This is a presentation I created for my ICBI 436 Industrial Enzymology, a Biotechnology course I took at Mahidol University International College (MUIC)
2. L-asparaginase
Background
• Source(s): E. coli, Pseudomonas aeruginosa
• Catalyzes the hydrolysis of L-asparagine to L-
aspartic acid + ammonia in leukemic cells
• Used to treat acute lymphocytic leukemia
• Mechanism of action: depletion of asparagine,
inhibition of protein synthesis, cell cycle
arrest in G1 phase, and apoptosis
4. L-asparaginase
Production
Slant Culture Preparation
1. Stock culture was maintained on tryptic soy
agar slants, containing tryptic casein Bios D
(1.5%), Soy peptone (0.5%), NaCl (0.5%),
and agar Bios U (1.5%)
2. Culture was incubated at 37 C for 18 h and
stored at 4 C
3. Around 48 h, single slant cultures were
transferred into 50 ml aliquots of defined
inducible medium
5. L-asparaginase
Production
4. Culture + medium were then placed in 250 ml
Erlenmeyer flasks and used as standard inocula (3
ml/flask)
Fermentation media preparation
5. Using, 10 g Soya bean meal of 0.4-0.8 cm particle
size were moistened with 10 ml of 0.01 M
phosphate buffer pH 7.4, and placed in 250 ml
Erlenmeyer flasks
6. The fermentation media were sterilized by
autoclaving for 15 min at a pressure of 15 Ib/inch to
raise the temperature to 121 o C
6. L-asparaginase
Production
Inoculation + Enzyme preparation
7. The flasks were inoculated with 3 ml of the
prepared bacterial suspension and incubated
under static conditions at 37 o C for 4 d.
8. The extracellular crude enzyme was prepared at
the end of the fermentation period by the addition
of 90 ml of a 0.01 M phosphate buffer pH 7 to the
fermented medium, shaking for 15 min followed by
centrifugation at 8,000 rpm for 20 min. The cell free
supernatant was used as the crude enzyme
preparation
8. Collagenase
Background
• Source: Clostridium histolyticum
• Catalyzes the hydrolysis of collagen
• Used to treat severe burns or skin ulcers
• Mechanism of action: Helps breakdown
collagen in damaged tissues within the skin
and helps the body generate new healthy
tissue.
9. Collagenase
Production
1. A stock culture of the bacterium NW4327 [4] was
maintained on solid Difco Marine Agar 2216 at 0–
4uC and sub-cultured every month. For large scale
enzyme preparation, the isolate was grown in 2 litres
of liquid medium (Difco Marine Broth 2216, pH 7.5
to 8.0) formulated in deionised and purified water
(,18 MV) using a MilliQ system (Millipore, Sydney,
Australia) and incubated in a rotary shaker set at
28uC and 100 rpm for 48 hrs.
10. Collagenase
Production
2. The cell mass was separated by centrifugation
(Beckman Coulter Avanti J26 XPI at 10,000 rpm
for 15 min using a JLA 16.250 rotor) and the
supernatant preserved for further purification by
storing at 0–4uC.
11. Collagenase
Purification
Ultrafiltration.
1. The crude culture filtrate was ultrafiltered
through a 50 kDa ultrafiltration membrane at room
temperature (,22–24uC) (Pall Corporation,
USA) and the retentate used for
chromatography. Ultrafiltration was conducted
in an Amicon Model 52 (Cell capacity 50.0 mL;
membrane diameter 43 mm) stirred
ultrafiltration cell with pressure applied using high
purity nitrogen gas (BOC Australia, Townsville,
Australia).
12. Collagenase
Purification
Semi-preparative molecular size exclusion
chromatography.
2. The ultrafiltration retentate was applied to a 16100
cm column packed with Superdex G200 (GE
Healthcare, Sydney, Australia) which was eluted
with 10 mM Tris HCl and 4 mM CaCl 2 , pH 9.0,
[10] flowing at 0.3 ml min 21 with fractions collected
every 13 min.
13. Streptokinase
Background
• Source(s): Streptococci
• Catalyzes the conversion of plasminogen (inactive) to
plasmin (active)
• Used to treat acute myocardial infarction
• Mechanism of action: creates an active complex
which promotes the cleavage of the Arg/Val bond in
plasminogen to form the proteolytic enzyme plasmin.
Plasmin in turn degrades the fibrin matrix of the
thrombus, thereby exerting its thrombolytic action.
This helps eliminate blood clots or arterial blockages
that cause myocardial infarction.
15. Streptokinase
Production
Extraction of streptokinase from H46A culture
1. Bacteria was cultured in TSA at 37 C
2. The pH of the culture was maintained at 7 during
incubation at 37°C for 8 hours by adding sterile
4% (w/v) glucose and 5.0 N NaOH
3. The culture was centrifuged for 25 minutes at
10,000 g
4. The supernatant was filtered through a 0.45 µm
cellulose acetate filter
16. Streptokinase
Production
5. After standing at 4°C overnight, the precipitate
was harvested by centrifugation at 4°C for 20
minutes at 12,000 g and dissolved in 1 ml of 10
mM Tris buffer, pH=8.0, and dialyzed against
repeated changes of the same buffer.
Preparation of plasminogen affinity column
6. Purified plasminogen was prepared from human
plasma by lysine Sepharose affinity
chromatography (21) . 500 ml of human plasma
was centrifuged at 3000 rpm for 1 hour at 4°C to
remove residual particles present in the plasma.
17. Streptokinase
Production
7. The supernatant then was diluted to one liter
in 0.003 M EDTA and passed through a 50 ml
lysine-Sepharose column at a rate of 70 ml per hour
and then washed with 0.3 M sodium phosphate at
pH=7.4. Upon elution with 0.3 M sodium
phosphate, 0.2 M EACA, pH= 7.4, fractions of 4
ml were collected.
8. The material was dialyzed against 0.1 M
NaHCO3, pH=8.3 and lyophilized. The purity of
plasminogen was confirmed by SDS- PAGE
18. Streptokinase
Production
8. Plasminogen was then added to the resin in 0.1
M NaHCO3 (pH=8.3), and after 2 hours at room
temperature, the resin was washed with 0.2 M
glycine, (pH=8.0), and then alternately for three
times with 0.1 M sodium acetate, 0.5 M NaCL,
(pH=4.0), and 0.1 M NaHCO3, (pH=8.3).
19. Streptokinase
Purification
1. 12 ml bed column of plasminogen coupled to
cyanogen bromide- activated sepharose 4B was
equilibrated with o.ol M Tris- HCl (pH=8.0)
2. The immobilized plasminogen was treated with
6ml of 0.5 mM NPGB in 0.01 M Tris-HCI, (pH=
8.0). The NPGB was initially dissolved in
dimethylformamide at a concentration of 250 mM.
3. The dialyzed extract of streptokinase passed
through column and the column was then
washed with 32 ml of buffer containing 0.01 M
Tris-HCI, I M NaCL, (pH= 8.0).
