[cDNA cloning and expression of thermostable chitinase from thermophilic fungus Thermomyces lanuginosus].
A chitinase-encoding gene from Thermomyces lanuginosus was cloned by Rapid Amplification of cDNA Ends (RACE) using degenerated oligonucleotide primers designed from N-terminal amino acid sequence and the conserved amino acid sequence. The cloned full-length cDNA named chit, 1500bp in size, contained an OFR with 442 amino acids. The gene chit has been registered in GenBank with accession number DQ092332. The alignment results of putative amino acids sequence showed the catalytic domain of chit was high homology with the catalytic domains of the other chitinases in family-18, contained 2 conserved motifs related with catalytic activity of chitinase. The recombinant plasmid was generated by inserting the ORF of mature protein Chit into expression vector pPIC9K, and transformed into Pichia pastoris GS115. The recombinant chitinase was secreted successfully with the expression level of 0.36mg/mL and its activity could reach to 2.261U/mL after methanol induction 6d. The recombinant chitinase exhibited optimum catalytic activity at pH 5.5 and 60 degrees C. The enzyme was stable at 50 degrees C and the half life time at 65 degrees C was 40min.
Guo
Run-fang
RF
Department of Environmental Biology, Shandong Agricultwural University, Tai' an 271018, China. runfangg@163.com
Li
Duo-Chuan
DC
D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology / Q000235:genetics*; D002688:Chitinases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D005656:Fungal Proteins; D002688:Chitinases