Urease: Difference between revisions
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===Process=== | ===Process=== | ||
Repeat: | #Repeat: | ||
# Combine 5% v/v '''acid buffer''' with solution | ## Combine 5% v/v '''acid buffer''' with solution | ||
# Add propanone dropwise until it becomes ''slightly'' cloudy | ## Add propanone dropwise until it becomes ''slightly'' cloudy | ||
# Stand overnight | ## Stand overnight | ||
# Centrifuge at 0° for 30 min | ## Centrifuge at 0° for 30 min | ||
# Filter, discard filtrate | ## Filter, discard filtrate | ||
#: The residue is crystals of urease. | ##: The residue is crystals of urease. | ||
# | # Until (the crystals are sufficiently homogeneous) | ||
# Dissolve residue in '''neutral buffer''' solution | # Dissolve residue in '''neutral buffer''' solution | ||
# Centrifuge | # Centrifuge | ||
# Retain clear fluid | # Retain clear fluid | ||
==Testing== | ==Testing== | ||
==Storage== | ==Storage== |
Revision as of 05:44, 2 August 2019
Urease is an enzyme that converts urea into ammonia.
Uses
Urease autocatalyzes the conversion of urea to ammonia
- CO(NH2)2 + H2O{urease2 NH3 + CO2}→
- Optimal catalytic activity occurs at approximately 30°C and pH of 7.4-8.0.
- In a perfectly pure sample, 1g of urease will produce 100g of ammonia nitrogen from urea in 5 minutes at 20°C.
Natural occurrence
- Bacteria and fungi produce urease
- Urease is present in several types of plant seeds (watermelon, pea, and bean)
Production
Extraction
Since urease is soluble in propanone, a solvent with a very high vapor pressure, most extractions salve biomass containing urease in propanone, filter and evaporate. The entire process is carried out at low temperatures, often 2-4°C
The source is typically freshly germinated seeds: pea seeds, dehusked watermelon seeds, or jack bean meal.
Catalytic solution
- Combine 160cc of dry propanone with 340cc of distilled water
- Mix in 100g of seed pulp
- Filter, discard residue
- Dry until all scent of propanone is gone
- (If necessary) add water to dissolve any precipitate
- The solution may be added to sources of urea to convert them to ammonia
Purification
Materials
- Citric acid buffer: Solution of trisodium citrate (Na3C6H5O7) with 4% w/w citric acid
- Phosphate neutral buffer: Solution of KH2PO4 with 150% w/w Na2HPO4
Process
- Repeat:
- Combine 5% v/v acid buffer with solution
- Add propanone dropwise until it becomes slightly cloudy
- Stand overnight
- Centrifuge at 0° for 30 min
- Filter, discard filtrate
- The residue is crystals of urease.
- Until (the crystals are sufficiently homogeneous)
- Dissolve residue in neutral buffer solution
- Centrifuge
- Retain clear fluid