Synthesis and high expression of chitin deacetylase from Colletotrichum lindemuthianum in Pichia pastoris GS115.
A gene, ClCDA, encoding chitin deacetylase from Colletotrichum lindemuthianum, was optimized according to the codon usage bias of Pichia pastoris and synthesized in vitro by overlap extension PCR. It was secretorily expressed in P. pastoris GS115 using the constitutive expression vector pHMB905A. The expression level reached the highest with 110 mg/l culture supernatant after 72 h of methanol induction, which comprised 77.27 U/mg chitin deacetylase activity. SDS-PAGE, mass spectrometry, and deglycosylation assays demonstrated that partial recombinant protein was glycosylated with an apparent molecular mass of 33 kDa. The amino acid sequences of recombinant proteins were confirmed by mass spectrometry.
Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, Hubei Province, P R China.
Lixin
L
Kang
Xiaomei
X
Chen
Chao
C
Zhai
Lixin
L
Ma
D000581:Amidohydrolases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D003062:Codon; D020231:Colletotrichum / Q000201:enzymology* / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D003062:Codon; D011994:Recombinant Proteins; D000581:Amidohydrolases; C019866:chitin deacetylase