Cloning, Sequence Analysis, and Expression in Escherichia coli of the Gene Encoding the Candida utilis Urate Oxidase (Uricase)

Koyama, Yasuji; Ichikawa, Toshio; Nakano, E.

doi:10.1093/oxfordjournals.jbchem.a021514

Cited by 47 publications

(30 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The combined use of fed-batch culture and pH-controlled strategy increased the expression level of uricase significantly. The extracellular uricase production of 52.3 U/ml (approximately 2.1 g/l of protein) was obtained, which is much higher than that produced by recombinant E. coli strains (about 6.1 U/ml or 238 mg/l; Koyama et al 1996;Li et al 2006).…”

Section: Discussionmentioning

confidence: 77%

See 1 more Smart Citation

Uricase production by a recombinant Hansenula polymorpha strain harboring Candida utilis uricase gene

Chen

Wang

et al. 2008

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

Uricase is an important medical enzyme which can be used to determine urate in clinical analysis, to therapy gout, hyperuricemia, and tumor lysis syndrome. Uricase of Candida utilis was successfully expressed in Hansenula polymorpha under the control of methanol oxidase promoter using Saccharomyces cerevisiae α-factor signal peptide as the secretory sequence. Recombinant H. polymorpha MU200 with the highest extracellular uricase production was characterized with three copies of expression cassette and selected for process optimization for the production of recombinant enzyme. Among the parameters investigated in shaking flask cultures, the pH value of medium and inoculum size had great influence on the recombinant uricase production. The maximum extracellular uricase yield of 2.6 U/ml was obtained in shaking flask culture. The yield of recombinant uricase was significantly improved by the combined use of a high cell-density cultivation technique and a pH control strategy of switching culture pH from 5.5 to 6.5 in the induction phase. After induction for 58 h, the production of recombinant uricase reached 52.3 U/ml (about 2.1 g/l of protein) extracellularly and 60.3 U/ml (about 2.4 g/l) intracellularly in fed-batch fermentation, which are much higher than those expressed in other expression systems. To our knowledge, this is the first report about the heterologous expression of uricase in H. polymorpha.

show abstract

Section: Discussionmentioning

confidence: 77%

“…The gene contains 909 base pairs without any intron and encodes a protein composed of 303 amino acid residues with a mass of 34,1463 Da (Koyama et al 1996). The C. utilis uricase does not require any cofactor for the enzymatic oxidation (Bonnete et al 2001).…”

Section: Introductionmentioning

confidence: 99%

Uricase production by a recombinant Hansenula polymorpha strain harboring Candida utilis uricase gene

Chen

Wang

et al. 2008

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

show abstract

“…This enzyme is found in mammals [1,2], plants [3], fungi [4], yeasts [5][6][7] and bacteria [8]. Uric acid, the primary end-product of purine metabolism, is present in biological fluids, including blood and urine [9].…”

mentioning

confidence: 99%

“…Only one uricase, from Bacillus sp. TB-90, was observed to have both high activity and thermostability over a wide range of pH values (pH [6][7][8][9].…”

mentioning

confidence: 99%

Modified colorimetric assay for uricase activity and a screen for mutant Bacillus subtilis uricase genes following StEP mutagenesis

Huang

2004

European Journal of Biochemistry

View full text Add to dashboard Cite

This study describes a modified colorimetric assay for uricase activity in flexible 96‐well microtiter plates using the uricase/uric acid/horseradish peroxidase/4‐aminoantipyrine/3,5‐dichloro‐2‐hydroxybenzene sulfonate colorimetric reaction. The utility of this assay was demonstrated in a screen for mutant uricase enzymes derived from the uricase gene of the thermophilic bacterium Bacillus subtilis by a modified staggered extension process (StEP) mutagenesis. An Escherichia coli library of StEP‐derived uricase mutant clones was screened yielding two identical active mutant uricase genes. Two motifs conserved in eukaryotic and prokaryotic uricases are highly conserved in the mutant uricase. The mutant uricase protein was found to exhibit high uricase activity (13.1 U·mg−1). Finally, the modified colorimetric method is much more efficient than the conventional ones and greatly reduces assay time from 4 days to less than 20 h.

show abstract

“…89 Vários estudos (NISHIMURA et al, 1979;KOYAMA;. Quando comparamos o grau de modificação atingindo pela BSA com o mPEG-pNP percebemos que esse polímero é menos reativo, porque podemos observar pelos poços 1, 2 e 3 que existe a formação de um conjugado em torno de 86 kDa e a BSA nativa livre (poço 1).…”

Section: (B)unclassified

Funcionalidade e caracterização das propriedades físico-químicas, biológicas e estruturais da uricase modificada por PEGlação.

Freitas¹

View full text Add to dashboard Cite

Cloning, Sequence Analysis, and Expression in Escherichia coli of the Gene Encoding the Candida utilis Urate Oxidase (Uricase)

Cited by 47 publications

References 26 publications

Uricase production by a recombinant Hansenula polymorpha strain harboring Candida utilis uricase gene

Uricase production by a recombinant Hansenula polymorpha strain harboring Candida utilis uricase gene

Modified colorimetric assay for uricase activity and a screen for mutant Bacillus subtilis uricase genes following StEP mutagenesis

Funcionalidade e caracterização das propriedades físico-químicas, biológicas e estruturais da uricase modificada por PEGlação.

Contact Info

Product

Resources

About