Improvement of Aspergillus sulphureus Endo-β-1,4-Xylanase Expression in Pichia pastoris by Codon Optimization and Analysis of the Enzymic Characterization

Abstract

The gene xynB from Aspergillus sulphureus encoding the endo-beta-1,4-xylanase was de novo synthesized by splicing overlap extension polymerase chain reaction according to Pichia pastoris protein's codon bias. The synthetic DNA and wild-type DNA were placed under the control of a glyceraldehyde-3-phosphate dehydrogenase gene promoter (GAP) in the constitutive expression vector plasmid pGAPzalphaA and electrotransformed into the P. pastoris X-33 strain, respectively. The transformants screened by Zeocin were able to constitutively secrete the xylanase in YPD liquid medium. The maximum yield of the recombinant xylanase produced by the synthetic DNA was 105 U ml(-1), which was about 5-fold higher than that by wild-type DNA under the flask culture at 28 degrees Celsius for 3 days. The enzyme showed optimal activity at 50 degrees Celsius and pH 5.0. The residual activity remained above 90% after the recombinant xylanase was pretreated in Na(2)HPO(4)-citric acid buffer (pH 2.4) for 2 h. The xylanase activity was significantly improved by Zn(2+). These biochemical characteristics suggest that the recombinant xylanase has a prospective application in feed industry as an additive.

High-level expression of improved thermo-stable alkaline xylanase variant in Pichia Pastoris through codon optimization, multiple gene insertion and high-density fermentation

“…The recombinant strains producing high-yield of xylanase will be better for industrial application with less downstream purification, so xylanase production will be cheaper and cost efficient2122. Several studies have reported the role of different host strains produce high level of exogenous xylanase, like Bacillus , Trichoderma reesei , Pichia pastoris , Saccharomyces cerevisiae and Escherichia coli 2324252627.…”

Thermostable Sites and Catalytic Characterization of Xylanase XYNB of Aspergillus niger SCTCC 400264

“…The hydrolysates of xylan by XYNB suggested that the enzyme had great value in the preparation of xylooligosaccharide [4,13,23]. Xylanase has not only broad application prospects in biotechnological and industrial applications such as animal feed, textile, paper industries, and functional xylooligosaccharides production [14,21], but also great potential in the bioconversion of lignocellulosic feedstocks to fuel-grade ethanol, which has attracted tremendous interests in the past few years. Two approaches have been successfully tried to obtain thermophilic adaption xylanase [24].…”

Codon optimisation improves the expression of Trichoderma viride sp. endochitinase in Pichia pastoris

“…The synthetic DNA and wild-type DNA were placed under the control of a glyceraldehyde-3-phosphate dehydrogenase (GAP) gene promoter in the constitutive expression plasmid pGAPzαA and separately electrotransformed into the P. pastoris X-33 strain. The maximal yield of the recombinant xylanase encoded by the synthetic DNA was 105 U/mL, which was approximately 5-fold higher than that of the xylanase encoded by the wild-type DNA under shaking-flask culture at 28°C for 3 d31. More interestingly, when the wild-type lipase gene ( lipJ08 ) from Candida rugosa was expressed in P. pastoris , no lipase activity was detected.…”