Molecular cloning and characterization of the gene encoding a fibrinolytic enzyme from Bacillus subtilis Strain A1

Abstract

A fibrinolytic metalloprotease gene from Bacillus subtilis has been cloned in Escheridria coli XL1-Blue and the bacterial expressed enzyme was purified. The nucleotide sequence of the cloned fibrinolytic enzyme gene revealed a single open reading frame of 1023 bp coding for 341 amino acids (M r 37708.21 Da). N-terminal amino acid sequencing of the fibrinolytic enzyme excreted from E. coli host cells revealed that the mature fibrinolytic enzyme consists of 288 amino acids (M r 31391.1 Da). The deduced amino acid sequence showed significant homology with Erwina carotovora neutral metalloprotease and Serratia marcescens minor metalloprotease by 65 and 58% amino acid sequence identity, respectively. The protein showed significant alignments with the conserved domain of catalytic activity and the a-helix domain in Bacillus anthracis thermolysis metalloprotease. The biochemical properties of the purified enzyme suggested that the enzyme is a fibrinolytic metalloprotease, which has optimal activity at pH 7.0 and 50°C.

Biochemical analysis of a fibrinolytic enzyme purified from Bacillus subtilis strain A1

“…1 and Table 1). The purified fibrinolytic enzyme was an approximately 28 kDa, which was slightly different from the 31 kDa metalloprotease BK II identified in the same strain (Jeong et al, 2004a). The fibrinolytic activities of the 28 kDa fibrinolytic enzyme, designated as Bacillokinase I (BK I), in the plasminogen-free Polyacrylamide gel electrophoresis of the fibrinolytic enzyme (BK I) purified from B. subtilis strain A1.…”

“…We previously reported a fibrinolytic enzyme (Bacillokinase II, BK II) from B. subtilis strain A1, which is a metalloprotease (Jeong et al, 2004a). In this paper, the fibrinolytic enzyme, which is distinguished from BK II and other known fibrionlytic enzymes with regard to biochemical characteristics, was found and characterized by biochemical and molecular analyses.…”

“…subtilis strain A1 producing a fibrinolytic enzyme was isolated from soil sediments in Korea (Jeong et al, 2004a). The strain was aerobically cultured at 37°C overnight using Luria Broth (LB) medium consisting of 1% bacto-tryptone (Sigma, USA), 1% sodium chloride, and 0.5% bacto-yeast extract (Sigma).…”

Fibrinolytic Bacterial Enzymes with Thrombolytic Activity

“…KA38. Jeong et al (2004) reported a fibrinolytic metalloprotease from Bacillus subtilis strain A1, with optimal activity at 50°C. Chiang et al (2005) reported that Wang et al (1999a) Vibrio vulnificus secretes a broad-specific metalloprotease capable of interfering with blood hemostasis.…”

Purification and Biochemical Characteristics of Fibrinolytic Enzyme from Streptomyces corcohrussi JK-20

“…have also been interested in the thrombolytic agent because of a high efficiency in the fibrinolytic reactions such as plasmin activation. A variety of extracellular and intracellular proteases including Subtlisin Carsberg [22], nattokinase [19], amylosachariticus [26], aprE [20], Subtilin J [8] and BK II [9] have been produced by the Bacillus spp.…”