High-level expression, purification, and characterization of non-tagged Aspergillus flavus urate oxidase in Escherichia coli

Li, Jianmin; Chen, Zhao; Hou, Lihua; Fan, Huahao; Weng, Shao-Jie; Xu, Chun’e; Ren, Jun; Li, Bing; Chen, Wei

doi:10.1016/j.pep.2006.02.003

Cited by 31 publications

(42 citation statements)

References 16 publications

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“…One unit of enzyme was defined as the amount of enzyme that produces 1.0 μmol of H 2 O 2 per minute under the standard assay conditions. This method was also confirmed by the method described by Li et al (2006) by following the disappearance of uric acid detected by a decrease in absorbance at 292 nm.…”

Section: Assay Of Uricase Activitymentioning

confidence: 66%

“…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%

“…Uricase from C. utilis, A. flavus, Bacillus. sp, and recombinant uricase from E. coli or S. cerevisiae are now commercially available, but the production procedures still have the problems with low productivity, complicated isolation, and purification procedure for clinical applications (Li et al 2006;Lotfy 2008). The methylotrophic yeast H. polymorpha has been proven to be an effective expression system for the production of heterologous proteins with medical application (Gellissen 2000;Gellissen et al 2005).…”

Section: Discussionmentioning

confidence: 99%

“…Microorganisms, such as bacteria, yeast, and filamentous fungi, are important sources of uricase. Genes encoding uricase have been cloned from A. flavus, A. nidulans, C. utilis, Arthrobacter globiformis, Cellulomonas flavigena and symbiont of Nilaparvata lugens (Suzuki et al 2004), and expressed in Escherichia coli (Li et al 2006). Uricase from A. flavus was expressed in the yeast S. cerevisiae in a soluble and active form (Leplatois et al 1992).…”

Section: Introductionmentioning

confidence: 99%

“…Uricase from A. flavus was expressed in the yeast S. cerevisiae in a soluble and active form (Leplatois et al 1992). However, the recombinant uricase from E. coli or S. cerevisiae still has the problem with low productivity and cost-effective separation for medical applications (Li et al 2006). As the methylotrophic yeast, H. polymorpha in particular has been studied extensively for its highly regulated biogenesis of peroxisomes, nitrate assimilation, and methanol metabolism.…”

Section: Introductionmentioning

confidence: 99%

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

Chen

Wang

et al. 2008

Appl Microbiol Biotechnol

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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.

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Section: Assay Of Uricase Activitymentioning

confidence: 66%

Section: Discussionmentioning

confidence: 77%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Chen

Wang

et al. 2008

Appl Microbiol Biotechnol

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Economic analysis of uricase production under uncertainty: Contrast of chromatographic purification and aqueous two-phase extraction (with and without PEG recycle)

Torres‐Acosta

Aguilar‐Yáñez

Rito‐Palomares

et al. 2015

Biotechnol Progress

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Uricase is the enzyme responsible for the breakdown of uric acid, the key molecule leading to gout in humans, into allantoin, but it is absent in humans. It has been produced as a PEGylated pharmaceutical where the purification is performed through three sequential chromatographic columns. More recently an aqueous two‐phase system (ATPS) was reported that could recover Uricase with high yield and purity. Although the use of ATPS can decrease cost and time, it also generates a large amount of waste. The ability, therefore, to recycle key components of ATPS is of interest. Economic modelling is a powerful tool that allows the bioprocess engineer to compare possible outcomes and find areas where further research or optimization might be required without recourse to extensive experiments and time. This research provides an economic analysis using the commercial software BioSolve of the strategies for Uricase production: chromatographic and ATPS, and includes a third bioprocess that uses material recycling. The key parameters that affect the process the most were located via a sensitivity analysis and evaluated with a Monte Carlo analysis. Results show that ATPS is far less expensive than chromatography, but that there is an area where the cost of production of both bioprocesses overlap. Furthermore, recycling does not impact the cost of production. This study serves to provide a framework for the economic analysis of Uricase production using alternative techniques. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:126–133, 2016

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Process Economics: Evaluation of the Potential of ATPS as a Feasible Alternative to Traditional Fractionation Techniques

Torres‐Acosta

Ruiz‐Ruiz

Benavides

et al. 2017

Food Engineering Series

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High-level expression, purification, and characterization of non-tagged Aspergillus flavus urate oxidase in Escherichia coli

Cited by 31 publications

References 16 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

Economic analysis of uricase production under uncertainty: Contrast of chromatographic purification and aqueous two-phase extraction (with and without PEG recycle)

Process Economics: Evaluation of the Potential of ATPS as a Feasible Alternative to Traditional Fractionation Techniques

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