Patent ID: 7776574

Claim:
A process for the preparation of purified thrombinase, having a molecular weight in the range of 31,000 to 32,000 Daltons, which comprises: (i) Culturing of cells of Bacillus sphaericus serotype H5a 5b in a culture medium consisting of 0.03 to 1.5% of yeast extract, 0.2 to 1.5% peptone, 1 to 1.6% sodium acetate, 0.3 to 0.5% beef extract, 0.2 to 0.5% sodium chloride, 0.5 to 1% Soya peptone, and 0.68% ammonium sulphate at a pH in a range of 7.2 to 8, (ii) Removing the cultured cells by cross flow filtration using 0.22 mμ filter to obtain a cell supernatant, (iii) Subjecting the cell supernatant to two step ultra filtration: a. with a first ultra filtration of the cell supernatant using 100,000 MW (Molecular Weight) cut off membrane to obtain a filtrate, and b. with a second ultra filtration of the filtrate using 10,000 MW cut off membrane to obtain a retentate, (iv) Salting out the retentate with ammonium sulphate in a concentration in the range of 20 to 40% to obtain a precipitate, (v) Subjecting the precipitate to dialysis, (vi) Re-precipitating the dialyzed precipitate using ice-cold acetone to obtain a re-precipitated precipitate, (vii) Reconstituting the re-precipitated precipitate in buffer, (viii) Decolorizing the reconstituted precipitate, and then dialyzing and lyophilizing to obtain a lyophilized precipitate, (ix) Subjecting the lyophilized precipitate to ion exchange chromatography and to gel filtration chromatography to obtain a fraction showing fibrinolytic activity, and (x) Dialyzing the fraction showing fibrinolytic activity and lyophilizing to obtain purified thrombinase having a molecular weight in the range of 31,000 to 32,000 Daltons.