Purification and specificity of a cell-wall-associated proteinase from Lactobacillus helveticus CP790.
A cell-wall-associated proteinase from Lactobacillus helveticus CP790, grown in skim milk, was purified to homogeneity by DEAE ion exchange chromatography in the presence of EDTA. Its molecular weight was estimated to be 45,000 by SDS-PAGE and also by the recovery of proteinase activity only from this fraction of SDS-PAGE gel slices of crude extract. It had maximum activity at pH 6.5 and 42 degrees C. Since the activity was completely inhibited by phenylmethanesulfonyl fluoride (PMSF), it was suggested to be a serine-type proteinase. It preferentially hydrolyzed alpha- and beta-casein but did not hydrolyze kappa-casein by the purified enzyme. The 10 main peptides liberated from alpha-casein, and the 15 peptides liberated from beta-casein, were isolated by reversed phase HPLC. These peptides were hydrolyzed by HCl and their amino acid compositions were analyzed and identified. Many of peptides were located in the C-terminal parts of alpha- and beta-casein. Peptide bonds cleaved by the proteinase had no clear specificity but Leu-X, Phe-X, Ser-X, Lys-X, Glu-X, and Gln-X sequences frequently appeared.
Yamamoto
N
N
R&D Center, Calpis Food Industry Co., Ltd., Tokyo.
Akino
A
A
Takano
T
T
D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000378:metabolism; D002455:Cell Division; D002473:Cell Wall / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007778:Lactobacillus / Q000201:enzymology* / Q000254:growth & development; D008892:Milk / Q000382:microbiology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
D002364:Caseins; D010664:Phenylmethylsulfonyl Fluoride; D010450:Endopeptidases