Microbial Enzymes and Biotransformations Microbial Enzymes and ...

Microbial Proteases 171
3. Crude enzyme (dialyzed in water for 12 h at 4°C).
4. Trichloroacetic acid (TCA) (10% v/v).
5. 1 M NaOH.
6. Spectrophotometer.
2.5. Gelatin Zymography
1. Minigel apparatus.
2. Power supply (capacity 200 V, 500 mA).
3. Zymogram resolving gel: 7.5% polyacrylamide, 0.1% sodium dodecyl sulfate
(SDS), and 0.15% w/v copolymerized gelatin.
4. Laemmli sample buffer (5X): 60 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS,
14.4 mM 2-mercaptoethanol, and 0.1% bromophenol blue.
5. 2.5% Triton X-100.
6. Zymogram development solution: 0.2 M citrate phosphate buffer, pH 6.5.
7. Staining solution: 45% methanol, 10% acetic acid, and 0.1% Coomassie brilliant
blue R-250.
8. Destaining solution: 40% v/v methanol, 10% v/v acetic acid, and 50% distilled water.
2.6. Immunoblot Analysis
1. Electroblotting apparatus.
2. Power supply.
3. Nitrocellulose membranes (Bio-Rad, Hercules, CA).
4. Whatman 3 MM paper (Whatman, Kent, UK).
5. Transfer buffer: 20 mM Tris, 150 mM glycine, and 20% methanol.
6. Blocking solution: 3% bovine serum albumin in Tris-buffered saline.
7. Tris-buffered saline (TBS): 10 mM Tris-HCl, pH 7.5, and 150 mM NaCl.
8. Dilution buffer: 1% gelatin, 200 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 0.05%
Tween-20.
9. Antiguinea-pig-horseradish-peroxidase complex (Bio-Rad).
10. Color developing solution: Add 10 mL methanol to 1 mL of chloronaphthol solution
(30 mg/mL in methanol), and made up to 50 mL with TBS. Then add 30 µL
30% H 2 O 2 .
3. Methods
3.1. Isolation of Proteolytic Organisms
The organisms inhabiting protein-rich soil tend to utilize more amounts of
proteinaceous material by producing higher amounts of proteolytic enzymes.
To isolate proteolytic microorganisms, the following protocol can be used:
1. Suspend about 1 g of the soil sample in 5 mL sterile distilled water and mix vigorously.
2. Streak aliquots of the clear suspension onto nutrient agar medium containing 1%
casein.
3. Incubate for 48 h at 37°C.