De novo synthesis, constitutive expression of Aspergillus sulphureus beta-xylanase gene in Pichia pastoris and partial enzymic characterization.
The endo-beta-1, 4-xylanase gene xynA from Aspergillus sulphureus, encoded a lack-of-signal peptide protein of 184 amino acids, was de novo synthesized by splicing overlap extension polymerase chain reaction according to Pichia pastoris protein's codon bias. The synthetic DNA, composed of 572 nucleotides, was ligated into the downstream sequence of an alpha-mating factor in a constitutive expression vector pGAPzalphaA and electrotransformed into the P. pastoris X-33 strain. The transformed yeast screened by Zeocin was able to constitutively secrete the xylanase in yeast-peptone-dextrose liquid medium. The heterogenous DNA was stabilized in the strain by 20-times passage culture. The recombinant enzyme was expressed with a yield of 120 units/mL under the flask culture at 28 degrees C for 3 days. The enzyme showed optimal activity at 50 degrees C and pH 2.4-3.4. Residual activity of the raw recombinant xylanase was not less than 70% when fermentation broth was directly heated at 80 degrees C for 30 min. However, the dialyzed xylanase supernatant completely lost the catalytic activity after being heated at 60 degrees C for 30 min. The recombinant xylanase showed no obvious activity alteration by being pretreated with Na(2)HPO(4)-citric acid buffer of pH 2.4 for 2 h. The xylanase also showed resistance to certain metal ions (Na(+), Mg(2+), Ca(2+), K(+), Ba(2+), Zn(2+), Fe(2+), and Mn(2+)) and EDTA. These biochemical characteristics suggest that the recombinant xylanase has a prospective application in feed industry as an additive.
Cao
Yunhe
Y
National Key Laboratory of Animal Nutrition, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing, 100094, People's Republic of China.
Qiao
Jiayun
J
Li
Yihang
Y
Lu
Wenqing
W
D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D043364:Endo-1,4-beta Xylanases / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D005285:Fermentation; D015870:Gene Expression; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D017931:DNA Primers; D004271:DNA, Fungal; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases