Improvement of Aspergillus sulphureus endo-beta-1,4-xylanase expression in Pichia pastoris by codon optimization and analysis of the enzymic characterization.
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.
National Key Laboratory of Animal Nutrition, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing, 100193, People's Republic of China.
Yihang
Y
Li
Bo
B
Zhang
Xiang
X
Chen
Yiqun
Y
Chen
Yunhe
Y
Cao
D001230:Aspergillus / Q000201:enzymology*; D001483:Base Sequence; D003062:Codon / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013696:Temperature
D003062:Codon; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases