Purification and characterization of a novel thermostable phytase from the thermophilic<i>Geobacillus</i>sp. TF16

Dokuzparmak, Emre; Şirin, Yakup; Cakmak, Ummuhan; Ertunga, Nagihan Saglam

doi:10.1080/10942912.2016.1203930

Cited by 17 publications

(11 citation statements)

References 44 publications

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“…Similar monomeric enzymes were characterized from Lactobacillus sanfranciscensis (De Angelis et al 2003 ), Lactobacillus plantarum (Sumengen et al 2013 ), Enterobacter sp., Geobacillus sp. (Dokuzparmak et al 2017 ), and Bacillus subtilis (Jain et al 2018 ) with molecular masses of 50, 46, 42, 106.04, and 46 kDa, respectively. However, trimeric intracellular phytases with subunits of 72, 36, and 33 kDa were reported for Lactobacillus plantarum (Sumengen et al 2013 ).…”

Section: Resultsmentioning

confidence: 99%

Characterization of a putative metal-dependent PTP-like phosphatase from Lactobacillus helveticus 2126

Priyodip

Balaji

2023

Int Microbiol

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To date, there are very limited reports on sequence analysis and structure-based molecular modeling of phosphatases produced by probiotic bacteria. Therefore, a novel protein tyrosine-like phosphatase was characterized from L. helveticus 2126 in this study. The purified bacterial phosphatase was subjected to mass spectrometric analysis, and the identity of constructed sequence was analyzed using peptide mass fingerprint. The 3-D structure of protein was elucidated using homology modeling, while its stability was assessed using Ramachandran plot, VERIFY 3D, and PROCHECK. The bacterium produced an extracellular phosphatase of zone diameter 15 ± 0.8 mm on screening medium within 24 h of incubation. This bacterial phosphatase was highly specific towards sodium phytate as it yielded the lowest Km value of 299.50 ± 4.95 μM compared to other phosphorylated substrates. The activity was effectively stimulated in the presence of zinc, magnesium, and manganese ions thereby showing its PTP-like behavior. The phosphatase showed a molecular mass of 43 kDa, and the corresponding M/Z ratio data yielded 46% query coverage to Bacillus subtilis (3QY7). This showed a 61.1% sequence similarity to Ligilactobacillus ruminis (WP_046923835.1). The final sequence construct based on these bacteria showed a conserved motif “HCHILPGIDD” in their active site. In addition, homology modeling showed a distorted Tim barrel structure with a trinuclear metal center. The final model after energy minimization showed 90.9% of the residues in the favorable region of Ramachandran’s plot. This structural information can be used in genetic engineering for improving the overall stability and catalytic efficiency of probiotic bacterial phosphatases.

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Section: Resultsmentioning

confidence: 99%

Characterization of a putative metal-dependent PTP-like phosphatase from Lactobacillus helveticus 2126

Priyodip

Balaji

2023

Int Microbiol

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“…One phytase unit is defined as the amount of the enzyme releasing 1 μM inorganic phosphate per minute under the assay conditions. All samples were assayed in triplicate [ 42 ].…”

Section: Methodsmentioning

confidence: 99%

Characterisation of a soil MINPP phytase with remarkable long-term stability and activity from Acinetobacter sp.

Rix¹,

Sprigg²,

Whitfield³

et al. 2022

PLoS ONE

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Phylogenetic analysis, homology modelling and biochemical methods have been employed to characterize a phytase from a Gram-negative soil bacterium. Acinetobacter sp. AC1-2 phytase belongs to clade 2 of the histidine (acid) phytases, to the Multiple Inositol Polyphosphate Phosphatase (MINPP) subclass. The enzyme was extraordinarily stable in solution both at room temperature and 4°C, retaining near 100% activity over 755 days. It showed a broad pH activity profile from 2–8.5 with maxima at 3, 4.5–5 and 6. The enzyme showed Michaelis-Menten kinetics and substrate inhibition (Vmax, Km, and Ki, 228 U/mg, 0.65 mM and 2.23 mM, respectively). Homology modelling using the crystal structure of a homologous MINPP from a human gut commensal bacterium indicated the presence of a potentially stabilising polypeptide loop (a U-loop) straddling the active site. By employ of the enantiospecificity of Arabidopsis inositol tris/tetrakisphosphate kinase 1 for inositol pentakisphosphates, we show AC1-2 MINPP to possess D6-phytase activity, which allowed modelling of active site specificity pockets for InsP6 substrate. While phytase gene transcription was unaltered in rich media, it was repressed in minimal media with phytic acid and orthophosphate as phosphate sources. The results of this study reveal AC1-2 MINPP to possess desirable attributes relevant to biotechnological use.

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“…The enzyme was previously precipitated by using ammonium sulfate and purified with DEAE-Sepharose Fast Flow ion-exchange chromatography. [25] Immobilization of purified enzyme in chitosan particles by covalent binding…”

Section: Purification Of Geobacillus Sp Tf16 Phytasementioning

confidence: 99%

“…TF16 free phytase were 1.31 mM and 526.28 U/mg protein, respectively. [25] It was reported that K m and V max values were found to be 10 mM and 1 U/mg for immobilized avocado phytase, respectively. [37] Also, K m values were calculated to be 95 μmol/L (A. niger), 220 μmol/L (E. albertii), and 455 μmol/L −1 (rye).…”

Section: Kinetic Parameters Of Immobilized Enzymesmentioning

confidence: 99%

Dephytinization of food stuffs by phytase of Geobacillus sp. TF16 immobilized in chitosan and calcium-alginate

Şirin

Akatin

Çolak

et al. 2017

International Journal of Food Properties

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In this work, Geobacillus sp. TF16 phytase was separately immobilized in chitosan and Ca-alginate with the efficiency of 38% and 42%, respectively. These enzymes exhibited broad substrate specificity. Maximal relative phytase activity was measured at pH 5.0 and 95°C and pH 3.0 and 75°C for chitosan and Ca-alginate, respectively. The enzymes were highly stable in a wide pH and temperature range. Values of K m and V max were determined as 2.38 mM and 3401.36 U/mg protein for chitosan, and 7.5 mM and 5011.12 U/mg protein for Ca-alginate. The immobilized enzymes showed higher resistance to proteolysis. After 4 h incubation, hydrolysis capacities of chitosan-and Ca-alginate immobilized enzymes for soymilk phytate were calculated as 24% and 33%, respectively. The chitosan-and Ca-alginate immobilized phytases conserved its original activity after 8 and 6 cycles of reuse, respectively. The features of the enzymes were very attractive and they might be useful for some industrial applications.

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Purification and characterization of a novel thermostable phytase from the thermophilicGeobacillussp. TF16

Cited by 17 publications

References 44 publications

Characterization of a putative metal-dependent PTP-like phosphatase from Lactobacillus helveticus 2126

Characterization of a putative metal-dependent PTP-like phosphatase from Lactobacillus helveticus 2126

Characterisation of a soil MINPP phytase with remarkable long-term stability and activity from Acinetobacter sp.

Dephytinization of food stuffs by phytase of Geobacillus sp. TF16 immobilized in chitosan and calcium-alginate

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