Expression of Bacillus subtilis MA139 beta-mannanase in Pichia pastoris and the enzyme characterization.
The 1014 nucleotides long gene-encoding beta-mannanase from Bacillus subtilis strain MA139 was cloned using PCR. To obtain high expression levels in Pichia pastoris, the beta-mannanase gene was optimized according to the codon usage bias of P. pastoris and fused downstream of GAP promoter. The reconstituted plasmid pGAP-mann was transformed into P. pastoris X-33 strain to constitutively express beta-mannanase. When cultured at 28 degrees Celsius for 3 days protein yields up to 2.7 mg/mL was obtained with the enzyme activity of up to 230 U/mL. In comparison, wild-type gene product yielded 1.9 mg/mL and 170 U/mL, respectively indicating that the protein yield and enzyme activity were significantly improved by codon modification. After purification, the enzyme properties were characterized. The optimal activity was at pH 6.0 and 50 degrees Celsius. In the pH range of 3.0 to 9.0, beta-mannanase showed above 60% of its peak activity. Among the numerous ions tested copper significantly inhibited the enzyme activity. These results suggested that codon-optimized beta-mannanase expressed in P. pastoris could potentially be used as an additive in the feed for monogastric animals.
Qiao
Jiayun
J
College of Animal Science and Technology, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing, 100193, People's Republic of China.
Rao
Zhenghua
Z
Dong
Bing
B
Cao
Yunhe
Y
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013696:Temperature; D013997:Time Factors; D044902:beta-Mannosidase / Q000235:genetics / Q000378:metabolism*
D003062:Codon; D044902:beta-Mannosidase