Cloning, characterization, and expression of cDNA encoding a lipase from Kurtzmanomyces sp. I-11.
A cDNA clone of the lipase secreted by Kurtzmanomyces sp. I-11 was isolated from a cDNA library of this yeast by PCR screening using oligonucleotide primers designed on the basis of the partial amino acid sequence of the lipase. The cloned cDNA (lip1) encoded a hydrophobic protein of 484 amino acids, where the first 20 amino acids and the following 6 amino acid sequences were predicted to be the signal sequence for secretion and a pro-sequence, respectively. The deduced amino acid sequence of the Kurtzmanomyces lipase was most similar to Candida antarctica DSM 3855 lipase A (74% identity) and weakly to other lipases. The consensus pentapeptide (-Gly-X-Ser-X-Gly-) that forms a part of the interfacial lipid recognition site in lipases was conserved. A high level of lipase was produced by Pichia pastoris transformed with the lip1 cDNA, indicating that the cloned cDNA indeed encodes a lipase.
Kakugawa
Koji
K
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Kagamiyama, Hiashi-Hiroshima, Japan. kakugawa@syokuhim-kg.pref.hiroshima.jp
Shobayashi
Megumi
M
Suzuki
Osamu
O
Miyakawa
Tokichi
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D005658:Fungi / Q000201:enzymology* / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D011994:Recombinant Proteins; D008049:Lipase