Design and expression of a synthetic phyC gene encoding the neutral phytase in Pichia pastoris.
The 1074-bp phyCs gene (optimized phyC gene) encoding neutral phytase was designed and synthesized according to the methylotrophic yeast Pichia pastoris codon usage bias without altering the protein sequence. The expression vector, pP9K-phyCs, was linearized and transformed in P. pastoris. The yield of total extracellular phytase activity was 17.6 U/ml induced in Buffered Methanol-complex Medium (BMMY) and 18.5 U/ml in Wheat Bran Extract Induction (WBEI) medium at the flask scale, respectively, improving over 90 folds compared with the wild-type isolate. Purified enzyme showed temperature optimum of 70 degrees and pH optimum of 7.5. The enzyme activity retained 97% of the relative activity after incubation at 80 degrees for 5 min. Because of the heavy glycosylation the expressed phytase had a molecular size of approximately 51 kDa. After deglycosylation by endoglycosylase H (EndoH(f)), the enzyme had an apparent molecular size of 42 kDa. Its property and thermostability was affected by the glycosylation.
Zou
Li-Kou
LK
Laboratory of Veterinary and Biotechnology, Sichuan Agricultural University, Yan'an 625014, China.
Wang
Hong-Ning
HN
Pan
Xin
X
Xie
Tao
T
Wu
Qi
Q
Xie
Zi-Wen
ZW
Zhou
Wan-Rong
WR
D010832:6-Phytase / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D018387:Codon, Initiator; D004789:Enzyme Activation; D005813:Genes, Synthetic; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D018387:Codon, Initiator; D010940:Plant Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D010832:6-Phytase