Elsevier

Process Biochemistry

Volume 45, Issue 1, January 2010, Pages 88-93
Process Biochemistry

A novel fibrinolytic protease from Streptomyces sp. CS684

https://doi.org/10.1016/j.procbio.2009.08.010Get rights and content

Abstract

A fibrinolytic protease (FP84) was purified from Streptomyces sp. CS684, with the aim of isolating economically viable enzyme from a microbial source. SDS-PAGE and fibrin zymography of the purified enzyme showed a single protein band of approximately 35 kDa. Maximal activity was at 45 °C and pH 7–8, and the enzyme was stable between pH 6 and 9 and below 40 °C. It exhibited fibrinolytic activity, which is stronger than that of plasmin. FP84 hydrolyzed Bβ-chains of fibrinogen, but did not cleave Aα- and γ-chains. Km, Vmax and Kcat values for azocasein were 4.2 mg ml−1, 305.8 μg min−1 mg−1 and 188.7 s−1, respectively. The activity was suppressed by Co2+, Zn2+, Cu2+ and Fe2+, but slightly enhanced by Ca2+ and Mg+2. Additionally, the activity was slightly inhibited by aprotinin and PMSF, but significantly inhibited by pefabloc, EDTA and EGTA. The first 15 amino acids of N-terminal sequence were GTQENPPSSGLDDID. They are highly similar to those of serine proteases from various Streptomyces strains, but different with known fibrinolytic enzymes. These results suggest that FP84 is a novel serine metalloprotease with potential application in thrombolytic therapy.

Introduction

Cardiovascular diseases such as high blood pressure, acute myocardial infarction, ischemic heart disease, valvular heart disease, peripheral vascular disease, arrhythmias, stroke, etc. are the primary causes of death throughout the world. According to data of World Health Organization (WHO) in 2000, 29% of the total mortality rate in the world is caused by cardiovascular diseases [1]. Similarly, another report of WHO in 2006 shows about 18 million people die every year from cardiovascular diseases [2]. The fundamental pathophysiological process related to this devastating problem is the formation of fibrin (blood clots), which adheres to the unbroken wall of blood vessels. Fibrin is normally formed from fibrinogen by the action of thrombin (EC 3.4.21.5); it is lysed by plasmin (EC 3.4.21.7), which is activated from plasminogen by tissue plasminogen activator. In balanced condition, fibrin clots are hydrolyzed by plasmin to avoid thrombosis in blood vessels; however, in unbalanced condition as a result of pathophysiological disorders, the clots are not hydrolyzed, and thus thrombosis occurs. Urokinase-type plasminogen activator (EC 3.4.21.31), tissue plasminogen activator (t-PA), bacterial plasminogen activator streptokinase (EC 3.2.1.35) are typical thrombolytic agents used for therapeutic purposes [2]. Based on working mechanisms, thrombolytic agents are of two types: one is plasminogen activators and urokinase which activates plasminogen into active plasmin to degrade fibrin and the other is plasmin-like proteins which directly degrade fibrin [3]. Moreover, on the basis of catalytic mechanisms, microbial fibrinolytic enzymes are classified into three types: serine protease (e.g. nattokinase), metalloprotease (e.g. Armillaria mellea metalloprotease) and mixture of both serine and metalloprotease (e.g. protease from Streptomyces sp. Y405) [3]. Despite widespread uses, thrombolytic agents such as t-PA and urokinase are expensive, exhibit low fibrin specificity, and have undesired side effects such as gastrointestinal bleeding, resistance to repercussion and allergic reactions [4]. Therefore, continuous efforts have been focused in the search of safer and less expensive thrombolytic agents from diverse sources. Until recently, fibrinolytic enzyme with potential thrombolytic application have been purified from various sources such as fermented food, earthworms, mushrooms, snake venom and microbial sources [5]. From microbial sources, bacteria, actinomyces, fungi and algae are reported to produce fibrinolytic enzyme. Of them, only few reports are from Streptomyces origin [6], [7], [8].

Streptomyces sp. CS684 was isolated from Korean soil as an antibiotic producer [9] and the strain also produced a novel low molecular weight phospholipase D [10]. A profound proteolytic enzyme with fibrinolytic activity was found in the culture medium; therefore, we were interested in its potential biocatalytic application. Here, we describe purification and biochemical properties of a novel fibrinolytic enzyme, FP84, produced by Streptomyces sp. CS684.

Section snippets

Materials

Thrombin, fibrinogen and plasmin (all from human plasma), azocasein, Folin–Ciocalteu's phenol reagent and tyrosine were purchased from Sigma–Aldrich (St. Louis, MO, USA). Sepharose CL-6B was obtained from Pharmacia (Uppasala, Sweden) and Poros-HQ was from Applied biosystems (Foster city, CA, USA). Protease inhibitors pefabloc, aprotinin, EDTA were acquired from Roche Applied Science (Mannheim, Germany). All other chemicals and reagents used were of analytical grade.

Bacterial strain and culture conditions

Streptomyces sp. CS684 was

Enzyme purification and molecular weight

The purification of FP84 from the culture supernatant of Streptomyces sp. CS684 is summarized in Table 1. The enzyme was purified to homogeneity by a three-step procedure (Table 1 and Fig. 1), resulting in 10.5-fold purification and 5.4% activity recovery. An SDS-PAGE analysis gave a single band of FP84 corresponding to a molecular weight of 35 kDa (Fig. 2). In the activity staining (fibrin zymography) step, FP84 appeared as a single band corresponding to the same protein band appeared in

Discussion

It is indispensable to develop new thrombolytic agents since a variety of cardiovascular diseases and drawbacks in the typical thrombolytic agents are being increasingly reported [1], [18]. In this study, we described purification and characterization of a fibrinolytic enzyme, designated as FP84, from Streptomyces sp. CS684. Until recently, only few fibrinolytic enzymes have been reported from actinomycetes, a large group of organisms capable of producing several beneficial products including

Conclusions

A fibrino(geno)lytic enzyme, designated as FP84, was purified from Streptomyces sp. CS684. The enzyme degraded fibrin clot by direct fibrinolysis more efficiently than plasmin. It hydrolyzed Bβ-chains, but could not cleave Aα- and γ-chains of fibrinogen. The N-terminal amino acid residues of FP84 revealed almost 80% homology with serine proteases isolated from various Streptomyces strains, but showed low or no homology with the known fibrinolytic enzymes. The enzyme might be a novel type of

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