Cloning, expression, and fibrin (ogen)olytic properties of a subtilisin DJ-4 gene from Bacillus sp. DJ-4.
Previously, we purified a strong fibrinolytic enzyme (subtilisin DJ-4) from Bacillus sp. DJ-4 and characterized its enzymatic activity. Here, we cloned the gene subtilisin DJ-4, and determines its nucleotide sequence, which showed 97% identity with subtilisin BPN' from B. amyloliquefacens. Recombinant full-subtilisin DJ-4 (rf-subDJ-4) and mature-subtilisin DJ-4 (rm-subDJ-4) were expressed using a pET29 vector system, and their fibrin (ogen)olytic and plasminogen activator activities were studied. rf-subDJ-4 was found to have a higher stability to heat (60 degrees C) and to acidic conditions (pH 3.0-4.0) than the native subtilisin DJ-4 of Bacillus sp. DJ-4. The plasminogen activator activity of rf-subDJ-4 was 2.75 times greater than that of plasmin on a molar basis. And its specific activity (F/C, the ratio of fibrinolytic activity to caseinolytic activity) was 2.67 and 3.97 times higher than those of subtilisin BPN' and subtilisin Carlsberg, respectively. rf-subDJ-4 rapidly hydrolyzed the Aalpha-, Bbeta-, and gamma-chains of fibrinogen within 5 min. But, unlike subtilisin BPN' at a very low concentration (50 ng), the gamma-chain was not cleaved. On the other hand, rm-subDJ-4 did not show enzyme activity.
Choi
Nack-Shick
NS
Primate Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, Taejon 305-333, Republic of Korea.
Chang
Kyu-Tae
KT
Jae Maeng
Pil
P
Kim
Seung-Ho
SH
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D002364:Caseins / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005337:Fibrin / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism*; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010958:Plasminogen / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D020860:Subtilisin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
D002364:Caseins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D005337:Fibrin; D005340:Fibrinogen; D010958:Plasminogen; D020860:Subtilisin