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Int J Biol Macromol. 2017 Nov;104(Pt A):724-731. doi: 10.1016/j.ijbiomac.2017.06.025. Epub 2017 Jun 7.

Purification and partial characterization of a low molecular fibrinolytic serine metalloprotease C142 from the culture supernatant of Bacillus subtilis C142.

Author information

1
Department of Food Science and Biotechnology, Gwangju University, Gwangju, 61743, Republic of Korea.
2
Department of Pharmacy, College of Pharmacy, Mokpo National University, Muan, Jeonnam 58554, Republic of Korea.
3
College of Pharmacy, Keimyung University, 1095 Dalgubeoldaero, Dalseo-Gu, Daegu, 42601, Republic of Korea.
4
Department of Nursing, Dongshin University, Naju, Jeonnam 58245, Republic of Korea.
5
Department of Biochemical and Polymer Engineering, Chosun University, Gwangju, 61452, Republic of Korea.
6
Department of Food Science and Biotechnology, Gwangju University, Gwangju, 61743, Republic of Korea. Electronic address: jhshkl@gwangju.ac.kr.
7
Department of Pharmacy, College of Pharmacy, Mokpo National University, Muan, Jeonnam 58554, Republic of Korea. Electronic address: sscho@mokpo.ac.kr.

Abstract

Novel serine metalloprotease-like enzyme, C142 was purified from the culture supernatant of Bacillus subtilis C142. The C142 was purified to homogeneity by a two-step procedure with a 20.7-fold increase in specific activity and 0.9% recovery. The molecular mass of C142 was approximately 23.5kDa based on SDS-PAGE. The N-terminal amino acid sequence of the first 21 amino acids of C142 was AQSVPYGISQIKAPALHSQGY. Its optimum pH, optimum temperature, pH stability, and thermal stability were pH 6, 40°C, pH 6-8, and 20-35°C, respectively. C142 was strongly inhibited by PMSF and EGTA, suggesting that C142 was a serine metalloprotease-like enzyme. C142 showed the highest specificity toward the substrate for t-PA. The apparent Km, Vmax, and Kcat values of C142 toward H-d-Ile-Pro-Arg-pNA were determined as 0.34mM, 0.25mmolmg-1min-1, and 46.83s-1. C142 exhibited fibrinolytic activity, which is stronger than that of plasmin. C142 hydrolyzed Aα, and Bβ-chains of fibrinogen, but did not cleave γ-chains. C142 had antithrombotic effect in three animal models. C142 was devoid of hemorrhagic activity at a dose of 20,000FU/kg. Taken together, our results indicate that B. subtilis C142 produces a serine metalloprotease-like enzyme/fibrinolytic enzyme and this enzyme might be used as a new thrombolytic agent.

KEYWORDS:

Anticoagulant; Antithrombotic; Bacillus subtilis C142; Serine metalloprotease; Thromboembolism

PMID:
28600208
DOI:
10.1016/j.ijbiomac.2017.06.025
[Indexed for MEDLINE]

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