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Uricase, which is an enzyme that is involved in the metabolism of purines, has recently attracted a lot of attention in modern medicine. This article offers a comprehensive and detailed overview of the use of uricase in different fields of medicine. It describes what uricase is and its function in the metabolic process regarding uric acid and its relevance in medicine. New directions in the application of uricase in the treatment of diseases such as gout are explained. Furthermore, the article discusses the concerns and issues that research and clinical applications face when introducing uricase and attempting to enhance its efficacy and security. The conclusion brings the focus to uricase for it to be emphasized as a therapeutic agent of the 21st century with development and clinical trials exploration being necessary.

Keywords

Uricase Enzyme, Therapeutic Potential, Modern Medicine, Hyperuricemia, Gout, Biomarker, Metabolic Syndrome, Clinical Implementation, Efficacy and Safety, Clinical Trials

Introduction

Brief Overview of Uricase and Its Medical Applications

The therapeutic enzyme urate oxidase (UOX), sometimes referred to as uricase, is absent from humans' bodies naturally and was lost along the course of evolution (Tandon, 2021). Uricase, also known as urate oxidase, serves as a vital oxidoreductase enzyme in the body, catalyzing the conversion of uric acid (UA), the end product of purine metabolism, into the highly water-soluble compound, allantoin. This enzymatic process is crucial for maintaining balanced uric acid levels in the body. However, in humans and certain primates, including humans, the gene responsible for uricase production has undergone evolutionary silencing, leading to the accumulation of uric acid and subsequent hyperuricemia. This condition, characterized by elevated uric acid levels, can result in the formation of monosodium urate crystals in the joints, a hallmark of the inflammatory disease known as Gout. While uric acid possesses antioxidant properties that may have conferred evolutionary advantages, its excessive accumulation in humans necessitates therapeutic interventions (Dudala, 2023).  

The treatment of Gout revolves around two primary 

approaches. Firstly, there is the breakdown of uric acid into a more soluble form, facilitating its excretion from the body. Alternatively, uric acid production can be regulated through drugs such as allantoin, although hypersensitivity to this treatment has been reported in some individuals with Gout. Efforts are therefore directed towards harnessing the enzymatic activity of uricase to efficiently metabolize uric acid into the more readily excretable allantoin, offering a promising therapeutic avenue for managing hyperuricemia and its associated complications (Khade 2016; Garay, 2012).




Importance of Biotechnological Methods in Enhancing Uricase Production

Simple biotechnological methods can be utilized to enhance uricase production, providing great opportunities for the therapy of HU and its related diseases, including Gout, by modifying microbial systems for maximum production of uricase. 

Biotechnological approaches, along with their manipulation, can also use genetic engineering to boost the expression of uricase in microbial hosts. Once genetic modifications, such as the addition of gene-amplifying plasmids or promoter sequences, can be introduced into microbial strains, researchers can create uricase-yield high strains. This can generate a selective screening for uricase-secreting strains between candidate strains. Consequently, a higher-yield uricase can be produced by a microbial host and, thus, benefit to uricase production processes in terms of scalability.

Except for microbial systems, the diverse expression systems that are being used for the production of uricase through a biotechnological approach include plant and fungal systems. These systems come with special features such as post-translational modifications and capacities to fold the particular proteins, which, in turn, may augment and strengthen the performance and stability of uricase. This way it is understood that researchers, by taking advantage of each specific expression system, can indeed extend their options in trying to produce uricase and find more efficient ways of using it in the treatment of gout and associated diseases.

Purpose and Scope

Purpose

In a way, this review article's primary goal is to give an in-depth account of how the involvement of biotechnology in the improvement of producing uricase. Uricase, an enzyme of primary importance in the metabolism of purines, is responsible for changing uric acid to allantoin, thereby creating solubility. It is a highly dynamic biochemical process and is of extreme therapeutic significance, especially in the treatment of hyperuricemia-connected diseases, including Gout. Though the production of uricase has always posed functional challenges, the conventional techniques of uricase production have been confronted with serious challenges such as low yield, inability to upscale, and high cost of production. 

Against this background, the review seeks to provide a focused yet comprehensive analysis of how biotechnological inventions have sought to address these challenges in the production of uricase. Novel approaches like recombinant DNA technology, fermentation technology, enzymatic engineering, and downstream processing have not only improved the yield and purification of uricase but also the technology developed is highly scalable. 

As the current and prior advancements have been discussed in this review, it is becoming easier to understand how biotechnological innovation is driving the development of uricase forward. It also delves into multiple stages of biotechnology at work within each approach, covering gene cloning and expression, selection of suitable host organisms, optimization of fermentation conditions, and the different enzyme engineering tactics, thus allowing the reader a perception of the principles behind the advanced techniques in the field. 

Additionally, the need to look at another potential impact of these innovations is an aim of this review, of course in addition to examining the uricase production specifically. 




Scope 

To this end, the present review article covers a broad research area that aims at providing an in-depth analysis of the historical background of uricase including the various existing and emergent biotechnological strategies for uricase production, new developments in downstream processing, the existing challenges as well as the future of uricase production. Effectively infusing quite a number of extant researched and produced biotechnological processes that have been developed over the decades, the review explores various methods of boosting uricase production. 

To begin, the discussion provides a historical background of uricase extraction and purification processes and thus, highlights some of the major difficulties encountered by researchers in the conventional manufacturing process and the complications associated with manufacturing on a larger scale. I am therefore cognizant of how it goes ahead to break down the complexity of the biotechnological processes of uricase production starting with basic gene cloning and expression procedures and moving to advanced enzyme engineering techniques. 

This review article is intended to cover every aspect of this enzyme with concise, yet profound descriptions of various aspects of uricase production that may be useful for researchers, clinicians, and industry professionals working on uricase-based therapies. In this regard, the review aims to discuss tactical advances in the field and highlight remaining challenges and potential development prospects in order to contribute to further development and productive work in the sphere of biotechnology.

Historical Background of Uricase Production

Early Methods of Uricase Extraction and Purification

The history of uricase yields a chronicle of discovery Born out of scientific inquisitiveness and unimaginable technological advancement. During the early exploration of uricase, which dates back to the middle of the 20th century, researchers undertook pursuits to extract the enzyme and purify it from diverse biological organisms. These pioneering achievements provided the foundations for more future developments in enzymology and biotechnology. 

The initial attempts in purifying uricase involved the use of uricase-producing organisms including some bacterial and fungal species that were grown under controlled substrate conditions using nutrient-rich broth. After microbial growth, crude enzyme extracts were collected from cell lysates or culture supernatants using procedures like cell disruption or centrifugation sessions. These crude extracts actually consisted of proteins and some of them were uricase but the crude extract that had the enzyme in question needed to be purified.

The liver tissues of the pig were homogenized and the extracts tested were derived from minced pig liver treated 

With acetone, followed by washing and extraction of proteins with phosphate and borate buffers. The collected powder was then treated with barium acetate for precipitation, followed by elimination of overall impurities with ammonium sulfate, and lastly, the steps of filtration and change in pH. Finally, the uricase was collected from samples after passing through several cycles of purification with desired levels of solubility in alkaline buffers. 

In an attempt to salvage active uricase from the crude extracts the following issues arose: Firstly, the response of the enzyme was generally low, and secondly the intrinsic presence of other cellular ingredients. In order to isolate uricase from the preparations, various methods such as precipitation techniques, chromatography, and dialysis were used by researchers. These purification steps were taken in order to obtain the uricase enzyme in its active conformation free from any other associated proteins and other cellular components.

Challenges Faced In Traditional Production Methods

While employing uricase through traditional methods during the history of its production, there were several problems that lawmakers could not overcome for the practical application of this enzyme. These challenges arose from mismatch or inadequacy of extraction, purification, and storage methods, and characteristics of the uricase enzyme.

Low Yield and Purity: It was known that one of the largest problems of the previous methods of uricase production from natural sources is the low purity of the enzyme obtained at this stage. Animal tissues, blood, and microbial cultures which may be good sources for uricase often provided crude preparations that solely contained multiple protein species. There are various technical difficulties that come with purifying uricase from these extracts, mainly the low yield and relatively poor purity that come with the time-consuming processes of chromatography and dialysis.

Complexity of Purification Procedures: Another problem observed when purifying uricase from crude extracts was that previous work utilized sophisticated procedures to achieve this. Many of these procedures required sequential steps, through which the operator was trying to concentrate on the purification of particular sorts of impurities and to emphasize the enrichment of the uricase. Despite this, the efficiency of these purification steps was not homogenous hence worrying yields and low enzyme activity in the purified preparation were observed. 

Stability Issues: Uricase was also shown to have moderate stability specific environmental factors including; pH, Temperature, and Storage conditions. Another factor was concerned with the stability of uricase during purification, storage, and transportation so that the enzymatic activity and therapeutic value would be made available to the patient. 

Cost and Resource Intensiveness: Both of such approaches were time-consuming and expensive since, the initial process, involved culturing huge quantities of microbes or using animal tissues, followed by further purification steps. These methods included high costs and resource intensity due to which the methods could not be applied for large-scale production which further led to reducing the market accessibility of the uricase-based therapeutics (Dako, 2012).

Limited Scalability: There were also certain limitations that prohibited the large-scale production methods of traditional uricase; these include microbial expression systems. When it comes to genetic manipulations or improving production through stakes on a fermentation-based production process, microbial hosts were advantageously positioned. However, as the demand for production increased the difficulties of scaling up presented major concerns. Some of the problems that arose included the difficulties in the development of fermentation scale-up, downstream processing, and product recovery which made it challenging to ramp up production at the industrial level. 




Need for More Efficient and Scalable Production Techniques

The history of uricase production and its progress exists as an example of a growing demand for effective and highly reproducible production strategies suited for the effective treatment and diagnosis of various diseases and conditions by using the modern possibilities of uricase. The old ways used to extract and purify uricase were cumbersome, time-consuming processes that could only be performed on a small scale, a factor that has remained a major hurdle that called for new approaches.

Rising Demand: The occurrence of adverse effects attributable to hyperuricemia and its related complications like Gout indicates the need to develop more effective molecules and systems employing uricase in a diversity of diagnostic tests and scientific investigations. Due to the increasing use of phosphate, the traditional ways of production that utilized natural resources and simple purification were not adequate hence forcing a need for modern methods of production.

Cost and Resource Efficiency: The original procedures for the preparation of uricase involved many economic and time-consuming steps, such as the massive cultivation of microbial cultures or animal tissues and an elaborate purification process. Despite the significant potential for these methods in the creation of stabilized IS, the high costs and resources were a problem for further scaling up and commercialization of uricase-based therapies. As a result, a great demand for new and improved methods that would require lesser amounts of capital and resources to create products was stimulated to take place. 

Scalability: Another issue to be considered was the scale-up of uricase production methods especially when using microbes to synthesize the protein. Though microbial hosts had the benefit of being more genetically amenable to manipulation and easier to cultivate in large bioreactors through fermentation techniques, problems arose when the scales were to be escalated for industrial applications. Challenges concerning fermentation scale-up, downstream processing, and product recovery posed some of the challenges in developing larger-scale systems to feed the market.

Advances in Biotechnology: The advent of biotechnological tools and techniques, such as recombinant DNA technology, enzyme engineering, and high-throughput screening, revolutionized the field of uricase production. Recombinant DNA technology enabled the engineering of microbial hosts for high-level expression of uricase, circumventing the limitations associated with natural sources. Enzyme engineering facilitated the development of novel uricase variants with enhanced catalytic activity, stability, and expression levels, offering improved therapeutic efficacy and productivity. High-throughput screening techniques accelerated the discovery and optimization of uricase-producing strains and production processes, facilitating rapid advancements in the field.

Integration of Bioprocess Engineering: Bioprocess engineering played a crucial role in enhancing the efficiency and scalability of uricase production processes. Optimization of fermentation conditions, media composition, and bioreactor design enabled the development of robust and scalable production platforms capable of meeting industrial-scale demands. Integration of online monitoring systems allowed real-time assessment of key parameters, enabling precise control and optimization of production conditions, further enhancing productivity and cost-efficiency. 

The historical background of uricase production underscores the need for more efficient, cost-effective, and scalable production techniques to meet the growing demand for uricase-based therapeutics and diagnostic tools. Advances in biotechnology, enzyme engineering, and bioprocess engineering have paved the way for the development of innovative production approaches that promise to address these challenges and unlock new opportunities for the treatment and management of hyperuricemia-related disorders (Dako, 2012).The advent of biotechnological tools and

Approaches to Improve Uricase Production

Humans suffer from many diseases including gout, hyperuricemia, renal failure,  and diabetes all because the uricase is not functioning properly. Uricase is responsible for its uricolytic activity but unluckily uricase enzymes are inactive in humans due to the genetic mutation termed frameshift mutation. So in order to combat the above diseases, uricase enzyme must be synthesized.




From Bacteria

In literature, some of the bacteria that produce uricase involve Bacillus fastidious, Mucor hiemalis, Streptomyces exfoliates, Bacillus thermocatenulatus, Pseudomonas aeruginosa Gliocladium viride, Aspergillus welwitschiae, Bacillus cereus, Enterobacter cloacae, etc.




Uricase Producing Bacterium from Canal Water

A bacterium-producing strain of uricase was isolated from canal water near the dairy. Agar medium plates were made, and the sample was transferred to it, ingredients included peptone, Beef extract, NaCl, and Agar 2%, supplemented with uric acid (Chen, 2016).

After incubation for 24 hours, the zone of clearance was examined. The bacterium produces the extracellular uricase enzyme, which causes the uric acid added in the agar medium to degrade and cause the zone development. This strain is named as DSS001. 




Recombinant DNA Technology to Improve Uricase Production

In this study, the main aim is to make a genetically engineered bacteria-producing uricase and check its effect on a rat model suffering from hyperuricemia (Abdel, 2005).

DNA shuffling was created which refers to the recombination of equivalent genes from natural homologous families rather than random gene mutation. It is a potent strategy for molecular-directed evolution in vitro that builds a library of chimeras by homologous sequence recombination and combines advantageous mutations for specific genes. Briefly, two or more parental genes are broken by DNase I, and the fragments of varying lengths are reassembled in PCR. The fragments anneal when there is sufficient sequence identity, resulting in full-length versions of the original gene with inherited mutations from numerous templates (Anupama, 2000). resulting in more human-like chimeric uricase.




Gene Cloning and Expression of Uricase

One hundred twenty-seven clinical strains of P. aeruginosa were isolated from collections collected at Mansoura University hospitals in Dakhla, Egypt. The initial screening of uricase among various clinical specimens was carried out by inoculating the test organisms. 

The uricase enzyme level was estimated using the uricase plate assay technique. The chromosomal DNA of P. aeruginosa Ps43 was isolated using a genomic DNA extraction kit.

The resulting expression construct was termed pU3. Western blot examination of the expressed protein was carried out. The uricase plate assay method was used to quantitatively estimate uricase production (Chen, 2008).

Advantages: Recombinant DNA has emerged as an important research tool in biological science, biochemistry, and the broader life sciences community. Recombinant DNA is employed to acquire a better understanding of illness formation and thus the creation of remedies. Furthermore, recombinant DNA (rDNA) provides the foundation for the production of recombinant antibodies, allowing for significant advances in the treatment of a variety of medical problems.

Recombinant uricase production achieved 52.3 U/ml extracellularly and 60.3 U/ml intracellularly in fed-batch fermentation. which are much higher than those expressed in other expression systems (Nyborg, 2016).

Limitations: The destruction of native species in the habitat where genetically modified species are introduced. Robust plants can possibly produce robust weeds that are difficult to control.

Fermentation Processes

The improvement of fermenting circumstances, especially physical and chemical parameters, is crucial in the development of any fermentation process since it affects the operation's economics and practicability (Abdel-Fattah, 2005).




Optimization of Culture Conditions for Uricase Production

The Plackett-Burman screening strategy was used to identify the most relevant parameters influencing enzyme production. Then the Box-Behnken design was used to estimate the optimal level of each significant parameter for maximum uricase production.




Use of Microbial Hosts for Large-scale Production

Several studies have looked into how different microbial hosts can be used to produce uricase on a big scale. One discovered that hypoxanthine induced Candida utilis to produce some quantities of uricase. Similarly, another one adjusted the conditions for Bacillus cereus uricase synthesis, attaining high enzyme activity while employing poultry feces as a cheaper source. One isolated and characterized uricase from Penicillium purpurescens, describing its high enzyme stability and activity. Scientists also identified uricase-producing bacteria from chicken, with Bacillus cereus strain DL3 having the maximum uricolytic activity. These investigations reveal that microbial hosts have the potential to produce uricase on a massive scale.




Advancements in Downstream Processing

Downstream processing, we all know that it is refers to a process that involves the recovery and purification of the product. It is a step being involved in the biosynthesis of products. Processing downstream processing guarantees the effective recovery of items that are more useable. In order to preserve the qualities of the product and achieve high yield and purity, downstream activities are crucial. In this process, we will study only the process of separating and purifying the end products (Guajardo, 2023).

In downstream processing, Cells are usually extracted from the perfusion bioreactor. Processes of product recovery, product purification, and product formulation are involved. The choices for continuous chromatographic separations are significant (Croughan, 2015).

Purification Technique for Uricase

Purification aims to remove impurities, concentrate desired proteins, and transfer them to a stable environment for their intended purpose (Pustake, 2022).

Production and optimization of uricase from Gliocladium and Gliomastix species are already defined above (Torres?Acosta, 2015), moving towards the purification of uricase.

Pure uricase from Pseudomonas has been examined for diagnostic purposes since it demonstrates high activity and thermostability over a wide range of temperatures (Nyborg, 2016).




The Three-Phase Partitioning Method

The crude enzyme in this investigation was purified using a three-phase partitioning method, using ammonium sulfate and t-butanol. The supernatant obtained by the centrifugation of broth was then made to 30, 50, and 70% (w/v) saturations using ammonium sulfate. Then t-butanol was added and a ratio of 1:0.5, 1:1, 1:1.5 (v/v) ratio of crude enzyme i.e. uricase to butanol was to be obtained.

Phase separation after centrifugation leads to the separation. Phases were analyzed for uricase activity. The phase which shows the highest level of specific gravity was chosen. Uricase was obtained from the interface. So the best process that leads to maximum uricase recovery was chosen resulting in 80.109%  of uricase at 30°C  with a uricase to t-butanol ratio of 1:1(v/v)  (Torres?Acosta, 2015).




Aqueous Two-phase Systems (ATPS)

The liquid-liquid fractionation method known as aqueous two-phase system (ATPS) has attracted attention due to its enormous potential for the extraction, purification, and enrichment of proteins, membranes, viruses, enzymes, nucleic acids, and other biomolecules (Konak, 2014).

Aqueous two-phase systems (ATPS) have been shown to yield remarkable levels of pharmaceutical purity and recovery recently.

By using this method, Uricase is purified without the need for chromatography. Through process integration and intensification, ATPS has been shown to be a less costly and time-consuming procedure than chromatography, saving resources (Iqbal, 2016).




Purification from Bacillus Subtilis

Using ammonium sulfate precipitation and ion-exchange chromatography uricase is purified from the species Bacillus Subtilis (Pustake, 2022).




Using Chromatographic Method

Urate, xanthine, oxonate, and cyanurate, uricase inhibitors, were used as ligands to produce bio affinity to promote uricase chromatographic separation. Only derivatized purines exhibited inhibitory features, whereas derivatized s-triazines appeared to lose absorption ability. The use of chromatography on a bio affinity support resulted in a greater yield and refinement of the Uricase than typical chromatographic methods (El?Naggar, 2019).

Formulation and Storage Stability of Uricase

Lipid Vesicles Loaded with Uricase

Uricase was enclosed in the alkaline liquid phase of the lipid vesicle, which increased the durability of its tetrameric form and retained its activity (Tan, 2010).

The long-term stability of the uricase enzyme in lipid vesicles was substantially higher than that of free uricase at 4°C (El?Naggar, 2019).




Pegylation of Uricase

Specific to the site PEGylation of uricase employing methoxy polyethylene glycol maleimide (mPEG-mal) via thiol PEGylation process is a successful way to overcome the disadvantage of its brief plasma half-life in blood and also to boost its therapeutic potency (Tan, 2020).




Stable Formulations

Uricase-containing lipid vesicles (UOXLVs) were successfully synthesized using the reverse-phase evaporation process, and their properties were characterized. 




Long-term Storage Considerations

The journey to maintaining uricase's activity begins with understanding the multifaceted storage conditions that can either preserve its therapeutic potential or lead to its rapid degradation. 

Temperature: Typically, enzymes are stored at low temperatures to slow down the rate of degradation. The ideal temperature for uricase storage has been found to be just above freezing, where enzymatic reactions are sufficiently slowed down without the risk of ice crystal formation. This delicate balance ensures the enzyme remains in a state conducive to long-term storage, without compromising its structural integrity or catalytic activity.

pH: Maintaining the correct pH is achieved through the use of buffer solutions, which resist changes in pH upon the addition of acids or bases. These buffers help to create a stable environment for uricase, protecting it from fluctuations that could lead to premature denaturation.




Uricase Enzyme Therapy: An Overview

Approved Formulations: Raburicase and pegloticase are two uricase formulations approved for clinical use. Raburicase is typically used for the short-term management of tumor lysis syndrome, while pegloticase is employed for the long-term treatment of refractory gout (Yang, 2012).

Innovations in Uricase Therapy

Enhancing Uricase Efficacy through PEGylation: 

Monomethoxyl-poly(ethylene glycol) (mPES)is a crucial component of pegloticase, which is a human UA-degrading enzyme utilized in gout therapy. mPEG is modification that is hoped to increase the therapeutic properties of the uricase to ensure that it would provide symptomatic relief of gout. Nevertheless, although the employment of peg-containing compounds in the pegloticase has some benefits associated, there are some challenges that come along with it, particularly the immunogenicity. Immunogenicity is the likelihood that the immune system will recognize mPEG as a foreign object, whether it be an active recognition or passive detection that results in an immune response against it. According to evidence, there is an occurrence of approximately 20% of patients who are undergoing this pegloticase treatment for 3 months display immunological reactions towards mPEG antigen. These graft vs host responses might actually undermine the effectiveness of the treatment in some patients and cause side effects in others. In an attempt to prevent the immunogenic display of mPEG, researchers and developers are looking at alternate strategies in which uricase-based therapies can be given to people with refractory gout. A promising strategy can be achieved by the application of PEG optimization methods that eliminate the immunogenicity of the mPEG derivative and make it more effective. PEGylation is a process of covalently linking PEG molecules with the uricase enzyme. It helps in improving the pharmacokinetic properties of the enzyme along with prolongation of its circulation. Through the planning and mapping of suitable reactive amino acid residue sites on native uricases on which to incorporate site-specific PEGylation to minimize the possibility of the modified enzyme provoking an immune response but at the same time preserving the therapeutic activity of the poison. These approaches including the rational design and the evolutionary biotechnology products engineering of uricases on a molecular level form the basis of this process. These endeavors look at the possibilities of steric hindrance, thermality, and selectivity of the enzymes, assisting in increasing their effectiveness in resistant gout. Pharmacodynamics demonstration and experimentation plan the safer creation of uricase formulations. The studies will explore the interactions of PEGylated uricases with the immune system and develop an efficient dosage that can control the adverse reaction occurrence but still be beneficial (Chiu, 2021).

Molecular Engineering: With the help of site-directed mutagenesis, it is possible to manipulate enzymes’ molecular structure to create better performance characteristics. As an example, the two species of microorganisms, A. globiforms, and Bacillus sp. are sources of urate oxidases. TTP-90 was engineered through protein engineering, which took into account the thermostability of the protein. Details were that we stabilized the inter-subunit interactions via a mechanism of incorporating disulfide bonds which in turn resulted in improved thermal stability. In this case, the mutation sustains the structural form and net activity of the enzyme amid elevated temperatures, a critically important factor in the context of drug development and medical treatment (Pierzynowska, 2020).

Emerging Formulations: ALLN-346 is posed as a potent new drug formulation that can help in managing the cases of refractory gout and hyperuricemia-related diseases. Consequently, ALLN-346 does not include ULTs and instead, is focused on addressing the issue of urate degradation in the intestines as this pathway has been revealed to be the key one in the case of CKD. The resultant ALLN-346 formulation through the design of engineered urate oxidase activity rationally tailored for proteolytic stability and proving efficacious in the gastric microenvironment outcomes in the proof of concept studies which are notably in the URKO mice models. The studies showed a striking diminution in uric acid levels in plasma and urine without any significant increment in uric acid excretion, thereby indicating that curing hyperuricemia as well as any renal burden of urate could be possible. If a human clinical trial of ALLN-346 proves its validity further, then this oral enzyme therapy can be seen as one of the therapies that can change the course of the treatment for hyperuricemias. If further validated in human clinical trials, ALLN-346 holds significant promise as an oral enzyme therapy capable of reshaping the therapeutic landscape for hyperuricemic disorders, offering hope for improved outcomes and quality of life for patients facing refractory gout and related conditions (Mirzaeinia, 2017).

Biotechnological Challenges in Uricase Stability and Functionality

Protein Stability and Additives: Protein stability plays a major role in immobility and durability of given enzyme therapeutics. Nevertheless, the lack of stability (including one of the drug enzymes such as uricase) represents the major obstacle to their use in practice. In the efforts to counter this problem, different methods come in handy with the introduction of stabilizing additives. As one of the aims of the study mentioned, the genetic engineering of E. coli BL21 (DE3) bacteria led to the expression of the uricase gene from Aspergillus flavus. The recombinant protein of interest came out as the uricase protein, which was then purified using Ni-NTA agarose chromatography, hence displaying a successful protein expression and purification. In spite of the fact that the purified enzyme exhibited rather narrow temperature stability (half-life in 30 minutes at 40°C), the study demonstrated that it was still highly active. In order to increase the stability of the gels, glucose, sorbitol, and glycerol were the additives utilized. From these, glucose turned out to be the one showing the biggest protection result, doubling the enzyme's lifetime parameter. This additional stability may be due to the specific character of the glucose surface charges which helps keep the uricase enzyme structural. Therefore, the study highlights the significance of protein stability one of the factors setting effective of additive addition, particularly glucose, in stabilizing the enzyme uricase, which is a potential therapeutic interest (Selvaraj, 2017).

Production and Optimization Studies: If we focus on the enzyme-producing Pseudomonas aeruginosa strain, some trials are being conducted to amplify the uricase production and activity optimization. Factors such as pH, temperature, and the provision of carbon and nitrogen sources were varied to establish the key role of each of these in uricase production. The obtained results demonstrated a significant enzyme yield estimation as 73U/ml which implied the successful enzyme generation. An optimum temperature of 45°C and pH value of 5 coupled with physiochemical conditions highlight the role of abiotic factors in boosting enzyme function. The purpose of that was to see whether there is a new technique that could be developed in order to detect microbes that produce uricase and estimate the activity of the enzyme, the ability which uricase has to dissolve uric acid. This suggested approach developed a new, suitable, sensitive, and facile screening method for such issues where it has applications across different medical fields. 

Innovations in Uricase Therapy

Oral Uricase Delivery

The administration of the recombinant uricase through oral delivery has the potential of being therapeutic in the management of the conditions associated with hyperuricemia such as gout and the dysfunction of the kidney. However, a number of strategies have been developed to thwart the challenge of immunogenic recombinant uricase causing problems in human patients due to the lack of endogenous enzymes. In this way, the functional ancestral uricase was encased with reverse, non-infectious Q? capsids to ensure the stability and maintenance of the active enzyme. The encapsulated uricase was closely attached to its catalytic activity, showing its potential as a therapeutic drug. They were found effective by oral administration which suppressed the key symptoms of kidney dysfunction in uricase knock-out mice by lowering the serum uric acid levels. Histological examination of kidney biopsy tissues from treated mice exhibited protection against the deteriorative influences of uric acid, supporting the idea of intervention in renal failure resulting from high uric acid levels. In essence, the coding of recombinant uricase and the oral delivery approach to therapy could be a providential method for interventions in conditions where uric acid is elevated, offering hope for improved patient outcomes and renal function (Tran, 2023).

Immunomodulation Techniques: The joint use of immunomodulators, such as methotrexate, with pegloticase, has been shown to be a successful strategy in reducing the levels of antidrug antibodies and hence improving the treatment response as well as the decline in the incidence of infusion reactions. Indeed this approach is a revolutionary biotechnology for the reduction of uricase-based therapies immunogenicity especially for the refractory gout treatment. Using pegloticase in combination with immunomodulators like methotrexate can lead to greater treatment effectiveness without as many adverse consequences, which in turn can increase the probability of people with uncontrolled gout receiving long-term therapy and improve patient outcomes (Schlesinger, 2022).




Engineering for Reduced Immunogenicity

Protein Engineering: Protein engineering has proved to play a key role in the establishment of novel uricase enzymes with improved characteristics, which brings new hope to patients who have problems with the treatment of diseases, like refractory gout. Human beings and higher primates possess anomalous aristofangin enzyme deficiency, and as such, they require novel methods of treatment in order to regulate progressively rising concentrations of urea and prevent gout and other conditions. Conventional uricase treatments include intra-muscular Krystexxa®, but its hainmunogenicity has been a cause for concern and has made other alternatives the only option. Emerging challenges around improving the quality of the already existing enzyme are linked to the adopted innovative technique of protein engineering that allowed for the development of a uricase with increased functional capacity.  Unlike uricase, which functions as the only enzyme capable of degrading uric acid, hundreds of different uricase sequences were properly aligned in order to identify some of the potential sequence liabilities and to improve protein properties. Via this mechanism, the determination was to identify a single lead uricase candidate that had encouraging features including soluble expression, neutral pH stability, high-level expression in Escherichia coli, thermal stability, and exceptional enzymatic performance. Particularly, the cysteine residue was engineered for Cysteine site-specific polyethylene glycol (PEG) conjugation and hereafter efficient attaching with polyethylene glycol (PEG) chains. Studies have confirmed the over 95% PEGylation efficiency, with the PEGylated uricase enzymatic activity retained both in the litter media at neutral pH, in human serum, and in animal models. Positively, once the PEGylated uricase was applied to the canines, they had an 85% reduction in the plasma uric acid levels after receiving just one subcutaneous injection, revealing the potential of this therapeutic agent. This non-immunogenic, PEGylated uricase represents a pioneering principle in the therapy of gout due to diminishing the main disadvantages of the treatment nowadays, promising substantial improvements for the patients (Nyborg, 2016).

Potential and Efficacy in Animal Models

Canine Studies: The innovative development of a tryptic nitricase with important properties such as soluble expression, neutral pH, high production in Escherichia coli, thermal stability, and high enzyme activity have paved the way for this research. Human cells' uricases’ alignment, over 200 almost identical, gave researchers the idea of identifying a lead sequence promising minimal immunogenicity potential. Furthermore, the connectivity of the cysteines was deliberately constructed within the lead sequence to achieve site-directed PEGylation that improves the stability and pharmacokinetic attributes of the enzyme. The PEGylated uricase infused into the canine significantly reduced the serum uric acid level by a staggering 85% following a single subcutaneous injection. This will help prove how this PEGylated, non-immunogenic uricase can be helpful in handling hyperuricemia and other uric acid-related disease in human patients. The outcomes of this canine study can shed light on the therapeutic benefits of this novel treatment course and propel new studies and trials of this therapy in humans who experience gout and other related diseases (Nyborg, 2016).




Cost-effectiveness and Scalability of Biotechnological Methods

ATPE can be considered a novel approach for uricase recovery that, compared to the existing technology, has potential in terms of cost-effectiveness and decreasing the time it takes to manufacture the product. This way becomes even more efficient through a feature that allows it to reuse some of the crucial parts, which addresses the environmental concern related to solid waste. A comprehensive financial assessment was done through BioSolve software which compared different uricase manufacturing strategies ranging from traditional column chromatography to ATPE and ATPE with PEG recycling. The result was a revealing of the key parameters including in the sensitivity analysis and the subsequent Monte Carlo analysis done to determine their effect on the production costs. Data analysis showed that while ATPE can be cheaper than other methods, such as chromatography, still there are some situations where the cost of production of both technologies will be the same, which suggests that recycling PEG does not bring a great change to the cost. Instead of traditional chromatography, using an aqueous two-phase system (ATPS) could actually lead to the saving of a massive $4000 per gram of uricase that is produced.

Such savings are mainly developed from low capital investment, lower consumable costs, and employee reduction. Bacterial urease activity is being optimized with corn steep liquor (CSL) as one of the low-cost options. CCD and ANN models will be used in the investigation of CLS concentration, urea concentration, fermentation time, and the effects of nickel supplementation on urease yield. The performance capacity of ANN has proved better than CCD for predicting ammonia yield. Urease activity is the highest (3. 6 mM urea min^-1) under the ideal conditions. It indicates that the CSL-urea medium is a suitable alternative to yeast extract nutrient medium in terms of the cost factor (Clavijo, 2019; Khade, 2018; Dudala, 2023).

Clinical Trials and Treatment Outcomes

Phase III Clinical Trials: From clinical studies with the pegloticase drug, only 42% of the patients responded well to treatment, and the presence of drug antibodies reduced the effectiveness.

Biweekly Infusions: They were put on the pegloticase drug or control group and got the pegloticase therapy or placebo every two weeks. The principal endpoint was keeping probationary serum urate levels lower than 6. The study period must be three months, during which the patient must be 0 mg/dL for at least 80% of the time.

Antibody Monitoring: Testing of anti-pegliclase, PEG, and uricase antibodies was fulfilled using previously tested enzyme-linked immunosorbent assay (ELISA) and blood samples were collected repeatedly during the trial (Schlesinger, 2022).

Challenges and Considerations in Uricase Therapy

Antibody Development and Response Rates

High Incidence of Antibodies: There is a prominent concern in the context of uricase therapy, that is, the development of anti-treatment antibodies. Research indicates that pegloticase antibody titer (IgG) of 41% is very common in patients.

Impact on Treatment Efficacy: The presence of these antibodies and the fact that they remain in a nonresponder status makes it possible with the low pegloticase serum concentrations. This leads to the well-known phenomenon that every patient is different, therefore reducing the overall effectiveness of therapy in controlling uric acid levels.

Increased Risk of Infusion Reactions: Besides that, with the development of antibodies infusion reactions occur with a higher risk, and it can change the way NbT cells are administered and affect patients' safety (Guttmann, 2017).

Administration and Stability Challenges

Safe and Efficient Administration: Uricase-based therapies to become the main course of treatment is currently regarded as the most significant impediment, and this is because its way of administration, with respect to both safety features and effectiveness, should be found. The enzyme must be delivered through a method that allows it to be specific, that ensures it reaches the organ in which it is required, and that decreases the negative effects it could have.

Stability during Storage and Transportation: Indeed, keeping uricase instant during the storage and transport stages is the first important parameter for long-term storage and one major prerequisite. The enzyme is required not to change the potency through the whole synthesizing cycle till the patient gets it, and also it needs to be non-inactivated, which means none of its parts must get destroyed (Yang, 2012).




Side Effects and Downstream Interactions of Antibiotics

Managing Side Effects: Side effects are what we consider carefully in developing uricase tools. Patients may suffer side effects which must be tackled well meaning patient continuity and safety.

Efficacy in Complex Biological Environments: Another substantial challenge is that maintaining the uricase enzyme, the one that is responsible for breaking down the uric acid will always be bypassed in case other biological molecules and conditions of the body are found on the scene. This involves comprehensive knowledge of enzyme interactions as well as the establishment of formulations that adjust to the tasking environment (Roman, 2023).




Genomic Approaches to Uricase Integration

CRISPR-Cas9 and Uricase Gene: The technology known as CRISPR-Cas9 has become the subject matter of discussion owing to its ability to cure or even reverse diseases that have particular genetic mutations with the recent occurrence in the human genome. The moral issue of its applications of manipulating genetic outcomes is there but there is room for its potential in supplying human beings with metabolites or prophylactic molecules for long-term health. Among various ways is the introduction of a uricase gene functional with the CRISPR/CAS9 system in the human genome. This enzymatically active protein in the uricase gene has a function of either significantly reducing or even eliminating the high uric acid levels. While the uricase enzyme is mostly present in the peroxisomes, cells with this gene deliver a real triumph by effectively handling the exogenous uric acid levels. The thing that makes this very special is that these are genetically modified to pave the way to the next phase of human evolution. It implies the initial one among the primate ape lineage of having a functional enzyme encoded in the genome for the last 10 million years. This achievement makes the way open for treating diseases related to high uric levels, such as gout, hypertension, and fatty liver. The outlook is even more encouraging as cells containing the uricase enzyme could be used as a prophylactic to protect against the detrimental effects of hyperuricemia. Through CRISPR technology to induce uricase genes in the human genome, researchers aim to pursue work for better metabolic health and disease resistance in the future (De 2021).




Genetic Approaches for Improving Uricase Functionality

Cloned Bacillus subtilis BS04 uricase gene into pET28a (+) vector. The purified recombinant protein (56.63 kDa) showed optimal activity at pH 9.0, 45°C, with 4.97 U/mg specific activity. The activity enhanced by 137.12% with Mn^2+, inhibited by 95.42% with Cu^2+. Cys-directed mutants analyzed for pH and temperature dependence showed impaired activity compared to native uricase. Mutant C489A exhibited higher alkali resistance and thermostability, suggesting a link between cysteine substitution and increased thermal resistance, aiding in enzyme therapeutic development (Zhu, 2021).

Emerging Trends and Technologies in Uricase Production

Isolation and Optimization of Uricase Production from Poultry Soil Bacteria

A study involved the identification and enhancement of uricase production from bacteria that is obtainable in poultry soil. First, three bacterial species were chosen and then the end product formation was assayed visually on the utrate agar plate (the clear gradient zone) employing media wells. A number of species isolated from this source were observed to possess such uricolytic nature and among them, Bacillus cereus strain DL3 was found to be the most active and the identification of its genetic nature was done using sequencing techniques. Thereafter, the study was conducted in order to find out how long the enzyme took to produce and the way to maximum yield of the enzyme using the best medium. After optimization, the two identified crucial sources are carboxymethylcellulose and asparagine, which provide carbon and nitrogen, respectively. Being an enzyme on the surface of cells, uricase works best at pH 7. 0, a concentration of inducer of 2, 0 g/L, and 5% inoculum size were used. On the contrary, during these circumstances, the uricolytic activity was greatly enhanced and it was the maximum that reached 15. 43 U/mL achieved. Additionally, its efficacy was studied in varied pH and temperature conditions with hyperuricemia and gout diseases in mind. With the results, its expected applicability is now evident (Nanda, 2014).




Innovative Screening Method for Uricase Production

The enzyme, uricase, was obtained with pseudomonas aeruginosa which was subjected to different pH, temperature, carbon, and nitrogen sources leading to 73 U/ml. The maximum uricase activity of 45°C and pH 5 resulted in the optimal functioning of the enzyme. The objective was to design a new technique for detecting microbial uricase production levels and determining the enzyme production yield. The researchers employed the principle that uric acid dissolves as a uricase unites with it thus stimulating the increase in uricase production. This means is stated as highly specific, low cost, uncomplicated, and sensitive for uricase screening and estimation (Selvaraj, 2017).




Broader Implications and Future Directions

Research efforts are continuously being made to find and produce drugs that prevent a wide array of pathogenic mechanisms involved in gout. This comprises the investigation of the efficacy of interleukin-1 inhibitors, inflammasome inhibitors, as well as novel urate-lowering agents like uricosurics and xanthine oxidase inhibitors. The combination of uricase therapy with other innovative treatments, like urate transport inhibitors and anti-inflammatory agents, is being studied in order to find the best approach to managing gout. Using more sophisticated biotechnological platforms, one can engineer molecules that act as the therapeutic uricase for gout treatment, helping in better management of this painful disease. 

References

• Abdel-Fattah, Y. R., Saeed, H., Gohar, Y. M., & Elbaz, M. (2005). Improved production of Pseudomonas aeruginosa uricase by optimization of process parameters through statistical experimental designs. Process Biochemistry, 40(5), 1707–1714. https://doi.org/10.1016/j.procbio.2004.06.048

• Anupama, & Ravindra, P. (2000). Value-added food: Biotechnology Advances, 18(6), 459–479. https://doi.org/10.1016/s0734-9750(00)00045-8
• Chen, J., Jiang, N., Wang, T., Xie, G., Zhang, Z., Li, H., Yuan, J., Sun, Z., & Chen, J. (2016). DNA shuffling of the uricase gene leads to a more "human-like" chimeric uricase with increased uricolytic activity. International Journal of Biological Macromolecules, 82, 522–529. https://doi.org/10.1016/j.ijbiomac.2015.10.053
• Chen, Z., Wang, Z., He, X., Guo, X., Li, W., & Zhang, B. (2008). Uricase production by a recombinant Hansenula polymorpha strain harboring Candida utilis uricase gene. Applied Microbiology and Biotechnology, 79(4). https://doi.org/10.1007/s00253-008-1472-8
• Chiu, Y., Hsu, T., Huang, C., & Hsu, C. (2021). Molecular Elucidation of a Urate Oxidase from Deinococcus radiodurans for Hyperuricemia and Gout Therapy. International Journal of Molecular Sciences, 22(11), 5611. https://doi.org/10.3390/ijms22115611
• Clavijo, V., Torres‐Acosta, M. A., Vives, M., & Rito‐Palomares, M. (2019). Aqueous two-phase systems for the recovery and purification of phage therapy products: Recovery of salmonella bacteriophage ϕSan23 as a case study. Separation and Purification Technology, 211, 322–329. https://doi.org/10.1016/j.seppur.2018.09.088

• Croughan, M. S., Konstantinov, K., & Cooney, C. L. (2015). The future of industrial bioprocessing: Batch or continuous? Biotechnology and Bioengineering, 112(4), 648–651. https://doi.org/10.1002/bit.25529

• Dako, É., Bernier, A., Dadié, A., & Jankowski, C. K. (2012). The Problems Associated with Enzyme Purification. In InTech eBooks. https://doi.org/10.5772/33307
• Dako, É., Bernier, A., Dadié, A., & Jankowski, C. K. (2012b). The Problems Associated with Enzyme Purification. In InTech eBooks. https://doi.org/10.5772/33307
• Dudala, S. S., Venkateswarulu, T., A, V. N., & Dulla, J. B. (2023). Modeling and optimization of uricase production from a novel Pseudomonas mosselii using response surface methodology and artificial neural network. Biomass Conversion and Biorefinery. https://doi.org/10.1007/s13399-023-04468-3
• Dudala, S. S., Venkateswarulu, T., Alugunulla, V. N., Krupanidhi, S., & Dulla, J. B. (2023). Enhanced uricase production using novel Escherichia marmotae strain (DJDSS001): Characterization and optimization. Biocatalysis and Agricultural Biotechnology, 48, 102649. https://doi.org/10.1016/j.bcab.2023.102649
• El‐Naggar, N. E., Haroun, S. A., El-Weshy, E. M., Metwally, E. A., & Sherief, A. A. (2019). Mathematical modeling for bioprocess optimization of a protein drug, uricase, production by Aspergillus welwitschiae strain 1–4. Scientific Reports, 9(1). https://doi.org/10.1038/s41598-019-49201-1
• Garay, R. P., El-Gewely, M. R., Labaune, J., & Richette, P. (2012). Therapeutic perspectives on uricases for gout. Joint Bone Spine, 79(3), 237–242. https://doi.org/10.1016/j.jbspin.2012.01.004
• Guajardo, N., & Schrebler, R. (2023). Upstream and downstream bioprocessing in enzyme technology. Pharmaceutics, 16(1), 38. https://doi.org/10.3390/pharmaceutics16010038
• Guttmann, A., Krasnokutsky, S., Pillinger, M. H., & Berhanu, A. (2017). Pegloticase in gout treatment - safety issues, latest evidence and clinical considerations. Therapeutic Advances in Drug Safety, 8(12), 379–388. https://doi.org/10.1177/2042098617727714
• Iqbal, M., Tao, Y., Xie, S., Zhu, Y., Chen, D., Wang, X., Huang, L., Peng, D., Sattar, A., Shabbir, M. a. B., Hussain, H., Ahmed, S., & Zhang, Y. (2016b). Aqueous two-phase system (ATPS): an overview and advances in its applications. Biological Procedures Online, 18(1). https://doi.org/10.1186/s12575-016-0048-8
• Khade, S., Srivastava, S. K., & Tripathi, A. D. (2016). Production of clinically efficient uricase enzyme induced from different strains of Pseudomonas aeruginosa under submerged fermentations and their kinetic properties. Biocatalysis and Agricultural Biotechnology, 8, 139–145. https://doi.org/10.1016/j.bcab.2016.09.005
• Khade, S., Srivastava, S. K., Kumar, K., Sharma, K., Goyal, A., & Tripathi, A. D. (2018). Optimization of clinical uricase production by Bacillus cereus under submerged fermentation, its purification and structure characterization. Process Biochemistry, 75, 49–58. https://doi.org/10.1016/j.procbio.2018.09.010
• Konak, Ü. İ., Turhan, İ., & Certel, M. (2014). Chromatographic methods in protein purification. Akademik Gida, 12(2), 79–87. https://www.cabdirect.org/cabdirect/abstract/20143348265
• Mirzaeinia, Somayyeh & Pazhang, Mohammad & Imani, Mehdi. (2017). Investigation of the stability of uricase from Aspergillus flavus and its stabilization by Glucose. Biotechnology Tarbiat Modares University. 8. 50-60.
• Nanda, P., & Babu, P. J. (2017). Solid phase PEGylation of Uricase. Materials Today: Proceedings, 4(9), 10494–10497. https://doi.org/10.1016/j.matpr.2017.06.407
• Nyborg, A. C., Ward, C., Zacco, A., Chacko, B., Grinberg, L., Geoghegan, J. C., Bean, R., Wendeler, M., Bartnik, F., O’Connor, E., Gruia, F., Iyer, V., Feng, H., Roy, V., Berge, M., Miner, J. N., Wilson, D. M., Zhou, D., Nicholson, S. M., . . . Baca, M. (2016). A Therapeutic Uricase with Reduced Immunogenicity Risk and Improved Development Properties. PloS One, 11(12), e0167935. https://doi.org/10.1371/journal.pone.0167935
• Nyborg, A. C., Ward, C., Zacco, A., Chacko, B., Grinberg, L., Geoghegan, J. C., Bean, R., Wendeler, M., Bartnik, F., O’Connor, E., Gruia, F., Iyer, V., Feng, H., Roy, V., Berge, M., Miner, J. N., Wilson, D. M., Zhou, D., Nicholson, S. M., . . . Baca, M. (2016b). A Therapeutic Uricase with Reduced Immunogenicity Risk and Improved Development Properties. PloS One, 11(12), e0167935. https://doi.org/10.1371/journal.pone.0167935
• Nyborg, A. C., Ward, C., Zacco, A., Chacko, B., Grinberg, L., Geoghegan, J. C., Bean, R., Wendeler, M., Bartnik, F., O’Connor, E., Gruia, F., Iyer, V., Feng, H., Roy, V., Berge, M., Miner, J. N., Wilson, D. M., Zhou, D., Nicholson, S. M., & Baca, M. (2016c). A Therapeutic Uricase with Reduced Immunogenicity Risk and Improved Development Properties. PloS One, 11(12), e0167935. https://doi.org/10.1371/journal.pone.0167935
• Pierzynowska, K., Deshpande, A., Mosiichuk, N. M., Terkeltaub, R., Szczurek, P., Salido, E., Pierzynowski, S., & Grujić, D. (2020). Oral treatment with an engineered uricase, ALLN-346, reduces hyperuricemia, and uricosuria in urate Oxidase-Deficient mice. Frontiers in Medicine, 7. https://doi.org/10.3389/fmed.2020.569215
• Pustake, S. O., Bhagwat, P., Pillai, S., & Dandge, P. B. (2022). Purification and characterisation of uricase from Bacillus subtilis SP6. Process Biochemistry, 113, 55–61. https://doi.org/10.1016/j.procbio.2021.12.010
• Roman, Y. M. (2023). The Role of Uric Acid in Human Health: Insights from the Uricase Gene. Journal of Personalized Medicine, 13(9), 1409. https://doi.org/10.3390/jpm13091409
• Roman, Y. M. (2023). The Role of Uric Acid in Human Health: Insights from the Uricase Gene. Journal of Personalized Medicine, 13(9), 1409. https://doi.org/10.3390/jpm13091409
• Schlesinger, N., Padnick‐Silver, L., & LaMoreaux, B. (2022). Enhancing the Response Rate to Recombinant Uricases in Patients with Gout. BioDrugs, 36(2), 95–103. https://doi.org/10.1007/s40259-022-00517-x
• Schlesinger, N., Padnick‐Silver, L., & LaMoreaux, B. (2022b). Enhancing the Response Rate to Recombinant Uricases in Patients with Gout. BioDrugs, 36(2), 95–103. https://doi.org/10.1007/s40259-022-00517-x
• Selvaraj, C., & Vasan, P.T. (2017). Screening, production and optimization of Uricase from P. aeruginosa. European Journal of Biotechnology and Bioscience, 5, 57-61.
• Tan, Q., Wang, N., Yang, H., Zhang, L., Liu, S., Chen, L., Liu, J., Zhang, L., Hu, N., Zhao, C., & Zhang, J. (2010). Characterization, stabilization and activity of uricase loaded in lipid vesicles. International Journal of Pharmaceutics, 384(1–2), 165–172. https://doi.org/10.1016/j.ijpharm.2009.09.036
• Tan, Q., Wang, N., Yang, H., Zhang, L., Liu, S., Chen, L., Liu, J., Zhang, L., Hu, N., Zhao, C., & Zhang, J. (2010). Characterization, stabilization and activity of uricase loaded in lipid vesicles. International Journal of Pharmaceutics, 384(1–2), 165–172. https://doi.org/10.1016/j.ijpharm.2009.09.036
• Tandon, S., Sharma, A., Singh, S., Sharma, S., & Sarma, S. J. (2021). Therapeutic enzymes: Discoveries, production and applications. Journal of Drug Delivery Science and Technology, 63, 102455. https://doi.org/10.1016/j.jddst.2021.102455
• Torres‐Acosta, M. A., Aguilar‐Yáñez, J. M., Rito‐Palomares, M., & Titchener‐Hooker, N. J. (2015). Economic analysis of uricase production under uncertainty: Contrast of chromatographic purification and aqueous two-phase extraction (with and without PEG recycle). Biotechnology Progress, 32(1), 126–133. https://doi.org/10.1002/btpr.2200
• Tran, L., Das, S., Zhao, L., Finn, M. G., & Gaucher, E. A. (2023). Oral Delivery of Nanoparticles Carrying Ancestral Uricase Enzyme Protects against Hyperuricemia in Knockout Mice. Biomacromolecules, 24(5), 2003–2008. https://doi.org/10.1021/acs.biomac.2c01388
• Yang, X., Yuan, Y., Chen, Z., & Liao, F. (2012b). Uricases as therapeutic agents to treat refractory gout: Current states and future directions. Drug Development Research, 73(2), 66–72. https://doi.org/10.1002/ddr.20493
• Zhu, T., Chen, H., Yang, L., Liu, Y., Li, W., & Sun, W. (2021). Characterization and Cys-directed mutagenesis of urate oxidase from Bacillus subtilis BS04. Biologia, 77(1), 291–301. https://doi.org/10.1007/s11756-021-00941-4