Gene cloning, expression and characterization of novel phytase from Obesumbacterium proteus

Zinin, Nickolay V; Serkina, A. V.; Gelfand, Mikhail S.; Шевелев, А. Б.; Sineoky, Sergei P

doi:10.1016/j.femsle.2004.05.051

Cited by 16 publications

(15 citation statements)

References 32 publications

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“…There have been many reports on the cloning and expressions of phyA, phyC and appA phytases (Chen et al, 2004;Rodriguez et al, 2000;Kim et al, 1999;Zinin et al, 2004;Rodriguez et al, 1999;Wang et al, 2005;Wu et al, 2004a;2004b;. Recently phyA was site-mutated to phyA m and phyCs was synthesized without altering the protein sequence of phyC according to the Pichia pastoris codon usage bias because of the low productions of wild-type phyA and phyC phytases in P. pastoris, and we overexpressed them in yeast as an active and extracellular enzyme (Chen et al, 2005;Zou et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

Expression, purification and characterization of a phyA m -phyCs fusion phytase

Zou

Wang

Pan

et al. 2008

J. Zhejiang Univ. Sci. B

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Abstract:The phyA m gene encoding acid phytase and optimized neutral phytase phyCs gene were inserted into expression vector pPIC9K in correct orientation and transformed into Pichia pastoris in order to expand the pH profile of phytase and decrease the cost of production. The fusion phytase phyA m -phyCs gene was successfully overexpressed in P. pastoris as an active and extracellular phytase. The yield of total extracellular fusion phytase activity is (25.4±0.53) U/ml at the flask scale and (159.1±2.92) U/ml for high cell-density fermentation, respectively. Purified fusion phytase exhibits an optimal temperature at 55 °C and an optimal pH at 5.5~6.0 and its relative activity remains at a relatively high level of above 70% in the range of pH 2.0 to 7.0. About 51% to 63% of its original activity remains after incubation at 75 °C to 95 °C for 10 min. Due to heavy glycosylation, the expressed fusion phytase shows a broad and diffuse band in SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). After deglycosylation by endoglycosidase H (EndoH f ), the enzyme has an apparent molecular size of 95 kDa. The characterization of the fusion phytase was compared with those of phyCs and phyA m .

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Section: Introductionmentioning

confidence: 99%

Expression, purification and characterization of a phyA m -phyCs fusion phytase

Zou

Wang

Pan

et al. 2008

J. Zhejiang Univ. Sci. B

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“…The enrichment of OTUs related to Pseudomonas and Rahnella is likely due to the previously described phosphate solubilizing activities of related genera isolated from rhizosphere and U-contaminated sediments [22], [31], [51]. The Obesumbacterium -related OTU has not been described as a common phosphate solubilizing isolate but characterization of an encoded phytase in Obesumbacterium proteus suggests related strains may be capable of organophosphate hydrolysis [58]. The genera Arsenophonus , Photorhabdus , Brenneria , and Pectobacterium contain species that have been described as symbionts or plant pathogens but to date have not been shown to enhance phosphate solubilization [59]–[62].…”

Section: Discussionmentioning

confidence: 90%

Microbial Community Responses to Organophosphate Substrate Additions in Contaminated Subsurface Sediments

Martinez

Beazley

et al. 2014

PLoS ONE

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BackgroundRadionuclide- and heavy metal-contaminated subsurface sediments remain a legacy of Cold War nuclear weapons research and recent nuclear power plant failures. Within such contaminated sediments, remediation activities are necessary to mitigate groundwater contamination. A promising approach makes use of extant microbial communities capable of hydrolyzing organophosphate substrates to promote mineralization of soluble contaminants within deep subsurface environments.Methodology/Principal FindingsUranium-contaminated sediments from the U.S. Department of Energy Oak Ridge Field Research Center (ORFRC) Area 2 site were used in slurry experiments to identify microbial communities involved in hydrolysis of 10 mM organophosphate amendments [i.e., glycerol-2-phosphate (G2P) or glycerol-3-phosphate (G3P)] in synthetic groundwater at pH 5.5 and pH 6.8. Following 36 day (G2P) and 20 day (G3P) amended treatments, maximum phosphate (PO4 3−) concentrations of 4.8 mM and 8.9 mM were measured, respectively. Use of the PhyloChip 16S rRNA microarray identified 2,120 archaeal and bacterial taxa representing 46 phyla, 66 classes, 110 orders, and 186 families among all treatments. Measures of archaeal and bacterial richness were lowest under G2P (pH 5.5) treatments and greatest with G3P (pH 6.8) treatments. Members of the phyla Crenarchaeota, Euryarchaeota, Bacteroidetes, and Proteobacteria demonstrated the greatest enrichment in response to organophosphate amendments and the OTUs that increased in relative abundance by 2-fold or greater accounted for 9%–50% and 3%–17% of total detected Archaea and Bacteria, respectively.Conclusions/SignificanceThis work provided a characterization of the distinct ORFRC subsurface microbial communities that contributed to increased concentrations of extracellular phosphate via hydrolysis of organophosphate substrate amendments. Within subsurface environments that are not ideal for reductive precipitation of uranium, strategies that harness microbial phosphate metabolism to promote uranium phosphate precipitation could offer an alternative approach for in situ sequestration.

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“…Interestingly, the substrate affinity as well as the catalytic efficiency ( k cat / K m ) are indeed higher than many HAPs [Fugthong et al, 2010;Gu et al, 2009;Huang et al, 2009a;Kim et al, 2003;Luo et al, 2007;Pandee et al, 2011;Rodriguez et al, 1999;Sajidan et al, 2004;Shi et al, 2008;Vats and Banerjee 2005;Wu et al, 2014;Zinin et al, 2004] and non-HAP phytases [Gulati et al, 2007;Hmida-Sayari n/a n/a n/a 2.1 2.1 0.7 0.8 n/a n/a n/a 2-Naphthyl phosphate 28.1±3.3 n/a n/a n/a n/a 4.9 n/a n/a n/a n/a n/a n/a n/a Glucose-6-phosphate The phytase residual activity, measured at pH 2.5 and 37°C, is shown as the percentage defining the activity as 100% for the phytate. For the phosphorohydrolytic activities of other HAP phytases, the number of decimal places as well as standard deviations (if available) were kept the same as in the cited publications.…”

Section: Discussionmentioning

confidence: 99%

A Novel Phytase Derived from an Acidic Peat-Soil Microbiome Showing High Stability under Acidic Plus Pepsin Conditions

Tan¹,

Wu²,

Xie³

et al. 2016

Microb Physiol

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Four novel phytases of the histidine acid phosphatase family were identified in two publicly available metagenomic datasets of an acidic peat-soil microbiome in northeastern Bavaria, Germany. These enzymes have low similarity to all the reported phytases. They were overexpressed in Escherichia coli and purified. Catalytic efficacy in simulated gastric fluid was measured and compared among the four candidates. The phytase named rPhyPt4 was selected for its high activity. It is the first phytase identified from unculturable Acidobacteria. The phytase showed a longer half-life than all the gastric-stable phytases that have been reported to date, suggesting a strong resistance to low pH and pepsin. A wide pH profile was observed between pH 1.5 and 5.0. At the optimum pH (2.5) the activity was 2,790 μmol/min/mg at the physiological temperature of 37°C and 3,989 μmol/min/mg at the optimum temperature of 60°C. Due to the competent activity level as well as the high gastric stability, the phytase could be a potential candidate for practical use in livestock and poultry feeding

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Gene cloning, expression and characterization of novel phytase from Obesumbacterium proteus

Cited by 16 publications

References 32 publications

Expression, purification and characterization of a phyA m -phyCs fusion phytase

Expression, purification and characterization of a phyA m -phyCs fusion phytase

Microbial Community Responses to Organophosphate Substrate Additions in Contaminated Subsurface Sediments

A Novel Phytase Derived from an Acidic Peat-Soil Microbiome Showing High Stability under Acidic Plus Pepsin Conditions

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