RT-PCR amplification of a Rhizopus oryzae lactate dehydrogenase gene fragment

No amino acid or DNA sequence information in sequence databases was found f or a fungal lactate dehydrogenase (LDH) isozyme. Highly conserved regions i n the lactate dehydrogenase enzymes of all taxonomies are found to be beta alpha beta nucleotide binding and substrate binding sites, also catalysis/a ctive site. The conserved regions were selected as PCR primer target region s. The degenerate primers were designed according to the codon usage, deter mined by analyzing a number of different genes of Rhizopus species. A fragm ent of the gene (ldh), coding for similar to 72% of the lactate dehydrogena se enzyme from Rhizopus oryzae, was amplified using degenerate primers by R everse Transcriptase-Polymerase Chain Reaction (RT-PCR). The size of the am plified fragment containing beta alpha beta nucleotide binding site, substr ate binding site and catalysis/active site is found to be about 700 bp. The reported degenerate PCR primers and the amplification conditions may lead to the cloning of the lactate dehydrogenase gene of R. oryzae, which is an important organism due to its utilization in lactic acid and enzyme product ions in industrial scales. (C) 2001 Elsevier Science Inc. All rights reserv ed.