Attican Journal of Microbiology Research Vol. 6(30), pp. 6914-5923, 9 August, 2012 ‘Available online at htip:/iwmwacademicjournals.orgiAJMR Ol: 10.5897/AJMR12.218 ISSN 1996-0808 ©2012 Academic Journals Full Length Research Paper Characterization of urease enzyme from marine bacterium Klebsiella species Senthil Balan S."*, Fazila Fathima” and Jayalakshmi S." Centre of Advanced Study (CAS), Marine Biology, Annamalai University, Parangipettai, Cuddalore District, Tamil Nadu, India *Jamal Mohammed College, Bharathidasan University, Trichy, Tamil Nadu, India, ‘Accopled 3 Api, 2012 The rapidly increasing importance of enzyme urease applications has drawn attention to the need of enzyme study. It prompted the present study to hunt a promising bacterial strain with the desired nature from the Porto Novo coast. Urease enzyme is used in diagnostic kits for measuring blood urea, removal of urea from alcoholic beverages, urease conductomaetric biosensors for detection of heavy ‘metal ions, etc. In our study, urease enzyme produced by marine bacteria, was isolated, identified and characterized. Samples of water and sediment from Porto Novo coast were taken for analysis. After that, sediment sample showed more number of urease producing bacteria when compared to water sample. Urease producing bacteria were identified as Klebseilla spp, Proteus spp, Lactobacillus spp and Streptococcus spp, from that urease producer Klebseilla spp showed maximum urease production through phenol hypochlorite assay. The potential strain was optimized with their physiochemical parameters showing maximum urease production at 48 h incubation, pH 7, temperature 35°C, salinity 20 ppt producing 1.7, 1.72, 1.67 and 1.72 U/ml, respectively. An optimization with different carbon and itrogen source showed maximum at 0.7% glucose with 2.05 U/ml and 0.7% peptone with 2.15 Ulml. Mineral supplements 0.03% sodium acetate, 0.04% potassium dihydrogen phosphate, 0.04% nickel sulphate and 0.04% magnesium sulphate showed maximum production of 2.18, 2.27, 2.37 and 2.4 U/ml, respectively. Urea, the enzyme substrate showed maximum production at 0.3% with 2.25 U/ml urease production, Final optimization with inoculum size showed maximum production with increased volume of inoculum with 50 ml of 6 - 8 x 10’ cell/ml to 500 ml of fermentation medium, showed 2.65 Ulml of urease on 36 h of incubation. Urease enzyme was produced in fermentation medium with the above optimized condition and showed a production of 2.8 U/ml. Then the enzyme was partially purified using dialysis membrane after being fractionated with 60% ammonium sulphate at 5°C. The characterization of urease enzyme from marine bacterium Klebsiella species and the optimization of various physico- chemical factors for maximum urease production and its activity stand as a ready reference for more elaborate work of this line in future. The results of the present study will be a base line data for the application of this urease. Key words: Klebsiella spp, marine bacterium, urease enzyme. INTRODUCTION Marine environment has been polluted with numerous Modern agriculture practices introduce ferilizers like urea chemicals from industrial, agricultural, urban activities. into the aquatic environment. When these pollutants persist in the environment, total heterotrophic bacteria (THB) population adopt to produce various enzymes including urease hence the present study. There are *Corresponding author. E-mail: senthilsenthibalan@yahoo.co.in. many applications for the enzyme urease (that is,) it can Tel +919486198685, be used as a diagnostic tool to detect the blood urea(Smith et al, 1993), to remove urea from alcoholic beverages (Fujinawa and Dela, 1990) and in urease conductometric biosensors for detection of heavy metal ions, biocalefication (Sarda et al, 2009), etc. Urease (urea amidohydrolase, EC 3.5.1.5) catalyzes the hydrolysis of urea into ammonia and carbamate (Karplus et al., 1997) and they play an important rae in nitrogen metabolism (Ferrero et al, 1986). Its a nickel-containing metalloonzyme first isolated from seeds of tho jack bean plant in 1926 (Dixon et al., 1975). Urease is widely distributed in nature and is detected in microorganisms, plants and animals (Tabatabai, 1994) and it is also implicated in the virulence of various human and animal pathogens (Mobley otal, 1995). More number of microbial source for this enzyme including bacteria such as Lactobacillus ruminis, corynebacterium lium, Lactobacilus fermentum, Lactobacillus reuteri (Kakimoto et al., 1989; Kakimoto and Suzuki, 1992) Bacilus pasteuri (Achal et al, 2009), Enterobacter sp (Yang et al., 2008) Klebseilla spp (Rao etal, 1993) and fungi such as Aspergilus niger (Smith et al, 1993), Aspergillus nidulans (Mackay and Pateman, 1980; Creaser and Porter, 1985), Rhizipus oryzae (Farley and Santosa, 2002) have been well characterized. When microorganisms utiize urea in urea medium, ammonia is formed during the incubation which makes these media alkaline. Consequently urease production can be detected using pH indicators. Although urea is the major substrate of urease, the enzyme is capable of hydrolyzing ‘ther substrates such as. acetamide, formamide, N- methylurea and semicarbazide (Dixon et al, 1980). Urea hydrolysis has been widely used for the classification and identification of microorganisms, especially members of the family Enterobacteriaceae, Pseudomonads, HaemopIhitis spp and other Gram-negative bacteria (Qadri etal, 1984). ‘The present study focus on the production of urease by « Kiebsiela spp isolated from a sediment sample of Porto Novo coast, India. Klebsiella is a rod-shaped Gram- negative bacteria with a prominent polysaccharide-based capsule, facultave anaerobic, non motile bacteria and oxidase-negative. Klebsiella organisms occur in soil and water and on plants, and some strains are considered a part of the normal flora of the human gastrointestinal tract. The genus is named for German physician and bacteriologist Edwin Klebs. This strain of Klebseilla spp could be used for the production of urease, grow readily under laboratory conditions. The enzyme showed high efficiency on hydrolysis of urea through phenol hypochlorite assay, The objects of this study to characterize the urease enzyme produced by a marine bacterium Klebsiella sp. MATERIALS AND METHODS Collection of samples Water and sediment samples were taken from mouth of the Porto Balan etal. 5915 Novo coastal area (laude 11°29'N; long 79°46'E) during Decemiver 2009 to January 2010. Samples were transferred to the Taboratory in an lee box maintained at 4°C ‘or further study. Water sample Surface water samples were collected in pre stetlized bottles allowing enough air space inside ¢0 a8 to faciltate thorough mixing. Precautonary measures were taken to minimize the contamination Sediment sample Sediment samples were collected using a sterle spatula. The central portions of the collected samples were aseptically transferred info sterile polythene bags. Isolation of urease producing marine bacteria (primary sereening) Water and sediment sample were serially luted using 50% aged ‘sea walor as diluent and plated on urea agar (Christensen medium) containing urea -20 g, peptone -1 g, KH;PO, - 2.0 g, glucose - 1.0, 4g, NaCl - 5.0 g, agar- 15.0 9, 50% aged sea water -1000 mi and Phenol red indicator- 0,072 g pH 6.8 0.2 at 25°C and the plates ‘were incubated at 37°C for 48 h (Atlas, 1946) Selection of potential bacteria through phenel hypochlorite assay (secondary screening) The axenic cultures were individually tested for their urease producing potential using Phenol hypochlorite assay, Axenic Cultures were cultured on urea Broth and incubated for 72 h. The broth was centiluged after incubation period and the supernatant was tested for lis urease activity. Identification of potential strains ‘The axenic cultures producing urease enzyme during screening ‘were identified using Bergy’s Manual of Determinative Bacteriology (Buchanan and Gibbons, 1974). Optimization of physico-chemical parameters for better enzyme production Cultural conditions tke incubation period, pH, temperature, salinity and nutritional factors fike carbon, ritrogen and mineral salts were tested forthe potential strains. Experiments were done by adopting search technique that is, varying parameters one at a time, were conducted in 250 mi’ Erlenmeyer flasks containing urease production medium and every varying parameters were carried out in triplicates and the average values were taken into account. The range of parameter achieved by one stop was fixed in subsequent experiments. Various physico-chemical parameters were optimized with the base of that production medium 2% D-glucose, 1% peptone, 0.5% yeast extrac, 0.2% KHsPO., 0.5% NaCl, 0.2% NaAe, and 0.5% urea, 0.005% MnSO., and 0.005% NISO. with pH 55 (Vang eta, 2008), Incubation period Incubation was carted out for a time period ranging from 8 to 64h5916 Afr. J, Microbiol. Res, With 8h interval and the urea production was estimated. pH To determine the optimal pH for maximum urease, the production was tested ranging from 5 to B with the Interval of pH 05. Temperature To know the optimal temperature for maximum urease, the production ranging between 20 to 40°C with the interval of °C. Salinity ‘The effect of varying salt concentrations was checked betwsen 5 to 50 ppt with the interval ofS ppt since the strain is of marine origin, Carbon sources ‘The effect of alfferent carbon sources Ike glucose, fructose, lactose, mannitol, slateh, sucrose, mallose with varying ‘conceniration 0.1% to 1% were ested for urease production. ‘Nitrogen sources Strains were cultured with diferent nitrogen sources like peptone, yeast extract, beef extract, ammonium ritrate and ammonium ‘Sulphate with different concentration of 0.tto 1% were checked for urease praduction ‘Mineral supplements Effect of diferent ionic supplements tke sodium acetate, potassium dhycrogen phosphate, nickel sulphate and manganese sulphate used in media with varying concentration 0.1 to 1% were checked {for maximum urease production. Urea concentration Varying the concentration of enzyme substrate urea ranging from 0.1 to 7% was tested, Inoculum size ‘Optimization of inoculum size with varying concentrations of ‘addition 10 to 50 ml af 6-8 > 10" celisimlis one important factor for maximizing urease production and time conception by earlier production Phenol-hypochilorite assay ‘The urease activity was measured by phenol-hypochlorite assay (Weatherburn, 1967). The reactions wore done in micro tubes Containing 100 lof sample, 500 pl of 60 mM urea and 500 ul of 100 mM potassium phosphate buffer (pH 8.0) giving a total volume (of 1-1 ml. The reaction mixture was incubated at 37°C for 30 min in ‘a shaking water bath, The reaction was stopped by transfering 50 Ul of reaction mixture to the tubes containing 500 ul of phenol Sodium nitroprusside solution (0.05 g sodium ritroprusside + 1g phenol/100 mi distiled water). 600 jl of akaline hypochlorite (3.56 NasHPO. + 1 mi sodium hypochlorite + 100 mi cistiled water) was. added to the tubes, and incubated at room temperature for 30 min. Finally, the optical density of the colour complex was measured at 1620 nim against the blank (500 ml phenol nitroprusside sodium = 500 ml sodium hypochlorite + 50 mi distiled water) in a spectrophotometer and compared to a standard curve prepared with (NH,):S0.. Contols used for the enzyme reactions were Feaction mixture without substrale and reaction mixture without incubation, One unit of urease activity was defined as the amount of, enzyme liberating 1 mg NH from urea per minute, under the above assay conditions (Smith et a, 1993). Inoculum preparation Potential axenic culture of Klebsella spp was optimized for inoculum density on optimized production medium in 60 of 250 ml Erlenmeyer's conical flask and incubated at optimized time and temperature, Bulk production For mass scale production, 500 mi of optimized urease production medium was prepared in 7 lie Eenmeyer's conical flask. Aller sterilization prepared inoculum was inoculated and incubated for 48 h. In intervals of 4 afer 12 h of incubation production medium was tested for urease production using phenol-hypochiorte assay, Partial purification and lyophilisation ‘Aller incubation period, the mass scale culture was collected and Cenirfuged at 10.000 rpm for 15 min, and the cel free supematant was collected. The cell fee supernatant was precipitated with the addition of ammonium sulphate to 60% saturation at §°C and kept fer aver night. The precipitated particles again centrifuged at 10,000, rom for 15 min and the pellet was collected. The pellet was suspended in a minimum volume of phosphate buffer (pH 7) and dialysed using dialysis membrane against four changes ofthe same Phosphate buffer. Then the partially purified enzyme was Iyophilized, In between, each of the above process, urease enzyme ‘was checked for its activily by pheno-hypochlorite assay anc compared with the standard values. RESULTS AND DISCUSSION Urease producing bacteria was pursuit on water and sediment samples of Porto Novo coast, India during December 2009 to January 2010. The samples were serially diluted and plated on urea agar with pH 6.5 with Phenol red pH indicator and incubated at 37°C for 48 h. After incubation, colonies showed pink colour are urease producers (that is) confirm hydrolysis of urea resulting ammonia, which forms an alkaline environment. The Urease producing strains were pure cultured using phase streaking plate technique on the same media urea agar and the axenic cultures were lyophilized and were used {or further studies. Among the samples, sediment sample harboured more number of urease producing bacteria. The viable count of urease producing bacteria was ranged nearly negligible in water and was in the range of 2.8 * 10°- 8 x 10" CFUlg in sediment. The strains wereBalan etal. 5917 ‘Table 1. Percentage composition of urease producing bacteria in sediment Identified urease producing bacterial strain Presence of urease producing bacteria in urea agar plate (%) ‘Klobsolla spp 78 Proteus spp 13 Lactobacillus spp 6 ‘Streptococcus spp 3 Biochemically identified Number of same genus Percentage of individual genus KKiebseila spp 25 78 Proteus spp 4 13 Lactobacillus spp 2 6 Streptococcus spp 4 3 Total numberof urease producing bacteria was isolated through primary screening method from both water and sediment sample were 31 Figure 1. Zone formed by Klebsialla spp on urea agar. ah teh Bah Urease activity (U/ml/min) 32h 40h ——«aBSC* GHC Incubation Time Figure 2. Effect of diferent incubation period on urease production. identified by biochemical methods with the help of Bergy’s Manual of Determinative Bacteriology. The isolated strains were identified as Klebsiella spp, Proteus ‘spp, Lactobacillus spp and Streptococcus spp. Out of that Klebsoilla spp was found to be a dominating (Table 1) forming 78%, The others forms compared to others were Proteus spp, Lactobacillus spp and Streptococcus spp, respectively constituting about 13, 6 and 3% (Table 1).. These axenic strains were grown in urea broth and after incubation at 37°C for 48 h with pH 6.5, the broth was centrifuged and the cell free supernatant was used for phenol hypochlorite assay. Klebsiella spp showed a maximum urease activity of 1.75U/ ml followed by Proteus spp, Lactobacillus spp and Streptococcus spp, respectively. Hence Klebseilla spp was selected as the ‘most potential strain (Figure 1) and further studies were5918 Afr. J. Microbiol. Res, Effect of pH on urease production Urease activity (U/ml/min) 65 7 75 8 pH Figure 3. Etfect of diferent parameters of pH on urease production. Effect of temperature on urease production Ls Urease activity (U/ml/min) 20 25 30 35 40 Temperature (°C) Figure 4. Effect of cfferent parameters of temperature on urease production. ‘Table 2. Statistical measurements of diferent Incubation period on urease production ime inte Urease (Vim 8 0.08 16 0.18 24 047 32 0.89 40 146 48 17 56 1.56 64 4.07 done in this strain. The result was supported by the research carried out by Yamaguchi et al. (1999) and Rao et al. (1993) who characterized urease enzyme in Klebsiella spp. When Klebsiella strain was optimized for various physico-chemical parameters of potential bacteria, Kiebseilla spp for maximum urease production, 48 h of incubation period was found to be ideal for maximum urease production of 1.7 Ulml (Figure 2 and Table 2) initially with production medium while completing all other optimization of physiochemical parameters the incubation period was once second checked for maximum production was 2.65 Uiml at 36 h, Similarly Yang et al (2008) reported 36 h was ideal incubation time for urease: production with Enterobacter spp. In our study pH 7 was found to be optimum at which 1.72 Ulml (Figure 3 and Table 3) for maximum urease production, However Suzuki et al. (1979) reported as pH of 4 and 8 were optimum, respectively for Lactobacillus fermentum and Bacillus muttiacidus. The optimization of temperature showed a maximum urease production of1.67 Uiml at 35°C (Figure 4 and Table 4) which was supported by Yang et al. (2008) who reported the same in Enterobacter spp. As the samples were of marineBalan etal. 5919 Effect of salinity on urease production in > — © in Urease activity (U/ml/min) © SN 10 «15 «20 25 Salinity (ppt) 30°35 « 404550. Figure 5. Eifect of diferent parameters of salinity on urease production ‘Table 3. Staistical measurements of different parameters of pH on urease production Table 5. Statistical measurements of different parameters of salinity on urease production pH internal Urease activity (Uimlimin) Salinity parameter (ppt) __Urease activity (Uimllmin) 5 0.17 5 0.54 55 0.35 10 0.83 6 078 8 1.55 6s 112 20 172 7 172 Py 1.84 75 151 30 151 09. 35 1.33 40 1 45 o7 50 027 Table 4. Statistical measurements of different parameters of {temperature on urease production jure (°C) interval Urease activity (Uiml/min 20 1.09) 28 1.29 30 152 38 1.87 40 1.33 ‘origin, Klebseilla spp was optimized for salinity also and 20 ppt was found to be ideal (1.72 Uiml (Figure § and Table 5)). Among carbon sources, glucose showed maximum urease production at 0.7% (2.05 Ulm! (Figure 6 and Table 6)). However Ruth et al (1998) and Yang et al (2008) used 2% of glucose with both growth and fermentation medium. Regarding nitrogen sources, peptone (0.7%) showed maximum urease production (2.15 Ulm! (Figure 7 and Table 7)). However Ghasemi et al, (2004) recorded yeast extract as the best nitrogen source for urease production by fungi Aspergillus niger. In the present study mineral supplement using NaAc, KH,PO,, NiSO,and MgSO, attempted. The result showed that 0.03% of sodium acetate, 0.04% of potassium dihydrogen phosphates, 0.04% of nickel Sulphate and 0.04% of magnesium sulphate gave 2.18, 2.27, 237 and2.4 Uiml (Figure 8 and Table 8) of enzyme activity. Research of Yamaguchi et al. (1999) supported the need for optimization of nickel reported that urease enzyme possesses a dinuclear Ni active site. However Bakhtiari et al. (2008) reported that 13.3 g of KH,PO, and 0.5 g of MgSO, 5H20 was the optimum concentration for urease production by Aspergillus niger PTCC5011, The enzyme Substrate urea was optimized from 0.1 to 1.0% and 0.3% of urea resulted in 2.25 Uiml (Figure 9 and Table 9) when substrate concentration was increased further it inhibited the growth of the organism as well as the urease5920 Afr. J. Microbiol. Res, 2.5 + Glucose = Fructose 2 Lactose Mannitol —*- Starch —* Sucrose Urease activity (U/ml/min) 0 —— Maltose 01 0.2 0.3 04 05 0.6 0.7 08 0.9 1.0 Different carbon source (%) Figure €. Effect of diferent carbon source on urease production. ‘able 6, Statistical measurements of diferent carbon source on urease prosucon Different carbon sources ferent carbon Sources Glucose Fructose Lactose Mannitol Starch Sucrose Maltose o4 08 08 oa 067 ou 055 087 oz wn o77 058 029 0.49 os7 029 03 125 0.99 or 0.99 os7 0.89 429 oa 1a 123 0.98 127 079 um 156 os 165 144 11 145 098 134 478 08 129 169 129 wT 123 187 189 o7 205 187 133 189 145, 189 2 08 2 19 148 1.98 187 1.99 185 09 191 187 145, tat 156 tat tat 1 183. 171 431 477 151 477 187 =z 2s z —s—Peptone, E 4 3g? —s— Yeast Extract 5 15 4 Beef Extract 2 i =e 14 Ammonium = nitrate 2 054 —s— Ammonium 2 sulphate s 0 5 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Figure 7. Elfect of diferent nitrogen source on urease production Different nitrogen source (%)Balan etal. 5921 ‘Table 7. Statistical measurements of lifferent nitrogen source on urease production Different aivogan sources pocigag Veastenvact Best envact Ammonium nivale Ammonium sulphate at different parameter si of 09 O78 om oat on 02 136 oe or 078 ort 05 {56 29 ost oor ose oa in 138 W 19 un os 0 tn ta iat iar os {98 tor er 5 tar o7 2s 208 33 ts iss 08 2 2 ‘ee im in 0 201 180 et ter 7 1 ‘9 ini is8 a7 168 = 3 205 Sodium acetate 23 [| SS 5 Potassium dihydrogen 5 10s phate. Eus RRC Miphate 4 Magnesium sulphate 2 05 gz 0 2 0.01 0.02 0.03 0.04 0.05 Different mineral source (%) ‘Table 8, Statistical measurements of cifferent minerals source on urease production, Different mineral sources sogium acetate Potassium dehydrogenate Nickel sulphate Magnesium sulphate at different phosphate 0.07 187 7.89 137 Ta 0.02 1.91 24 2.09 221 0.03 218 221 227 234 0.04 1.98 227 237 24 0.05 1.56 201 224 221 production. Similarly Suzuki et al. (1978) used 0.3% of urea in the screening media for urease activity. Finally after all the required physico-chemical parameters were optimized, the inoculum size was optimized with the addition of 10 to 50 ml of 6 - 8 x 10" cell/m! in inoculum preparation to increasing the inoculum concentration the production was increased with decreasing incubation Period, 2.65 U/ml urease formed from 36 h incubation period (Figure 10 and Table 10). Inoculum size of optimized 6 - 8 x 10 7 cellim! was prepared with optimized physiochemical parameters in Urease production medium in 50 of 250 ml Erlenmeyer's conical flask. After incubation period of 24 h inoculums was transferred to 500 of 1000 ml Erlenmeyer’s conical flask. After incubation, the urease activity was found to be 2.8 Ulml, The whole cultured broth was centrifuged at 10,000 rpm for 15 min and the supematant was fractionated with 60% ammonium sulphate at §°C. which5922 Afr. J. Microbiol. Res. Effect of urea on urease production es g BE o2 z — Ss a 205 g 0 £ 01 02 03 04 O05 06 08 09 1.0 Urea (%) Figure 9, fect of urea concentration on urease production, ‘Table 9, Statistical measurements of urea concentration on urease production 02 03 oa 0s 06 o7 os 09 244 225 2.01 1.87 un 187 1.48 1.35 1.25 Effect of inoculum cell density on urease production x ee —— now in Urease activity (U/ml/min) © okey 10mi 20ml 30ml 40ml 50ml Inoculum size (6 - 8 107) Figure 10, Effect of diferent inoculums size on urease productionBalan etal. 5923 ‘Table 10. Statistical measurements of diferent inoculums size on urease production. Different ineculums 10 20 30 40 50 was allowed for overnight to form precipitation. Then the precipitated medium was centrifuged against 10,000 rpm for 15 min and the pellet was collected and dissolved in phosphate buffer at pH 7. The pellet was dialysed using dialysis membrane with the same buffer four times. Then the partially purified extracellular urease was lyophilized. Conclusion In this study, an industrial important enzyme: urease, isolated from Klebsiella spp of Porto Novo coast, India is shown to have the ability to produce respectable quantity for its applications. The maximum production of the ‘enzyme was optimized and partially purified with ammonium sulphate precipitation and dialysis; enzyme activity was checked and shown increased during the procedure without any denaturation or loss of ‘activity.Further purification and characterization of the enzyme are under study. The present study shows that urease enzyme can be industrially produced using Klebsiella strain isolated from marine environment. ACKNOWLEDGMENT ‘The authors gratefully acknowledge Prof and Dean Dr S. T. Balasubramanium, Faculty of Marine Science, Annamalai University’ for supporting our work by Providing necessary lab facilities during the study period. 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