Purification and properties of a fibrinolytic enzyme from Bacillus subtilis

Abstract

A fibrinolytic enzyme obtained from B. subtilis was purified, using DEAE-cellulose column chromatography, and gel filtration on Sephadex G-100. The preparation was homogeneous as tested by gel filtration on Sephadex G-200, and disc electrophoresis.The molecular weight of this enzyme was 29.400 estimated by gel filtration on Sephadex G-100. The optimum pH for enzyme activity was 7.2. Copper ions significantly increased enzyme activity, while Zn++ and Mn++ caused marked inhibition.Microbial proteases are well-known enzymes, several investigators have explored this field and studied the production of these enzymes. A review about characterization and methods for their production and isolation was presented by KEAY (1971).Proteolytic enzymes produced by bacteria, e.g. by B. I n a previous study we reported the production of a fibrinolytic enzyme from a strain of Bacillus subtilis (FODA and FAYEK 1975). For further characterization it was necessary to have this enzyme in a pure form. This study presents a method for purification and some properties of the enzyme. Materials and methodsOrganism and growth conditions: A strain of Bacillus subtilis from the NRC collection was used throughout this study. Details of growth medium and enzyme production were as described by FODA and FAYEK (1975). At the end of the growth period, bacterial cells were removed by centrifugation and the cell free supernatant was dialyzed against 0.03 M phosphate buffer a t 4 "C.Enzyme concentration : Small aliquots of crude enzyme preparation after dialysis were concentrated by dialysis against sucrose. The concentrated enzyme solution was than dialyzed against cold 0.1 M phosphate buffer pH 7.0 to eliminate sucrose. DEAE-cellulose column chromatography : DEAE-cellulose was pretreated as recommended by PETERSON and SOBER (1962) and equilibrated with 0.025 M phosphate buffer pH 7.0. After packing in a column (50 x 1.7 om), the concentrated enzyme was placed on the top, and eluted with a stepwise gradient ranging from 0.0-1.0 M sodium chloride in 0.025 M phosphate buffer, p H 7.0. The flow rate was 30 ml/hour. The effluent was collected in samples of 5 ml, transmittance a t 260 and 280 nm was recorded. The protein concentration in each fraction was then calculated using the nomograph developed by ADAMS (1942). Gel filtration on Sephadex G-100: The gel was allowed to swell for 3 days in excess water, and then equilibrated for another 2 days a t 4 "C in 0.05 M phosphate buffer pH 7.0, after which it was packed in a column (1.5 x 60 cm). Collected active fractions from the DEAE-cellulose column were concentrated against sucrose before applying to the top of the Sephadex column, the flow rate was 25 ml/hour. and the effluent has collected in 5 ml samples. The transmittance at 260 and 280 nm was recorded. Test of purity and homogeneity: A) Pooled active fractions irom the Sephadex 6-100 column were placed on the top of a Sephadex G-200 column (50 x 0.8 om), equilibrated and developed

Physiological Functionality and Enzyme Activity of Biomass fromPichia anomalaGrown on Ginseng-Steaming Effluent

“…The fibrinolytic activity was assayed by the method of Fayek et al (1980) Each sample of 0.5 ml was added to 3 ml of a substrate solution (0.6% fibrin in 0.1M McIlvaine buffer, pH 7.0) and incubated at 40℃ for 10 min. The reaction was stopped by adding 3 ml of 0.4M TCA for 30 min.…”

Nutritional Characteristics and Physiological Functionality of Lipase Inhibitor-Containing Desmodium oxyphyllum DC. Extracts

“…AChE 저해 활성(%)={1-(시료구/대조구)}×100 혈전 용해 활성은 ㎕당 0.1 unit의 thrombin을 함유한 평판 배지에 pH 7.0의 인산완충용액에 용해시킨 0.6%의 fibrinogen 을 주입하여 고형화 시켰다. 여기에 시료 25 ㎕를 함유한 paper disk를 놓고 37℃에서 6시간 반응시킨 후 투명환의 크 기를 측정하여 혈전 용해 활성을 ㎜로 표시하였다(Fayek & El-Sayed 1980).…”