Production and genetic regulation of an amylase in Lactococcus lactis

Over 100 lactococcal strains isolated from their natural niche cow-milk were tested for production of amylolytic enzymes. Two of the tested strains were found to produce an amylase. One of them, Lactococcus lactis IBB500 was used for biochemical and genetic studies. We observed that L. lactis IBB500 strain started to produce the extracellular amylase in the BHI broth at the end of the logarithmic phase of growth. The maximal amount of the enzyme was detected at the early stationary phase. It demonstrated wide temperature optimum with at least 50% of its activity found in the range between 25 degreesC and 60 degreesC. The highest activity was observed at 50 OC. Moreover, production of the amylase was induced to different levels by various sugars e.g.: starch, maltose, maltotriose, pullulan etc. When glucose was added to the medium repression of the enzyme production was observed. Genetic studies showed that amylase gene was located on a 30 kb plasmid. This gene was cloned and expressed in Lactococcus lactis. Analysis of a partial DNA sequence of the region containing the gene indicated that it could encode a polypeptide homologous to various amylases like izoamylase from Pseudomonas amyloderamosa, pullulanase from Thermus sp. IM6501, pullulanase precursor from K. pneumonie, termostable pullulanase from Bacillus stearothermophilus and pullulanase from Bacteroides thetaiotaomicron.