Cloning, Site-Directed Mutagenesis And High-level Expression Of Xylanase Gene From Aspergillus Niger

The endo-1,4-(3-D-xylanase as the crucial enzyme component of xylanolytic enzyme systems shows great potential in development of industry processes, which was widely used for different fields, such as animal feeding, food additive industry, paper and pulp industry, medicine and bioconversion of lignocellulosic waste to ethanol.The gene xynB of endo-1,4-xylanase was cloned from Aspergillus niger nl-1 by using PCR based on the conserved 5'terminal and 3'terminal amino acid sequences of several Aspergillus niger xynB genes. The sequencing result showed that the full-length gene contained 745 bp, including an intron of 67 bp which has high similarity with other xylanase genes. From the sequence analysis, it would appear the enzyme belonged to the family 11 of glyoside hydrolases.Four recombinant plasmids including pTrc-99a-xynB, pTrc-99a-xynB (S), pET-20b-xynB and pET-20b-xynB (S) were successfully contrasted and expressed in the different E.coli stains by inducing with IPTG. And the recombinant protein expressed in pTXBC was highest,with the maximum intracellular activity of 299 IU/L in 4 h and total activity of 650 IU/L after 10 h. Compared to the vector pET-20b, pTrc-99a and the native signal peptide was more effective for xylanase expression.The xynB gene was inserted into the Pichia pastoris expression vector and transformed into Pichia pastoris GS115, KM71H and X33 competent cells by electroporation. The clones was selected and detected for induction expression. The maximum activity of 526 IU/mL was obtained with the a-factor signal peptide in Pichia pastoris GS115. To further improve the expression level of recombinant xylanase in Pichia pastoris, the xynB gene was optimized and synthesized by the overlapping extension PCR based on the codon usage bias of Pichia pastoris. The activity of the recombinant xylanase reached to 1408 IU/mL induced after a 14-day cultivation in shake flasks. Furthermore, the maximum xylanase production was 20424.2 IU/mL with 0.584 mg/mL total soluble protein in a 3 L fermentor.A homology modeling of xynB was constructed by SWISS-MODEL and BLAST to change the optimal pH. The constructs XynB-80 (N80D), XynB-117 (A160Q/A161P/Q164K +D117N) and XynB-164 (A160Q/A161P/Q164K) were created by site-directed mutagenesis. The properties of mutational enzymes were analysed and showed the mutation XynB-80 decreased the optimal pH from 5.0 to 4.5 with the acid pH stability. And the optimal activity pH of mutation XynB-117 raised from 5.0 to 5.5, but had no effect for the mutation XynB-164 which had a better thermostability.The effect of Arginines, disulphide bridge and hydrophobic interaction between N-terminal region in the structure of xylanase were studied to increased the thermostability of enzyme. The optimal temperature of the mutation XynB-5Arg and XynB-77 were increased from 45℃to 50℃, with the thermostability increased in the presence of substrate. And the optimal temperature of the mutation XynB-144 was also increased by 5℃, which was created at 55℃for 10 min with higher activity compared to the wild enzyme. The apparant temperature optima for the mutation XynB-45 was significantly improved to 55℃. After incubation at pH 5.0,80℃for 20min, the residual activity of the mutation xylanase was over 60%. These enzyme properties suggest that this enzyme has great potentiality.