NA

WE CLAIM
1. A process for preparation of thrombolytic enzyme, thrombinase from the culture filtrate of Bacillus sphaericus serotype H5a 5b in the culture medium consisting of 0.03 to 1.5% of yeast extract with one or more of constituents: 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% soyapeptone, and 0.68% ammonium sulphate, wherein the process comprises steps of cell removal by cross flow filtration using 0.22µ filter, subjecting the cell supernatant thus obtained to two step ultra filtration using 1,00,000MW (Molecular Weight) out off membrane followed by ultra filtration of the filtrate thus obtained using 10,000MW cut off membrane, rejecting the filtrate, salting out the retente with ammonium sulphate, subjecting it to dialysis, reprecipitating it using ice-cold acetone, reconstituting in buffer, decolorizing by using modified CDR (cell debris remover) treatment, as herein described, dialyzing, lyophilizing and purification firstly by preferably ion exchange chromatography followed by gel filtration chromatography, dialysing the fraction showing fibrinolytic activity and lyophilisng to obtain purified thrombinase having a molecular weight of 31,700.
2. A process for preparation of thrombinase as claimed in claim 1 wherein pH of culture medium is 6.5 to 7.5.
3. A process for preparation of thrombinase as claimed in claim 1 wherein ammonium sulphate is taken preferably in quantity 20 to 40%.
4. A process for preparation of thrombinase as claimed in claim 1 wherein
buffer used is Tris 0.01 to 0.05 M, pH 8.0.
5. A process for preparation of thrombinase as claimed in claim 1 where
said gel filtration chromatography is carried out using preferably
sephacryl S-200.
6. A process for preparation of, thrombinase as claimed in claim 1 wherein
ice-cold acetone and crude enzyme, are in ratio of 1:1 to 1:1.5 (v/v)
7. A process for preparation of thrombolytic enzyme, thrombinase as
claimed in claim 1 wherein said modified CDR (cell debris remover)
treatment comprises:-
(a) making a bed of CDR using, for example, buchner funnel and
layering crude enzyme over it;
(b) applying vacuum to the receiving container and eluting with Tris 0.01
to 0.05 M pH 8.0 containing 0 1 to 0.5 M NaCl to elute fibrinolytic
enzyme bound to CDR;
8. A process for preparation of thrombinase as substantially described and
illustrated herein.