Enzymatic analysis of native and recombinant fibrinolytic enzyme from bacillus cereus 13BN isolated from belacan
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Date
2016
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Universiti Teknologi Malaysia
Abstract
Fibrinolytic enzymes from natural sources have now attracted more attention than ever because they are less costly and have less undesirable side-effects as compared to modern chemical thrombolytic agents. A bacterial isolate designated as Bacillus cereus 13BN having excellent fibrinolytic enzyme was isolated from fermented shrimp paste, belacan. The enzyme has a dual mechanism of fibrin degradation; it directly degrades fibrin plus it can also act as a tissue type plasminogen activator (t-PA), which converts plasminogen to plasmin and consecutively degrades fibrin. T-PA also participates in a wide variety of connective tissue matrix alterations and upregulated by proinflammatory cytokines which can act as antidepressants. In silico analyses indicated that the enzyme belonged to the typical subtilisin-like serine protease group with a conserved region of a catalytic triad; Asp226, His298 and Ser658. Subtilisin-like 13BN gene was successfully cloned into pET22b (+) vector and then transformed into E. coli BL21 (DE3) to give recombinant BL-21(DE)-(pET22b(+)(SL-13BN). The vector carries an N-terminal pelB signal sequence for periplasmic translocation and a C-terminal His•Tag® for easy purification of the protein. Recombinant enzyme RSL-13 BN was over-expressed as extracellular enzyme (EX) at 41% of total fibrinolytic activity, besides been expressed as periplasmic enzyme (PE) at 56 % and solubilized inclusion bodies (SIB) at 3 %. Under optimized condition where cell mass was propagated by induction with 1 mM IPTG at 30 ºC for 16 hours, 14,616 UL-1 total activity by RSL-13BN could possibly be achieved. This is four fold higher than the extracellular native enzyme SL-13BN (3,468 UL-1). Purification of extracellular SL-13BN enzyme was preceded with ammonium sulphate precipitation, followed by anion exchange and gel filtration chromatography. Alternatively, following ammonium sulphate precipitation, single-step purification was performed for extracellular RSL-13BN enzyme using affinity chromatography utilizing His????-TrapTM Ni-NTA Sepharose column. Both native and recombinant enzymes have equivalent biochemical characteristics. These enzymes are serine proteases with plasminogen activator potential and have a molecular weight of 80kDa. Their strong fibrinolytic activity was verified through fibrin clot degradation pattern on SDS-PAGE gel. The proteolytic action of SL-13BN and RSL-13BN differ from the typical human plasmin in that they can completely hydrolyze whole fibrinogen and fibrin clots without the aid of other proteolytic enzymes.The optimum temperature for fibrinolysis for both was 50°C but their optimum pH differed slightly; pH 7.4 for SL-13BN and pH 7 for RSL-13BN. The activity of both enzymes was enhanced by Ca2+, Na+, K+, Mg2+ and Mn2+, but inhibited by Ag+, Co2+, Cu2+, Fe2+, Fe3+, Hg+, Zn2+, PMSF, EDTA and SDS. Purified SL-13BN had a Vmax of 2.665 U mg-1min-1and Km of 0.5722 mg mL-1 and purified RSL-13BN had a Vmax of 2.642 U mg-1min-1and Km of 0.5776 mg mL-1 towards fibrin. In conclusion, this study showed that RSL-13BN enzyme can be over-produced, acting as t-PA which can provide many medical benefits, easily purified as well as can hydrolyze fibrin and fibrinogen as a whole without the help of other proteolytic enzymes. This makes the enzyme a potential anticoagulant substitute for the treatment of thrombosis-linked diseases
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Thesis (PhD. (Biosciences))
Keywords
Biosciences and medical engineering