Ureases (EC 3.5.1.5), functionally, belong to the superfamily of amidohydrolases and phosphodiesterase. Nickel containing metalloenzyme. Ureases are found in numerous bacteria, fungi, algae, plants, and some invertebrates, as well as in soils, as a soil enzyme. Not synthesized by animals. James B. Sumner in 1926, Noble Prize in Chemistry in 1946. Urease catalyzes the hydrolysis of urea to from ammonia and Carbon dioxide

1. Isolation of Enzyme and
their assay
Gopal Khodve M.S.Pharmacology & Toxicology
1

2. Urease
• Ureases (EC 3.5.1.5), functionally, belong to
the superfamily of amidohydrolases and
phosphodiesterase.
• Nickel containing metalloenzyme.
• Ureases are found in numerous bacteria, fungi, algae,
plants, and some invertebrates, as well as in soils, as a soil
enzyme.
• Not synthesized by animals.
• James B. Sumner in 1926, Noble Prize in Chemistry in
1946. 2

3. • Molecular Weight:-480 kDa or 445 kDa for jack bean
urease.(hexamer)
• Optimum pH:-7.4
• Optimum Temperature:- 60 °C
• Enzymatic Specificity:- Urea & Hydroxyurea
• Inhibitors:-Heavy Metals(Pb),Fluorine
• Catalysis hydrolysis of urea to CO2 & NH3
Characteristic
3

4. • It is also used in the diagnostic kits for the determination of urea
concentration in the blood, and biosensor of hemodialysis for urea
detection in the blood.
• Urease can be used in dermatological products as well as for
nonsurgical debridements of nails.
• Ureases has very wide application in the beverage industry to reduce
the urea in the alcohol.
• Due to these facts, the urease requirement in the industry is
increasing day by day, that can only be fulfilled by large scale
fermentation.
Applications of Urease
4

5. Isolation
• Isolation of urease from various Source Plants, Bacteria and
intestinal microorganisms.
• Urease can also be produced by bacteria but their production ratio is
lower as compared to fungi. Among the filamentous fungi,
Aspergillus niger is the best strain for the production of the
commercial urease enzyme.
5

6. 6

7. Materials and method
7
Inoculum preparation

8. continue
8
Production and harvesting of urease

9. Assay of urease
The reaction mixture was kept in a shaking
bath at
37 °C for 30 min. After the addition of 500 μL of
phenol-sodium nitroprusside solution
02
03
01
Na2HPO4 To 100 μL of the sample, 500
μL of urea and potassium phosphate
buffer (pH 8) were added
to 50 μL of the reaction mixture, it was again
kept
for 30 min at 37 °C. The optical density of the
color complex was measured by a
spectrophotometer at 630 nm
For the enzyme activity assay, Weatherburn (1967) method was
adopted with slight modification, i.e. Na2HPO4 was used in place
of
NaOH and the time for color development increased from 20 to
30 min.

10. • Alkaline phosphatase (EC 3.1.3.1), or basic phosphatase, is
a homodimeric protein enzyme of 86 kilodaltons.
• ALP has the physiological role of dephosphorylating compounds. The enzyme is
found across a multitude of organisms, prokaryotes and eukaryotes alike, with the
same general function.
• In humans, it is found in many forms depending on its origin within the body – it
plays an integral role in metabolism within the liver and development within the
skeleton.
• Due to its widespread prevalence in these areas, its concentration in the
bloodstream is used by diagnosticians as a biomarker in helping determine
diagnoses such as hepatitis or osteomalacia.
10
Alkaline phosphatase

11. • Each monomer contains five cysteine residues, two zinc atoms and one
magnesium atom crucial to its catalytic function, and it is optimally active
at alkaline pH environments.
• The enzyme catalyze the hydrolysis of monoesters in phosphoric acid which
can additionally catalyze a transphosphorylation reaction with large
concentrations of phosphate acceptors.
• Typical uses in the lab for alkaline phosphatases include removing phosphate
monoesters to prevent self-ligation, which is undesirable during plasmid DNA
cloning
11
Characteristics

12. • The enzyme alkaline phosphatase is an important serum analyte and its
elevation in serum is correlated with the pressure of bone, liver, and other
diseases.
• The analysis of the isoenzymes of alkaline phosphatase is an aid in diagnosing
liver and/or bone disease, especially the high molecular weight isoenzymes that
appear in cholestatic liver disease.
• In humans, alkaline phosphatase is present in all tissues throughout the body,
but is particularly concentrated in the liver, bile duct, kidney, bone, intestinal
mucosa and placenta.
• In the serum, two types of alkaline phosphatase isozymes predominate: skeletal
and liver.
12
Applications

13. • Alkaline phosphatase (EC 3.1.3.1, ALP) is hydrolase that has been
implemented to remove phosphate groups from many types of
molecules, such as nucleotides, proteins, and alkaloids.The process of
removing the phosphate group is dephosphorylation.
• Many bacteria produce ALP, such as Escherichia coli, Bacillus species,
Mycobacterium smegmatis, Thermotogamaritime, Haloarcula
marismortui, especially Gram-negative bacteria starved of inorganic
phosphate. LAB is permitted for processing in food raw materials
13
Isolation

14. 14
Step 1
Step 2
Step 3
Step 4
The
lyophilized
Lactobacillus
casei
Materials
Preparation of
crude enzyme
.
Ammonium
sulfate
precipitation
Alkaline
phosphate
purification
Materials & Process

15. The medium was inoculated with Lactobacillus casei. 355 at 37 C for 24 h in
Erlenmeyer flasks, and harvested by centrifugation at 10 000 g for 10 min at 4 C.
The cell pellet was washed three times with carbonate–bicarbonate buffer (10 mmol
L1, pH 10.5), resuspended in carbonate–bicarbonate buffer and agitated for 1 min.
The mixture was sonicated 10 times (10 s at 15 s interval), as described in a previous
study, and centrifuged at 10 000 g and 4 C for 15 min.20 The supernatant was
collected for subsequent enzyme purification.
15
Materials & Purification

16. Ammonium sulfate precipitation
The crude enzyme solution was added ammonium sulfate to the degree of 80%
saturation, and was kept at 4 C overnight.
Subsequently, the precipitate was then collected by centrifugation at 12 000 g for
10 min at 4 C. The protein was dissolved in minimal amount of Tris–HCl buffer (50
mmol L1, pH 7.4), and dialyzed against the same buffer for 24 h at 4 C.
16

17. ALP Purification
The concentrated crude enzyme was loaded onto DEAE Sepharose column
(16 200 mm) pre-equilibrated with 50 mmol L1 Tris–HCl buffer (pH 7.4).
The column was washed with buffer until no protein was detected in the
elutriate.Then it was eluted using a gradient of 0–1 mol L1 NaCl in the same
buffer at Flow rate as 2 mL min1
Fractions of 5 mL were collected and analyzed for protein content and
enzyme activity.
Fractions containing ALP were pooled, dialyzed overnight, and lyophilized.
17

18. The lyophilized enzyme was dissolved in Tris–HCl buffer (50 mmol L1, pH 7.4).
The enzyme solution from the previous step was processed on a column with
Superdex 75 (10 300mm) equilibrated previously with Tris–HCl buffer.
Elution was performed with the same buffer at a flow rate 0.8 mL min1.
Fractions containing ALP were pooled, dialyzed against Tris– HCl buffer and
lyophilized.
Purified enzyme Isolated
18
Conti.

19. The assay method was first described by King and Armstrong, modified by Ohmori, Bessey, Lowry, and Brock,
and later improved by Hausamen et al. In 1983.
The International Federation of Clinical Chemistry (IFCC) recommended a standardized method for the
determination of alkaline phosphatase using an optimized substrate concentration and 2-amino-2-methyl-1-
propanol as buffer, plus the cations magnesium and zinc.
Colorimetric assay in accordance with a standardized method.In the presence of magnesium and zinc ions, p
nitrophenyl phosphate is cleaved by phosphatases into phosphate and p-nitrophenol.
The p-nitrophenol released is directly proportional to the catalytic ALP activity.
It is determined by measuring the increase in absorbance at 409 nm.
19
Assay of ALP

20. 20
References
1)Purification and characterization of alkaline phosphatase from lactic acid bacteria
Yu-Hao Chu,ab Xin-Xin Yu,ab Xing Jin,ab Yu-Tang Wang,ab Duo-Jia Zhao,ab
Po Zhang,ab Guang-Mei Sunab and Ying-Hua Zhang *ab
2)G. Gotthard, J. Hiblot, D. Gonzalez, M. Elias and E. Chabriere, PLoS One, 2013, 8,
e7799511.
3) R. Iyer, B. Iken and A. Damania, Environ. Microbiol. Rep., 2013, 5, 787–798.
4)J. P. Verma, D. K. Jaiswal and R. Sagar, Rev. Environ. Sci. Bio/ Technol., 2014, 13,
429–466.
5) K. Kawahara, A. Tanaka, J. Yoon and A. Yokota, J. Gen. Appl. Microbiol., 2010,
56(3), 249–255.
6) R. R. M. Thengodkar and S. Sivakami, Biodegradation, 2010, 21(4), 637–644