Urease: Difference between revisions

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[[Category: NOWA]]Urease is an enzyme that converts [[urea]] into ammonia.
Urease is an enzyme that converts [[urea]] into ammonia.
==Uses==
==Uses==
Urease autocatalyzes the conversion of [[urea]] to [[ammonia]]
Urease autocatalyzes the conversion of [[urea]] to [[ammonia]]
Line 8: Line 8:
* Bacteria and fungi produce urease
* Bacteria and fungi produce urease
* Urease is present in several types of plant seeds (watermelon, pea, and bean)
* Urease is present in several types of plant seeds (watermelon, pea, and bean)
==Hazards==
==Character==
* For  jack bean urease<ref>{{cite web|url=http://www.toyobousa.com/enzyme-URH-201.html|title=UREASE from Jack bean}}</ref>
** Optimal temperature: 60 &deg;C
** Optimal pH 6.0
==Production==
==Production==
===Extraction===
===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&deg;C
The source is typically freshly germinated seeds: pea seeds, dehusked watermelon seeds, or jack bean meal.
The source is typically freshly germinated seeds: pea seeds, dehusked watermelon seeds, or jack bean meal.
Ten grams of germinated seeds of pisum sativum were pasted in a mortar and pestle and then suspended in 40 mL of 20% chilled acetone (−20°C). Occasional stirring for 3 h was required. Double layer cheese cloth was used for filtrating of the suspension. After 15 minutes of centrifuging of the filtrate, the supernatant was isolated and used as “crude extract”.
The “crude extract” was adjusted to 50% saturation by addition of acetone (chilled to −20°C) under constant and gentle stirring. The resulting precipitate was centrifuged, collected, dissolved in minimum volume of pre-cold 50 mM phosphate buffer (pH = 7.4), and finally dialyzed against the same buffer for 24 h. The resulting solution was then centrifuged for 10 min and the clear supernatant was designated as “crude enzyme solution” <ref>{{cite pub
|last1=El-Hefnawy
|first1=Mohamed E
|last2=et al
|title=Extraction, purification, kinetic and thermodynamic properties of urease from germinating Pisum Sativum L. seeds.
|publication=BMC biochemistry
|volume=15
|year=2014
|doi=10.1186/1471-2091-15-15
|url=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4121304/
|courtesy=NIH}}</ref>
====Catalytic solution====
====Catalytic solution====
# Combine 160cc of dry [[propanone]] with 340cc of distilled [[water]]
# Combine 160cc of dry [[propanone]] with 340cc of distilled [[water]]
Line 34: Line 28:
==Purification==
==Purification==
===Materials===
===Materials===
: '''Citric acid buffer''': Solution of trisodium citrate ({{#Chem:Na3C6H5O7}}) with 4% w/w [[citric acid]]
: '''Citric acid buffer''': Solution of '''sodium citrate''' with 4% w/w [[citric acid]]
: '''Phosphate neutral buffer''': Solution of {{#Chem:KH2PO4}} with 150% w/w {{#Chem:Na2HPO4}}
: '''Phosphate neutral buffer''': Solution of {{#Chem:KH2PO4}} with 150% w/w {{#Chem:Na2HPO4}}


===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&deg; for 30 min
# Centrifuge at 0&deg; 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)
# IF (the crystals are sufficiently homogeneous) THEN exit loop
# Dissolve residue in '''neutral buffer''' solution
# Dissolve residue in '''neutral buffer''' solution
# Centrifuge
# Centrifuge
# Retain clear fluid
# Retain clear fluid


==Testing==
==Storage==
==Disposal==
==See Also==
==See Also==
* [[urea]]
* [[urea]]
* [[urine]]
* [[urine]]
* [[ammonia]]
* [[ammonia]]
* {{cite pub
|first1=G.|last1=Gorin
|first2=C.C.last2=Chin
|publication=Biochim.Biophys.Acta.
|title=Urease. V. Some observations on the procedure for its isolation.
|volume=99
|pages=418
|year=1965}}
* {{cite pub
|first1=H.G.|last1=Schlegel
|first2=H.|last2=Kaltwasser
|publlication=Methods of Enzymatic Analysis
|title=Urease
|volume=2
|pages=1081
|year=1962}}
*{{cite pub
|first1=J.B.|last1=Sumner
|first2=D.B.|last2=Hand
|publication=J.Am.Chem.Soc.
|title=THE ISOELECTRIC POINT OF CRYSTALLINE UREASE
|volume=31
|pages-=1255
|year=1929}}
==References==
==References==
<references/>
<references/>
 
[[Category:Complex Organics]][[Category:NOWA]]
[[Category:Complex Organics]]

Latest revision as of 15:34, 11 May 2020

Urease is an enzyme that converts urea into ammonia.

Uses

Urease autocatalyzes the conversion of urea to ammonia

CO(NH2)2 + H2O
{urease
→
}
2 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)

Hazards

Character

  • For jack bean urease[1]
    • Optimal temperature: 60 °C
    • Optimal pH 6.0

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

  1. Combine 160cc of dry propanone with 340cc of distilled water
  2. Mix in 100g of seed pulp
  3. Filter, discard residue
  4. Dry until all scent of propanone is gone
  5. (If necessary) add water to dissolve any precipitate
  6. The solution may be added to sources of urea to convert them to ammonia

Purification

Materials

Citric acid buffer: Solution of sodium citrate with 4% w/w citric acid
Phosphate neutral buffer: Solution of KH2PO4 with 150% w/w Na2HPO4

Process

Repeat:

  1. Combine 5% v/v acid buffer with solution
  2. Add propanone dropwise until it becomes slightly cloudy
  3. Stand overnight
  4. Centrifuge at 0° for 30 min
  5. Filter, discard filtrate
    The residue is crystals of urease.
  6. IF (the crystals are sufficiently homogeneous) THEN exit loop
  7. Dissolve residue in neutral buffer solution
  8. Centrifuge
  9. Retain clear fluid

See Also

  • urea
  • urine
  • ammonia
  • Gorin, G. (1965) "Urease. V. Some observations on the procedure for its isolation.".
    Biochim.Biophys.Acta. 99; pp418. 
  • Schlegel, H.G.; Kaltwasser, H. (1962) "Urease".; pp1081.
  • Sumner, J.B.; Hand, D.B. (1929) "THE ISOELECTRIC POINT OF CRYSTALLINE UREASE".
    J.Am.Chem.Soc. 31 

References

  1. ↑ UREASE from Jack bean
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