Uricase Production Research Articles

Streptomyces griseorubens asa microbial cell factory for extracellular uricase production and bioprocess optimization using statistical approach.

Uricase is a bio-drug used to reduce urate accumulation in gout disease. Thus, there is a continuous demand for screening soil samples derived from a variety of different sources in order to isolate a strain that possesses a high potential for producing uricase. Streptomyces sp. strain NEAE-5 demonstrated a significant capacity for uricase production was identified based on the physiological, morphological and biochemical characteristics, as well as 16S rDNA sequencing analysis. Using a Plackett-Burman statistical design, the impact of eighteen process factors on uricase production by Streptomyces griseorubens strain NEAE-5 was investigated. Using central composite design, the most important variables that had a favourable positive impact on uricase production by Streptomyces griseorubens strain NEAE-5 were further optimized. It is clear that the morphological and chemotaxonomic features of Streptomyces sp. strain NEAE-5 are typical for the Streptomyces genus. Phylogenetic analysis indicated that Streptomyces sp. strain NEAE-5 belongs to the genus Streptomyces and closely related to Streptomyces griseorubens which it has a 95-96% identity in 16S rDNA gene sequencing. Accordingly, the strain is proposed to be identified as Streptomyces griseorubens strain NEAE-5. The three factors that had the significant positive impacts on uricase production were uric acid, hypoxanthine, and yeast extract. As a result, the best conditions for achieving the highest experimental uricase production by Streptomyces griseorubens strain NEAE-5 after central composite design were (g/L): uric acid 6.96, glycerol 5, hypoxanthine 5.51, MgSO4.7H2O 0.1, KNO3 2, CaCl2 0.5, K2HPO4 0.5, NaCl 0.5, yeast extract 1.08. In addition, the period of incubation is seven days, pH 7.5 and 37°C with an inoculum size of 2mL (105cfu/mL) /100mL medium. After optimization, the obtained uricase activity was 120.35 U/mL, indicating that the Streptomyces griseorubens strain NEAE-5 is a potent uricase producer and that the statistical approach used for optimization was appropriate.

Proteolysis-resistant extracellular uricase from the sponge-derived Streptomyces rochei

This study aimed to identify proteolysis-resistant extracellular uricase by screening three hundred marine microorganisms, including bacteria, actinobacteria and filamentous fungi. The isolate, demonstrating resistance to proteolysis and producing extracellular uricase with relatively high activity was identified as Streptomyces rochei 6SM85. The enzyme was purified approximately 3.6 fold, yielding 1.23%, using ion exchange chromatography. The molecular weight of the purified uricase was estimated to be approximately 14 kDa by SDS-PAGE. The optimum temperature and pH for uricase were determined to be 35 °C and 8–9, respectively. It was demonstrated that uricase retained up to 46% of its activity after half an hour at 40 °C and exhibited higher activity at neutral and basic pH compared to acidic pH. Its activity increased in the presence of 1 mM Mg2+, Na+, and EDTA, while it decreased by 86% in the presence of Cu2+. This study represents the first report on the production and characterization of extracellular proteolysis-resistant uricase from marine-derived Streptomyces.

Microbial Uricase and its Unique Potential Applications

Abstract The uricase enzyme yields allantoin, hydrogen peroxide, and carbon dioxide by catalyzing the oxidative opening of the purine ring in the urate pathway. This enzyme is important for biochemical diagnosis and reduces toxic urate accumulation during various diseases (hyperuricemia, gout, and bedwetting). Direct urate oxidase injection is recommended in renal complications-associated gout and to prevent chemotherapy-linked hyperuricemia disorders. Thus, uricase is a promising enzyme with diverse applications in medicine. Microbial production of uricase is featured by high growth rates, cost-effective bioprocessing, and easy optimization of the medium. Microbes produce the enzyme extracellular or intracellular. Extracellular uricase is preferred for biotechnological applications as it minimizes time, effort, and purification processes. This review provides insights into uricase-producing microbes, bacterial uric acid degradation pathways, degrading enzymes, and uricase-encoding genes. Furthermore, aspects influencing the microorganisms’ production of the uricase enzyme, its activity, and its purification procedure are also emphasized. Cell disruption is mandatory for intercellular uricase production, which elevates 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, as well as a simple purification methodology. Furthermore, uricase gene-carrying recombinant probiotic microorganisms could become an effective tool for gout treatment.

Maximization of uricase production in a column bioreactor through response surface methodology-based optimization

Uricase (EC 1.7.3.3) is an oxidoreductase enzyme that is widely exploited for diagnostic and treatment purposes in medicine. This study focuses on producing recombinant uricase from E. coli BL21 in a bubble column bioreactor (BCB) and finding the optimal conditions for maximum uricase activity. The three most effective variables on uricase activity were selected through the Plackett–Burman design from eight different variables and were further optimized by the central composite design of the response surface methodology (RSM). The selected variables included the inoculum size (%v/v), isopropyl β-d-1-thiogalactopyranoside (IPTG) concentration (mM) and the initial pH of the culture medium. The activity of uricase, the final optical density at 600 nm wavelength (OD600) and the final pH were considered as the responses of this optimization and were modeled. As a result, activity of 5.84 U·ml−1 and a final OD600 of 3.42 were obtained at optimum conditions of 3% v/v inoculum size, an IPTG concentration of 0.54 mM and a pH of 6.0. By purifying the obtained enzyme using a Ni-NTA agarose affinity chromatography column, 165 ± 1.5 mg uricase was obtained from a 600 ml cell culture. The results of this study show that BCBs can be a highly effective option for large-scale uricase production.

New uricase producing Stenotrophomonas isolate recovered from a hot well in western south Egypt: Strain identification, Statistical optimization of enzyme production and In vitro application in gout treatment

Uricase, an enzyme catalyzing the breakdown of uric acid into more soluble compounds, holds significant therapeutic potential in managing hyperuricemia and associated conditions like gout. The current study intended to bioprospect new uricase producers in one of rarely studied ecological niches (ground water well that delivers water from 1500 m depth with temperature 50 °C in Al Qasr Oasis, New Valley Governorate, Egypt). Eight isolates were screened for uricase production, and the most potent was identified using phenotypic and genotypic methods as Stenotrophomonas maltophilia with 99% similarity. This is the first record about the ability of a member in the genus Stenotrophomonas to produce uricase, and was thus targeted in subsequent optimization studies. The initial activity of 7.20 U/mL was obtained in medium No. 2 after testing enzyme production in five different media. The uricase production was then enhanced using statistical design and modeling. Plackett-Burman design (PBD) was used to screen and identify the parameters influencing uricase activity. Four of the nine investigated factors were the most significant and were further optimized using Box-Behnken design (BBD). After two optimization cycles using PBD and BBD, the yield was improved to 20.60 and 25.67 U/mL, respectively, indicating a significant 3.5-fold improvement in the enzyme yield. The uricase was partially purified by ammonium sulphate 75% with 2.58-fold increase in activity (final recovered activity of 87.5%). Under polarized light microscopy, the prepared monosodium urate crystals clumps degraded at different levels of the obtained enzyme which encourages its future application in gout treatment.

Development of a continuous fermentation process for the production of recombinant uricase enzyme by Pichia pastoris.

Recent studies in the biopharmaceutical industry have shown an increase in the productivity and production efficiency of recombinant proteins by continuous culture. In this research, a new upstream fermentation process was developed for the production of recombinant uricase in the methylotrophic yeast Pichia pastoris. Expression of recombinant protein in this system is under the control of the AOX1 promoter and therefore requires methanol as an inducing agent and carbon/energy source. Considering the biphasic growth characteristics of conventional fed-batch fermentation, physical separation of the growth and induction stages for better control of the continuous fermentation process resulted in higher dry-cell weight (DCW) and enhanced recombinant urate oxidase activity. The DCW and recombinant uricase activity enzyme for fed-batch fermentation were 79g/L and 6.8u/mL. During the continuous process, in the growth fermenter at a constant dilution rate of 0.025h-1 , DCW increased to 88.39g/L. In the induction fermenter, at methanol feeding rates of 30, 60, and 80mL/h, a recombinant uricase activity was 4.13, 7.2, and 0u/mL, respectively. The optimum methanol feeding regime in continuous fermentation resulted in a 4.5-fold improvement in productivity compared with fed-batch fermentation from 0.04u/mL/h (0.0017mg/mL/h) to 0.18u/mL/h (0.0078mg/mL/h).

High yield expression and purification of Aspergillus flavus uricase cloned in Pichia pink™ expression system.

In this research, for the first time, A. flavus uricase gene was cloned in pPink-UOX plasmid under strong alcohol oxidase promoter of Pichia pink expression system after codon optimization. After selecting the best uricase producing clone with an activity of 0.7U/ml at the Flask level, a 5-L fermenter was used to increase the expression of the enzyme. Within 60h, the fermentation process produced 1500g of biomass from 4L of semi defined culture media and expressed 2.5g/L of the enzyme. The purity of recombinant uricase production using three consecutive DEAE Sepharose, CM Sepharose and Phenyl Sepharose columns was above 99%, which was confirmed by SDS-PAGE and RP-HPLC analyses. Size exclusion chromatography analysis showed that the purified enzyme has comparable heterogeneity to the Rasburicase. The yield of recombinant uricase production in this study was 63% and its specific activity was 24U/mg. The high expression of recombinant uricase in the Pichia pink strain and the increased enzyme activity compared to the standard sample indicate the potential of therapeutic and diagnostic applications of recombinant uricase in the present study.

Modeling and optimization of uricase production from a novel Pseudomonas mosselii using response surface methodology and artificial neural network

Modeling and optimization of uricase production from a novel Pseudomonas mosselii using response surface methodology and artificial neural network

Extra-cellular production of uricase through the sec-type secretion system in Escherichia coli

A sec-type protein secretion is an alternative approach to recombinant protein production, where protein expression and purification are simultaneously achieved. In this study, uricase (urate oxidase, EC 1.7.3.3) was fused with a sec-type Epr signal peptide to achieve the extra-cellular uricase production in Escherichia coli MG1655. In shake-flask experiments, the enzyme activity and the specific activity in the extra-cellular fraction reached 0.73 ± 0.05 U/mL and 3.17 ± 0.22 U/mg-protein, respectively. When a 3-L bioreactor was used, the extra-cellular fraction exhibited the enzyme activity of 0.74 ± 0.00 U/mL and the specific activity of 2.75 ± 0.35 U/mg-protein. It was estimated that 89 % and 72 % of total enzyme activity as produced were found in the extra-cellular fraction in the shake flask and the 3-L bioreactor, respectively. The uricase secretion in E. coli was further supported by SDS-PAGE. In contrast, no uricase activity was found when Bacillus subtilis 168 was used as the host. This study demonstrates the efficacy of protein secretion using the Epr signal peptide in E. coli MG1655. Finally, this study presents that acidic pH results from the acetate production could be detrimental to extra-cellular uricase production.

Enhanced uricase production using novel Escherichia marmotae strain (DJDSS001): Characterization and optimization

Uricase is a diagnostic and therapeutic enzyme used to detect the levels of purine end product namely uric acid in the biological samples and it also promotes the conversion of uric acid to a soluble allantoin. In this study, a potential extracellular uricase-producing Escherichia sp. was isolated from canal water near dairy and molecular characterization using16 S rRNA sequence was performed and the data showed 98.95% similarity with Escherichia marmotae. Morphological and biochemical characteristics of the isolated bacterium were determined. The effect of medium components on uricase production was studied and optimized medium enhanced the uricase production to 27.47 U/ml ± 0.44 which is 1.37 fold greater than initial uricase production. The enzyme was purified using ammonium sulphate precipitation followed by ion exchange chromatography and the resulted enzyme is stable over wide pH range from 6.0 to 9.0 and temperatures ranging from 30 °C to 37 °C retaining more than 94% activity. The enzyme activity and specific activity of the purified enzyme were 40.02 U/mL and 87.76 U/mg respectively with an overall purification fold of 4.04 and the yield obtained is 14.57%.

Medium optimization for extracellular urate oxidase production by a newly isolated Aspergillus Niger

Urate oxidase is a peroxisomal enzyme with four equal subunits that convert uric acid to allantoin, a more soluble metabolite for excretion. The usage of uricase as a drug in medicine is to treat hyperuricemia. Many microorganisms have been used for uricase production such as Streptomyces exfoliates, Pseudomonas aeruginosa, and Aspergillus flavus. In this study, soil samples were collected and then cultured in a screening medium including uric acid as the sole carbon source. Samples with the higher ability of uricase production were selected for enzyme assay. Enzyme activity was measured by spectrophotometry and the sample with the maximal uricase activity was identified as Aspergillus niger and selected for further studies. According to the results of experiments, the optimized temperature for enzyme production by Aspergillus niger was determined to be 35±2°C. The best carbon and nitrogen source was glucose and NH4NO3, and the highest enzyme activity was observed in the presence of Cu2+ ion.

Isolation and Characterization of Uricase Produced from Chicken Liver

Uricase is an enzyme that degrades uric acid into allantoin. One of the uricase sources is obtained from chicken species (Gallus gallus domesticus) liver which are broiler and native chicken. This study aims to determine the maximum uricase activity in broiler and native chicken liver. The uricase activity was obtained by measuring the uric acid concentration as uricase substrate using spectrophotometric method and wavelength at 291 nm. Uricase isolation was carried out into extraction process, ammonium sulfate fractionation (0-60% saturation of ammonium sulfate), and dialysis. During isolation process, centrifugation speed was also optimized to obtain the maximum uricase crude extract and uricase activity. The molecular weight of uricase was also determined by SDS PAGE. The result showed that the highest uricase activity remained using centrifugation speed of 15,000 rpm. The optimum uricase fraction for broiler chicken liver was obtained at 20-40% saturation of ammonium sulfate with uricase activity was 1.854 x 10-2 U/mg, and the uricase fraction for native chicken liver was obtained at 40-60% saturation of ammonium sulfate with uricase activity was 2.496 x 10-2 U/mg. The optimum fraction for uricase production and isolation is carried out to the dialysis process. The optimum uricase activity of broiler chicken liver crude extract was 4.921 x 10-4 U/mg, the uricase fraction was 3.989 x 10-3 U/mg, and the dialysate was 5.120 x 10-3 U/mg. While the native chicken liver crude extract was 2.980 x 10-4 U/mg, the uricase fraction was1.415 x 10-2 U/mg, and the dialysate was 1.753 x 10-2 U/mg. The molecular weight of the uricase was around 35 kDA according to the SDS PAGE result.

Heterologous expression of recombinant urate oxidase using the intein-mediated protein purification in Pichia pastoris.

The potential of urate oxidase (uricase) for clinical use has been highlighted because of its role in lowering the blood uric acid levels for the treatment of tumor lysis syndrome. In the present study, the codon-optimized synthetic gene of Aspergillus flavus uricase was fused to the Mxe GyrA intein and chitin-binding domain. The construct was inserted into pPICZA and pPICZαA vectors and electroporated into Pichia pastoris GS115 for the cytosolic and secretory expression. Transformants were screened on gradients of Zeocin up to 2000μg/ml to find multi-copy integrants. For both constructs, colonies with more resistance were screened for the highest uricase producers by enzyme assay. PCR analysis confirmed successful cassettes insertion into the genome and Mut + phenotype. The gene copy index was determined to be two and five for cytosolic and secretory strains, respectively. Productivity of the cytosolic and secretory strains was found to be 0.74 and 0.001 U/ml culture media in order while the cytosolic recombinant enzyme accounted for about 6% of total proteins. One-step purification of the expressed uricase was done with the aid of the chitin affinity column, followed by DTT induction for intein on-column cleavage. The yield of 40.8mg/L and K m of 0.22mM was obtained for intracellular expression. It seems that the intracellular production of uricase can indeed serve as an effective alternative to secretory expression. Moreover, this is the first report considering cytosolic production of uricase using the intein-mediated protein purification in the methylotrophic yeast, P. pastoris.

A new uricase from Bacillus cereus SKIII: Characterization, gene identification and genetic improvement

Twenty-five microbial isolates were investigated for uricase production on uric acid medium. All isolates were obtained from Jeddah, Saudi Arabia. The highest uricase producer was identified as Bacillus cereus SKIII. Using glucose peptone broth at pH 7.5, incubation temperature 30 °C for 3 days with shaking of 150 rpm were the best conditions for maximum enzyme production. Glucose and peptone were the best carbon and nitrogen sources. The molecular weight of the purified enzyme was34.5 KDa, and isoelectric point was 7.9. The optimum pH and temperature were pH 8.0 and 35 °C, respectively. It was stable at 35 °C for 60 min, but thermally inactivated at 60 °C after 60 min Its enzymatic activity was enhanced by Mg2+, Ca2+,Fe2+, Mn2+, Zn2+ions and inhibited by Co2+, Na+, Hg2+, Ag+ ions and EDTA at 1 mM. Uricase production was enhanced using UV mutation and the obtained mutant produced six times higher than the original isolate. An amplicon 900 bp of uricase gene (Pucl) was sequenced (accession number MF417635). No remarkable difference was noticed in B. cereus SKIII and SKm mutant nucleotide sequences. In conclusion, SKIII and SKm are promising strains in uricase production for biotechnological applications.

Structural insights into the catalytic mechanism of lovastatin hydrolase

Structural insights into the catalytic mechanism of lovastatin hydrolase

Mathematical modeling for bioprocess optimization of a protein drug, uricase, production by Aspergillus welwitschiae strain 1\u20134

Microbial uricase is effective protein drug used to treat hyperuricemia and its complications, including chronic gout, also in prophylaxis and treatment of tumor lysis and organ transplants hyperuricemia. Uricase is commonly used as diagnostic reagent in clinical analysis for quantification of uric acid in blood and other biological fluids. Also, it can be used as an additive in formulations of hair coloring agents. A newly isolated strain, Aspergillus sp. 1–4, was able to produce extracellular uricase on a medium containing uric acid as inducer. Phylogenetic analysis based on ITS region sequence analysis and phenotypic characteristics showed that Aspergillus sp. strain 1–4 is closely related to Aspergillus welwitschiae and its nucleotide sequence was deposited in the GenBank database and assigned sequence accession number MG323529. Statistical screening using Plackett-Burman design with 20 runs was applied to screen fifteen factors for their significance on uricase production by Aspergillus welwitschiae. Results of statistical analysis indicated that incubation time has the most significant positive effect on uricase production followed by yeast extract and inoculum size with the highest effect values of 13.48, 5.26 and 4.75; respectively. The interaction effects and optimal levels of these factors were evaluated using central composite design. The maximum uricase production was achieved at incubation time (5 days), yeast extract (2 g/L) and inoculum size (4 mL/50 mL medium) are the optimum levels for maximum uricase production (60.03 U/mL). After optimization, uricase production increased by 3.02-folds as compared with that obtained from the unoptimized medium (19.87 U/mL).

Statistical media optimization for the production of clinical uricase from Bacillus subtilis strain SP6

In this study, a potent uricase producing organism was isolated by a thorough screening and identified as Bacillus subtilis strain SP6 by using 16s rDNA sequencing. Response surface methodological optimization was employed for the enhanced production of uricase from newly isolated Bacillus subtilis strain SP6. In media optimization studies, Plackett Burman (PB) design was used for the selection of the critical media components; which were further optimized using central composite design (CCD). Lactose, soya peptone, uric acid and FeSO4.7H2O were found to be the critical factors influencing the enzyme production. Optimum uricase production with these factors was deduced using central composite design. Significant level of the factors were 12.2 g/L of lactose, 12.79 g/L of soya peptone, 2.55 g/L of uric acid and 0.00325 g/L FeSO4.7H2O. Use of statistical optimization upsurges uricase yield from 1.2 U/ml to 15.87 U/ml enhancing the overall production by 13.23 fold; which confirms that the model is effective for process optimization.

Production and Characterization of Recombinant Wild Type Uricase from Indonesian Coelacanth (L. menadoensis) and Improvement of Its Thermostability by In Silico Rational Design and Disulphide Bridges Engineering.

The ideal therapeutic uricase (UOX) is expected to have the following properties; high expression level, high activity, high thermostability, high solubility and low immunogenicity. The latter property is believed to depend largely on sequence identity to the deduced human UOX (dH-UOX). Herein, we explored L. menadoensis uricase (LM-UOX) and found that it has 65% sequence identity to dH-UOX, 68% to the therapeutic chimeric porcine-baboon UOX (PBC) and 70% to the resurrected ancient mammal UOX. To study its biochemical properties, recombinant LM-UOX was produced in E. coli and purified to more than 95% homogeneity. The enzyme had specific activity up to 10.45 unit/mg, which was about 2-fold higher than that of the PBC. One-litre culture yielded purified protein up to 132 mg. Based on homology modelling, we successfully engineered I27C/N289C mutant, which was proven to contain inter-subunit disulphide bridges. The mutant had similar specific activity and production yield to that of wild type (WT) but its thermostability was dramatically improved. Up on storage at −20 °C and 4 °C, the mutant retained ~100% activity for at least 60 days. By keeping at 37 °C, the mutant retained ~100% activity for 15 days, which was 120-fold longer than that of the wild type. Thus, the I27C/N289C mutant has potential to be developed for treatment of hyperuricemia.

Screening of some Fungal Isolates for their Potentialities for Uricase Production and Optimization of the Factors Affecting the Production

Screening of some Fungal Isolates for their Potentialities for Uricase Production and Optimization of the Factors Affecting the Production

Optimization of clinical uricase production by Bacillus cereus under submerged fermentation, its purification and structure characterization

Abstract The uricase production from Bacillus cereus after statistical optimization of process variables designed by L18 orthogonal array gave enhanced enzyme activity by 13% (29.7 U/mL) as compared with un-optimized medium (26.3 U/mL). The optimized process variables used for scale up of uricase production in a bioreactor gave 39.7 U/mL of uricase activity resulting 51% increase as compared with unoptimized medium. The Luedeking-Piret model classified the uricase production from Bacillus cereus as mixed-growth associated. The culture supernatant having uricase activity (7.7 U/mg) on purification by (NH4)2SO4 precipitation followed by anion-exchange and size-exclusion chromatography resulted 87 U/mg giving 11-fold purification. The purified uricase can be used for treating gout disease. The de novo amino acid sequence analysis of purified uricase by MALDI-TOF MS showed 301 amino acids and molecular mass of 34.4 kDa, which was corroborated by 35 kDa from SDS-PAGE. The phylogenetic tree generated by homologous amino acid sequences displayed the clustering of Bacillus uricase with fungal uricase. The secondary structure analysis of uricase by circular dichroism showed 18.6% α-helices and 27.4% β-strands. These results were corroborated by secondary structure prediction analysis by PsiPred. The homology modelling of uricase displayed the conserved T-fold structure.