Urease

ABSTRACT


Urease is well studied enzyme and its importance is also well known .In this studies novel source of the enzyme is reported i.e. Lagenaria siceraria. Enzyme was studied from the dried seeds, and tried to purify it .The different parameters of the partially purified enzyme was studied like effect of enzyme concentration, substrate concentration ,effect of pH , effect of temperature , effect of metal ion . Urease was purified in different steps like acetone precipitation, ammonium sulphate fractionation, dialysis, Bio-gel p-100 chromatography, PAGE etc. The study also included the comparism of enzyme activity of this source with previous well known source Jack bean in crude and in acetone precipitated fraction .and it shown the urease from Lagenaria siceraria has highest activity Inhibition of the enzyme haring the clinical significance.
Therefore in this study plant Tinospora cordifolia was studied for the inhibitory property the different solvent extract of tinospora cordifolia where tried to test inhibitory property. The (diethyl ether) extract shown the higher inhibitory action. The different concentration of dichloromethane extract of Tinospora cordifolia at different substrate concentration was study to check the type of inhibition.









INTRODUCTION

The enzymes are the reaction catalyst of biological systems .They are protein nature. The existence of enzyme has been known for well over a century the first enzyme obtained in pure form by James b. Sumner of Cornell university . Sumner was able to isolate and crystallized the enzyme urease from jack bean. His work was earning him noble prize 1947.
About so year later it was shown to be the first nickel containing Enzyme (Dixon 1975).
Urease is protein synthesized by bacteria ,fungi and plants Urease (urea aminohydrolase) is found in large amount in jack bean (Canavalia ensiformis), soybean (Glycine max) and other members of leguminasae ,as well as in wide variety of human tissues(the gastric mucosa ,liver, kidney and erythrocytes).There are many microbial sources of these enzyme including bacteria such as Lactobacillus ruminis, Lactobacillus fermentum ,Lactobacillus ruteri ,helicobacter pylori ,Campylobacter pylori and fungi such as Rhizopus oryzae.
The properties and activities of urease (Urea aminohydrolase) was found to be different for various sources .Urease (Urea aminohydrolase), EC (3.5:1.5) is nickel containing enzyme ,that catalyses the hydrolysis of urea in to ammonia and carbon dioxide (Andrews et al,1986; Hausinger 1993 ; Park and Hausinger ; Pearson et al ,1997).

H2N--C--NH2+H2O  NH3+H2N—C--OH

H2N—C—OH+H2O  NH3+H2CO3
Urease has much industrial application in diagnostic kits for measuring urea, in alcoholic beverages as a urea reducing agent (Fujiyama and Della, 1990; Fujiyama and ouch, 1991)
ROLE OF UREASE IN PLANT
There are three enzymes involved in the urea metabolism in plants: Arginase, Urease and Glutamine synthetase .The primary role of urease is to allow the organisms to use external or internally generated urea as a nitrogen source (Mobley and Hausinger, 1989, Mobley et al , 1995 ).Significant amounts of plant nitrogen flow through urea . Nitrogen present in urea is unavailable to the plant unless hydrolyzed by urease. Urease play important role in germination and seedlings nitrogen metabolism. (Zonia et al. in 1955)

UREASE IN PARENCHYMA
There is rapid synthesis of urease in the activity dividing meristematic cells. This synthesis continues during the period of cell elongation although at decreasing rate, the amount of urease of cell reaching at a maximum at the end of the elongation period .After this period, there is a decomposition of urease, which continues down to a certain level.


UREASE: DEFENSIVE ACTION:
The defensive role of urease that is involvement in protection against plant pathogens (due to production of toxic ammonia) has been studied by Polacco and Holland in 1944.
Jack bean urease and soybean urease are used as a insecticides Jack bean urease and soybean urease are highly toxic to cotton strainer bug oysdercus Peruvians in feeding trails.

UREASE: IN UREA METABOLISM

The primary environment role of urease is to allow organism to use externally and internally generated urea .The nitrogen present in urea is unavailable to plant unless hydrolyzed by urease. The ubiquitous urease found in all plant tissue is responsible for recycling the metabolically derived urea. Now a days urease is used along with the nitrogen fertilizers e.g. urea, because urease is only enzyme that able to recapture the nitrogen from urea in plant.

UREASE INHIBITOR-PREVENT STONE FORMATION IN HUMAN URINE
Inhibition of the enzyme having the clinical significance.Urease hydrolyzes urea, leading to hyperammonuria and the alkalinization of urine, which causes hypersaturation with respect to struvite and calcium phosphate , with consequent crystallization of sturuite and Aptite. Urinary stone formed with infection by Proteus mirabilis in vitro (Griffith, D. P, D. M musher and J .W.Campbell .(1973) in human urine The high urease activities were detected in urine of the patients with urinary stones caused by infection the formation of such urinary stones was prevented by treatment with urease inhibitor .

PROPERTIES OF UREASE –

• Systematic name – Urea aminohydrolase
• Molecular weight – 480 KDa or 545 KDa for jack bean uerease ( calculated mass from the amino acid sequence )
• Composition – Monomeric (a) urease can polymerize to form six unit polymers of about three million Daltons
• Optimum pH – 6.0 – 10.
• Stability – dry at 4`c 12 months
• Thermal stability – below 70`c
• Enzymatic specificity – urea and hydroxyl urea
• Inhibitors – heavy metals, suramine, thiocynate.
• Urease unit – one urease unit is defined as the amount of enzyme, which hydrolyze one micromole of urea per minute at room temperature.
PROTEIN STRUCTURE OF UREASES-
The plant and fungal urease are homo-algometric proteins (consist of identical subunits), while the bacterial urease are multimers formed from a complex of two or three subunit (Mobley et al 1995, Tonge and Niwa1997) . Significant amino – acid similarities were observed between all known urease. Jack bean urease exists as a homotrimer able to aggregate to a homohexamer (Hirai et al.1993).Bacterial ureases posses structures similar to the jack bean urease.

MODE OF ACTION OF UREASE
Urea is absorbed from solution by urease and then dissociated in to ammonia and cyanic acid in it’s unstable form. The ammonia is absorbed by the enzymes, the cyanic acid remains in the solution. In aqueous solution the cyanic acid is rapidly hydrolyzed in to ammonia and carbonic acid.
The purpose of present study was to study urease from seeds of Lagenaria Siceraria, isolation, purification and characterization of urease and some physicochemical parameters were tested and inhibition of urease from novel plant inhibitor was studied.
MATERIALS AND METHOD-

Different sources used-
To search the urease activity, different plant sources have been used are –
* Tur – cajanus Cajan
* Harbhara – cicer orietinum
* Gulmoher – Delonix regia
* Babul – Accacia nikotica
* Tarvad – Cassia auriculata
* Bottle ground – Lagenaria siceraria
* Jack bean – Canavalia ensiformis
Seeds from aboves plant collected, dried and powdered and used for the extraction of urease
Extraction –

1 gm of sample powder dissolved in 10 ml 1 Milimolar EDTA – Mercaptanol containing distilled water and allowed it for half hour. Aboves mixture was centrifuged at 10000 rpm for 10 min. at 4`c . The supernatants were used as crude enzyme.

CHEMICALS

1. Urea 50 Mm
2. Potassium Phosphate buffer 100 Mm ( PH – 8.0 )
3. Alkaline hypochlorite
(3.56 gm of disodium hydrogen phosphate + 1.00 ml
Of sodium hypochlorite, make it up to 100 ml Distilled water)
4. Phenol – sodium – nitro prosside.
(0.05 gm sodium nitroprosside + 1.00 gm phenol make it up to 100 ml with distilled water)
5. Bio- gel P100
6. Polyacrilamide
7. TEMED
8. Tris- Hcl buffer9
9. Dialysis bag
10. Ammonium sulphate
11. 1 Mm EDTA and 1 Mm Mercaptanol -->200 ml
12. Acetate buffer 0.1 M PH 4 -5
13. Carbonate
14. Chilled Acetone
15. Biuret reagent
16. BSA
17. Diethyl ether, chloroform petroleum ether, cyclohexane

Instrument used

1. Cold centrifuge
2. Colorimeter
3. Spectrophotometer


Enzyme Assay


The activity of urease from different screened seed sample has been measured by the release of ammonia and carbondixoide from urea over fixed interval of time. In semiqantitative urease assay, the enzyme activity is observed in supernatant after centrifugation, activity was measured by Weather burn method (1967). The reaction were carried out in eppendropes tubes containing 100 micro liter of enzyme, 500 micro liter of substrate(urea),500 micro liter of 100 Mm potassium phosphate buffer ( pH 8 ) in total volume of 1.1ml .The reaction mixture was incubated at 37 for 30 min . The reaction was stopped by transferring 0.5ml of reaction mixture in to separate tubes containing 1.5ml of phenol-sodium nitro prosside solution , in that mixture 1.5 ml of alkaline hypochlorite solution was added and incubated at room temperature for 30 min . Finally O.D of blue color complex was taken at 600 nm on colorimeter and compared to standard curve prepared with ammonium sulphate standard graph. One unit of Urease defined as amount of enzyme librating one micromole of ammonia from urea per minute under standard condition .
The maximum activity of urease obtained from the seeds of Lagenaria siceraria (Bottle guard) all over screened sample including well known source Jack bean.


Lagenaria siceraria

Common name – Bottle guard, Bhopala

Family - Cucurbitacae

Photographs of dried seeds of bhopala —

Protein Determination

Protein concentration present in the crude urease of L. siceraria (Bhopala ) was determined by Biuret method at 540nm .

The principle is based on that ,-CO-NH- group of protein react with copper ions of biuret reagent , form blue color complex in alkaline medium .

Protein content of partially purified urease by gel chromatography were estimated by specrophotometrically at 280nm .

Purification of urease

Purification of crude urease was carried out by several methods -

Acetone precipitation
Ammonium sulphate fractionation
Dialysis
Gel filtration –Bio gel P100


Acetone precipitation

PRINCIPLE-

“ Solubility of protein depends on, among other things ,the dielelectric constant of the solution. Precipitation of protein can be induced by lowering the effective dielectric constant of the medium this can be induced by adding water soluble solvent such as acetone to an aqueous protein solution”.

Acetone precipitation of L.siceraria crude Urease solution was done by drop wise addition of chilled acetone at 0 c with continuous stirring .Acetone addition was continued till complete precipitation of protein, then it was kept in freeze for 2 hours ,supernant was decanded and residue was kept in freeze to evaporate acetone completely .
This residue was dissolve in phosphate buffer of PH 8 and used as partial purified enzyme for further analysis .

Ammonium sulphate fractionation –

PRINCIPLE –

“ The solubility of protein depends on among the other things , the salt concentration in the solution. .At low concentration in the solution. At low concentration, the present of Salt enhancing the solubility of protein commonly known as salting in. Further increase in salt concentration, protein start to precipitate because of less
Water is not sufficient to solubalized it .This phenomenon of protein precipitation”.
.Ammonium sulphate has been most widely used because it have high solubility and relatively less inexpensive.
Ammonium precipitation was carried out 0c ice bath. 30% saturation was by adding 3.52 gm of ammonium sulphate powder in 20 ml of enzyme supernatant after centrifugation at 10000 rpm ,4c for 10 minute, supernatant and residue both was tested for the activity of enzyme
, activity of Urease observed in supernatant. Then 60% saturation was made by adding 3.46 gm of ammonium sulphate salt in to previous supernatant of 30% saturation . It was kept overnight and centrifuged next day at 10000rpm, 4c for 10 min. Urease activity was observed in supernatant, then for 90%saturation , 3.8 gm of ammonium sulphate was added and kept it overnight for extraction at +4c . Next day after centrifugation , urease activity was observed in residue .Residue was dissolved in buffer solution for its activity and followed it for further purification.

DIALYSIS -

Dialysis is commonly used for removing salts from the proteins to avoid this interference . Protein solution desalted by using dialysis bag have property to allow to the compounds with small molecular weight like salt not large compounds like proteins from dialysis bag.
The 60% and 90% fractions from ammonium sulphate precipitation was dialysis in 0.1M phosphate buffer
PH 8 .
Repeated change of dialysis fluid was helpful to reducing the salt concentration inside the bag .

GEL CHROMATOGRAPHY

Purification of urease were carried out chromatographically by using Bio -Gel P100 column with 0.25M phosphate buffer .
1 ml crude urease sample was loaded on well
packed column of Bio gel p100 in 0.25M potassium phosphate buffer .Depending on the nature of the molecular weight of loaded compound ,it excluded from gel. Higher molecular weight comes first through the column. Fraction was collected after eluting 15ml bed volume. Depending on the molecular weight of Bottle guard urease , it come in to fractions , each fraction was assayed for urease activity.

PAGE - Poly Acryl amide Gel Electrophoresis

PRINCIPLE -

“PAGE is an important technique for the separation of proteins, based on the migration of charged proteins in an electric field .The poly acryl amide gel act as molecular sieve, slowing the migration of proteins approximately in the proportion to their charge to mass ratio”.

Different urease sample obtained from novel plant source i.e. from Bottle guard crude sample acetone precipitated, 60% ammonium sulphate fraction, was loaded in different well on the top of the gel .The protein (enzymes) move in to gel according to their molecular weight, when an electric field was applied. The gel minimizes convention currents caused by small temperature gradients as well as the protein movement other than those induced by the electric field. When the band of enzyme was reached at the bottom
of gel ,the gel was removed from the electrophoresis apparatus and visualized by treating it with Comassive Brilliant blue, which was bind to protein but not gel.
Each band shows presence of protein or protein subunit. Enzyme having smaller molecular weight was moved faster than enzyme having large molecular weight.



FACTORS AFFECTING ON THE ACTIVITY OF PARTIALLY PURIFIDE ENZYME ----
The following factor were investigated for their effect on the activity of partially purified Bottle guard urease –

Different temperature according to Bongaerts et al (1978), different volumes of partially purified enzyme on the activity,

Effect of different concentrations of substrate (urea) on the activity of enzyme.

Effect of different time intervals during incubation and effect of different metal salts according to Kimikasu et al (1967).

Rates of hydrolysis of urea were measured for urease preparation in the presence of urease inhibitor.

UREASE INHIBITOR ---
Rates of urea hydrolysis were measured colorimetric ally for urease preparation of Lagenaria siceraria isolate in the presence of urease inhibitor from novel plant source “Tinospora cardifolia”. The various concentration of inhibitors present in reaction mixture was varies from different solvent extract of Tinospora cardifolia were shows the inhibitory property , the dichloromethane extract shows the higher inhibitory action . Dichloromethane extract of T. cordifolia at different substrate concentration was studied to check the type of inhibition .





RESULT AND DISSCUION

Urease activity screened from different sources including well known source jack bean .The maximum urease activity observed in Langenaria siceraria (bhopala) which is unknown and have higher activity than well known source of urease i.e. jack bean.
The activity of different screened sources showed in Table No 1.–
As Bhopala urease showed maximum activity, it was used for further purification and characterization.


Table no -1 Screening of urease activity from different plant sources.


Sr.No.
Plant source
Enzyme activity micromole of NH4 released)
1
Canavalia enciformis –jack bean 75
2 Cajanus cajan –Tur 150
3
Acacia Arabica –Babul

09
4 Delonix regia –Gulmohar
24
5
Cicer orietinum- Harbhara
04
6 Lagenaria siceraria- bottle guard
174

7

Cassia auriculata- tarvad

09


Purification of urease.

The supernatant of crude urease enzyme obtained after the centrifugation was subjected to purification procedure consisting of different steps i.e. acetone precipitation, ammonium fractionation dialysis gel chromatography etc. The specific activity of crude bhopala extract was 2666.0 U per mg per ml. By using acetone purification fold increase in the specific activity of urease was manifested while recovered activity was ----%.From the elution profile of gel chromatography as shown in Fig 1, it can be seen that L.siceraria urease was eluted from the column in to two peaks. The first peak (A) was observed in the fraction (29-37) while the second peak (B) was observed in the fraction (38-45).

Protein content of partially purified enzyme—
Protein content of urease from jack bean &Bhopala by acetone precipitation enzyme and crude extract was determined by Biuret method .From table no 2 , the specific activity of urease from jack bean and Bhopala was792U & 2666.0 U per mg respectively.

Characterization of partially purified L. siceraria urease ------
Effect of enzyme concentration –
There was continuous increase in the activity of urease by increases its volume in the reaction mixture. This was true up to 250 micro liter of enzyme volume shown in Fig 2. Further increase in the enzyme volume, activity remains constant.
Effect of substrate concentration ---

The optimum urea concentration (acting substrate) in the reaction mixture was 800 Mm. Beyond rapidly decrease in the activity of enzyme as shown in Fig 3. A lower urease activity was observed in ammonium –grown cells than in the presence of urea of nitrate (Collier et al, 1999).





Effect of temperature ----

The temperature stability was determined after incubation of enzyme at various temperatures ranging from 10, 20----70 .The optimum temperatures was obtained at 30 Fig 4.


Effect of PH –

The partially pure enzyme was stable in the rages between PH 6 to 12 in two different buffer systems (0.1M phosphate buffer& .0.1M carbonate bicarbonate buffer). The optimum PH was obtained at PH 10. Below or above this PH value an irreversible inactivation was observed Fig 5


Effect of metal salt on enzyme activity-

The table 3 indicate the inhibition of tested Urease by different metal salts – Sodium ,potassium , magnesium, cobalt, calcium , zinc, ferrous salts shows the inhibition of urase while the salt of mercury shows complete inhibition of urease as shown Fig 6,in agreement with that obtained by Rai and Singh (1987)and Mobley et al ,(1995).

Effect of incubation time on enzyme activity –

Urease in reaction mixture was incubated at different time intervals i.e. 00 , 10, 20--------70 min.Optium activity was observed at 30 min, above and below this value enzyme showed decreased activity ; while urease completely inaction at 70 mjn time interval as shown in Fig 7.

Table 3 Effect of metal salt on enzyme activity Conc-10-3M


Metal
Activity of urease (U per ml)
Nacl 11.39
KCL 7.8
Mgso4 5.8
Znso4 1.4
Cocl2 1.4
Cacl2 1.0
Fecl2 1.4
Hgcl2
00
















Urease inhibition

To determine the usefulness of specific inhibitor in reducing or eliminating enzyme activity, we determined rates of urea hydrolysis in the presence of various concentrations of compounds listed in Table No 3 and 4.The inhibition of urease activity by competatitive and noncompetitive inhibitors and divalent cation chelation relevated that mercury chloride salts and Tinospora cordifolia extract in different solvent systems is strong inhibitor of urease.
Urease inhibition was studied by plant Tinospora cordifolia. Different solvent systems were used for T. cardifolia, extraction including Chloroform, petroleum ether cyclohexane and diethyl ether etc. Among which the maximum inhibition of urease is obtained by diethyl ether solvent extraction systems.
Percent inhibition of urease by different inhibitor concentration is shown in Table No 5



References
1 Andrews, R.K., A. Dexter R.L.Blakely and B Zener, 1986.Jack bean urease (EC 3.5.1.5.).On inhibition of urease by amides and esters of phosphoric acid. J Am. Chem.soc, 108:7124-7125.

2 Archer, D.B. and J.F. peberdy, 1997. The molecular biology of secreted enzyme production by fungi.Crit.Rev.Biotechnol, 17:273-306.

3 Weatherburn, M.W, 1967. Phenol hypochlorite reaction for determination of ammonia. Anal.Chem,39:971-203

4 Fujinawa, S.B.G. and T.P. Dela, 1990.Application of acid urease to reduction of urea in commercial wines . Am. J.Enol. Viticul. 91: 350-354.

5 Fumuyiwa, OO and C.S Ouch, 1991.Modification acid urease activity by fluoride ions and mallic acid in wines .Am J.Enol .Victual, 91:350-359.

6 Hausinger, RP, 1993 .Biochemistry of nickel. Plenium publishing Crop. New York, USA.

7 Klimikasu, L, Y.Takehiko and F.Jukhiro, 1967.Studies on yeast uricase purification and some enzymatic properties of yeast uricase .Agric Biol.Chem, 31:1256 – 1264.

8 Farley, P.C. and S. Santosa, 2002.Regulation of expression of Rhizopus Oryzae uricase and uriase enzymes. Can J. Microbial. 48 :1104 – 1108.

9 Mobley, H.L.T. and R.P. Hausinger, 1989. Microbial ureases: significance, regulation and molecular characterization .Microbial .rev, 53:85 –108.

10 Pearson, M.L. Michel, R Hausinger, PKarplus, 1997.structures of Cys 319 variants and Acehydroxamate inhibited Klebsiella aerogenes urease.Biochemistry, 36:113 -117.

11 Rai, a. k., 1989. Purification and properties of urease from a
cyanobacterium anabena doliolum.
FEMS.Microbial.Lett, 61:319 -322.

12. Smith, P.T, A.K.J.Douglas and N. Goodman, 1993.
Isolation of urease from Aspergellus niger.
J. Gen. Microbiol.139:957 -962.

13. Sachiko Takeb et al.1984. Stone formation by Ureaplasma
urealyticum in human urine and its prevention by Urease
Inhibitor.Microbilogy J. 20:869 – 873.

14. .Harry L.T. Mobely, Manuel J. Cortesia ,1988. Characterization of urease from Campylobacter pylori
J.Clinical Microbiology. 16:831 – 836.

15. J. Jayraman.Laboratory Manual in Biochemistry.









INDEX

1. Abstract
2. Introduction
3. Materials and methods

*Materials – Chemicals and instruments used
* Methods —

Enzyme extraction

Enzyme purification by –

Acetone precipitation
Ammonium sulphate fractionation
Dialysis
Gel chromatography – Bio gel p100
PAGE

Characterization-

Effect of enzyme conc
Effect of substrate conc
Effect of temp
Effect of PH
Effect of time
Effect of metal salts

Study of enzyme inhibitor

4. Result and Discussion
5. References.









Fig 1 Effect of Substrate concentration on enzyme activity




Fig 2 Effect of pH on enzyme activity



Fig 3 Effect of temperature on enzyme activity




Fig 4 Effect of incubation period on enzyme activity





Fig 5 Effect of various salts (10-3 mM) on enzyme activity




Fig 6 Inhibition of urease at various concentration of Tinospora cordifolia




Fig 7 Effect of Enzyme concentration on enzyme activity