Indian J Med Res 126, November 2007, pp 459-464

Production & purification of a fibrinolytic enzyme (thrombinase)

from Bacillus sphaericus

K. Balaraman & G. Prabakaran

Vector Control Research Centre (ICMR), Puducherry, India

Received June 2, 2006

Background & objectives: Treatment of thromboembolic vascular disease has relied on anticoagulants.
However, recognition that lysis of preformed fibrin could be accomplished in vivo by a process
involving the conversion of inactive plasminogen to active plasmin enzyme led to an alternative
enzyme-based approach. The drugs used for this therapy are called the fibrinolytic enzymes. In this
study we attempted the production, purification and characterization of fibrinolytic enzyme from
Bacillus sphaericus.
Methods: The seed was prepared in nutrient yeast salt medium (NYSM) in shake flask and organism
was produced in 100 l pilot fermentor. Biomass was separated by centrifugation and crude protein
was prepared by ammonium sulphate precipitation. Purification was done by ion exchange
chromatography using Q sepharose followed by gel filtration chromatography using Sephacryl S-
300. Molecular weight was determined through HPLC. Fibrinolytic activity was assayed by fibrin
plate method.
Results: The production method yielded 64 mg/l of the crude enzyme and after purification it was
6.3 mg/l. The molecular weight of the compound was 18.6 kDa.
Interpretation & conclusions: The enzyme exhibited similar fibrinolytic activity as that of
streptokinase, on fibrin plates that were devoid of plasminogen, suggesting that its fibrinolytic action
is independent of plasminogen and it is not a plasminogen activator.

Key words Bacillus sphaericus - fibrin clots - fibrinolytic - protease - thrombinase

Earlier days treatment of thromboembolic vascular
disease was relied on the use of anticoagulants, such as
warfarin (coumarin) and heparin to inhibit the formation
of fibrin clots. However, recognition that lysis of
preformed fibrin could be accomplished in vivo by a
process involving the conversion of inactive plasminogen
to active plasmin enzyme led to an alternative enzyme-
based approach. Fibrinolytic or thrombolytic agents
convert plasminogen to plasmin, lyse the clot by breaking
459
down the fibrin contained in a clot. Currently, several
thrombolytic agents such as streptokinase, urokinase,
prourokinase, reteplase (r-PA), alteplase (t-PA), reptilase,
brinase and anisoylated purified streptokinase activator
complex (APSAC) are available for clinical use1. All
these thrombolytic agents still suffer significant
shortcomings, including requirement of large therapeutic
dose, short plasma half-life, limited fibrin specificity,
reocclusion and bleeding complications2.
460 INDIAN J MED RES, NOVEMBER 2007
Fibrinolytic enzymes have been purified from many
plant, animal and microbial sources. Fibrinolytic
enzymes from microbial sources have been reported
from various species of Bacillus3-8, Pseudomonas9,
Staphylococcus 10 , Alteromonas 11 , Coryneform
bacteria12, Penicillium13, Aspergillus14-16, Fusarium17,18,
Trichotecium19, Actinomyces20,21, Streptomyces22,23 and
Esherichia coli24. In the present study we attempted the
isolation of a novel fibrinolytic enzyme, named as
thrombinase, from the cultures of Bacillus sphaericus.
This is the first report of a fibrinolytic enzyme from B.
sphaericus and preliminary toxicology studies indicated
that it is safe to mammals25,26.
Material & Methods
Organism: B. sphaericus of H5a5b serotype obtained from
the culture collection of Vector Control Research Centre
(VCRC), Puducherry code named as VCRC B42, was
used for this study.
Seed culture preparation: First stage seed was taken
from the lyophilized seed lot and the spores were
suspended in 1 ml of nutrient yeast salt medium
(NYSM) broth27. A loopful of the spore suspension was
inoculated to NYSM agar slopes and incubated for 2
days at 30o C. Then a loopful of culture from the slope
culture was transferred to 10 ml NYSM broth taken in
a boiling tube and incubated on a shaker at 200 rpm at
30oC for 7 h. Three ml of first stage seed was transferred
to 60 ml of NYSM broth held in 250 ml Erlenmeyer
flask and incubated on a shaker at 200 rpm at 30oC for
7 h. Thirty ml of second stage seed was transferred to
600 ml of NYSM broth held in 2000 ml Erlenmeyer
flask and incubated on a shaker at 200 rpm at 30oC for
7 h.
Fermentation procedure: The production medium
(NYSM) (60 l) was charged into a 100 l bioreactor and
sterilized at 121oC /15 psi for 20 min. The third stage
seed was inoculated at 2 per cent level (v\v) and the
fermentation was carried out for 24 h under the
following conditions – temperature: 30 o C, pH: 7.4,
dissolved oxygen: 40 per cent saturation, and agitation:
200 rpm. Foam was controlled with silicone antifoam
agent.
Assessment of dry cell mass production: At every 3 h
interval, samples of 100 ml were collected and
centrifuged at 15000 x g for 20 min. The supernatant
was discarded, the cell pellet was lyophilized, and dry
weight was determined. This was done till 24 h of
fermentation.
Purification of the fibrinolytic enzyme: Fermentation
was stopped when the culture sporulated completely,
usually after 24 h. Cell harvesting was carried out by
tangential flow filtration using 0.45 µ HVLP membrane
cassettes installed in a Pellicon Ultrafiltration System
(Millipore, USA). One litre of the cell free supernatant
was subjected to two step ultrafiltration (UF). At the
first stage, the supernatant was subjected to UF with
30,000 nominal molecular weight cut-off limit (NMWL)
PTTK cassette (Millipore, USA). Filtrate obtained from
the first step was further subjected to UF with 10,000
NMWL PTGC cassette. The retentate was subjected to
ammonium sulphate (50% w/v) precipitation by
constant overnight stirring at 4 oC. The precipitate
formed was centrifuged at 8000 x g for 30 min and the
precipitate obtained was dissolved in minimum amount
of 50 mM Tris-HCl buffer, pH 7.2. The dark brown
solution thus obtained was dialysed at 4o C for 24-48 h
against 10 mM Tris-HCl buffer, pH 7.2. Cell debris
remover (CDR, Whatman Ltd, England), a modified
cellulose (anionic), was used for decolourization of the
dialysed crude prior to chromatographic purification.
Pharmacia K-26 columns with end fittings were used
in all chromatographic separations. Addition of sample
to the column, elution of proteins, maintenance of
constant flow rate of 2 ml/min and effluent monitoring
at 254 nm were performed using a Shimadzu LC-6A
Model (Shimadzu, Japan) system. Constant time
fractions (5 min/fraction) were collected with the help
of a fraction collector (Haake Buchler, model-100,
USA).
Ion exchange chromatogray: Q Sepharose (Pharmacia,
Sweden) was used for anion exchange in column (bed
dimension 2.6 x 30 cm), which was equilibrated with
10 mM Tris-HCl buffer, pH 8.0. The lyophilized
decolourised fraction was dissolved in the above buffer,
dialysed extensively against the same buffer for 24 h
and then loaded to the column (5 ml/run). Elution was
carried out in a step-wise manner at a flow rate of 2 ml/
min at regular intervals (5 min/fraction). The first step
of elution was carried out with the column buffer
containing 0.1 M NaCl and the second elution was with
the same buffer containing 0.5 M NaCl. All the
individual fractions were analysed for protease activity
on azo albumin and fibrinolytic activity on fibrin clot.
The active fraction obtained after anion exchange
chromatography was further purified by gel filtration
chromatography.
Gel filtration chromatography: Sephacryl- S 300
(Pharmacia, Sweden) was used for gel filtration
 BALARAMAN & PRABAKARAN: PRODUCTION OF FIBRINOLYTIC ENZYME FROM B. SPHAERICUS
chromatography. A column (2.6 X 36 cm) was
equilibrated with 50 mM Tris-HCl buffer containing
0.1 M NaCl, pH, 7.5. The active fraction was dissolved,
dialysed, and loaded onto the column (2 ml/injection)
and eluted with the same buffer. Fractions were
collected at flow rate of 2 ml/min at regular intervals (5
min/fraction). Column operation and monitoring were
similar to that described under ion-exchange
chromatography.
Determination of molecular weight by HPLC:
Molecular weight determination of the protein was
carried out by HPLC on GF-250 column. Analytical
conditions were: column- Zorbax-bioseries GF250,
guard column- Zorbax-Diol-bioseries, flow rate- 2 ml/
min, mobile phase: Na 2HPO 4 0.2 M, pH- 7.0, and
detector- UV 254. Typically, the elution order or
retention time obtained for a given molecule is inversely
proportional to the logarithm of its molecular size.
Molecular size is related to molecular weight. The
standard proteins used were RNase-A (13700 kDa),
ovalbumin (43000 kDa), albumin (67000 kDa), ferritin
(443000 kDa), and thyroglobulin (669000 kDa).
SDS-PAGE: SDS-PAGE was carried out with the SDS-
tris-glycine system (discontinuous) of Laemmli 28.
Polyacrylamide slab gel of 160x140x1.5 mm (length x
width x thickness) dimension was used. Enzyme protein
(1.0 mg/ml) dissolved in Tris-HCl buffer (pH 6.8)
containing 0.5 per cent each of SDS and
mercaptoethanol was kept in a boiling water bath for 3
min in a tightly stoppered vial. Fifty microlitres of this
sample was loaded on the gel. For molecular weight
determination, the standard proteins used were soya
bean trypsin inhibitor (20,100 kDa), carbonic anhydrous
(29,000), ovalbumin (43,000), bovine serum albumin
(68,000) and phosphorylase b (97,000).
Assay of fibrinolytic activity: The original fibrin plate
method of Astrup and Mullertz 29 with slight
modification was used for measuring the fibrinolytic
activity of the test preparation along with streptokinase.
Petridishes containing 9 ml of 0.2 per cent fibrinogen
solution (pH 7.8) were placed on a horizontal glass plate.
To each of these petridishes, 0.2 ml of plasminogen (10
units) was also added and mixed well. Clotting was
induced by the addition of 0.2 ml of thrombin solution
(20 units). In order to speed up the clotting process, the
plates were incubated at 37oC for 15 min. Plates were
prepared afresh every time. Known quantities of the
enzyme solution and standard were placed as small
droplets on the surface of the fibrin clot. The plates
461
o
were then incubated at 37 C for 2 h and visually
inspected for liquefaction. The area of digested fibrin
was considered as a quantitative measure of the
fibrinolytic activity of the enzyme.
Assay for protease activity: Protease level was
determined by the method of Li and Yousten30 using
0.5 per cent (w/v) of azoalbumin in 0.1 M morpholino
propane sulphanic acid (pH 7.0) as the substrate. One
unit of protease was considered as that amount of
enzyme which produced an absorbance increase of 0.01,
under the assay conditions of pH 7.0, temperature
o
30 C, and incubation time 1 h.
Results & Discussion
The biomass produced at different hours of growth
ranged from 3.0 to 4.83 g/l. It reached the maximum at
15 h of growth and thereafter it declined due to
sporulation (Fig. 1). Ultrafiltration with 30,000 and
10,000 NMWL membrane cassettes resulted in the
recovery of 77 per cent protease activity from the culture
filtrate and 20-fold reduction in volume. The crude
enzyme obtained after ammonium sulphate precipitation
and decolourisation suggested that the UF could retain
the specific molecular weight proteins.
Anion exchange chromatography separated the
decolourised fraction into two major peaks, i.e., bound
and free. Though both the fractions showed proteolytic
activity, only the free fraction I showed fibrinolytic
activity. Fraction I was pooled and lyophilized after
dialysis. About 50 per cent of the protease activity was
lost with the bound fraction. The bound fraction was
not processed further. Further purification of this
fraction was carried out using gel filtration
chromatography.
Fig. 1. Growth curve of B. sphaericus in terms of dry biomass
weight (g/l).
462 INDIAN J MED RES, NOVEMBER 2007

Gel filtration chromatography showed two major An overall purification of 91-fold was achieved by
fractions. Fraction I was weakly proteolytic whereas this procedure. The production method yielded 64 mg/l
fraction II showed proteolytic and fibrinolytic activity of the crude enzyme after ammonium sulphate
(Fig. 2). HPLC on GF-250 column showed single peak precipitation and 6.3 mg/l of the purified enzyme after
for the fibrinolytic fraction II. Fraction II was pooled, down stream processing. The purification procedure
dialysed and lyophilized. The molecular weight of the adopted is well suited for industrial applications and
purified fraction was 18.6 kDa as determined by HPLC can be scaled up to any level. Summary of the overall
on GF-250 column (Fig. 3). Again, the purified fraction purification steps involved is presented in the Table.
showed a single band with a molecular weight of 18
Fibrinolytic enzymes, including plasminogen
kDa on SDS-PAGE (Fig. 4).
activators have been reported from various microbes3-24.
However, except for streptokinase, none of them are
practically in use as a thrombolytic agent. Streptokinase
Fibrinolytic is a bacterial protein from beta haemolytic E. coli24. It
does not have a direct fibrinolytic activity and the
therapeutic action is via the activation of blood
plasminogen to the clot dissolving plasmin.
Absorbance 254 nm

0 40 80 120 140 180

Retention Time (min )

Fig. 2. Purification of fibrinolytic protease by gel filtration on

Sepharcyl S-300 column.*Fibrinolytic and proteolytic activity.

A 2 B
Absorbance 254 nm

3
4
5

3 4 5 6 7 8 3 4 5 6 7 8

Retention time (min)

Fig. 3. Molecular weight determination by HPLC on GF-250 Fig. 4. SDS-PAGE profile of fibrinolytic protease from
column (A) Profile of protein molecular weight marker, (B) Profile B. sphaericus (Lane 1- crude protein from culture filtrate; Lane 2-
of purified fibrinolytic protease. molecular weight marker; Lane 3-purified thrombinase).
 BALARAMAN & PRABAKARAN: PRODUCTION OF FIBRINOLYTIC ENZYME FROM B. SPHAERICUS 463

Table. Summary of the purification process of fibrinolytic protease (thrombinase) from B. sphaericus
Step Volume Total activity Total protein Specific activity Recovery Purification
(ml) (Units) (mg) (Units/mg) (%) (fold)
1. Culture filtrate 1000 106000 2214 47 100 1
2. Ultrafiltration
a. 30,000 NMWL cut-off 950 98250 1817 54 92 1.2
(filtrate)
b. 10,000 NMWL cut-off 50 81250 175 462 77 10
(retentate)
3. Ammonium sulphate 2.5 71665 64 1111 67 24
fraction
4. Decolorised 2.5 62500 41 1506 59 32
fraction
5. Ion exchange 3.75 31562 18 1765 30 38
chromatography
6. Gel filtration 3.75 26828 6.3 4258 25 91
chromatography

(a) (b)

Fig. 5. Fibrinolytic activity of thrombinase (a) Positive result of thrombinase (B & D) and streptokinase (A & C) activity (positive control),
(b) Negative result without thrombinase (C & D) or streptokinase (A & B) (negative control).

The results of the fibrinolytic assay are presented in
(Fig. 5 a & b). Zone A and C represented 10 and 20 units
of streptokinase. Zone B and D represented 20 and 40 µl
of the purified fibrinolytic protease. The results showed
that 1 mg protein of the purified material contained
fibrinolytic activity equivalent to that of 5 lakh units of
streptokinase. The nature of fibrinolytic activity of the
protease on fibrin plates was similar to that of
streptokinase. The nature of fibrinolytic activity of the
protease on fibrin plates was similar to that of
streptokinase. The RBC’s released from the blood clot
by the action of fibrinolytic protease were normal in
appearance under microscopic observation. Its
fibrinolytic activity was greater than the commercially
available streptokinase and hence it has got potential use
in the treatment of myocardial infarction. Thrombinase,
the fibrinolytic protease obtained from B. sphaericus,
may play a vital role in saving the humanity from blood
clot related diseases with its unique mode of action and
less side effects in future, provided it passes through the
toxicological tests and clinical trials successfully.
Acknowledgment
Authors thank Dr P.K. Das, formerly Director, VCRC,
Puducherry, for his support and critical suggestions, and the
technical staff of the Division of Product Development, VCRC,
for their assistance.
464 INDIAN J MED RES, NOVEMBER 2007
References
1. Cadroy Y, Haarkeer LA. Thrombosis. In: Antonaccio MJ,
editor, Cardio vascular pharmacology, 3rd ed. New York:
Raven Press; 1990. p. 515.
2. Reddy DS. Newer thrombolytic drugs for acute myocardial
infarction. Indian J Exp Biol 1998; 36 : 1-15.
3. Yu R, Qi H, Zhang T, Wu WT. Preliminary studies on in vitro
and in vivo thrombolytic activities of thrombolytic enzyme
from an induced Bacillus subtilis strain. Sichuan Da Xue Xue
Bao Yi Xue Ban 2005; 36 : 93-6.
4. Cherkessova GV, Nesterova NG, Fetisova ZS. The
thrombolytic activity of Baccillus mesentricus at different
conditions of nitrogen nutrition. Mikrobiologiia 1989; 58 :
915-9.
5. Fayek KI, El-Sayed ST. Some properties of two purified
fibinolytic enzymes from Bacillus subtilis and B. polymyxa. Z
Allg Mikrobiol 1980; 20 : 383-7.
6. Vyrbornykh CN, Landau NS, Egorov NS. Biosynthesis of
fibrinolytic enzymes with different mechanisms of action by
microorganisms of the genus Bacillus. Mikrobiologiia 1990;
59 : 782-9.
7. Egorov NS, Iudina TG, Loriia ZHK, Kreier VG. Fibrinolytic
activity of several variants of Bacillus thuringiensis. Prikl
Biokhim Mikrobiol 1979; 15 : 416-20.
8. Peng Y, Huang Q, Zhang RH, Zhang YZ. Purification and
characterization of a fibrinolytic enzyme produced by Bacillus
amyloliquefaciens DC-4 screened from douchi, a traditional
Chinese soybean food. Comp Biochem Pysiol B Biochem Mol
Biol 2003; 134 : 45-52.
9. Imshenetskii AA, Demina NS, Lysenko SV, Evdokimova MD.
Fibrinolytic activity of bacteria from Pseudomonas genus.
Prikl Biokhim Mikrobiol 1991; 27 : 845-9.
10. Berdzulishvili EM, Afanas’eva TI, Alergant AP. Fibrinolytic
activity of pathogenic staphylococci of different origins. Lab
Delo 1973; 6 : 332-4.
11. Demina NS, Veslopolova F, Gaenko GP. The marine bacterium
Alteromonas piscicida-a producer of enzymes with
thrombolytic action. Izv Akad Nauk SSSR Biol 1990; 3 :
415-9.
12. Egorov NS, Landau NS, Milovanova II. Fibrinolytic activity
in mono- and mixed cultures of coryneform bacteria. Nauchnye
Doki Vyss Shkoly Biol Nauki 1982; 10 : 86-90.
13. Andreeva NA, Ushakova VI, Egorov NS. Study of proteolytic
enzymes of various strains of Penicillium lilacinum in relation
to their fibrinolytic activity. Mikrobiologiia 1972; 4 : 417-22.
14. Larcher G, Bouchara JP, Annaix V, Symoens F, Chabasse D,
Tronchin G. Purification and characterization of a
fibrinogenolytic serine proteinase from Aspergillus fumigatus
culture filtrate. FEBS Lett 1992; 10 : 65-9.
Reprint requests: Dr K. Balaraman, Formerly Deputy Director (SG), Vector Control Research Centre (ICMR)
Indira Nagar, Puducherry 605006, India
e-mail: drkbraman@gmail.com
15. Ushakova VI, Dal’ko LD, Egorov NS. Study of the protease
complex synthetized by Aspergillus kanagawaensis in relation
to its fibrinolytic activity. Nauchnye Doki Vyss Shkoly Biol
Nauki 1974; 8 : 93-7.
16. Klocking HP, Markwardt F. Thrombolytic and
pharmacodynamic properties of Aspergillus ochraceus
protease. Farmakol Toksikol 1975; 38 : 341-9.
17. Abdel-Fattah AF, Ismail AS, Saleh SA. Purification and
properties of two fibrinolytic enzymes from Fusarium
oxysporum N.R.C.1. Zentralbl Mikrobiol 1993; 148 : 123-8.
18. El-Aassar SA. Production and properties of fibrinolytic
enzyme in solid state cultures of Fusarium pallidooroseum.
Biotechnol Lett 1995; 17 : 943-8.
19. Stepanova TSN, Maksimova RA, Iulikova EP, Silaev AB,
Andreenko GV. Fractionation of a preparation of fibrinolytic
enzymes “tricholysin” formed from Trichotecium roseum Lk.
ex Fr. on carboxymethyl-sephadex C-50. Prikl Biokhim
Mikrobiol 1976; 12 : 407-10.
20. Egorov NS, Kochetov GA, Khaidarova NV. Isolation and
properties of the fibrinolytic enzyme from the Actinomyces
thermovulgaris cultural broth. Mikrobiologiia 1976; 45 : 455-9.
21. Egorov NS, al-Nuri MA Krivova AIu. Optimization of the
nutrient medium for Actinomyces spheroides-producer of
proteolytic enzymes with fibrinolytic activity. Mikrobiologiia
1976; 45 : 607-13.
22. Egorov NS, Kochetov GA, Khaidarova NV. Physicochemical
properties of a fibrinolytic enzyme from Streptomyces
thermovulgaris culture broth. Nauchnye Doki Vyss Shkoly Biol
Nauki 1986; 4 : 89-94.
23. Chitte RR, Dey S. Potent fibrinolytic enzyme from a
thermophilic Streptomyces megasporus strain SD5. Lett Appl
Microbiol 2000; 31 : 405-10.
24. Malke H, Ferretti JJ. Streptokinase: cloning, expression and
excretion by Escherichia coli. Proc Natl Acad Sci USA 1984;
81 : 57-61.
25. Balaraman K, Kuppusamy MA. Process for the production of
THROMBINASE, a blood clot dissolving enzyme from
Bacillus sp. Indian patent no. 232/DEL/91 dt 21-3-91.
26. Balaraman K, Kuppusamy MA. Process for the preparation
of thrombinase. US Patent 1995; 5 : 434 059.
27. Myers PS, Yousten AA. Localization of a mosquito larval toxin
of Bacillus sphaeicus 1593. Appl Environ Microbiol 1980;
39 : 1205-19.
28. Laemmli UK. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature (Lond) 1970;
227 : 680-5.
29. Astrup T, Mullertz S. The fibrin plate method for estimating
fibrinolytic activity. Arch Biochem 1952; 40 : 346-51.
30. Li E, Yousten AA. Metalloprotease from Bacillus
thuringiensis. Appl Microbiol 1975; 30 : 354-61.