Abstract: Two lipase nucleotide sequences were cloned from DNA and total RNA of R. miehei 3.4960 by PCR and RT-PCR, respectively. The sequencing results showed that the nucleotide sequence from the DNA of R. miehei 3.4960 was composed of five introns and six extrons. The lengths of five introns were 75, 58, 64, 73 bp and 59 bp, which were located at the 600－674, 929－986, 1076－1139, 1227－1299 and 1382－1440 sites of nucleotide sequence, respectively. There were five-site mutations between the nucleotide sequence from total RNA of R. miehei 3.4960 and mRNA (EMBL Accession No. A02536) from R. miehei, which could result in one amino acid substitution. The RML gene was successfully expressed in S. cerevisiae MT8-1. The characteristics of RML showed that its optimal temperature and pH were 45 ℃ and 8.6, respectively. RML activity was inhibited by Cu2+ and Fe2+, and activated by Ca2+ and Mg2+. The RML activity was higher for substrates with long chains, especially a substrate with a chain length of C12, but it was very low for substrates with short chains.

Key words: lipase, gene cloning, expression, catalytic characterization