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ISOLATION, SCREENING AND PRODUCTION STUDIES OF URICASE PRODUCING BACTERIA FROM POULTRY SOURCES a
Pooja Nanda & P. E. Jagadeesh Babu
a
a
Department of Chemical Engineering , National Institute of Technology Karnataka, Srinivasnagar , Surathkal , Karnataka , India Accepted author version posted online: 26 Nov 2013.Published online: 11 Jul 2014.
To cite this article: Pooja Nanda & P. E. Jagadeesh Babu (2014) ISOLATION, SCREENING AND PRODUCTION STUDIES OF URICASE PRODUCING BACTERIA FROM POULTRY SOURCES, Preparative Biochemistry and Biotechnology, 44:8, 811-821, DOI: 10.1080/10826068.2013.867875 To link to this article: http://dx.doi.org/10.1080/10826068.2013.867875
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Preparative Biochemistry & Biotechnology, 44:811–821, 2014 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6068 print/1532-2297 online DOI: 10.1080/10826068.2013.867875
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ISOLATION, SCREENING AND PRODUCTION STUDIES OF URICASE PRODUCING BACTERIA FROM POULTRY SOURCES
Pooja Nanda and P. E. Jagadeesh Babu Department of Chemical Engineering, National Institute of Technology Karnataka, Srinivasnagar, Surathkal, Karnataka, India
& Uricase (urate oxidase EC 1.7.3.3) is a therapeutic enzyme that is widely used to catalyze the enzymatic oxidation of uric acid in the treatment of hyperuricemia and gout diseases. In this study, three bacterial species capable of producing extracellular uricase were isolated from a poultry source and screened based on the size of the clear zone using a uric acid agar plate. The bacterial species capable of producing uricase with the highest uricolytic activity was identified as Bacillus cereus strain DL3 using a 16SrRNA gene sequencing approach. The time-course study of uricase production was performed and the medium was optimized. Carboxymethylcellulose and asparagine were found to be the best carbon and nitrogen sources. Maximum uricolytic activity was observed at pH 7.0 with an inducer concentration of 2.0 g=L. Inoculum size of 5% gave maximum uricolytic activity. The maximum uricolytic activity of 15.43 U=mL was achieved at optimized conditions, which is 1.61 times more than the initial activity. Further, enzymatic stability was determined at different pH and temperature. Keywords Bacillus cereus strain DL3, optimization, uricase
INTRODUCTION Uricase (urate oxidase EC 1.7.3.3) is a therapeutic enzyme that belongs to the class of oxidoreductases; it catalyzes the oxidation of uric acid to allantoin, carbon dioxide, and hydrogen peroxide[1] and also plays a vital role in the purine metabolic pathway.[2] Uric acid, which is the substrate for microbial uricase production, is an end product of purine metabolism in humans. Uric acid is also produced in the body as a result of tumor lysis syndrome.[3] The accumulation of uric acid in the blood plasma results in a hyperuricemic condition that is commonly known as the disease called gout. It can be cured by intravenous administration of uricase enzyme, Address correspondence to P. E. Jagadeesh Babu, Department of Chemical Engineering, National Institute of Technology Karnataka, Srinivasnagar, Surathkal, Karnataka 575 025, India. E-mail: [email protected]
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which can convert uric acid crystals to allantoin. Allantoin is 5 to 10 times more soluble than uric acid and it can be easily eliminated through kidneys.[4] Microbial uricase also finds application as a clinical reagent for the determination of blood and serum uric acid concentrations,[5] as a uric acid biosensor in its immobilized form,[6] as the protein drug Rasburicase for treatment of hyperuricemia,[7] and infrequently as a component of hair coloring reagents.[8] Though many organisms including animals, plants, and microbes have the ability to produce uricase, the uricase-producing microbial sources have an edge over the other sources owing to their higher growth rates, easy medium optimization, and cost-effective bioprocessing. The literature reveals that more than half of poultry excrement contains 63 to 87% uric acid, which is the sole source of nitrogen for the aerobic bacterial action.[9,10] Aerobic digestion of poorly soluble uric acid indicates the possibility of presence of uricase-producing microorganisms in poultry excrement. In this study, we report the isolation and screening of uricase-producing bacterial species from poultry deep litter samples. Further, the medium for uricase production was optimized through shake-flask studies, and the stability of the produced uricase was analyzed at various pH levels and temperatures. MATERIALS AND METHODS Samples From Poultry Farms Samples of poultry deep litter and excrement were brought to the laboratory in an ice pack from a poultry farm located in Mulky, Karnataka, India. One gram of sample was added to 100 mL of sterilized nutrient broth and incubated for 48 hr at 30 C at 150 rpm in an incubator shaker. After 48 hr of growth, 1 mL of turbid broth was inoculated into a selective medium. The composition of the selective medium is as follows (g=L): sucrose, 20.0; uric acid, 2.0; dipotassium hydrogen phosphate, 1.0; magnesium sulfate, 0.5; ferrous chloride, 0.01; sodium chloride, 0.5. One hundred parts per million of amphotericin (antifungal agent) was used in the selective medium to suppress possible fungal growth. The pH of the medium was adjusted to 6.5 using 0.1 M NaOH solution. The cultures that showed satisfactory growth in 24 hr were transferred to fresh selective medium. After three consecutive transfers, 24-hr-old turbid broth was serially diluted and spread plated on a nutrient agar plate having 1.5 g=L of uric acid and incubated at 35 C for 24 hr. Discrete colonies showing clear zones due to production of extracellular uricase were picked up and preserved at 4 C on nutrient agar slants. Later, individual colonies were streaked for isolation on the uric acid agar plates and were subjected to gram staining. The pure cultures thus obtained were preserved in 20% glycerol stock for further use.
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Inoculum Development and Production Medium A loop full of the bacterial strain from the uric acid agar slant was transferred to 50 mL of inoculum development medium. The inoculum development medium had the following composition (g=L): sucrose, 20.0; uric acid, 1.0; dihydrogen potassium phosphate, 1.0; magnesium sulfate, 0.5; sodium chloride, 0.5; and ferrous sulfate heptahydrate, 0.01, with uric acid as a sole nitrogen source and inducer for uricase production. The pH of the inoculum development medium was adjusted to 7.0 using 0.1 N NaOH. For further studies, 24-hr-old inoculum with an inoculum size of 2% was used. One milliliter of this bacterial culture was then inoculated into 50 mL of the production medium, which had the following composition (g=L): peptone, 10.0; sucrose, 20.0; dihydrogen potassium phosphate, 1.0; magnesium sulfate, 0.5; sodium chloride, 0.5; ferrous sulfate heptahydrate, 0.01; and uric acid, 1.5. It was then incubated for 24 hr at 32 C at 250 rpm. Fermentation broth samples of 5 mL were drawn from the production medium every 4 hr until 60 hr of incubation and centrifuged at 5000 rpm for 10 min. The supernatant was used as crude enzyme solution and was analyzed for uricase activity. The bacterial biomass was determined by measuring the absorbance of the broth at 510 nm against a blank. Enzyme Assay The enzymatic assay was carried out by the method described by Mahler et al.[11] Three milliliters of 20 mM sodium borate buffer, pH 9.0, was added to 75 mL of 3.57 mM uric acid solution and 20 mL of cell free supernatant (used as crude enzyme), at 25 C. Blank solution was separately prepared by adding 20 mL of buffer solution instead of the cell-free supernatant. The blank and the test solutions were incubated at 25 C for 10 min. The reduction in the uric acid concentration in the test sample was measured using an ultraviolet (UV)-visible spectrophotometer at 293 nm. The difference between the absorbance of the test and blank was equivalent to the decrease in the uric acid concentration during the enzymatic reaction. Thus, 1 unit (U) of enzyme activity was defined as the amount of uricase required to convert 1 mmol uric acid into allantoin per minute at 25 C and at a pH 9.0, considering the millimolar extinction coefficient of uric acid (e) at 293 nm as 12.6 mM 1 cm 1. Medium Optimization Since uric acid was used as inducer, its effect on the production of uricase was optimized by measuring the uricase activity at different uric acid concentrations, ranging from 0 to 3.5 g=L. Different carbon sources like glucose, glycerol, maltose, lactose, starch, and carboxymethylcellulose
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sodium salt were used to find the best carbon source for the production of highly active uricase. Different nitrogen sources like yeast extract, casein hydrolysate, urea, asparagine, ammonium chloride, sodium nitrate, ammonium nitrate, and ammonium tartarate were tested. Carbon and nitrogen sources were added at a concentration of 10 g=L, while carboxymethylcellulose was used at a concentration of 2 g=L. The initial medium pH was optimized in the range of 4.0 to 10.0 using NaOH and HCl, and the uricase activity was measured after 20 hr of incubation, using the prior optimized parameters. Further, the dependency of uricase on the biomass was examined by varying the amount of bacterial culture from 1% to 8%. Uricase Stability Optimum pH for the stable and maximum uricolytic action of produced uricase was found by using 0.1 M buffer solution with pH ranging from 6.0 to 10.0. For maintaining pH 6–7, sodium phosphate buffer, for pH 8–9, borate buffer, and for pH 10, Tris-HCl buffer were used. The optimal temperature for the stable and maximum uricolytic activity was determined by carrying out the enzymatic reaction of uricase in 0.1 M borate buffer (pH 8.5) at various temperatures ranging from 30 C to 60 C for 15 min, and immediately after this the enzyme activities were determined. The bacterial species that produced uricase with maximum uricolytic activity and that had maximum stability was identified to be Bacillus cereus strain DL3 by 16S rRNA gene sequencing. The sequence data have been submitted to the NCBI GenBank database under accession number KC755041.1. RESULTS AND DISCUSSION Isolation and Screening of Uricase-Positive Microorganism Microbes were screened based on the width of clear zone formed on uric acid agar plates. Figure 1 shows the bacterial isolates on the uric acid agar plate with its clear zone. From Figure 1, it has been inferred that the
FIGURE 1 Three bacterial isolates on a uric acid agar plate with the clear zones formed due to the production of extracellular uricase.
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bacterial species DL3 has produced uricase with maximum uricolytic activity, which was characterized by bigger zones of clearance. Table 1 shows the gram characteristic of the three isolates and their uricase activity, which was performed in the selective medium. From the table, it has been observed that the bacterial species DL3 is a gram-positive rod with a maximum uricolytic activity of 8.9 U=mL. The isolate DL3 was further identified as Bacillus cereus using 16SrRNA gene sequencing procedure. Figure 2 shows the time course for uricase production using Bacillus cereus strain DL3 in the production medium. An S-shaped growth pattern with the stationary phase after 20 hr of incubation and a decrease in the biomass production at 48 hr of incubation were observed for Bacillus cereus strain DL3. The maximum uricase activity was observed to be 9.55 U=mL at 20 hr of incubation in the production medium. Effect of Inducer Concentration on Uricase Activity In this study, uric acid is used as an inducer to stimulate uricase production. The enzyme induction by uric acid is justified by the fact that uric acid, being a sparingly soluble low-molecular-mass compound, undergoes rapid cellular uptake, leading to the production of uricase with higher activity. The effect of inducer concentration on the production of uricase was studied and the results are shown in the Figure 3. At a concentration of 2 g=L uric acid, maximum uricase activity of 10 U=mL was observed. A further increase in the uric acid concentration reduced the uricase activity, which could be attributed to substrate inhibition by the enzyme. Similar results were reported in the literature in terms of optimal uric acid (inducer) concentration for different microbes like Microbacterium sp. (0.3 g=L),[12] Bacillus thermocatenulatus (0.3 g=L),[13] Proteus vulgaris 1357 (0.2 g=L), Proteus vulgaris B-317-C (0.15 g=L), and 1 g=L for both Streptomyces graminofaciens and Streptomyces albidoflavus.[14] Effect of Various Carbon and Nitrogen Sources Carbon and nitrogen are the vital sources for the growth of the microorganism and also influences the production of uricase. Figure 4 and Figure 5 show the effect of various carbon and nitrogen sources on the TABLE 1 Gram Characteristics and Uricoltyic Activities of the Isolates Bacterial Isolate
Gram Character and Shape
DL 1 DL 2 DL 3
Gram-negative cocci Gram-negative small rods Gram-positive rods
Uricase Activity (U=mL) 2.89 4.67 8.96
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FIGURE 2 Time course of uricase production.
production of uricase in terms of its activity. It has been observed that carboxymethylcellulose, maltose, and lactose have a considerable positive effect on the uricase activity in comparison to sucrose, whereas the other carbon sources like glucose, glycerol, and starch did not enhance the uricase production. Further, it was also observed that the carbon sources have slight effect on biomass. Similarly, the uricase production and biomass were significantly affected by the presence of nitrogen sources, as shown in Figure 5. In this study, four organic and four inorganic nitrogen sources were used, among which organic nitrogen sources were found to have higher influence on uricase and biomass production compared to the inorganic nitrogen sources. In particular, maximum uricase activity was achieved with asparagine as a nitrogen source. Yeast extract and ammonium nitrate yielded maximum biomass with reduced uricase activity. The observations agree with the findings by Khucharenphaisan and Sinma in 2011,[15] Lofty in 2007,[13] and Zhou et al. in 2005[12] on uricase-producing bacteria.
FIGURE 3 Effect of inducer concentration on uricase activity.
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FIGURE 4 Effect of different carbon sources on biomass formation and uricase activity.
Effect of Initial pH of the Production Medium Figure 6 indicates the effect of initial pH of the production medium on uricase activity. The medium consisted of maltose and asparagine as carbon and nitrogen sources, respectively, and with an inducer concentration of 2 g=L. It has been observed that the maximum uricolytic activity of 13.88 U=mL was achieved at an initial pH of 7.0. The results are in concordance with the findings of other researchers on the optimal initial pH, like that of Bacillus thermocatenulatus at an initial pH of 7.0,[13] Micrococcus roseus at pH 7.5,[16] Saccharopolyspora sp at pH 7.0,[15] and Microbacterium sp. at pH 7.5.[12] Effect of Inoculum Size on Uricase Activity Figure 7 shows the effect of inoculum size on the uricase activity. As the inoculum size was increased from 2% to 5%, uricase activity increased from 7.34 U=mL to 12.65 U=mL. Further increase in the inoculum size reduced the uricase activity, but resulted in an increase in biomass. It could be
FIGURE 5 Effect of different nitrogen sources on biomass formation and uricase activity.
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FIGURE 6 Effect of initial pH of the production medium on uricase activity.
concluded that uricase production is biomass independent. The reduction in the uricase activity at higher inoculum size could be due to the fast depletion of micronutrients in the production medium. A similar observation was made by Chen et al. in 2008,[17] while working with a recombinant Hansenula polymorpha strain harboring Candida utilis uricaseencoding gene. They have concluded that the uricase production is biomass independent and the optimum inoculum size as 6%. Table 2 indicates previously reported uricase-positive bacterial species and their uricolytic activities. Determination of Optimum pH and Temperature for Uricase Activity Figure 8 shows the effect of pH on the stability of uricase in terms of its activity. The enzyme produced by Bacillus cereus strain DL3 showed maximum stability at pH 8.5 in a 0.1 M borate buffer at 30 C. At the optimal pH of 8.5, uricase from Bacillus cereus strain DL3 retained a maximum
FIGURE 7 Effect of inoculum size on uricase activity.
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TABLE 2 Previously Reported Uricase Positive Bacterial Species
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Bacterial Species Bacillus thermocatenulatus Saccharopolyspora sp. Microbacterium sp. Pseudomonas aeruginosa Enterobacter cloaca Arthobacter globiformis Micrococcus luteus Proteus vulgaris Streptomyces graminofaciens Pseudomonas sp. Streptomyces sp. Corynebacterium uratoxidans Streptomyces cyanogenus Bacillus cereus DL3
Uricase Activity
Reference
1.25 U=mL 0.21 U=mL 1.0 U=mL 7.1 U=mL 6.6 U=mL 22 U=mg 1 U=mL 8.7 U=mg 8 U=mg 9 mU=ml 2.25 U=g wet cells 1.2 U=mL 12.3 U=mg 15.43 U=mL
[12] [14] [15] [18] [19] [20] [21] [14] [14] [22] [23] [24] [25] Present study
FIGURE 8 Effect of pH on uricase activity.
FIGURE 9 Effect of temperature on uricase activity.
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activity of 13.2 U=mL in a borate buffer for about 1 hr. It was also observed that at pH 8.0 and 9.0the enzyme still retained 12.45 U=mL and 11.64 U=mL of activity, respectively. Figure 9 indicates the effect of temperature on the stability of uricase, and it was found that uricase thus produced was not thermostable. It retained its maximum uricolytic activity at 30 C, when incubated in borate buffer of pH 8.5 for 1 hr. These conditions of pH and temperature can be used for the storage of uricase from Bacillus cereus strain DL3. Khucharenphaisan and Sinma (2011)[15] have reported the optimal pH and temperature for uricase from Saccharopolyspora sp to be 8.5 and 37 C, respectively, in a 50-mM borate buffer. CONCLUSION Three bacterial species capable of producing extracellular uricase were isolated from poultry deep litter and excrement samples. The bacterial species producing extracellular uricase with maximum uricase activity was identified to be Bacillus cereus strain DL3 through a 16SrRNA gene sequencing procedure. The time-course studies for uricase production were performed and it was observed that the uricase production was not biomass dependent and has an activity of 9.55 U=mL at 20 hr of incubation in the lag phase. Process parameters like initial medium pH, inducer concentration, various carbon and nitrogen sources, and inoculum size were optimized. Optimum initial medium pH was found to be 7.0, inducer concentration to be 2.0 g=L, and inoculum size to be 5%. Carboxymethylcellulose and asparagine were found to be the best carbon and nitrogen sources, respectively, for Bacillus cereus strain DL3. At the optimal conditions just described, maximum uricase activity was observed to be 15.43 U=mL, which is 1.63 times higher than the activity of the nonoptimized process. REFERENCES 1. Brogard, J.M.; Counaros, D.; Frankhauser, J.; Stahl, A.; Stahl, J. Enzymatic Uricolysis: A Study of the Effect of Fungal Urate Oxidase. Eur. J. Clin. Biol. Res. 1972, 17, 890–895. 2. Wu, X.; Wakamiya, M.; Vaishnav, S.; Geske, R.; Montgomery, C.; Jones, P.; Bradley, A.; Caskey, T. Hyperuricemia and Urate Nephropathy in Urate Oxidase Deficient Mice. Proc. Natl. Acad. Sci. USA 1994, 91, 742–746. 3. Cammalleri, L.; Malaguarnera, M. Rasburicase Represents a New Tool for Hyperuricemia in Tumor Lysis Syndrome and in Gout. Int. J. Med. Sci. 2007, 4(2), 83–93. 4. Pui, C.H.; Jeha, S.; Irwin, D.; Camitta, B. Recombinant Urate Oxidase (Rasburicase) in the Prevention and Treatment of Malignancy-Associated Hyperuricemia in Pediatric and Adult Patients: Results of a Compassionate-Use Trial. Leukemia 2001, 15, 1505–1509. 5. Adamek, V., Kralova, B.; Suchova, M.; Valentova, O.; Demnerova, K. Purification of Microbial Uricase. J. Chromatogr. A 1989, 497, 268–275. 6. Fatma, A. An Amperometric Biosensor for Uric Acid Determination Prepared From Uricase Immobilized in Polyaniline-Polypyrrole Film. Sensors 2008, 8, 5492–5500.
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7. Colloc’h, N.; Hajji, M.E.; Bachet, B.; L’Hermite, G.; Schiltz, M.; Prange, T. Crystal Structure of the Protein Drug Urate Oxidase-Inhibitor Complex at 2.05A˚ Resolution. Nat. Struct. Biol. 1997, 4, 947–952. 8. Nakagawa, S.; Oda, H.; Anazawa, H. High Cell Density Cultivation and High Recombinant Protein Production of Eschericia coli Strain Expressing Uricase. Biosci. Biotechnol. Biochem. 1995, 59, 2263–2267. 9. Tinsley, J.; Novakowski, T.A. The Composition and Manorial Value of Poultry Excreta, Straw Dropping Composts and Deep Litter. Exp. J. Deep Litter. J. Sci. Food. Agric. 1959, 10, 224–232. 10. Schefferle, H.E. The Decomposition of Uric Acid in Built Up Poultry Litter. J. Appl. Bacteriol. 1965, 28, 412–420. 11. Mahler, H., Hubscher, G.; Baum, H. Studies on Uricase: I. Preparation, Purification and Properties of a Cuproprotein. J. Biol. Chem. 1955, 216, 625. 12. Zhou, X.; Ma, X.; Sun, G.; Li, X.; Guo, K. Isolation of a Thermostable Uricase Producing Bacterium and Study on Its Enzyme Production Conditions. Process Biochem. 2005, 40, 3749–3753. 13. Lotfy, W.A. Production of a Thermostable Uricase by a Novel Bacillus thermocatenulatus Strain. Bioresource Technol. 2008, 99, 699–702. 14. Azab, A.E.; Magda, M.A.; Mervat, F.F. Studies on Uricase Induction in Certain Bacteria. Egypt. J. Biol. 2005, 7, 44–54. 15. Khucharoenphaisa, K.; Sinma, K. Production and Partial Characterization of Uric Acid Degrading Enzyme From a New Source Saccharopolyspora sp. Pak. J. Bio. Sci. 2011, 14(3), 226–231. 16. Olivieri, R.; Eugenio, F.; Pierluigi, C.; Ludwig, D. Uricase Production Method. U.S. Patent 4,389,485, 1983. 17. Chen, Z.; Wang, Z.; Xiuping, H.; Xuena, G.; Weiwei, L.; Borun, Z. Uricase Production by a Recombinant Hansenula polymorpha Strain Harboring Candida utilis Uricase Gene. Appl. Microbiol. Biotechnol. 2008, 79(4), 545–554. 18. Yasser, R.A.F.; Saeed, H.M.; Goharc, Y.M.; El-Bazb, M.A. Improved Production of Pseudomonas aeruginosa Uricase, by Optimization of Process Parameters Through Statistical Experimental Designs. Process Biochem. 2005, 40, 1707–171. 19. Machida, Y.; Nakanishi, T. Purification and Properties of Uricase From Enterobacter cloacae. Agric. Biol. Chem. 1980, 44, 2811–2815. 20. Nobutoshi, K.; Keisuke, S.; Takao, M.; Masaki, T.; Hitoshi, K.; Kazue, T. Chemiluminometric Determination of Uric Acid in Plasma by Closed Loop FIA With a Coimmobilized Enzyme Flow Cell. Anal. Sci. 2000, 16, 1203–1205. 21. Snoke, R.E.; Huge, A.R.; Richey, C.T. Production of Uricase From Micrococcus luteus. U.S. Patent 4,062,731, 1977. 22. Bachracuh, U. The Aerobic Breakdown of Uric Acid by Certain Pseudomonads. J. Gen. Microbiol. 1957, 17, 1–11. 23. Watanabe, Y.; Juichiro, Y.; Mayumi, F. Studies on the Formation of Uricase by Streptomyces. Agric. Biol. Chem. 1969, 33(9), 1282–1290. 24. Sugisaki, Z.; Norihiko, W.; Hirokazu, K. Process for Producing Uricase. U.S. Patent 3,767,533, 1973. 25. Ohe, T.; Watanabe, Y. Purification and Properties of Urate Oxidase From Streptomyces cyanogenus. J. Biochem. 1981, 89(6), 1769–1776.
Preparative Biochemistry and Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb20
ISOLATION, SCREENING AND PRODUCTION STUDIES OF URICASE PRODUCING BACTERIA FROM POULTRY SOURCES a
Pooja Nanda & P. E. Jagadeesh Babu
a
a
Department of Chemical Engineering , National Institute of Technology Karnataka, Srinivasnagar , Surathkal , Karnataka , India Accepted author version posted online: 26 Nov 2013.Published online: 11 Jul 2014.
To cite this article: Pooja Nanda & P. E. Jagadeesh Babu (2014) ISOLATION, SCREENING AND PRODUCTION STUDIES OF URICASE PRODUCING BACTERIA FROM POULTRY SOURCES, Preparative Biochemistry and Biotechnology, 44:8, 811-821, DOI: 10.1080/10826068.2013.867875 To link to this article: http://dx.doi.org/10.1080/10826068.2013.867875
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Preparative Biochemistry & Biotechnology, 44:811–821, 2014 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6068 print/1532-2297 online DOI: 10.1080/10826068.2013.867875
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ISOLATION, SCREENING AND PRODUCTION STUDIES OF URICASE PRODUCING BACTERIA FROM POULTRY SOURCES
Pooja Nanda and P. E. Jagadeesh Babu Department of Chemical Engineering, National Institute of Technology Karnataka, Srinivasnagar, Surathkal, Karnataka, India
& Uricase (urate oxidase EC 1.7.3.3) is a therapeutic enzyme that is widely used to catalyze the enzymatic oxidation of uric acid in the treatment of hyperuricemia and gout diseases. In this study, three bacterial species capable of producing extracellular uricase were isolated from a poultry source and screened based on the size of the clear zone using a uric acid agar plate. The bacterial species capable of producing uricase with the highest uricolytic activity was identified as Bacillus cereus strain DL3 using a 16SrRNA gene sequencing approach. The time-course study of uricase production was performed and the medium was optimized. Carboxymethylcellulose and asparagine were found to be the best carbon and nitrogen sources. Maximum uricolytic activity was observed at pH 7.0 with an inducer concentration of 2.0 g=L. Inoculum size of 5% gave maximum uricolytic activity. The maximum uricolytic activity of 15.43 U=mL was achieved at optimized conditions, which is 1.61 times more than the initial activity. Further, enzymatic stability was determined at different pH and temperature. Keywords Bacillus cereus strain DL3, optimization, uricase
INTRODUCTION Uricase (urate oxidase EC 1.7.3.3) is a therapeutic enzyme that belongs to the class of oxidoreductases; it catalyzes the oxidation of uric acid to allantoin, carbon dioxide, and hydrogen peroxide[1] and also plays a vital role in the purine metabolic pathway.[2] Uric acid, which is the substrate for microbial uricase production, is an end product of purine metabolism in humans. Uric acid is also produced in the body as a result of tumor lysis syndrome.[3] The accumulation of uric acid in the blood plasma results in a hyperuricemic condition that is commonly known as the disease called gout. It can be cured by intravenous administration of uricase enzyme, Address correspondence to P. E. Jagadeesh Babu, Department of Chemical Engineering, National Institute of Technology Karnataka, Srinivasnagar, Surathkal, Karnataka 575 025, India. E-mail: [email protected]
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which can convert uric acid crystals to allantoin. Allantoin is 5 to 10 times more soluble than uric acid and it can be easily eliminated through kidneys.[4] Microbial uricase also finds application as a clinical reagent for the determination of blood and serum uric acid concentrations,[5] as a uric acid biosensor in its immobilized form,[6] as the protein drug Rasburicase for treatment of hyperuricemia,[7] and infrequently as a component of hair coloring reagents.[8] Though many organisms including animals, plants, and microbes have the ability to produce uricase, the uricase-producing microbial sources have an edge over the other sources owing to their higher growth rates, easy medium optimization, and cost-effective bioprocessing. The literature reveals that more than half of poultry excrement contains 63 to 87% uric acid, which is the sole source of nitrogen for the aerobic bacterial action.[9,10] Aerobic digestion of poorly soluble uric acid indicates the possibility of presence of uricase-producing microorganisms in poultry excrement. In this study, we report the isolation and screening of uricase-producing bacterial species from poultry deep litter samples. Further, the medium for uricase production was optimized through shake-flask studies, and the stability of the produced uricase was analyzed at various pH levels and temperatures. MATERIALS AND METHODS Samples From Poultry Farms Samples of poultry deep litter and excrement were brought to the laboratory in an ice pack from a poultry farm located in Mulky, Karnataka, India. One gram of sample was added to 100 mL of sterilized nutrient broth and incubated for 48 hr at 30 C at 150 rpm in an incubator shaker. After 48 hr of growth, 1 mL of turbid broth was inoculated into a selective medium. The composition of the selective medium is as follows (g=L): sucrose, 20.0; uric acid, 2.0; dipotassium hydrogen phosphate, 1.0; magnesium sulfate, 0.5; ferrous chloride, 0.01; sodium chloride, 0.5. One hundred parts per million of amphotericin (antifungal agent) was used in the selective medium to suppress possible fungal growth. The pH of the medium was adjusted to 6.5 using 0.1 M NaOH solution. The cultures that showed satisfactory growth in 24 hr were transferred to fresh selective medium. After three consecutive transfers, 24-hr-old turbid broth was serially diluted and spread plated on a nutrient agar plate having 1.5 g=L of uric acid and incubated at 35 C for 24 hr. Discrete colonies showing clear zones due to production of extracellular uricase were picked up and preserved at 4 C on nutrient agar slants. Later, individual colonies were streaked for isolation on the uric acid agar plates and were subjected to gram staining. The pure cultures thus obtained were preserved in 20% glycerol stock for further use.
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Inoculum Development and Production Medium A loop full of the bacterial strain from the uric acid agar slant was transferred to 50 mL of inoculum development medium. The inoculum development medium had the following composition (g=L): sucrose, 20.0; uric acid, 1.0; dihydrogen potassium phosphate, 1.0; magnesium sulfate, 0.5; sodium chloride, 0.5; and ferrous sulfate heptahydrate, 0.01, with uric acid as a sole nitrogen source and inducer for uricase production. The pH of the inoculum development medium was adjusted to 7.0 using 0.1 N NaOH. For further studies, 24-hr-old inoculum with an inoculum size of 2% was used. One milliliter of this bacterial culture was then inoculated into 50 mL of the production medium, which had the following composition (g=L): peptone, 10.0; sucrose, 20.0; dihydrogen potassium phosphate, 1.0; magnesium sulfate, 0.5; sodium chloride, 0.5; ferrous sulfate heptahydrate, 0.01; and uric acid, 1.5. It was then incubated for 24 hr at 32 C at 250 rpm. Fermentation broth samples of 5 mL were drawn from the production medium every 4 hr until 60 hr of incubation and centrifuged at 5000 rpm for 10 min. The supernatant was used as crude enzyme solution and was analyzed for uricase activity. The bacterial biomass was determined by measuring the absorbance of the broth at 510 nm against a blank. Enzyme Assay The enzymatic assay was carried out by the method described by Mahler et al.[11] Three milliliters of 20 mM sodium borate buffer, pH 9.0, was added to 75 mL of 3.57 mM uric acid solution and 20 mL of cell free supernatant (used as crude enzyme), at 25 C. Blank solution was separately prepared by adding 20 mL of buffer solution instead of the cell-free supernatant. The blank and the test solutions were incubated at 25 C for 10 min. The reduction in the uric acid concentration in the test sample was measured using an ultraviolet (UV)-visible spectrophotometer at 293 nm. The difference between the absorbance of the test and blank was equivalent to the decrease in the uric acid concentration during the enzymatic reaction. Thus, 1 unit (U) of enzyme activity was defined as the amount of uricase required to convert 1 mmol uric acid into allantoin per minute at 25 C and at a pH 9.0, considering the millimolar extinction coefficient of uric acid (e) at 293 nm as 12.6 mM 1 cm 1. Medium Optimization Since uric acid was used as inducer, its effect on the production of uricase was optimized by measuring the uricase activity at different uric acid concentrations, ranging from 0 to 3.5 g=L. Different carbon sources like glucose, glycerol, maltose, lactose, starch, and carboxymethylcellulose
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sodium salt were used to find the best carbon source for the production of highly active uricase. Different nitrogen sources like yeast extract, casein hydrolysate, urea, asparagine, ammonium chloride, sodium nitrate, ammonium nitrate, and ammonium tartarate were tested. Carbon and nitrogen sources were added at a concentration of 10 g=L, while carboxymethylcellulose was used at a concentration of 2 g=L. The initial medium pH was optimized in the range of 4.0 to 10.0 using NaOH and HCl, and the uricase activity was measured after 20 hr of incubation, using the prior optimized parameters. Further, the dependency of uricase on the biomass was examined by varying the amount of bacterial culture from 1% to 8%. Uricase Stability Optimum pH for the stable and maximum uricolytic action of produced uricase was found by using 0.1 M buffer solution with pH ranging from 6.0 to 10.0. For maintaining pH 6–7, sodium phosphate buffer, for pH 8–9, borate buffer, and for pH 10, Tris-HCl buffer were used. The optimal temperature for the stable and maximum uricolytic activity was determined by carrying out the enzymatic reaction of uricase in 0.1 M borate buffer (pH 8.5) at various temperatures ranging from 30 C to 60 C for 15 min, and immediately after this the enzyme activities were determined. The bacterial species that produced uricase with maximum uricolytic activity and that had maximum stability was identified to be Bacillus cereus strain DL3 by 16S rRNA gene sequencing. The sequence data have been submitted to the NCBI GenBank database under accession number KC755041.1. RESULTS AND DISCUSSION Isolation and Screening of Uricase-Positive Microorganism Microbes were screened based on the width of clear zone formed on uric acid agar plates. Figure 1 shows the bacterial isolates on the uric acid agar plate with its clear zone. From Figure 1, it has been inferred that the
FIGURE 1 Three bacterial isolates on a uric acid agar plate with the clear zones formed due to the production of extracellular uricase.
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bacterial species DL3 has produced uricase with maximum uricolytic activity, which was characterized by bigger zones of clearance. Table 1 shows the gram characteristic of the three isolates and their uricase activity, which was performed in the selective medium. From the table, it has been observed that the bacterial species DL3 is a gram-positive rod with a maximum uricolytic activity of 8.9 U=mL. The isolate DL3 was further identified as Bacillus cereus using 16SrRNA gene sequencing procedure. Figure 2 shows the time course for uricase production using Bacillus cereus strain DL3 in the production medium. An S-shaped growth pattern with the stationary phase after 20 hr of incubation and a decrease in the biomass production at 48 hr of incubation were observed for Bacillus cereus strain DL3. The maximum uricase activity was observed to be 9.55 U=mL at 20 hr of incubation in the production medium. Effect of Inducer Concentration on Uricase Activity In this study, uric acid is used as an inducer to stimulate uricase production. The enzyme induction by uric acid is justified by the fact that uric acid, being a sparingly soluble low-molecular-mass compound, undergoes rapid cellular uptake, leading to the production of uricase with higher activity. The effect of inducer concentration on the production of uricase was studied and the results are shown in the Figure 3. At a concentration of 2 g=L uric acid, maximum uricase activity of 10 U=mL was observed. A further increase in the uric acid concentration reduced the uricase activity, which could be attributed to substrate inhibition by the enzyme. Similar results were reported in the literature in terms of optimal uric acid (inducer) concentration for different microbes like Microbacterium sp. (0.3 g=L),[12] Bacillus thermocatenulatus (0.3 g=L),[13] Proteus vulgaris 1357 (0.2 g=L), Proteus vulgaris B-317-C (0.15 g=L), and 1 g=L for both Streptomyces graminofaciens and Streptomyces albidoflavus.[14] Effect of Various Carbon and Nitrogen Sources Carbon and nitrogen are the vital sources for the growth of the microorganism and also influences the production of uricase. Figure 4 and Figure 5 show the effect of various carbon and nitrogen sources on the TABLE 1 Gram Characteristics and Uricoltyic Activities of the Isolates Bacterial Isolate
Gram Character and Shape
DL 1 DL 2 DL 3
Gram-negative cocci Gram-negative small rods Gram-positive rods
Uricase Activity (U=mL) 2.89 4.67 8.96
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FIGURE 2 Time course of uricase production.
production of uricase in terms of its activity. It has been observed that carboxymethylcellulose, maltose, and lactose have a considerable positive effect on the uricase activity in comparison to sucrose, whereas the other carbon sources like glucose, glycerol, and starch did not enhance the uricase production. Further, it was also observed that the carbon sources have slight effect on biomass. Similarly, the uricase production and biomass were significantly affected by the presence of nitrogen sources, as shown in Figure 5. In this study, four organic and four inorganic nitrogen sources were used, among which organic nitrogen sources were found to have higher influence on uricase and biomass production compared to the inorganic nitrogen sources. In particular, maximum uricase activity was achieved with asparagine as a nitrogen source. Yeast extract and ammonium nitrate yielded maximum biomass with reduced uricase activity. The observations agree with the findings by Khucharenphaisan and Sinma in 2011,[15] Lofty in 2007,[13] and Zhou et al. in 2005[12] on uricase-producing bacteria.
FIGURE 3 Effect of inducer concentration on uricase activity.
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FIGURE 4 Effect of different carbon sources on biomass formation and uricase activity.
Effect of Initial pH of the Production Medium Figure 6 indicates the effect of initial pH of the production medium on uricase activity. The medium consisted of maltose and asparagine as carbon and nitrogen sources, respectively, and with an inducer concentration of 2 g=L. It has been observed that the maximum uricolytic activity of 13.88 U=mL was achieved at an initial pH of 7.0. The results are in concordance with the findings of other researchers on the optimal initial pH, like that of Bacillus thermocatenulatus at an initial pH of 7.0,[13] Micrococcus roseus at pH 7.5,[16] Saccharopolyspora sp at pH 7.0,[15] and Microbacterium sp. at pH 7.5.[12] Effect of Inoculum Size on Uricase Activity Figure 7 shows the effect of inoculum size on the uricase activity. As the inoculum size was increased from 2% to 5%, uricase activity increased from 7.34 U=mL to 12.65 U=mL. Further increase in the inoculum size reduced the uricase activity, but resulted in an increase in biomass. It could be
FIGURE 5 Effect of different nitrogen sources on biomass formation and uricase activity.
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FIGURE 6 Effect of initial pH of the production medium on uricase activity.
concluded that uricase production is biomass independent. The reduction in the uricase activity at higher inoculum size could be due to the fast depletion of micronutrients in the production medium. A similar observation was made by Chen et al. in 2008,[17] while working with a recombinant Hansenula polymorpha strain harboring Candida utilis uricaseencoding gene. They have concluded that the uricase production is biomass independent and the optimum inoculum size as 6%. Table 2 indicates previously reported uricase-positive bacterial species and their uricolytic activities. Determination of Optimum pH and Temperature for Uricase Activity Figure 8 shows the effect of pH on the stability of uricase in terms of its activity. The enzyme produced by Bacillus cereus strain DL3 showed maximum stability at pH 8.5 in a 0.1 M borate buffer at 30 C. At the optimal pH of 8.5, uricase from Bacillus cereus strain DL3 retained a maximum
FIGURE 7 Effect of inoculum size on uricase activity.
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TABLE 2 Previously Reported Uricase Positive Bacterial Species
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Bacterial Species Bacillus thermocatenulatus Saccharopolyspora sp. Microbacterium sp. Pseudomonas aeruginosa Enterobacter cloaca Arthobacter globiformis Micrococcus luteus Proteus vulgaris Streptomyces graminofaciens Pseudomonas sp. Streptomyces sp. Corynebacterium uratoxidans Streptomyces cyanogenus Bacillus cereus DL3
Uricase Activity
Reference
1.25 U=mL 0.21 U=mL 1.0 U=mL 7.1 U=mL 6.6 U=mL 22 U=mg 1 U=mL 8.7 U=mg 8 U=mg 9 mU=ml 2.25 U=g wet cells 1.2 U=mL 12.3 U=mg 15.43 U=mL
[12] [14] [15] [18] [19] [20] [21] [14] [14] [22] [23] [24] [25] Present study
FIGURE 8 Effect of pH on uricase activity.
FIGURE 9 Effect of temperature on uricase activity.
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activity of 13.2 U=mL in a borate buffer for about 1 hr. It was also observed that at pH 8.0 and 9.0the enzyme still retained 12.45 U=mL and 11.64 U=mL of activity, respectively. Figure 9 indicates the effect of temperature on the stability of uricase, and it was found that uricase thus produced was not thermostable. It retained its maximum uricolytic activity at 30 C, when incubated in borate buffer of pH 8.5 for 1 hr. These conditions of pH and temperature can be used for the storage of uricase from Bacillus cereus strain DL3. Khucharenphaisan and Sinma (2011)[15] have reported the optimal pH and temperature for uricase from Saccharopolyspora sp to be 8.5 and 37 C, respectively, in a 50-mM borate buffer. CONCLUSION Three bacterial species capable of producing extracellular uricase were isolated from poultry deep litter and excrement samples. The bacterial species producing extracellular uricase with maximum uricase activity was identified to be Bacillus cereus strain DL3 through a 16SrRNA gene sequencing procedure. The time-course studies for uricase production were performed and it was observed that the uricase production was not biomass dependent and has an activity of 9.55 U=mL at 20 hr of incubation in the lag phase. Process parameters like initial medium pH, inducer concentration, various carbon and nitrogen sources, and inoculum size were optimized. Optimum initial medium pH was found to be 7.0, inducer concentration to be 2.0 g=L, and inoculum size to be 5%. Carboxymethylcellulose and asparagine were found to be the best carbon and nitrogen sources, respectively, for Bacillus cereus strain DL3. At the optimal conditions just described, maximum uricase activity was observed to be 15.43 U=mL, which is 1.63 times higher than the activity of the nonoptimized process. REFERENCES 1. Brogard, J.M.; Counaros, D.; Frankhauser, J.; Stahl, A.; Stahl, J. Enzymatic Uricolysis: A Study of the Effect of Fungal Urate Oxidase. Eur. J. Clin. Biol. Res. 1972, 17, 890–895. 2. Wu, X.; Wakamiya, M.; Vaishnav, S.; Geske, R.; Montgomery, C.; Jones, P.; Bradley, A.; Caskey, T. Hyperuricemia and Urate Nephropathy in Urate Oxidase Deficient Mice. Proc. Natl. Acad. Sci. USA 1994, 91, 742–746. 3. Cammalleri, L.; Malaguarnera, M. Rasburicase Represents a New Tool for Hyperuricemia in Tumor Lysis Syndrome and in Gout. Int. J. Med. Sci. 2007, 4(2), 83–93. 4. Pui, C.H.; Jeha, S.; Irwin, D.; Camitta, B. Recombinant Urate Oxidase (Rasburicase) in the Prevention and Treatment of Malignancy-Associated Hyperuricemia in Pediatric and Adult Patients: Results of a Compassionate-Use Trial. Leukemia 2001, 15, 1505–1509. 5. Adamek, V., Kralova, B.; Suchova, M.; Valentova, O.; Demnerova, K. Purification of Microbial Uricase. J. Chromatogr. A 1989, 497, 268–275. 6. Fatma, A. An Amperometric Biosensor for Uric Acid Determination Prepared From Uricase Immobilized in Polyaniline-Polypyrrole Film. Sensors 2008, 8, 5492–5500.
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7. Colloc’h, N.; Hajji, M.E.; Bachet, B.; L’Hermite, G.; Schiltz, M.; Prange, T. Crystal Structure of the Protein Drug Urate Oxidase-Inhibitor Complex at 2.05A˚ Resolution. Nat. Struct. Biol. 1997, 4, 947–952. 8. Nakagawa, S.; Oda, H.; Anazawa, H. High Cell Density Cultivation and High Recombinant Protein Production of Eschericia coli Strain Expressing Uricase. Biosci. Biotechnol. Biochem. 1995, 59, 2263–2267. 9. Tinsley, J.; Novakowski, T.A. The Composition and Manorial Value of Poultry Excreta, Straw Dropping Composts and Deep Litter. Exp. J. Deep Litter. J. Sci. Food. Agric. 1959, 10, 224–232. 10. Schefferle, H.E. The Decomposition of Uric Acid in Built Up Poultry Litter. J. Appl. Bacteriol. 1965, 28, 412–420. 11. Mahler, H., Hubscher, G.; Baum, H. Studies on Uricase: I. Preparation, Purification and Properties of a Cuproprotein. J. Biol. Chem. 1955, 216, 625. 12. Zhou, X.; Ma, X.; Sun, G.; Li, X.; Guo, K. Isolation of a Thermostable Uricase Producing Bacterium and Study on Its Enzyme Production Conditions. Process Biochem. 2005, 40, 3749–3753. 13. Lotfy, W.A. Production of a Thermostable Uricase by a Novel Bacillus thermocatenulatus Strain. Bioresource Technol. 2008, 99, 699–702. 14. Azab, A.E.; Magda, M.A.; Mervat, F.F. Studies on Uricase Induction in Certain Bacteria. Egypt. J. Biol. 2005, 7, 44–54. 15. Khucharoenphaisa, K.; Sinma, K. Production and Partial Characterization of Uric Acid Degrading Enzyme From a New Source Saccharopolyspora sp. Pak. J. Bio. Sci. 2011, 14(3), 226–231. 16. Olivieri, R.; Eugenio, F.; Pierluigi, C.; Ludwig, D. Uricase Production Method. U.S. Patent 4,389,485, 1983. 17. Chen, Z.; Wang, Z.; Xiuping, H.; Xuena, G.; Weiwei, L.; Borun, Z. Uricase Production by a Recombinant Hansenula polymorpha Strain Harboring Candida utilis Uricase Gene. Appl. Microbiol. Biotechnol. 2008, 79(4), 545–554. 18. Yasser, R.A.F.; Saeed, H.M.; Goharc, Y.M.; El-Bazb, M.A. Improved Production of Pseudomonas aeruginosa Uricase, by Optimization of Process Parameters Through Statistical Experimental Designs. Process Biochem. 2005, 40, 1707–171. 19. Machida, Y.; Nakanishi, T. Purification and Properties of Uricase From Enterobacter cloacae. Agric. Biol. Chem. 1980, 44, 2811–2815. 20. Nobutoshi, K.; Keisuke, S.; Takao, M.; Masaki, T.; Hitoshi, K.; Kazue, T. Chemiluminometric Determination of Uric Acid in Plasma by Closed Loop FIA With a Coimmobilized Enzyme Flow Cell. Anal. Sci. 2000, 16, 1203–1205. 21. Snoke, R.E.; Huge, A.R.; Richey, C.T. Production of Uricase From Micrococcus luteus. U.S. Patent 4,062,731, 1977. 22. Bachracuh, U. The Aerobic Breakdown of Uric Acid by Certain Pseudomonads. J. Gen. Microbiol. 1957, 17, 1–11. 23. Watanabe, Y.; Juichiro, Y.; Mayumi, F. Studies on the Formation of Uricase by Streptomyces. Agric. Biol. Chem. 1969, 33(9), 1282–1290. 24. Sugisaki, Z.; Norihiko, W.; Hirokazu, K. Process for Producing Uricase. U.S. Patent 3,767,533, 1973. 25. Ohe, T.; Watanabe, Y. Purification and Properties of Urate Oxidase From Streptomyces cyanogenus. J. Biochem. 1981, 89(6), 1769–1776.
