This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Introduction
Uricase, a peroxisomal (oxidoreductase) enzyme, catalyzes the oxidative opening of the purine ring in the urate pathway to yield allantoin, hydrogen peroxide, and carbon dioxide (Roman 2023). Uricase is important for the biochemical diagnosis of uric acid in the serum and biological fluids (urine). Biosensors can readily detect uric acid more precisely and accurately than other methods (Aafaria et al. 2022). It also alleviates the accumulation of toxic urate during various diseases (hyperuricemia, bedwetting, and gout). Uricase absence in some individuals could be attributed to metagenes, which prematurely terminate the translation process (Roman 2023). Direct urate oxidase injection is also suggested to treat renal complications-associated gout and prevent chemotherapy-linked hyperuricemia disorders (Cho et al. 2023). Microbes, animals, and plants can produce uricase. However, microbial production offers higher growth rates, cost-effective bioprocessing, and convenient optimization of the medium (Wan et al. 2023). The reported microbial uricase enzymes mainly include intracellular enzymes released via cell disruption. Therefore, extracellular enzyme production is crucial to reduce yielding time and purification. This review elaborates on the uricase-producing microorganisms, bacteria-based uric acid degradation pathway, uricase activity affecting factors, microbial production and purification, and its applications. Cell disruption is mandatory for intracellular uricase synthesis and purification, which enhances the production cost. Identifying extracellular uricase-synthesizing microbial strains and optimizing conditions is highly advisable. Moreover, uricase gene-carrying recombinant probiotic microbes could emerge as an efficient gout treatment strategy.
Uric acid: chemical structure and biosynthesis
Uric acid (C5H4N4O3) is a heterocyclic compound of carbon, hydrogen, nitrogen, and oxygen (Fig. 1). It forms different salts and ions such as acid urates, urates, and ammonium acid urate (El Ridi et al. 2017).
Fig. 1.
Chemical structure of uric acid.
The liver and small intestine produce heterocyclic uric acid (7,9-dihydro-1H-purine-2,6,8(3H)-trione) that has a molecular weight of 168 Da. The diet contains a low urate concentration, whereas food purines are the main source of uric acid synthesis by producing new purine bases or internal breakdown of purine bases (Fauci et al. 2012). Hepatic uric acid generation and intestinal and renal excretion depend on multiple variables involving complicated metabolic mechanisms. Different enzymes can convert purine nucleic acids (guanine and adenine) into uric acid (Chaudhary et al. 2013). Two types of mechanisms initially convert Adenosine monophosphate (AMP) into inosine such as (a) deaminase-based removal of amino group to form inosine monophosphate (IMP) followed by nucleotidase-based dephosphorylation to generate inosine nucleoside, or (b) nucleotidase-based removal of phosphate group to form adenosine followed by deamination to produce inosine nucleoside. Nucleotidase also transforms guanine monophosphate (GMP) into guanosine nucleoside. Then, xanthine-oxidase (XO) oxidizes hypoxanthine to form xanthine, whereas guanine deaminase deaminates guanine to form xanthine. Xanthine oxidase further oxidizes xanthine into the final product known as uric acid. Fig. 2 demonstrates the enzymatic pathway of purine degradation. Uric acid is a weak acid at physiologic pH with a pKa value of 5.8. Uric acid mostly exists in the form of urate salt (Jin et al. 2012), and its crystal formation is enhanced with the high blood concentration of urate.
Uric acid, a urine component, is a metabolic breakdown product of purine nucleotides (guanine and adenine) and a derivative of proteins. Uric acid is a dominant natural antioxidant plasma factor that activates immunity responses in certain illnesses; however, gout and joint infections (chronic and acute) are associated with uric acid. High concentrations of blood uric acid can cause gout and other medical complications such as diabetes and kidney stone (ammonium acid urate) formation. The liver is the main uric acid-producing organ, along with the intestinal wall, endothelium of kidneys, and blood vessels (Yeum et al. 2004). Uric acid plays important physiological functions, and the body re-absorbs almost 90% of uric acid (Maiuolo et al. 2016; Roman 2023). Antioxidant activity is an essential feature of uric acid that could eliminate half of the blood plasma’s free oxygen radicals (ROS) (Sautin and Johnson 2008; Roman 2023). Recent reports have highlighted uric acid-based initiation of inflammatory processes to facilitate tissue repair in addition to ROS removal (Nery et al. 2015). Lower blood uric acid levels might lead to mutations in renal carriers and blood cells (Sugihara et al. 2015). Contrarily, some studies have linked excess uric acid with kidney and cardiovascular diseases (Oberbach et al. 2014; Hammad et al. 2015; Roman 2023).
Uric acid is also known to protect the nervous system against various diseases. Autoimmune diseases such as lichen planes, Parkinson’s disease, and pemphigus vulgaris are linked to lower uric acid levels (Bakhtiari et al. 2017; Kuwabara et al. 2023). Excessive protein intake or alleviated kidney-based excretion of uric acid results in increased uric acid levels in the blood (Bobulescu and Moe 2012; Xu et al. 2017). Higher uric acid levels damage the kidney’s surface cells to cause their weak physiological activity, leading to chronic kidney diseases, particularly in Type 2 diabetes patients and individuals suffering from chemical intensification diseases (Xiao et al. 2015; Kim et al. 2015; Kuwabara et al. 2023). The uric acid accumulation in kidneys commonly results in kidney stone formation (Fathallah-Shaykh and Cramer 2014; Jalal 2016). Higher uric acid levels could also stimulate some autacoids and hormones to cause high blood pressure, whereas infiltration into smooth heart muscles leads to various cardiovascular illnesses (Kanbay et al. 2013; Kuwabara et al. 2023).
Uric acid degrading microorganisms
The first microbial uricase was isolated from a fungus (Neurospora crassa) in 1957 (Perez-Ruiz et al. 2014). Later, it was discovered that various bacteria could degrade uric acid as well including Pseudomonas aeruginosa, Bacillus thermocatenulatus, Microbacterium sp., Arthrobacter globiformis, Nocardia farcinica, Escherichia coli, Bacillus subtilis, and Bacillus fastidious (Saeed et al. 2004; Suzuki et al. 2004; Zhou et al. 2005; Lotfy 2008; Xu et al. 2022; Chen and Li 2023). The uricase enzyme production in Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Alcaligenes, and Bacillus thermocatenulatus is extracellular whereas its production is intracellular in Microbacterium, Proteus vulgaris, Streptomyces albidoflavus, Streptomyces graminofaciens, and Streptomyces exofolitus (Zhou et al. 2005; Ali et al. 2013; Chen and Li 2023). Lactic acid bacteria (Pediococcus species, Bifidobacterium, Leuconostoc, Lactobacillus, and Enterococcus) can degrade uric acid as their intake in rats/mice suppressed serum uric acid level (Ogawa 2006; Li et al. 2023; Negm El-Dein et al. 2023). Different soil fungi (Fusarium, Helminthosporium, Spondilocladium, Curvularia, Stemphylium, Aspergillus, Geotrichum, Penicillium, Mucor, Rhizopus, Alternaria, and Chaetomium) are capable of producing intracellular uricase at high rates, particularly in the presence of urea or uric acid as the sole nitrogen source (Geweely and Nawar 2011; Ali et al. 2013; Rajagopalan et al. 2017; Moradpour et al. 2022), Table I, lists some of the significant uricase-producing microorganisms.
The process of purine breakdown to uric acid is often conserved among organisms. However, uric acid degradation products could vary between species depending on the active catabolic enzymes, which can degrade or excrete uric acid in the peroxisomes (Lee et al. 2013). As a result of the degradation process, xanthine is the first intermediate product of all purine bases. Xanthine dehydrogenase degrades xanthine in the cytosol to generate urate. It is imported into the peroxisome and undergoes uricase-based oxidation to form 5-hydroxyisourate, which is converted to S-allantoin via 2-oxy-4-hydroxy-4-carboxy-5-ureidoi imidazoline by a functional allantoin synthase (Gabison et al. 2010). Urate oxidase evolution (allantoicase, uricase, and allantoinase) might be the reason behind varying degradation end-products of uric acid in different microorganisms. Most microorganisms can completely break down uric acid to ammonia through nitrogen catabolic enzymes (Marzluf et al. 1997). Allantoate amidinohydrolase (allantoicase) in certain bacteria and fungi hydrolyze allantoate to generate s-ureidoglycolate and urea (Marzluf et al. 1997) (Fig. 3).
Fig. 3.
The varying end products of purine metabolism in different species due to differential catabolic enzymes in the pathway (Lee et al. 2013).
Uric acid degrading enzymes
Uricase enzymes (protein) possess the features of cofactors, they catalyze various biochemical reactions, and their deficiency could lead to different diseases. Uricase is often used in biochemical diagnosis, blood uric acid detection, and industrial processes (El Ridi et al. 2017; Tandon et al. 2021). Therapeutic enzymes differentiate from other drugs due to their relationship with pathogens, such as activating or inhibiting a specific reaction and direct association with the diseasecausing substrate via its deposition in the body (Meletis and Barker 2005). Therapeutic enzymes could be biologically extracted from fungi, plants, and bacteria or synthesized in the laboratory (Babashamsi et al. 2009).
Therapeutic uricase (urate oxidase) is generally not detected in humans. However, an RNA study has revealed uricase production in human liver cells (Kratzer et al. 2014). Uricase (urate oxidase, EC 1.7.3.3, oxidoreductase) carries out purine metabolism and activates uric acid oxidation into soluble allantoin. It is present in most vertebrates except higher apes and humans, where it became non-functional due to point mutation during evolution and formed a redundant protein (Wu et al. 1989). Uricase is localized in various microorganisms, including Proteus mirabilis, Escherichia coli, and Bacillus pasteurii (Cheristians et al. 1986; Rando et al. 1990; Nakagawa et al. 1996). Microbacterium, Candida tropicalis, Pseudomonas aeruginosa, Streptomyces albosriseolus, and Bacillus thermocatenulatus can produce extracellular uricase after the optimization of culture media (Zhou et al. 2005; Abdel-Fattah et al. 2005; Lofty 2008). Uricase enzyme, containing four subunits, attracts and converts uric acid to hydrogen peroxide and allantoin through four identical type 2 copper binding sites (Fig. 4) (Wu et al. 1989). Several other enzymes, including xanthine oxidase, can also degrade uric acid by inhibiting a uric acid pathway reaction (hydrolysis). Xanthine oxidase is combined with different drugs for an efficient uric acid analysis (Li et al. 2005).
Pseudomonas and other probiotics lactic acid bacteria produce other uric acid degrading proteins (Kanmani et al. 2013). Uricase catalyzes the in-vivo uric acid oxidation to generate CO2 and allantoin in the presence of oxygen. The reduction of oxygen could also produce hydrogen peroxide (Fig. 5). Different types of thermostable microbial uricase enzymes are used in uric acid detection, which can sustain a wide range of pH (5, 6, 8, and 9) (Li et al. 2005; Ravichandran et al. 2015).
Fourteen functional genes have been discovered, which encode enzymes/proteins of the purine catabolic pathway. Xanthine dehydrogenase functioning requires the expression of five genes (pucE, pucD, pucC, pucB, and pucA), whereas two genes (pucM and pucL) encode uricase, and two genes (pucK and pucJ) encode uric acid transport system. The pucI, pucH, and pucF genes encode allantoin permease, allantoinase, and allantoate amidohydrolase. During a study, the pucR-mutant Bacillus subtilis expressed the lowest activity among all tested genes, indicating that PucR regulates puc gene expressions (Hafez et al. 2017). All 14 genes except pucI are located in a chromosomal gene cluster at 284–285° and participate in six transcription units. Uric acid, allantoic acid, and allantoin compounds regulate PucR for puc genes’ expression (Argyrou et al. 2001). The utilization of uric acid initiates virulence factors (urease and capsule) synthesis in fatal meningitis-associated Cryptococcus neoformans that potentially regulate the host’s immune response during the infection. Uricases (aquatic vertebrate and microbial) are mostly soluble and are found in bacterial cytoplasm or yeasts’ peroxisome (Kratzer et al. 2014).
Uricase activity affecting factors
Multiple factors affect uricase activity, mainly pH and temperature. Ravichandran et al. (2015) reported an optimum pH (8) and temperature range (25–45°C) for uricase activity. They further noted an almost 50% decrease in uricase activity at 60°C after exposure for one hour (Chohan and Becker 2009). Contrarily, Geweely and Nawar (2011) have reported an optimum temperature of 35°C for Aspergillus niger-based uricase. Heavy metals serve as cofactors for some enzymes, but their large quantities could also inhibit enzyme activities (Suzuki et al. 2004; Witkowska et al. 2021). Nelson (2005) reported copper-based uricase inhibition, but Ravichandran et al. (2015) noted enhanced (140%) enzyme activity in response to copper stimulation. Chohan and Becker (2009) revealed enzyme stimulation by ethylenediaminetetraacettic acid (EDTA) (1 molar), whereas the same concentration inhibited the activity of ERW. Thus, unknown elements could contribute to enzyme activation and inhibition, varying in different bacteria (Ravichandran et al. 2015).
Uricase production and purification
Different carbon and nitrogen sources in the culturing media of uricase-producing microorganisms can influence uricase production. Uricase production with different carbon sources can be arranged as sucrose > glucose > cellulose > starch > maltose. Peptone is known for its higher uricase yield than other nitrogen sources (yeast extract, beef extract, and ammonium nitrate) (Pfrimer et al. 2010).
Extracellular uricase
Microorganisms and higher plants produce more uricase, whereas humans cannot produce this enzyme, which leads to purine breakdown-associated uric acid accumulation in the body (Hafez et al. 2017). Uricase alleviates hyperuricemia, whereas plant or human uricase stimulates immune responses (Roman 2023). Therefore, bacterial uricase is a therapeutic agent that removes excessive uric acid from the body (Abdel-Fattah et al. 2005). Proteus mirabilis, Bacillus pasteurii, and Escherichia coli are known to secrete uricase enzymes (Rando et al. 1990; Nakagawa et al. 1996; Hafez et al. 2017). Pseudomonas aeruginosa, Tropical Candida, Thermobacilli, Albosriseolus, and Microbacterium have been reported to yield extracellular uricase in the optimized media (Zhou et al. 2005; Abdel-Fattah et al. 2005; Anderson and Vijayakumar 2011). Extracellular uricase activity was assessed on a solidified medium by following the agar plate assay method, in which uric acid served as an inciting agent. The uric acid screening medium was comprised of sucrose (20 g/L), magnesium sulfate heptahydrate (0.5 g/L) sodium chloride (0.5 g/L), uric acid (3 g/L), di-potassium hydrogen phosphate (1 g/L), agar (15 g/L), and ferrous sulphate (0.01 g/L) (El-Naggar et al. 2019). The pH was adjusted to 6.8, and plates were incubated for 5–7 days at 30°C. A clear zone around the colony confirmed uricase production (Fig. 6).
Fig. 6.
A clear zone indicating Alcaligenes faecalis-secreted uricase on uric acid (0.3%) – supplemented BT medium.
Intercellular uricase
Fungal or bacterial growth is not directly associated with uric acid production. However, different fungi and bacteria can utilize uric acid as the only source of nitrogen (Baumgardner 2016). During a study, a starter culture of lactic acid bacteria (1%) was grown in uric acid (0.2%)-supplemented PGY broth to obtain intracellular uricase. The culture was incubated at 37°C for 24 hours. After fermentation, centrifugation (3000 rpm, 20 minutes) was carried out at 4°C to separate the supernatant, followed by bacterial cell-based stability testing of intracellular uricase (Carevic et al. 2015). Intracellular uricase production was noted in Microbacterium spp., S. albidoflavus, P. vulgaris, and S. graminofaciens (Zhou et al. 2005; Azab et al. 2005). The addition of uric acid into the growth media induced the uricase production. However, various influencing factors (heavy metals, temperature, and pH) are needed to disturb the cell to obtain intracellular uricase (Bongaerts et al. 1978). Generally, intracellular uricase production in the gastrointestinal system is considered more stable (O’Connel and Walsh 2007; Pugin et al. 2022).
Uricase purification from different microorganisms
Uricase purification is necessary to achieve its higher yield, which has been carried out through various approaches.
Precipitation by ammonium sulfate
Different concentrations of ammonium sulphate (20, 40, 60, and 70% w/v) can be used to separate uricase from secreted proteins-containing supernatant. Briefly, solid ammonium sulphate is slowly added to the culture filtrate on an ice bath with gentle stirring until the required ammonium sulphate saturation is reached. Then, the mixture is left overnight at 4°C followed by centrifugation at low temperature (8000 rpm, 30 minutes, 4°C) (Saeed et al. 2004; Ram et al. 2015).
Removal of ammonium sulfate salts
The Removal of ammonium sulphate is performed by dissolving the precipitate in Tris-HCl (0.01 M, 10 ml) buffer (pH 8.5). The solution is dialyzed overnight in ultra-pure distilled water/buffer (1 L) using a dialysis tube. Then, the concentrated dialyzed cell-free supernatant is subjected to the column chromatography technique (Saeed et al. 2004; Ram et al. 2015).
Uricase purification through ion exchange chromatography on DEAE-cellulose
A DEAE-cellulose-containing column is equilibrated with Tris-HCl (10 mM) buffer (pH 8.5). Dialyzed and concentrated cell-free supernatant is applied to it for uricase purification. The column is washed thrice with the Tris-HCl (10 mM) buffer (pH 8.5). The bound proteins are eluted in the same buffer with a linear NaCl gradient (0–0.3 M), and collected fractions are analyzed at 280 nm using a UV spectrophotometer, whereas enzyme activity and protein concentration are detected at 293 nm (Saeed et al. 2004; Ram et al. 2015).
Uricase purification with gel filtration column
Potassium phosphate (50 mM) buffer (pH 8.2) is used to equilibrate the Superdex 200 HR-containing gel filtration column (Amersham Pharmacia Biotech, Germany). Ammonium sulfate-based partially purified uricase is dialyzed and applied to this column. The same buffer is used to elute uricase, and all fractions (0.5 ml) with high uricase activity are concentrated using an ultrafiltration membrane (YM 10) or following the lyophilized method. The concentrated fractions are stored at -20°C (Jianguo et al. 1994; Saeed et al. 2004; Ram et al. 2015). According to Laemmli et al. (1970), the molecular weight of the purified enzyme should be determined by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) under denaturation conditions to verify the purification stages of uricase. The determination of uricase’s molecular weight involves comparing its electrophoretic mobility with that of marker proteins.
Applications of uricase
Uricase (Urate Oxidase, EC 1.7.3.3) is a diagnostic enzyme to measure uric acid (urate) levels in the body. It also synthesizes medicines (pegloticase and rasburicase) for hyperuricemia treatment (Yang et al. 2012). Uricase is crucial for the human body as it disintegrates uric acid into allantoin, carbon dioxide, and hydrogen peroxide through oxidation. Generally, uric acid is excreted through the kidneys, but its blood solubility is extremely low (6.8 mg/L) (Ravichandran et al. 2015; Mei et al. 2022). Higher blood uric acid levels are associated with uric acid nephrolithiasis, gout, cardiovascular disease, hyperuricemia, diabetes, renal failure, and tumor lysis syndrome (Ganson et al. 2005; Roman 2023). The uricase enzyme remains inactive in humans because of frameshift mutation during evolution. Therefore, uricase synthesis from other sources is vital to counter associated disorders (Pawar et al. 2018).
Diagnostic role of uricase in clinical analysis
There are diverse applications of uricase enzyme, but its most important role is treating uric acid accumulation-related illnesses (nervous system, heart) (Hafez et al. 2017). It is commonly applied to assess blood uric acid levels in the blood. Moreover, it is also combined with a 4-amino-antipyrine-peroxidase system to determine uric acid levels in other biological fluids (Cheung et al. 2020). Rasburicase is frequently used to treat organ transplants and tumor lysis-associated hyperuricemia. Uricase is also a common additive of commercial hair coloring agents (Cheung et al. 2020).
Biosensor for bimolecular applications
Monitoring uric acid levels in urine and blood is necessary for the disease diagnosis. There are different methods of uric acid estimation, such as mass fragmentography, enzyme electrode, colorimetry, radiochemical-HPLC, fluorescent sol-gel, commercial uric acid kits, and chemiluminescence (Domagk and Schlicke 1968; Martinez-Pérez et al. 2003; Zhang et al. 2004; Bio-Assay Systems 2007; Chu et al. 2012). The colorimetric method is a simple, specific, and sensitive approach that employs peroxidase and uricase (Zhou et al. 2005). However, these enzymes are expensive, which makes this assay more costly than other methods. Different biosensing procedures have also been devised, which involve uricase immobilization on electrode surface using ZnO nanorods, polyaniline-polypyrrole film, polyaniline, ZnO nano-flakes, and polypyrrole nano-electrode (Uchiyama and Sakamoto 1997; Zhang et al. 2004; Arora et al. 2007; Arslan 2008; Yang et al. 2012)
A transducer converts the energy alterations during the interaction of biological elements (protein, antibody, and enzyme) and an analyte into a quantifiable signal (Ravichandran et al. 2015). Modern miniature microelectronics are featured with lower cost, better processing power, and enhanced analytical efficiency, which broaden their potential applications. Cellular interactions, enzymatic contacts, antibody-antigen interactions, nucleic acid connections, and artificial bioreceptor-based interactions are common biological recognition elements. Mass-sensitive, optical, and electrochemical transducers are frequently utilized for signal quantification (Javadi et al. 2018).
Agricultural applications of uricase
Biological compounds are known to enhance the quality of agricultural commodities. Soil microorganisms release vital secretions for better soil fertility, however, these microbial secretions could be hindered by different environmental factors (Javadi et al. 2018; Imran et al. 2021). Therefore, producing fungal and bacterial enzymes has been investigated to improve soil conditions and agricultural production at a reduced cost. The free uricase enzyme is utilized as calcium carbonate precipitate in the soil to promote soil mechanics by initiating urea breakdown (Hamdan et al. 2013).
Uricase-based detection of heavy metal water contamination
Heavy metals-containing toxic compounds are known to reduce enzyme activity, which is often used as a parameter to detect heavy metal contamination in water. The activity of uricase also decreases at varying levels in the presence of Hg2+ > Ag’+ > Cu2+ > Ni2+ > Cd2+ > Zn2+ > Co2+ > Fe2+ > Pb2+ > Mn2+. Therefore, it is a toxic compound detector in water samples (Zhylyak et al. 1995).
Uricase application in nanomaterial manufacturing
The importance of nanomaterials has significantly increased with diverse medical, agricultural, and industrial applications. Therefore, producing environment-friendly, low-cost, and stable industrial nanomaterials is being widely investigated worldwide. In this regard, enzyme applications in nanomaterial synthesis have become quite popular during the last decade (Durán et al. 2014; Adelere and Lateef 2016). Canavalia ensiformis-isolated uricase has produced Pt, Au, and Ag nanoparticles as a stabilizing and reducing agent. Similarly, the catalytic urease has been employed to synthesize core-shell ZnO nanomaterials at an ambient temperature. Exposed enzyme residue (Cys592) facilitates the synthesis of metal alloys and metallic nanoparticles (Sharma et al. 2013). During the process, the Zn2+ binds with negatively charged surface urease at pH 9 through a weak bond reaction to form intermediate zinc hydroxide. Further, zinc hydroxide dehydration under basic conditions yields ZnO on the precipitating enzyme’s surface through the ‘salting out’ effect (Makarov et al. 2002).
Conclusion and future perspectives
This review elaborates on uricase-producing microorganisms, bacterial uric acid degradation pathways, degrading enzymes, and uricase-encoding genes. Moreover, the uricase activity affecting factors, microbial uricase production, and uricase purification and applications are also discussed. Cell disruption is mandatory for intercellular uricase production, elevating production costs. Therefore, extracellular uricase-producing microbial strains should be investigated, and production factors should be optimized. Future techniques for obtaining extracellular enzymes should feature reduced time and effort and a simple purification methodology. Furthermore, uricase gene-carrying recombinant probiotic microorganisms could become an effective tool for gout treatment.
