title: Design and expression of a synthetic phyC gene encoding the neutral phytase in Pichia pastoris.
abstract: 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.
authors.affiliation: Laboratory of Veterinary and Biotechnology, Sichuan Agricultural University, Yan'an 625014, China.
authors.forename: Li-Kou
authors.initials: LK
authors.lastname: Zou
authors.forename: Hong-Ning
authors.initials: HN
authors.lastname: Wang
authors.forename: Xin
authors.initials: X
authors.lastname: Pan
authors.forename: Tao
authors.initials: T
authors.lastname: Xie
authors.forename: Qi
authors.initials: Q
authors.lastname: Wu
authors.forename: Zi-Wen
authors.initials: ZW
authors.lastname: Xie
authors.forename: Wan-Rong
authors.initials: WR
authors.lastname: Zhou
mesh_terms: 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
chemical_list: D018387:Codon, Initiator; D010940:Plant Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D010832:6-Phytase