Purification and characterization of two extracellular alkaline proteases from a newly isolated obligate alkalophilic Bacillus sphaericus.
Two novel extracellular serine proteases were purified to homogeneity from the cell-free culture filtrate of an obligate alkalophilic Bacillus sphaericus by a combination of ultrafiltration, ammonium sulfate precipitation and chromatographic methods. The enzymes showed similar substrate specificities, but differed in hydrophobicity and molecular mass. Protease A was a monomeric protease with a relative molecular mass (M(r)) of 28.7 kDa, whereas protease B, with a M(r) of 68.0 kDa, apparently consisted of smaller subunits. The purified protease A had a specific activity on hemoglobin of 5.1 U/mg protein compared to 40.9 U/mg protein in the case of protease B. Both proteases were most active on SAAPF-pNa, a substrate for chymotrypsin-like serine proteases. However, the K(m) values of these two proteases on SAAPF-pNa were higher than that for alpha-chymotrypsin, indicating a lower affinity of proteases A and B for this substrate compared to chymotrypsin. Unlike other Bacillus serine proteases, neither protease A nor B stained with Coomasie blue R-250, even with loading of a large amount of protein, and they stained poorly with the silver staining method. However, NH(2)-terminal amino acid sequencing of protease B revealed a high similarity with subtilisin Carlsberg (67% homology). Almost total inhibition of both proteases by PMSF, but very little/no inhibition by trypsin and chymotrypsin inhibitors (TPCK and TLCK) or thiol reagents (PCMB and iodoacetic acid), further supported the view that the enzyme belonged to the serine protease family.
Singh
J
J
Biochemical Engineering Research and Process Development Centre, Institute of Microbial Technology, Sector-39A, Chandigarh 160036, India.
Vohra
R M
RM
Sahoo
D K
DK
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012697:Serine Endopeptidases / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification* / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases