Purification and characterization of proteases from Bacillus amyloliquefaciens isolated from traditional soybean fermentation starter.

Bacillus amyloliquefaciens FSE-68 isolated from meju, a Korean soybean fermentation starter, was identified on the basis of biophysical tests and 16S rRNA gene sequence. A neutral metalloprotease (NPR68) and an alkaline serine-protease (APR68) were purified by ammonium sulfate precipitation and cation exchange chromatography and identified on the basis of their activities at different pH values and the selective protease inhibitors. The molecular weights of NPR68 and APR68 measured with ESI-MS were 32743 (+/- 0.8) and 27443 (+/- 0.5) Da, respectively. Against oxidized insulin chains, the NPR68 has a cleavage preference at the site where leucine is located as a P1' residue followed by phenylalanine, and the APR68 has broad specificity and favors leucine at the P1 site. These results indicate that the proteases are natural variants of subtilisin and bacillolysin.

[1]  H. Sumi,et al.  A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet , 1987, Experientia.

[2]  F. Kuo,et al.  Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK1. , 2000, Journal of agricultural and food chemistry.

[3]  S. H. Kim,et al.  Purification and Characterization of Subtilisin DJ-4 Secreted by Bacillus sp. Strain DJ-4 Screened from Doen-Jang , 2000, Bioscience, biotechnology, and biochemistry.

[4]  M. Sakaguchi,et al.  Purification and characterization of a soybean-milk-coagulating enzyme from Bacillus pumilus TYO-67 , 1999, Applied Microbiology and Biotechnology.

[5]  Seong-Jun Cho,et al.  Large-Scale Purification of Protease Produced by Bacillus sp. from Meju by Consecutive Polyethylene Glycol/Potassium Phosphate Buffer Aqueous Two-Phase System , 1999 .

[6]  A. Pihlanto-Leppälä,et al.  Impact of processing on bioactive proteins and peptides , 1998 .

[7]  Bokyung Kim,et al.  Effects of Meju Powder Supplementation on Lipid Metabolism in Rats Fed Hypercholesterolemic Diet , 1997 .

[8]  Shinyoung Lee,et al.  Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang , 1996, Applied and environmental microbiology.

[9]  H. Meisel,et al.  Bioactive peptides derived from milk proteins. Structural, physiological and analytical aspects. , 1995, Die Nahrung.

[10]  S. H. Chung,et al.  Purification of ACE inhibitor from soybean paste , 1994 .

[11]  A. Asada,et al.  Purification and characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan. , 1993, Biochemical and biophysical research communications.

[12]  M. Collins,et al.  Phylogenetic heterogeneity of the genus Bacillus revealed by comparative analysis of small‐subunit‐ribosomal RNA sequences , 1991 .

[13]  U. Geuß,et al.  Casein-resorufon, a new substrate for a highly sensitive protease assay , 1988 .

[14]  L. K. Nakamura.  Deoxyribonucleic Acid Relatedness of Lactose-Positive Bacillus subtilis Strains and Bacillus amyloliquefaciens , 1987 .

[15]  P. Matsudaira,et al.  Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. , 1987, The Journal of biological chemistry.

[16]  R. Pridmore.  New and versatile cloning vectors with kanamycin-resistance marker. , 1987, Gene.

[17]  P. Nedkov,et al.  Determination of the complete amino-acid sequence of subtilisin DY and its comparison with the primary structures of the subtilisins BPN', Carlsberg and amylosacchariticus. , 1985, Biological chemistry Hoppe-Seyler.

[18]  E. Ferrari,et al.  Replacement of the Bacillus subtilis subtilisin structural gene with an In vitro-derived deletion mutation , 1984, Journal of bacteriology.

[19]  A. O'donnell,et al.  Characterization of Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, and Bacillus amyloliquefaciens by Pyrolysis Gas-Liquid Chromatography, Deoxyribonucleic Acid-Deoxyribonucleic Acid Hybridization, Biochemical Tests, and API Systems , 1980 .

[20]  H. Zalkin,et al.  Extracellular and membrane-bound proteases from Bacillus subtilis , 1980, Journal of bacteriology.

[21]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[22]  M. Kame,et al.  Detergency and mechanism of soil removal in detergent-enzyme system , 1973, Journal of the American Oil Chemists' Society.

[23]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[24]  I. Svendsen,et al.  [11] The subtilisins , 1970 .

[25]  E. L. Smith,et al.  Subtilisin Carlsberg. V. The complete sequence; comparison with subtilisin BPN'; evolutionary relationships. , 1968, The Journal of biological chemistry.