Identification of two novel fibrinolytic enzymes from Bacillus subtilis QK02.
Two fibrinolytic enzymes (QK-1 and QK-2) purified from the supernatant of Bacillus subtilis QK02 culture broth had molecular masses of 42,000 Da and 28,000 Da, respectively. The first 20 amino acids of the N-terminal sequence are AQSVPYGISQ IKAPALHSQG. The deduced protein sequence and its restriction enzyme map of the enzyme QK-2 are different from those of other proteases. The enzyme QK-2 digested not only fibrin but also a subtilisin substrate, and PMSF inhibited its fibrinolytic and amidolytic activities completely; while QK-1 hydrolyzed fibrin and a plasmin substrate, and PMSF as well as aprotinin inhibited its fibrinolytic activity. These results indicated QK-1 was a plasmin-like serine protease and QK-2 a subtilisin family serine protease. Therefore, these enzymes were designated subtilisin QK. The sequence of a DNA fragment encoding subtilisin QK contained an open reading frame of 1149 base pairs encoding 106 amino acids for signal peptide and 257 amino acids for subtilisin QK, which is highly similar with that of a fibrinolytic enzyme, subtilisin NAT (identities 96.8%). Asp32, His64 and Ser221 in the amino acid sequence deduced from the QK gene are identical to the active site of nattokinase (NK) produced by B. subtilis natto.
Ko
Ju Ho
JH
Institute of Molecular Virology, College of Life Science, Wuhan University, Wuhan, Hubei, PR China 430072.
Yan
Jun Peng
JP
Zhu
Lei
L
Qi
Yi Peng
YP
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D005337:Fibrin / Q000378:metabolism; D005341:Fibrinolysin / Q000235:genetics / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D015183:Restriction Mapping; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D020860:Subtilisin / Q000235:genetics / Q000378:metabolism
D005343:Fibrinolytic Agents; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D005337:Fibrin; D012697:Serine Endopeptidases; D020860:Subtilisin; D005341:Fibrinolysin