Uricase (Urate oxidase, EC 18.104.22.168) catalyzes the oxidative opening of the
purine ring of urate to yield alantoin, carbondioxide and hydrogen peroxide.
It has vast and beneficial uses both in vitro and in vivo. Urate
is a final oxidation product of purine catabolism (Bertrand
et al., 2008). Determining the urate concentration in blood and urine
is required for the diagnostic of gout as urate accumulation. It is a causative
factor of gout in humans. Uricase is useful for enzymatic determination of urate
in clinical analysis by coupling with 4-aminoantipyrine-peroxidase system (Gochman
and Schmitz, 1971). It can be also used as protein drug for treatment of
hyperucicemia, as Rasburicase (Bomalaski and Clark, 2004;
Haidari et al., 2008).
Many organisms including plant and microorganisms are able to produced uricase.
To date, pure cultures of bacteria capable of producing uricase that have been
documented are only Pseudomonas aeruginosa (Frank
and Hahn, 1955), P. acidovorans (Sin, 1975),
Arthrobacter globiformis (Nobutoshi et al.,
2000), Bacillus subtilis (Hunag and Wu, 2004),
Nocardia farcinica (Ishikawa et al., 2004)
and Microbacterium sp. (Zhou et al., 2005;
Kai et al., 2008). But very limited research
has been directed towards uricase production from termite actinomycetes. The
genus Saccharopolyspora contains twenty described species. Saccharopolyspora
is a well know producer of macrolide antibiotic such as erythromycin and spinosad.
However, the production of uricase has not been reported.
The aim of the present study was to investigate the ability of Saccharopolyspora sp. PNR11 as a novel uricase producer. In addition, fermentation medium composition and relevant conditions were tested to optimized uricase enzyme productivity.
MATERIALS AND METHODS
This research project was conducted from January 2010 to December 2010 at Faculty of Science and Technology, Phranakhon Rajabhat University, Thailand.
Microorganism: Saccharopolyspora sp. PNR11 in this study was isolated from gut of termites in genus of Termes collected from Sakaerat Environmental Research Station in Nakhon Ratchasima province, Thailand. The culture was maintained on Yeast extract-Malt extract (ISP2) agar. Based on its partial 16S rDNA sequence and morphological characters, strain PNR11 was classified to the member of genus Saccharopolyspora.
Medium: The composition of pre-culture medium was 1% peptone, 1% glucose, 0.1% K2HPO4, 0.05% MgSO4•7H2O and 0.05% NaCl. The pH was adjusted to 7.0. For production medium, 1% uric acid was added into pre-culture medium and 1% maltose was used as a carbon source.
Culture condition for bacteria: The strain from a slant was transferred to a 500 mL Erlenmeyer flask containing 50 mL of sterilized pre-culture medium. It was then incubated in a rotary shaker operating at 180 rpm at 30°C for 48 h. At the end of incubation, 1 g of wet weight was transferred to another 500 mL Erlenmeyer flask containing 50 mL of production medium and used for the study of the fermentation condition. The uricase production was evaluated each 6 up to 36 h. Uricase activity and intracellular protein were performed. The data of experiments were analyzed by GNU PSPP Statistical Analysis Sofware Release 0.6.2.
Effect of carbon and nitrogen source: For the effect of carbon sources on uricase production, the 500 mL Erlenmeyer flasks were prepared containing 50 mL of the basal medium (1% peptone, 0.1% K2HPO4, 0.05% MgSO4•7H2O, 1% uric acid and 0.05% NaCl) supplemented with 1% of different carbon sources (maltose, glucose, galactose, fructose, lactose, sucrose, soluble starch and glycerol). The absent of carbon source was set for a control experiment. Uricase assay was performed after 24 h cultivation.
To detect the effect of nitrogen sources on uricase production, the 500 mL Erlenmeyer flasks were prepared containing 50 mL of the basal medium containing 1% of maltose as a carbon source and supplemented with 1% of different nitrogen sources (peptone, tryptone, corn steep liquor, yeast extract, ammonium acetate, sodium nitrate and potassium nitrate) in order to determine their influences in the uricase production. Uricase assay was performed after 24 h cultivation.
Determination of temperature and pH on uricase production: Effect of
temperature on the enzyme production was studied in the suitable production
medium from above experiment at different temperature ranging from 20 to 37°C.
The effect of pH was also studied by adjusting the pH of the production medium
to different level ranging from pH 5.0 to 9.0.
Preparation of crude intracellular uricase: The cells were collected by centrifugation at 13,000 rpm for 5 min and washed twice time with 0.85% NaCl. The cells were suspended in 10 volumes of wet basis of 50 mM borate buffer (pH 7.0) and treated by an ultrasonic device to lease the enzyme. It was then centrifuged at 13,000 rpm for 5 min; the supernatant was used for analysis of uricase activity.
Enzyme assay: Uricase activity was measured from cell-free extract. The assay mixture contained 0.5 mL of enzyme solution in 50 mM borate buffer (pH 8.5) and 0.01% uric acid in a final volume of 3.0 mL. Incubation was carried out at 37°C for 5 min. The reaction was terminated by the addition of 200 μL of 20% KOH. The absorbance was measured at 293 nm using spectrophotometer. As a control, the solution of KOH was added to substrate before the addition of the enzyme solution. One unit of enzyme was defined as the amount of enzyme necessary to transform 1 μmol of uric acid into allantoin in 1 min at 37°C
Protein determination: Protein was estimated by determination of intracellular
protein. Cell mass was separated from the medium, disrupted and released protein
was determined by the method of Lowry et al. (1951)
using Bovine Serum Albumin (BSA) as a standard for calibration.
Determination of optimum pH and temperature of uricase activity: Investigation to find out the optimum pH for uricase activity was carried out in 50 mM buffers with various pH values ranging from pH 4.0 to 10.0. Acetate buffer was used in the pH range of 4.0 and 5.0. Phosphate buffer was used for pH 6.0 and 7.0. Borate buffer was used for pH 7.0, 7.5, 8.0, 8.5 and 9.0. Glycine buffer was used for pH 10.0 and then incubated at 37°C for 5 min. Amount of uricase activity was determined. To determine the pH stability of the enzyme, the enzyme was incubated in different pH as mentioned above at 4°C for 10 min. The remaining uricase activity was measured under standard assay procedure.
The optimum temperature for uricase activity was determined by incubating the
enzyme in 50 mM borate buffer (pH 8.5) at various temperatures (20-55°C)
for 5 min. Amount of uricase activity was determined. For stability, those reactions
were pre-incubated at various temperatures for 10 min. The remaining uricase
activity was measured under standard assay procedure.
RESULTS AND DISCUSSION
Uricase production: The uricase activity was measured in both the cultural
supernatant as well as the intracellular fluid. No uricase activity was found
in the supernatant while uricase was detectable in biomass. The uricase production
level obtained from Saccharopolyspora sp. PNR11 was determined when it
was culture in the medium containing 1% peptone, 1% maltose, 0.1% K2HPO4,
0.05% MgSO4 7H2O, 0.05% NaCl and 1% uric acid. The uricase
production rapidly increased during the first 18 h of cultivation, yielding
150 mU mL-1 and then slowly increased until it reached the maximum
activity of 168 mU mL-1 after 24 h cultivation. The enzyme activity
was decreased to 130 mU mL-1 after 30 h cultivation (Fig.
1). The intracellular protein of cell mass was increased which was related
to the enzyme production. The cultivation time of Saccharopolyspora sp.
PNR11to reach the maximum enzyme production was shorter than that of Microbacterium
ZZJ4-1 for 12 h (Zhou et al., 2005). Therefore,
Saccharopolyspora sp. PNR11 was considered as an intracellular uricase
producer. Most of the microbial uricase from Microbacterium ZZJ4-1 and
Streptomyces cyanogenus origin (Zhou et al.,
2005; Kai et al., 2008; Yokoyama
et al., 1988) are intracellular and cell disruption is necessary
in order to obtain the enzyme. However, in some microbial resource such as Bacillus
fastidiosus (Bongaerts et al., 1978),
Mucor hiemalis (Yazdi et al., 2006) and Pseudomonas
aeruginosa (Saeed et al., 2004) extracellular
uricase with no need of cell disruption have been reported.
||Time course of uricase and intracellular protein produced
by the Saccharopolyspora sp. PNR11 cultured at 30°C in a medium
containing maltose as the carbon source
Effect of carbon and nitrogen source: The influence of different carbon
sources was studied and the results presented in Fig. 2a.
The effect of carbon sources showed that the uricase production was most affected
by the addition of maltose with activity of 168 mU mL-1 where it
activity was approximately 148 and 151 mU mL-1 higher than that of
control (production medium without carbon source) and glucose as a carbon source,
respectively. This indicated that glucose could not stimulate uricase productivity.
However, low level of uricase activity was detected in intracellular fluid due
to the present of uric acid as an inducer. Moreover, the result also showed
that uricase obtained from Saccharopolyspora sp. PNR11 was inducible
enzyme. Addition of maltose also affected on uricase production of Mucor
hiemalis with the activity of 67% higher than control (Yazdi
et al., 2006).
The production medium having maltose as carbon source was used for nitrogen
source optimization. The effect of nitrogen source, several inorganic and organic
nitrogen sources were evaluated (Fig. 2b). Result showed that
simple nitrogen source had less positive effect on the enzyme production. Among
the various complex nitrogen source, yeast extract partially increased enzyme
activities up to 216 mU mL-1 that was 76 and 48 mU mL-1
higher than that of control and peptone, respectively. However, the enzyme activity
was detected in control experiment containing uric acid alone without any other
nitrogen source. This result showed that uric acid not only inducer but also
served as a nitrogen source for uricase production. It has been reported that
the highest uricase induction levels for Candida utilis (Jianguo
et al., 1994) and Mucor hiemalis (Yazdi
et al., 2006) were obtained in the medium mainly containing uric
acid. Addition of corn steep liquor, sodium nitrate, potassium nitrate and ammonium
acetate did not enhance the enzyme production comparing to control. Most complex
nitrogen sources significantly increased enzyme production. In general, uricase
production was far more enhanced by using the organic nitrogen than the inorganic
nitrogen. It was possible that organic nitrogen may contains most kinds of amino
acids for the growth of bacterium that could be metabolized directly by cells,
consequently promoting the uricase production (Lotfy, 2008).
Therefore, uric acid itself was sufficient to induce the enzyme production.
Optimum pH and temperature for uricase production: The effect of initial
pH on uricase production was performed on a fermentation medium containing maltose
and yeast extract as a suitable C and N source, respectively.
|| Effects of carbon source (a) and nitrogen source (b) on uricase
production by Saccharopolyspora sp. PNR11
The enzyme production was observed at initial pH of 5.0 to 9.0. Uricase activity
was rapidly increased during pH 5.0 to 7.0 of cultivation and achieved to maximum
level at pH 7.0 with the activity of 216 mU mL-1 (Fig.
3a). The productivities were slowly decreased at alkali condition ranging
of pH 8.0 to 9.0 in which was 16 and 81 mU mL-1 lower than that of
the maximum activity, respectively. The optimum pH for uricase production of
this strain was higher than that of uricase produced by Mucor hiemalis
(Yazdi et al., 2006) but lower than that of the
enzyme produced by Microbacterium sp. ZZJ4-1 (Zhou
et al., 2005) and Gliomastix gueg (Atalla
et al., 2009).
The study on influence of temperature on enzyme production was carried out
at various temperatures in the production medium (pH 7.0) containing maltose
and yeast extract. Optimum temperature for uricase production of Saccharopolyspora
sp. PNR11 was 30°C with the activity of 216 mU mL-1 (Fig.
3b). Moreover, it was found that the production of enzyme severely decreased
to 180 mU mL-1 when the cultures were grown at 37°C. This result
was similar to uricase production by Gliomastix gueg (Atalla
et al., 2009). This indicated that less efficient on the enzyme production
was due to high temperature that is inheritance behavior of this strain. In
addition, the incubation at high temperature affected to reduce the enzyme production
might cause of less thermostability of the enzyme.
Optimization of pH and temperature on uricase activity and stability: Optimum pH for the uricase activity of Saccharopolyspora sp. PNR11was pH 8.5 and the activities gradually decreased when the pH raised (Fig. 4a). Notice that the relative activity at pH 8.0 and 9.0, activities of the enzymes still exhibited 84 and 60%, respectively. For storage, these enzymes had full activity when kept at 4°C for 10 min in a range of pH 7.0 to 10.0 as shown in Fig. 4a. Thus, this enzyme could storage without lose its activity at high pH that would protect them from bacteria.
Optimum temperature to exhibit its activities of uricase of Saccharopolyspora
sp. PNR11 was 37°C incubating for 5 min (Fig. 4b).
However, the enzymes exhibited low activities at over temperature of 45°C
and 20% of its optimum activity was remained when incubated at 55°C. This
enzyme was stable up to 30°C for 10 min (Fig. 4b). When
the enzyme was kept at 37°C, this enzyme was denatured rapidly. At 55°C,
its activities remained only 9% of original activity. The optimum pH and temperature
of this enzyme was closed to other uricase produced by Streptomyces sp.
THPN 58 that were in range of temperatures of 35°C and pH 8.5 (Khucharoenphaisan
and Sinma, 2010). It could be observed that the enzymatic activity was notably
dropped at high temperature of 45°C. Norcardia farcinica also has
been reported to produce non-thermal stable uricase and rapidly lost most of
it activity at 60°C (Schiavon et al., 2000).
||Effect of initial pH (a) and temperature (b) on uricase production
in 24 h by Saccharopolyspora sp. PNR11
||Effect of pH (a) and temperature (b) on crude uricase activity
and stability from Saccharopolyspora sp. PNR11. For the effect of
pH, the reaction mixture was incubated at 37°C with various buffers
and for the temperature analysis the sample were incubate at each temperature
for 5 min in 50 mM borate buffer pH 8.5.
In contrast, Microbacterium ZZJ4-1 was an excellent thermostable uricase
producer; its enzyme was stable at 65°C for 30 min (Kai
et al., 2008).
The actinomycetes, Saccharopolyspora sp. PNR11 produced an intracellular uricase. By optimizing cultural condition, the highest level of uricase activity attained at 216 mU mL-1. The optimum pH and temperature of this enzyme was pH 8.5 and 37°C, respectively. This is the first report on the production of uricase from Saccharopolyspora. This strain might be considered as a candidate source for uricase production in the further studies.
The authors wish to thank Ms. Chortip Lorrungruang for helpful English review of this manuscript. This research was supported by Institute of Research and Development Phranakhon Rajabhat University, Thailand.