Biochemical characterization and in vitro digestibility assay of Eupenicillium parvum (BCC17694) phytase expressed in Pichia pastoris

Fugthong, Anusorn; Boonyapakron, Katewadee; Sornlek, Warasirin; Tanapongpipat, Sutipa; Eurwilaichitr, Lily; Pootanakit, Kusol

doi:10.1016/j.pep.2009.10.001

Cited by 24 publications

(31 citation statements)

References 27 publications

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“…Similar results have been reported with the P. pastoris-produced N-glycosylated Bacillus subtilis phytase that shows resistance to trypsin but not pepsin (41). In contrast, the N-glycosylated phytases derived from Aspergillus japonicus BCC18313, A. niger BCC18081, Eupenicillium parvum BCC17694, and Neosartorya spinosa BCC41923 are resistant to pepsin but not to trypsin (26)(27)(28). Our results indicated that the two functional N-glycosylation motifs, NXS/T, of Yersinia phytases contribute to the enzyme proteolytic resistance to pepsin but not trypsin ( Fig.…”

Section: Discussionsupporting

confidence: 84%

“…The N-deglycosylated and untreated enzymes were analyzed in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and stained with Coomassie brilliant blue R-250 (39). The N-glycosylation level of each phytase was determined by the proportion of the N-glycans being estimated to account for the molecular mass, i.e., (actual molecular weight Ϫ theoretical molecular weight) ϫ 100/actual molecular weight, as described previously (27). The protein band intensity was estimated by using ImageJ software (http://rsbweb.nih.gov/ij/).…”

Section: Methodsmentioning

confidence: 99%

“…Site-directed mutagenesis has confirmed that the N-glycosylation of scavenger receptor SREC-I at N289 confers resistance against trypsin digestion (25). Some N-glycosylated microbial phytases also exhibit resistance to degradation by pepsin (26)(27)(28). Thus, N-glycosylation represents a stabilizing factor against proteolytic cleavage by proteases (29).…”

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confidence: 99%

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N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

Niu

Luo

Shi

et al. 2016

Appl Environ Microbiol

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N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed.A s much as 80% of the total phosphorus in cereal grains, oilseeds, and legumes exists in the form of phytate (myo-inositol hexakisphosphate), one of the most important functional ingredients in animal feed (1). However, phytate usually forms indigestible complexes with mineral cations and proteins, thereby causing reductions in nutrient utilization (2, 3). The dietary phytate undigested by monogastric animals is also released into the environment, where it causes phosphorus pollution (4, 5).The enzyme phytase catalyzes the sequential release of inorganic phosphate and lower myo-inositol phosphates from phytate (6); thus, inclusion of exogenous phytases in animal feeds as enzyme additives can enhance the nutrient uptake, lower the feedstuff production costs, and protect the environment in regions where intensive animal farming is conducted (7,8). Numerous phytases have been identified from microorganisms, animals, and plants (9-11), but only a few of them are commercially produced (12). The extensive application of phytases is limited by enzyme lability and protease sensitivity under high processing temperatures and low physiological pHs. Therefore, improving phytase stability at low pHs and high protease concentrations is desirable.Pepsin, a monomeric protein composed of two ␤-barrel-like domains, represents the main proteolytic enzyme in gastric juice (13). The acidic residues D32 and D215 are mainly responsible for proteoly...

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Section: Discussionsupporting

confidence: 84%

Section: Methodsmentioning

confidence: 99%

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N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

Niu

Luo

Shi

et al. 2016

Appl Environ Microbiol

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“…Phytate's phosphate residues are chemically active and can chelate metal ions. Such phytate-metal ion complexes at neutral and basic soil pH values are insoluble and precipitate easily, making them unavailable for cellular metabolism (5,6). In contrast, at acidic pH values the phosphate groups become protonated, can be easily dissolved in water, and enter metabolism.…”

mentioning

confidence: 99%

Novel Glucose-1-Phosphatase with High Phytase Activity and Unusual Metal Ion Activation from Soil Bacterium Pantoea sp. Strain 3.5.1

Suleimanova

Beinhauer

Valeeva

et al. 2015

Appl Environ Microbiol

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Phosphorus is an important macronutrient, but its availability in soil is limited. Many soil microorganisms improve the bioavailability of phosphate by releasing it from various organic compounds, including phytate. To investigate the diversity of phytate-hydrolyzing bacteria in soil, we sampled soils of various ecological habitats, including forest, private homesteads, large agricultural complexes, and urban landscapes. Bacterial isolate Pantoea sp. strain 3.5.1 with the highest level of phytase activity was isolated from forest soil and investigated further. The Pantoea sp. 3.5.1 agpP gene encoding a novel glucose-1-phosphatase with high phytase activity was identified, and the corresponding protein was purified to apparent homogeneity, sequenced by mass spectroscopy, and biochemically characterized. The AgpP enzyme exhibits maximum activity and stability at pH 4.5 and at 37°C. The enzyme belongs to a group of histidine acid phosphatases and has the lowest K m values toward phytate, glucose-6-phosphate, and glucose-1-phosphate. Unexpectedly, stimulation of enzymatic activity by several divalent metal ions was observed for the AgpP enzyme. High-performance liquid chromatography (HPLC) and high-performance ion chromatography (HPIC) analyses of phytate hydrolysis products identify DL-myo-inositol 1,2,4,5,6-pentakisphosphate as the final product of the reaction, indicating that the Pantoea sp. AgpP glucose-1-phosphatase can be classified as a 3-phytase. The identification of the Pantoea sp. AgpP phytase and its unusual regulation by metal ions highlight the remarkable diversity of phosphorus metabolism regulation in soil bacteria. Furthermore, our data indicate that natural forest soils harbor rich reservoirs of novel phytate-hydrolyzing enzymes with unique biochemical features. P hosphorus is one of the most important macroelements of living cells. It is a crucial component of nucleic acids, highenergy compounds, phospholipids, and other molecules and is essential for normal plant growth. Many soil types are naturally low in inorganic phosphorus, and, as the pressure on agriculture to provide food for the growing human population intensifies, so does the need for a massive use of phosphate fertilizers. However, rock phosphate is a nonrenewable natural resource (1). Furthermore, the vast majority of applied phosphate fertilizer quickly interacts with soil components, leading to its rapid transformation into various organic or precipitated compounds inaccessible to plants. Thus, much attention has recently been paid to alternative sources of phosphorus in soil.Organic phosphorus compounds in soil account for 30 to 50% of total soil phosphorus and are often considered natural alternatives to the use of mineral phosphate fertilizer (2). Phytate is a molecule of myo-inositol with six phosphate residues (3). Phytate concentration varies from 3.9% to 25.3% of total extractable P in carbonate-free cambisol soils and calcareous chernozems, respectively (4), suggesting that in some cases phytate can be one of the major for...

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“…However, only several phytases reported to date were characterized as stable under gastric or acidic conditions Fugthong et al, 2010;Garrett et al, 2004;Huang et al, 2008;Singh and Satyanarayana, 2009]. To our knowledge, the Yersinia rohdei phytase shows the highest stability in acidic plus pepsin conditions among all the phytases that have been reported to date , and can retain nearly 30% residual activity after incubation in simulated gastric fluid (SGF) for 20 min.…”

Section: Introductionmentioning

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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Biochemical characterization and in vitro digestibility assay of Eupenicillium parvum (BCC17694) phytase expressed in Pichia pastoris

Cited by 24 publications

References 27 publications

N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

Novel Glucose-1-Phosphatase with High Phytase Activity and Unusual Metal Ion Activation from Soil Bacterium Pantoea sp. Strain 3.5.1

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

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