Determination of uric acid on continuous-flow (AutoAnalyzer II and SMA) systems with a uricase/phenol/4-aminophenazone color test.

Klose, Susanne; Stoltz, M.; Muñz, E.; Portenhauser, R

doi:10.1093/clinchem/24.2.250

Cited by 38 publications

(6 citation statements)

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“…The urate oxidase activity was determined using a modified urate oxidase/phenol/4-aminoantipyrine colorimetric assay (Klose et al 1987 )). Briefly, uric acid can be specifically converted to allantoin and hydrogen peroxide by urate oxidase.…”

Section: Methodsmentioning

confidence: 99%

Cloning and expression of Aspergillus flavus urate oxidase in Pichia pastoris

et al. 2014

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Urate oxidase is an important enzyme with therapeutic and diagnostic applications. Rasburicase is a recombinant urate oxidase enzyme approved by FDA to use in the treatment of hyperuricemia conditions. Various hosts such as Saccharomyces cerevisiae, Hansenula polymorpha and Escherichia coli have been used to express the enzyme. Today, Pichia pastoris is considered as an important host for heterologous protein expression since it has beneficial characteristics such as strong promoters, simple scale up, post translational modifications, high cell density cultivation and simple genetic manipulation. In this study, Aspergillus flavus urate oxidase gene was cloned in pPICZαA expression vector and expressed in P. pastoris. The recombinant urate oxidase was expressed in secretory form and was confirmed through RT-PCR, SDS-PAGE analysis and western blotting. The enzyme activity was determined using a colorimetric assay. A production yield of 0.43 U/ml of culture supernatant was obtained.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-395) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Cloning and expression of Aspergillus flavus urate oxidase in Pichia pastoris

et al. 2014

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“…Uricase activity was assayed by a HRP-coupled reaction using horseradish peroxidase (10 mM), uric acid (1.5 mM), 4-AAP (12 mM), and phenol (60 mM) in 20 mM potassium phosphate buffer (pH 7.0) (Klose et al 1978). The amount of produced H 2 O 2 was measured using HRP catalyzing the oxidation of phenol in the presence of 4-AAP (Asad et al 2014).…”

Section: Enzyme Assaymentioning

confidence: 99%

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

Khaleghi

Asad

2021

3 Biotech

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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 2000 μg/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 40.8 mg/L and K m of 0.22 mM 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.

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“…However, when using these chemotherapy method may induce the side effects to the body. [7] Nephroprotective agents are the substances which possess protective activity against nephrotoxicity. Drugs like gentamicin, cisplatin, cyclosporine, Carbon tetrachloride are common source of acute kidney injury.…”

Section: Introductionmentioning

confidence: 99%

Nephroprotective Potential Compounds from Leaves Extracts of Andrographis Paniculata

Padmalochana¹

2017

TIJBMS

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Nephrotoxicity is the third most common problem of the renal system. Medicinal plants potentially useful for the treatment of severe renal disorders. In the present study, the effect of aqueous, ethanol and acetone extract of Andrographis paniculata leaves was evaluated by pretreating three groups of rat animals. Renal failure was induced by antibiotic drug gentamicin which was orally administered to group II animals and untreated control model was maintained. After oral administration for 10 days, serum levels of urea, uric acid, creatinine and total protein were assayed. All the biochemical parameters were significantly controlled (p<0.01 and p<0.001) on 10 th day after the treatment of aqueous, ethanol and acetone extracts as compared than the gentamicin induced nephrotoxic animals (p<0.05). Maximum nephroprotection was offered by the ethanol extract of A. paniculata leaves (p<0.001). The histological structure of renal was observed by staining with haemotoxylin-eosin viewed using light microscope. Gentamicin treated animals showed acute tubular necrosis due the injury in kidney. In aqueous, ethanol and acetone extract treated animals showed histopathological changes in renal revealed the nephroprotective activity of A. paniculata leaves. Finally, ethanol extract of A. paniculata was more suitable for nephroprotective action against gentamicin induced renal failure was evidenced by biochemical estimation and by restoration of histological changes of renal system.

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Determination of uric acid on continuous-flow (AutoAnalyzer II and SMA) systems with a uricase/phenol/4-aminophenazone color test.

Cited by 38 publications

References 20 publications

Cloning and expression of Aspergillus flavus urate oxidase in Pichia pastoris

Cloning and expression of Aspergillus flavus urate oxidase in Pichia pastoris

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

Nephroprotective Potential Compounds from Leaves Extracts of Andrographis Paniculata

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