99A-21 |
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E. SOERYAPRANATA1, J. R. Powers1, and G. U. Yuksel2. (1) Dept. of Food Science & Human Nutrition, Washington State Univ., 106 FSHN Bldg., PO Box 646376, Pullman, WA 99164-6376, (2) Dept. of Food Science & Toxicology, Univ. of Idaho, Agricultural Biotechnology Laboratory, Moscow, ID 83844-2312 Proteolysis of casein is important for development of flavor during ripening of cheese. However, bitterness can develop due to accumulation of certain peptides. Lactobacillus helveticus WSU-19 is used as adjunct culture to produce aged Cheddar-type cheese with little bitterness. We have previously shown the importance of general aminopeptidase N and endopeptidases of L. helveticus WSU-19 in the breakdown of bitter peptide, b-CN f193-209. Our long-range goal is to fully characterize the biochemistry and genetics of L. helveticus WSU-19 to determine the contribution of its peptidases to the development of cheese flavor. The objective of this study was to identify and clone selected peptidase genes from L. helveticus WSU-19. Identification of the putative pepN, pepE, pepO, and pepO2 genes from L. helveticus WSU-19 was accomplished using L. helveticus CNRZ-32 peptidase genes as probes high stringency Southern hybridization blots. With the goal of cloning pepE, hybridization-positive genomic DNA fragments were gel extracted and ligated to the pJDC9 vector DNA. The ligation mixtures were introduced into the electrocompetent Escherichia coli DH5a cells. Antibiotic- resistant transformants were screened using blue/white screening and colony hybridization. The plasmid DNA obtained from the hybridization-positive colonies were subjected to restriction enzyme analyses and DNA sequencing. Putative pepN, pepE, pepO, and pepO2 genes from L. helveticus WSU-19 were successfully identified. Two clones with the putative pepE gene were obtained. The complete sequencing of the putative pepE gene is in progress. Our current research deals with the cloning of the genes that encode for aminopeptidase N as well as endopeptidases O and O2. Understanding the enzymology of L. helveticus WSU-19 will allow more systematic development of improved adjunct cultures for the dairy industry. We are also interested in the inactivation of selected peptidases from L. helveticus WSU-19 using a recently developed electroporation protocol.
Session 99A, Biotechnology: General
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