Open AccessCloning and sequencing of the triacylglycerol lipase gene of Aspergillus oryzae and its expression in Escherichia coli
Author(s) -
Toida Jinichi,
Fukuzawa Mikio,
Kobayashi Gota,
Ito Kiyoshi,
Sekiguchi Junichi
Publication year - 2000
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2000.tb09223.x
Subject(s) - biology , complementary dna , biochemistry , aspergillus oryzae , lipase , microbiology and biotechnology , nucleic acid sequence , open reading frame , gene , peptide sequence , genomic dna , genomic library , diacylglycerol lipase , escherichia coli , molecular cloning , diacylglycerol kinase , enzyme , protein kinase c
Aspergillus oryzae produces at least three extracellular lipolytic enzymes, L1, L2 and L3 (cutinase, mono‐ and diacylglycerol lipase, and triacylglycerol lipase, respectively). We cloned the triacylglycerol lipase gene (provisionally designated tglA ) by screening a genomic library using a PCR product obtained with two degenerate oligonucleotide primers corresponding to amino acid sequences of L3 as probes. Nucleotide sequencing of the genomic DNA and cDNA revealed that the L3 gene ( tglA ) has an open reading frame comprising 954 nucleotides, which contains three introns of 47, 83 and 62 bp. The deduced amino acid sequence of the tglA gene corresponds to 254 amino acid residues including a signal sequence of 30 amino acids and, in spite of the difference in substrate specificity, it is homologous to those of cutinases from fungi. Three residues presumed to form the catalytic triad, Ser, Asp and His, are conserved. The cloned cDNA of the tglA gene was expressed in Escherichia coli , and enzyme assaying and zymography revealed that the cloned cDNA encodes a functional triacylglycerol lipase.