Cloning and sequencing of the triacylglycerol lipase gene of Aspergillus oryzae and its expression in Escherichia coli

Aspergillus oryzae produces at least three extracellular lipolytic enzymes, L1, L2 and L3 (cutinase, mono- and diacylglycerol lipase, and triacylglyce rol lipase, respectively). We cloned the triacylglycerol lipase gene (provi sionally designated tglA) by screening a genomic library using a PCR produc t obtained with two degenerate oligonucleotide primers corresponding to ami no 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 compri sing 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 th e difference in substrate specificity, it is homologous to those of cutinas es from fungi. Three residues presumed to form the catalytic triad, Ser, As p and His, are conserved. The cloned cDNA of the tglA gene was expressed in Escherichia coli, and enzyme assaying and zymography revealed that the clo ned cDNA encodes a functional triacylglycerol lipase. (C) 2000 Federation o f European Microbiological Societies. Published by Elsevier Science B.V. Al l rights reserved.