the enzymes by 2℃and 5℃for the ST4 and ST5, respectively.The modified enzymes ST4 and ST5 showed 65%and 75%of maximal activities after incubated for 15 min at 55℃compared to only 20%activity for wild-type enzyme.After incubated for 1 h at 55℃,ST4 and ST5 showed 50%and 65%of maximal activity compared to only 15%activity for wild-type enzyme.Having the good properties of XynⅡ,the mutants with higher thermostability are potentially useful in industrial applications.