Construction of uricase-overproducing strains of Hansenula polymorpha and its application as biological recognition element in microbial urate biosensor

Dmytruk, Kostyantyn V.; Smutok, Oleh; Dmytruk, Olena V.; Schuhmann, Wolfgang; Sibirny, Andriy А.

doi:10.1186/1472-6750-11-58

Cited by 7 publications

(3 citation statements)

References 33 publications

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“…However, the higher level of intracellular protein compared to the extracellular protein implicates a problem during the secretion of the recombinant protein into the culture medium. In a recent study by Dmytruk and colleagues, the recombinant urate oxidase activity expressed in H. polymorpha showed lower activity compared to the commercially available enzyme (Dmytruk et al 2011 ).…”

Section: Discussionmentioning

confidence: 98%

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: Discussionmentioning

confidence: 98%

Cloning and expression of Aspergillus flavus urate oxidase in Pichia pastoris

et al. 2014

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“…Human serum was centrifuged at 12,500 rpm for 15 min and diluted 100 times with 0.1 M PBS solution at pH 7.4. The same fold dilution was also performed with fresh urine samples to reduce the matrix effect of real samples without other pre-treatments [42]. The recovery of the spiked samples was found to be comparable with that of conventional HPLC techniques and commercialized EasyTouch meter, as shown in Figures 7 and 8, respectively.…”

Section: Real Physiological Sample Analysismentioning

confidence: 66%

Designed Mini Protein 20 Mimicking Uricase Encapsulated in ZIF-8 as Nanozyme Biosensor for Uric Acid Detection

et al. 2022

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This work presents the use of encapsulated mini protein 20 mimicking uricase (mp20)-zeolitic imidazolate framework-8 (ZIF-8) as a bioreceptor for the development of a nanozyme-based electrochemical biosensor for uric acid detection. The electrochemical performance of the biofunctionalized mp20@ZIF-8 on the reduced graphene oxide/screen-printed carbon electrode (rGO/SPCE) was investigated by optimizing operating parameters such as pH, deposition potential, and deposition time using a central composite design-response surface methodology (CCD-RSM). The quadratic regression model was developed to correlate the combination of each variable to the oxidation current density as a response. A significant effect on current response was observed under optimized conditions of pH of 7.4 at −0.35 V deposition potential and 56.56 s deposition time, with p < 0.05 for each interacted factor. The obtained coefficient of determination (R2) value of 0.9992 indicated good agreement with the experimental finding. The developed nanozyme biosensor (mp20@ZIF-8/rGO/SPCE) exhibited high selectivity in the presence of the same fold concentration of interfering species with a detection limit of 0.27 μM, over a concentration range of 1 to 34 μM. The practicality of the tailored biosensor in monitoring uric acid in human serum and urine samples was validated with high-performance liquid chromatography (HPLC) and a commercial uric acid meter. Hence, nanozyme-based is a promising platform that offers a rapid, sensitive, selective, and low-cost biosensor for the non-enzymatic detection of uric acid in biological samples.

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“…The H. polymorpha gcr1 mutants are commonly utilized as the bio-elements for Yeast-based biosensing. Some practical examples of these biosensors utilization include the detection of L-lactate (Smutok et al, 2007), urate (Dmytruk et al, 2011), formaldehyde (Sigawi et al, 2014), and D-lactate (Smutok et al, 2018).…”

Section: Why Use H Polymorpha As Host For Heterologous Expression?mentioning

confidence: 99%

Advances in Using Hansenula polymorpha as Chassis for Recombinant Protein Production

Manfrão-Netto

Gomes

Parachin

2019

Front. Bioeng. Biotechnol.

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The methylotrophic yeast Hansenula polymorpha , known as a non-conventional yeast, is used for the last 30 years for the production of recombinant proteins, including enzymes, vaccines, and biopharmaceuticals. Although a large number of reviews have been published elucidating the applications of this yeast as a cell factory, the latest was released about 10 years ago. Therefore, this review aimed at summarizing available information on the use of H. polymorpha as a host for recombinant protein production in the last decade. Examples of chemicals and virus-like particles produced using this yeast also are discussed. Firstly, the aspects that feature this yeast as a host for recombinant protein production are highlighted including the techniques available for its genetic manipulation as well as strategies for cultivation in bioreactors. Special attention is given to the novel genomic editing tools, mainly CRISPR/Cas9 that was recently established in this yeast. Finally, recent examples of using H. polymorpha as an expression platform are presented and discussed. The production of human Parathyroid Hormone (PTH) and Staphylokinase (SAK) in H. polymorpha are described as case studies for process establishment in this yeast. Altogether, this review is a guideline for this yeast utilization as an expression platform bringing a thorough analysis of the genetic aspects and fermentation protocols used up to date, thus encouraging the production of novel biomolecules in H. polymorpha .

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Construction of uricase-overproducing strains of Hansenula polymorpha and its application as biological recognition element in microbial urate biosensor

Cited by 7 publications

References 33 publications

Cloning and expression of Aspergillus flavus urate oxidase in Pichia pastoris

Cloning and expression of Aspergillus flavus urate oxidase in Pichia pastoris

Designed Mini Protein 20 Mimicking Uricase Encapsulated in ZIF-8 as Nanozyme Biosensor for Uric Acid Detection

Advances in Using Hansenula polymorpha as Chassis for Recombinant Protein Production

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