Cloning, expression, and fibrin (ogen)olytic properties of a subtilisin DJ-4 gene from Bacillus sp. DJ-4

Abstract

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 °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 Aα‐, Bβ‐, and γ‐chains of fibrinogen within 5 min. But, unlike subtilisin BPN′ at a very low concentration (50 ng), the γ‐chain was not cleaved. On the other hand, rm‐subDJ‐4 did not show enzyme activity.

Purification and Characterization of Two Novel Halotolerant Extracellular Proteases fromBacillus subtilisStrain FP-133

“…Based on the DDBJ database, the N-terminal amino acid sequence of expro-I showed 76% identity to pro-subtilisin from Bacillus sp. DJ-4, 19) under accession no. AY627764, whereas the Nterminal amino acid sequence of expro-II did not show any identity to those from other proteins.…”

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“…Although B. lehensis G1's proteases are difficult to express recombinantly, it does not seem unusual, as for subtilisin from Bacillus amyloliquefaciens DC-4 and Bacillus sp. DJ-4 [32,33] and nattokinase from B. subtilis [34] exhibit similar pattern of expression.…”

Molecular Cloning and Sequence Analysis of Two Distinct Halotolerant Extracellular Proteases fromBacillus subtilisFP-133

“…Previously reported Apr of Bacillus sp. DJ-4 9) and Npr of Bacillus nematocida 16) show high identity with Apr FP133 and Npr FP133 respectively. Although these recombinant mature proteins were also expressed in the E. coli transformant, the recombinant proteins were produced mainly as insoluble inclusion bodies and showed low protease activity.…”

“…The N-terminal amino acid residue corresponding to the N-terminus of purified Expro-I was located at position 108 of the deduced amino acid sequence, and Expro-I was synthesized as a preproprotein for subtilisins, as previously reported. 8,9) The sequence of the first N-terminal 30 residues of the 107 amino-acid-long preprotein region resembled a typical signal peptide sequence, with a short sequence containing two positively charged residues (Lys-Lys), followed by a long hydrophobic sequence and a potential peptidase cleavage site (Ala-X-Ala).…”

“…Although these recombinant mature proteins were also expressed in the E. coli transformant, the recombinant proteins were produced mainly as insoluble inclusion bodies and showed low protease activity. 9,16) Recombinant proteases Apr FP133 and Npr FP133 could not prepared in large enough amounts for further study, Expro-I and Expro-II of strain FP-133 were purified by previously reported procedures.…”