Activity of Escherichia coli, Aspergillus niger, and Rye Phytase toward

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Activity of Escherichia coli, Aspergillus niger and Rye Phytase towards Partially Phosphorylated myo-Inositol Phosphates Ralf Greiner J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03897 • Publication Date (Web): 20 Oct 2017 Downloaded from http://pubs.acs.org on October 22, 2017

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Journal of Agricultural and Food Chemistry

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Activity of Escherichia coli, Aspergillus niger and Rye Phytase towards Partially

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Phosphorylated myo-Inositol Phosphates

3 4

Ralf Greiner

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Max Rubner-Institut, Federal Research Institute of Nutrition and Food, Department of

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Food Technology and Bioprocess Engineering, Haid-und-Neu-Straße 9, 76131

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Karlsruhe, Germany

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*

To whom the correspondence should be addressed:

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R. Greiner

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Max Rubner-Institut, Federal Research Institute of Nutrition and Food

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Department of Food Technology and Bioprocess Engineering

19

Haid-und-Neu-Straße 9

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76131 Karlsruhe

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Germany

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Phone: +49 (0) 721 / 6625 300

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Fax: +49 (0) 721 / 6625 303

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e-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Kinetic parameters for the dephosphorylation of sodium phytate and a series of

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partially phosphorylated myo-inositol phosphates were determined at pH 3.0 and pH

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5.0 for three phytase preparations (Aspergillus niger, Escherichia coli, rye). The

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enzymes showed lower affinity and turnover numbers at pH 3 compared to pH 5

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towards all myo-inositol phosphates included in the study. The number and

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distribution of phosphate groups on the myo-inositol ring affected the kinetic

33

parameters. Representatives of the individual phytate dephosphorylation pathways

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were identified as the best substrates of the phytases. Within the individual phytate

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dephosphorylation pathways, the pentakisphosphates were better substrates

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compared to the tetrakisphosphates or phytate itself. E. coli and rye phytase showed

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comparable activities at both pH values towards the tetrakis- and trisphosphate,

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whereas A. niger phytase exhibited a higher activity towards the tetrakisphosphate. A

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myo-inositol phosphate with alternate phosphate groups was shown to be not

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significantly dephosphorylated by the phytases.

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KEYWORDS: dephosphorylation; kinetic parameters; myo-inositol phosphate esters; phytase

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INTRODUCTION

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Any phosphatase releasing at least one phosphate residue from phytate (myo-inositol

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hexakisphosphate), the most prevalent form of phosphate in grains and oil seeds, is

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called phytase1. While phytases have been identified in many pro- and eukaryotes,

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they have been most commonly characterised from fungi and bacteria . So far, four

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distinct classes of phytases have been reported in respect to the catalytic mechanism

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of phytate dephosphorylation; histidine acid phytases , β-propeller phytases , purple

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acid phytases4, and protein tyrosine phosphatase-like phytases5 In the last 15 years,

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phytases have become one of the most important feed supplements for monogastric

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animals, mainly swine and poultry6. The first commercialised phytase was of fungal

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origin6. However, from 2000 on a wave of research on bacterial phytases could be

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observed accompanied by a shift in screening for microorganisms producing high

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phytase activity towards microorganism producing phytases exhibiting interesting

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enzymatic properties such as broad acidic pH profile, thermal stability, catalytic

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efficiency, resistance to proteolysis and tolerance to low pH6. A further shift towards

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better phytases for the use as feed additives involved bioinformatics approaches

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including the identification of phytase-encoding genes in databases and optimising

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phytases by modification of existing enzymes through direct evolution and rational

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design7. Recently, the search for better phytases was extended to extremophiles8.

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Today approximately 70% of the diets for swine and poultry contain phytase and its

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market volume exceeds US$350 million per year9. The rapid growth of the phytase

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market resulted in a huge increase in competition and an increase in the number of

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commercialised phytase products.

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3

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Phytases, including the commercial enzymes, differ in properties relevant for their

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performance in animal feeds and digestive systems. Those properties include pH ACS Paragon Plus Environment


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profile, tolerance to low pH, pepsin tolerance, substrate specificity, and kinetic

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parameters (vmax, KM, kcat) for myo-inositol phosphate dephosphorylation. It is

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obvious that biological efficacy of a phytase under in vivo conditions is not linked to a

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single factor or enzymatic property. However, biochemical characterisation of the

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enzymes will help to better understand phytate dephosphorylation in the

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gastrointestinal tract of an animal. Enzymatic phytate dephosphorylation is a

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sequential and stepwise process1. Phytases are classified as 3-phytases (E.C.

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3.1.3.8), 5-phytases (E.C. 3.1.3.72) and 6-phytases (E.C. 3.1.3.26) based on the

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position of the first phosphate residue removed from the myo-inositol ring of phytate.

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The generated partially phosphorylated myo-inositol phosphate esters may be further

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dephosphorylated by a phytase1. However, information on the kinetic parameters for

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the enzymatic dephosphorylation of partially phosphorylated myo-inositol phosphate

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esters is very limited. Therefore, this study was performed to obtain additional data on

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the affinity of different phytases for and their catalytic efficiency towards several

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partially phosphorylated myo-inositol phosphate esters. The data obtained should help

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to better understand the differences in biological efficacies of phytases and to answer

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the question if simultaneous addition of a 3- and a 6-phytase to animal feed will result

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in a better phosphate release from dietary phytate than the addition of either a 3- or a

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6-phytase as argued by Stahl et al.10.

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MATERIALS AND METHODS

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Materials

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Natuphos® was obtained from BASF (Ludwigshafen, Germany) and QuantumTM from

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AB Enzymes (Darmstadt, Germany). Both enzymes were purified to apparent

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homogeneity

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Saccharomyces

according

to

Greiner

cerevisiae12,

et

al.11,12.

Escherichia

The

albertii13,


purified rye14

phytases and

from

Bacillus

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amyloliquefaciens15 were obtained as previously described. Phytic acid dodecasodium

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salt was purchased from Sigma-Aldrich (Taufkirchen, Germany). The sodium phytate

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was purified by anion-exchange chromatography on Q-sepharose and did not contain

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any inorganic phosphate. The amount of partially phosphorylated myo-inositol

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phosphate esters was below the detection limit of the HPLC system (2 nmol per myo-

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inositol phosphate ester).

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Preparation of partially phosphorylated myo-inositol phosphate esters

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In order to get access to pure partially phosphorylated myo-inositol phosphate esters,

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enzymatic dephosphorylation of sodium phytase was applied. All phytases used have

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been shown previously to generate only one myo-inositol pentakis-, one myo-inositol

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tetrakis-, and one myo-inositol trisphosphate isomer (Table 1). 100 µmol sodium

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phytate in 50 mM Na-acetate buffer, pH 5.0 (S. cerevisiae phytase), 50 mM Na-acetate

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buffer, pH 4.5 (E. albertii phytase), 50 mM Na-acetate buffer, pH 6.0 (rye phytase) or

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50 mM Tris-HCl buffer supplemented with 2 mM CaCl2, pH 7 (B. amyloliquefaciens

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phytase) were incubated with 0.4 U of the corresponding phytases in a final volume of

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40 mL at 37°C. After 150 min enzymatic reactions were terminated by incubation in a

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waterbath at 95°C for 10 min. 10 ml 0.2 M NH4-formate, pH 2.5 were used to dissolve

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the dry residues obtained after lyophilisation of the reaction mixtures. The solutions

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were applied to an anion-exchange column (Q-sepharose, 2.6 x 90 cm) operated at a

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flow rate of 2.5 mL min-1 and equilibrated with the same buffer. After washing the

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column with equilibration buffer (500 mL), elution of myo-inositol phosphate esters was

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performed with a linear gradient of NH4-formate, pH 2.5 (0.2 to 1.4 M, 2000 mL)

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collecting 10 mL fractions. The residues obtained by lyophilisation of 100 µL aliquots

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from even-numbered tubes were dissolved in 1.5 M sulfuric acid. To dephosphorylate

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the myo-inositol phosphate esters completely, the mixtures were treated for 90 min at ACS Paragon Plus Environment


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165°C. Thereafter, the liberated phosphate was quantified using NH4-molybdate.

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Fractions containing myo-inositol phosphate esters with the same number of

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phosphate residues were pooled and lyophilised until dryness. The residues obtained

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were dissolved in 10 mL water. To completely remove NH4-formate, lyophilisation and

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redissolving were repeated twice. HPLC systems as described by Sandberg and

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Ahderinne

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of the myo-inositol phosphate preparations, respectively. The purified partially

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phosphorylated myo-inositol phosphate esters did not contain other myo-inositol

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phosphate esters above the detection limit of the HPLC system (2 nmol per myo-

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inositol phosphate ester).

16

17

and Skoglund et al.

were applied to determine concentration and purity

133 134

Kinetic studies with individual myo-inositolphosphate isomers

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-1 50 µl of the respective phytases solution (10 U mL , determined at the individual buffer

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system) were added to 350 µl incubation buffer (either 0.1 mM glycine-HCl, pH 3.0 or

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0.1 mM Na-acetate, pH 5.0) containing the myo-inositol phosphate isomer

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(Ins(1,2,3,4,5,6)P6,

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Ins(1,2,5,6)P4, D-Ins(2,3,4,5)P4, D-Ins(2,4,5,6)P4, D-Ins(1,2,6), D-Ins(2,3,4)P3 and

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Ins(2,4,6)P3) in a serial dilution down to 0.015 mM of a concentrated stock solution (10

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mM). After 30 min at 37°C, the released phosphate was quantified using NH4-

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molybdate. Dephosphorylation was shown to be linear for the entire incubation time

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(data not shown). One phytase unit (U) was defined as the amount of enzyme that

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released 1 µmol of phosphate per minute under assay conditions. Blanks were

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prepared by adding the NH4-molybdate solution prior to the enzyme solution. Kinetic

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parameters (KM, kcat) were obtained by non-linear regression using DynaFit software

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version 4.0 (www.biokin.com). In order to calculate kcat, the following molecular masses

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were used: 65 kDa for the Natuphos® phytase18, 45 kDa for the QuantumTM

D-Ins(1,2,4,5,6)P5,

D-Ins(1,2,3,5,6)P5,


D-Ins(1,2,3,4,5)P5,

D-

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phytase19,20 and 67 kDa for the rye phytase14.

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Phosphate quantation

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400 µl of the mixtures of the phytase activity assay or suitably diluted hydrolysis

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mixtures were mixed with 1.5 mL freshly prepared solution consisting of 2 volumes

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acetone, 1 volume 2.5 M sulfuric acid and 1 volume 10 mM NH4-molybdate at 20°C.

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After vortexing, 100 µl of 1.0 M citric acid were added and the mixtures were

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centrifuged at 5,000g for 5 min to remove any cloudiness. Thereafter, absorbance at

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355 nm was measured. A calibration curve over the range of 5 to 600 nmol phosphate

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2 -1 was produced to quantify phosphate (ε = 8.7 cm nmol ).

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Statistical methods

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The Student’s t test was used for statistical comparison. A confidence level of 95%

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was used.

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RESULTS

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Two commercially available phytase preparations (Natuphos®: BASF, Aspergillus

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niger phytase; QuantumTM: AB Enzymes, Escherichia coli phytase) and a plant-

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derived phytase (rye) were included in the study. Their kinetic parameters (KM, kcat)

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for the dephosphorylation of sodium phytate and a series of partially phosphorylated

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myo-inositol phosphate esters were determined at pH 3.0 and pH 5.0 (Table 2). All

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three enzymes showed lower affinity and lower turnover numbers at pH 3 compared

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to pH 5 towards all myo-inositol phosphate esters used. It was shown that the

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number and distribution of phosphate groups on the myo-inositol ring affect the

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kinetic parameters. Among the myo-inositol phosphate esters with the same number

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of phosphate residues, representatives of the individual enzymatic phytate ACS Paragon Plus Environment


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dephosphorylation pathways were identified as the best substrates of the phytases

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studied. For the microbial phytases, an 8 to 80 fold difference in KM and an 8 to 25

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fold difference in kcat were observed for the dephosphorylation of the different

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pentakisphosphate isomers included in the study. The differences in the kinetic

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parameters for myo-inositol tetrakis- and trisphosphate dephosphorylation were

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determined to be significantly smaller: InsP4: KM 2-9 fold, kcat 1.6-10 fold; InsP3: KM:

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2.0-6.0 fold, kcat 1.2-1.6 fold. It was also shown that myo-inositol(2,4,6)trisphosphate

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was not significantly dephosphorylated by the microbial enzymes. Compared to the

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microbial phytases, the rye phytase exhibited significantly smaller differences in the

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kinetic parameters for myo-inositol penta- and tetrakisphosphate dephosphorylation:

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InsP5: KM 1.2-2.3 fold, kcat 3.6-4.9 fold; InsP4: KM 1.1-1.3 fold, kcat 1.7-1.9 fold. The

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differences

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dephosphorylation (rye phytase: InsP3: KM: 1.1-1.2 fold, kcat 1.7-2.0 fold) was shown

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to be comparable for all three phytases studied. As the two microbial phytases, the

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rye enzyme did not accept the myo-inositol(2,4,6)trisphosphate as a good substrate.

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Within the individual phytate dephosphorylation pathways (indicated in bold in Table

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1), the pentakisphosphate isomers were better substrates compared to the

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tetrakisphosphate isomers or phytate itself. E. coli and rye phytase showed

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comparable activities at both pH values towards the tetrakis- and trisphosphate

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esters, whereas A. niger phytase exhibited a higher activity towards the

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tetrakisphosphate ester compared to the trisphosphate ester.

in

the

kinetic

parameters

for

the

myo-inositol

trisphosphate

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DISCUSSION

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All three enzymes studied have been identified as histidine acid phytases11,14,21 and

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both microbial phytases are extensively used as animal feed supplements. The A.

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niger phytase represents a 3-phytase22, whereas the E. coli and rye phytase represent ACS Paragon Plus Environment

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a 6-phytase11 and a 4-phytase23, respectively. Even if biological efficacy of a phytase

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can only be fully determined by animal trials, their biochemical characterisation is

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essential to better understand phytate dephosphorylation in the gastrointestinal tract of

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an animal24. Up to now, the knowledge of the kinetics of the dephosphorylation of

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partially phosphorylated myo-inositol phosphate esters by phytases is rather limited.

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For several phytases myo-inositol pentakisphosphate was reported to be only a short-

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living intermediate during enzymatic phytate dephosphorylation at their individual pH

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optimum11,14,22. Furthermore, a significant reduction in dephosphorylation rate with time

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was observed resulting in a pronounced accumulation of myo-inositol phosphate

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esters with less than five phosphate residues on the myo-inositol ring in the reaction

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1 mixture . A. niger phytase for example was reported to accumulate considerable

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amounts of myo-inositol tris- and bisphosphate esters with sodium phytate as a

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substrate25 and the phytases from rye and E. coli were shown to accumulate myo-

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inositol tetrakis- and trisphosphate esters11,14. The above mentioned observations

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could be easily explained by the kinetic parameters for representatives of the individual

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enzymatic phytate dephosphorylation pathways (indicated in bold in Table 1) at pH

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5.0 (close to the pH optimum of the phytases studied) obtained in this study. For all

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three phytases, the pentakisphosphate isomers were better substrates compared to

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the tetrakisphosphate isomers or phytate itself. Furthermore, E. coli and rye phytase

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showed comparable activities towards the tetrakis- and trisphosphate esters,

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whereas A. niger phytase exhibited a higher activity towards the tetrakisphosphate

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ester compared to the trisphosphate ester. The lower turnover numbers or higher

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Michaelis-Menten constants for the dephosphorylation of myo-inositol phosphate

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esters with less than five phosphate residues on the myo-inositol ring is therefore one

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reason for the above mentioned reduction in dephosphorylation rate over time with

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sodium phytate as a substrate. A further factor is the increase in the concentration of ACS Paragon Plus Environment


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the dephosphorylation product phosphate with prolonged incubation times, because

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phosphate was shown to act as a competitive inhibitor of many phytases . Since it was

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reported, that determination of the enzymatic properties at pH 3.0 results in a

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significantly improvement of the value of the data in terms of prediction of the biological

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efficacy of a phytase , kinetic parameters for the dephosphorylation of the pure myo-

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inositol phosphate esters were also determined at pH 3.0. The general trends at pH

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3.0 and pH 5.0 were shown to be identical. At pH 3.0 however, the turnover numbers

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for the dephosphorylation of all myo-inositol phosphate esters included in the study

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were lower and their Michaelis-Menten constants higher compared to pH 5.0.

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Therefore, all these myo-inositol phosphate esters showed a lower affinity to the

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phytases and were dephosphorylated slower at pH 3.0 compared to pH 5.0.

1

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A simultaneous addition of phytases with different initiation sites to animal feed has

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been discussed to act additively or even synergistically in respect to phosphate release

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in the gastrointestinal tract of simple-stomached animals10,27-29. An intrinsic rye or

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wheat phytase and a supplemental A. niger phytase seem to act independently from

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each other in the stomach of growing pigs, because an additivity in their response on

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apparent phosphorus absorption was observed29. However, no synergistic effects have

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been observed so far from the simultaneous addition of phytases with different

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initiation sites10,27,28. The phytases could only act synergistically if the myo-inositol

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intermediates generated by one phytase are dephosphorylated faster by the other

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phytase than by the phytase generating the intermediates. This study showed

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however, that representatives of the individual enzymatic phytate dephosphorylation

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pathways were the best substrates at both pH values for all phytases studied. If the

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observation that the reaction intermediate generated by one phytase is always

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dephosphorylated slower by a phytase with a different initiation site is also applicable ACS Paragon Plus Environment

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for phytases not included in this study, a combination of phytases with different

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initiation sites will never have any advantage compared to the use of a single phytase

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as a feed supplement. However, this needs to experimentally verified.

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The difference in affinity and dephosphorylation rate among myo-inositol phosphate

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esters with the same number of phosphate residues was shown to be more

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pronounced for the myo-inositol pentakisphosphate isomers than for the myo-inositol

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tetrakis- and trisphosphate isomers as well as for the microbial phytases compared to

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the plant enzyme. Thus it could be concluded that phytases with broad substrate

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specificity such as the rye enzyme14 accept more likely different myo-inositol

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phosphate esters as a substrate than phytases with narrow substrate specificity such

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as the microbial enzymes11,22 included in the study. It was already shown that

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phosphatases in general are more likely accepting myo-inositol phosphate esters with

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a lower number of phosphate residues (4)1.

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Therefore, a combination of a phytase with an unspecific acid phosphatase

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dephosphorylating the myo-inositol phosphate intermediates accumulating by the

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action of the phytase (e.g. myo-inositol tetrakis- and/or -trisphosphates) faster than the

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phytase itself should result in an improvement of apparent phosphorus absorption in

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simple-stomached animals.

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Last but not least the study showed that myo-inositol(2,4,6)trisphosphate, a myo-

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inositol phosphate ester with alternate phosphate residues was not significantly

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dephosphorylated by all the three phytases studied. This observation is in excellent

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agreement with reports that histidine acid phytases prefer dephosphorylation of

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adjacent phosphate residues24.

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Acknowledgement

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The author want to thank Astrid Beinhauer and Petra Sämann for their excellent

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technical assistance.

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20. Tai, H.-M., Yin, L.-J., Chen, W.-C., Jiang, S.-T. 2013. Overexpression of

344

Escherichia coli phytase in Pichia pastoris and its biochemical properties. J.

345

Agric. Food Chem., 61, 6007-6015.

346

21. van Hartingsveldt, W., van Zeijl, C.M.J., Harteveld, G.M., Gouka, R.J.,

347

Suykerbuyk, M.E.G., Luiten, R.G.M., van Paridon, P.A., Selten, G.C.M.,

348

Veenstra, A.E., van Gorcom, R.F.M., van den Hondel, C.A.M.J.J. 1993.

349

Cloning, characterization, and overexpression of the phytase-encoding gene

350

(phyA) of Aspergillus niger. Gene, 127, 87-94.

351

22. Greiner, R., Gomes da Silva, L., Couri, S. 2009. Purification and

352

characterisation of an extracellular phytase from Aspergillus niger 11T53A9.

353

Braz. J. Microbiol., 40, 795-807.

354

23. Greiner, R., Alminger-Larsson, M. 2001. Stereospecificity of myo-inositol

355

hexakisphosphate dephosphorylation by phytate-degrading enzymes of

356

cereals. J. Food Biochem., 25, 229-248. ACS Paragon Plus Environment

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24. Greiner, R., Konietzny, U. 2015. Update on Characteristics of Commercial

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Phytases. Proceedings of the 2nd International Phytase Summit, Rome, Italy,

359

96-107.

360

25. Wyss, M., Brugger, R., Kronenberger, A., Remy, R., Fimbel, R., Oesterhelt, G.,

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Lehmann, M., van Loon, A.P.G.M. 1999. Biochemical characterization of fungal

362

phytases

363

properties. Appl. Environ. Microbiol., 65, 367-373.

(myo-inositol

hexakisphosphate

phosphohydrolase):

Catalytic

364

26. Greiner, R., Bedford, M. 2001. Phytase analysis, pitfalls and interpretation of

365

FTU for efficacy in the animal. In: Proceedings of the 1st International Phytase

366

Summit, Washington, D.C.

367

27. Augspurger, N.R., Webel, D.M., Lei, X.G., Baker, D.H. 2003. Efficacy of an E.

368

coli phytase expressed in yeast for releasing phytate-bound phosphorus in

369

young chicks and pigs. J. Anim. Sci., 81, 474-483.

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28. Gentile, J.M., Ronecker, K.R., Crowe, S.E., Pond, W.G., Lei, X.G. 2003,

371

Effectiveness of an experimental consensus phytase in improving dietary

372

phytate-phosphorus utilization by weanling pigs. J. Anim. Sci., 81, 2751-2757.

373

29. Zimmermann, B., Lantzsch, H.-J., Mosenthin, R., Biesalski, H.K., Drochner, W.

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2003. Additivity of the effect of cereal and microbial phytases on apparent

375

phosphorus absorption in growing pigs fed diets with marginal P supply. Anim.

376

Feed Sci. Technol., 104, 143-152.

377

30. Greiner, R., Farouk, A., Carlsson, N.-G., Konietzny, U. 2007. myo-Inositol

378

phosphate isomers generated by the action of a phytase from a Malaysian

379

waste-water bacterium. The Prot. J., 26, 577-584.

380

31. Greiner, R., Lim, B.L., Cheng, C.,Carlsson, N.G. 2007. Pathway of phytate

381

dephosphorylation by ß-propeller phytases of different origin. Can. J. Microbiol.,

382

53, 488-495. ACS Paragon Plus Environment


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384

Table 1

Enzymatic phytate dephosphorylation pathway of the phytases

385

used for the generation of the pure partially phosphorylated myo-

386

inositol phosphate esters

387

phytase source

enzymatic phytate dephosphorylation pathway

S. cerevisiae

D-Ins(1,2,4,5,6)P5, D-Ins(1,2,5,6)P4,

reference

12

D-Ins(1,2,6)P3, D-Ins(1,2)P2, Ins(2)P E. albertii

D-Ins(1,2,3,4,5)P5, D-Ins(2,3,4,5)P4,

30

D-Ins(2,3,4)P3, D-Ins(2,3)P2, Ins(2)P rye

D-Ins(1,2,3,5,6)P5, D-Ins(1,2,5,6)P4,

23

D-Ins(1,2,6)P3, D-Ins(1,2)P2, Ins(2)P B. amyloliquefaciens

D-Ins(1,2,4,5,6)P5, D-Ins(2,4,5,6)P4, D-Ins(2,4,6)P3

388


31


Page 18 of 22

18

389

Table 2

Kinetic parameters for enzymatic myo-inositol phosphate dephosphorylation

390 391 392

(A) A. niger phytase -1

KM [µM] InsPx isomer

pH 3.0

Ins(1,2,3,4,5,6)P6

131 ± 8

D-Ins(1,2,4,5,6)P5 D-Ins(1,2,3,5,6)P5 D-Ins(1,2,3,4,5)P5

kcat [s ] pH 5.0

a

82 ± 6b c

1745 ± 39

693 ± 32d b

32 ± 4

pH 3.0 a

19 ± 3b 1492 ± 28

c

394 ± 14d e

152 ± 9

a

451 ± 19b 22 ± 2

c

48 ± 5d 306 ± 8

e

321 ± 12

a

709 ± 23b 43 ± 2

c

89 ± 6d

96 ± 7

D-Ins(2,3,4,5)P4

451 ± 21e

271 ± 10f

61 ±

D-Ins(2,4,5,6)P4

823 ± 23f

647 ± 21g

39 ± 4d

D-Ins(1,2,6)P3

163 ± 17g

98 ± 7h

172 ± 11g

212 ± 13g

D-Ins(2,3,4)P3

417 ± 21h

267 ± 17f

149 ± 10a

142 ± 10h

1514 ± 41i

1123 ± 31i

24 ± 3c

40 ± 3c

393


5f

427 ± 15

e

D-Ins(1,2,5,6)P4

Ins(2,4,6)P3

72 ± 5

pH 5.0

92 ± 5d 59 ±

3f

Page 19 of 22


19

394

(B) E. coli phytase -1

KM [µM] InsPx isomer

pH 3.0

Ins(1,2,3,4,5,6)P6

227 ± 19

D-Ins(1,2,4,5,6)P5

532 ± 34b

kcat [s ] pH 5.0

a

pH 3.0

186 ± 13

a

506 ± 28b

c

1098 ± 41

d

39 ± 3

D-Ins(1,2,3,5,6)P5

1426 ± 39

D-Ins(1,2,3,4,5)P5

61 ± 5

D-Ins(1,2,5,6)P4

1745 ± 58e

D-Ins(2,3,4,5)P4

367 ± 21

D-Ins(2,4,5,6)P4

3023 ± 53g

f

h

pH 5.0

893 ± 51

a

168 ± 15b

c

94 ± 8

d

2261 ± 51

1509 ± 28e 307 ± 19

f

2462 ± 58g h

d

3017 ± 56

745 ± 28

f

98 ± 7c 511 ± 29

e

698 ± 31

D-Ins(2,3,4)P3

479 ± 27i

421 ± 19i

887 ± 25a

3031 ± 49g

874 ± 38k

45 ± 4g


247 ± 17b 127 ± 11

492 ± 34e

395

a

c

D-Ins(1,2,6)P3

Ins(2,4,6)P3

606 ± 41

1567 ± 47

c

d

578 ± 42e 1041 ± 39

f

132 ± 12c 639 ± 31

g

962 ± 49h 53 ±

5i


Page 20 of 22

20

396

(C) rye phytase -1

KM [µM]

kcat [s ]

InsPx isomer

pH 3.0

Ins(1,2,3,4,5,6)P6

412 ± 34

D-Ins(1,2,4,5,6)P5

509 ± 41b

D-Ins(1,2,3,5,6)P5

278 ± 21

c

205 ± 15

c

523 ± 37

c

641 ± 45

c

D-Ins(1,2,3,4,5)P5

428 ± 25

a

450 ± 34

b

109 ± 9

b

177 ± 15

d

D-Ins(1,2,5,6)P4

431 ± 26a

D-Ins(2,3,4,5)P4

494 ± 34

D-Ins(2,4,5,6)P4

501 ± 21b

D-Ins(1,2,6)P3

435 ± 32

D-Ins(2,3,4)P3

497 ± 37b

459 ± 35b

105 ± 6b

1876 ± 51d

1428 ± 42g

23 ± 2g

Ins(2,4,6)P3

pH 5.0 a

b

a

342 ± 26

pH 3.0 a

463 ± 34b

338 ± 22d 421 ± 25

e

432 ± 26e 366 ± 31

f

295 ± 23

pH 5.0 a

117 ± 9b

247 ± 16d 147 ± 13

e

141 ± 11e 205 ± 17

f

331 ± 21

a

131 ± 12b

322 ± 26a 171 ± 15

d

169 ± 10d 233 ± 17

e

138 ± 9b 36 ±

4f

397 398

50 µl of the respective phytases solution (10 U ml-1) were added to 350 µl incubation

399

buffer (either 0.1 mM glycine-HCl, pH 3.0 or 0.1 mM Na-acetate, pH 5.0) containing the

400

myo-inositol phosphate isomer in a serial dilution of a concentrated stock solution (10

401

mM). After 30 min at 37°C, the released phosphate was quantified using ammonium

402

molybdate. One phytase unit (U) was defined as the amount of enzyme that released 1

403

µmol of phosphate per minute under assay conditions. The DynaFit software version

404

4.0 (www.biokin.com) was used to determine kinetic parameters (KM, kcat). In order to

405

calculate kcat, the following molecular masses were used: 65 kDa for the Natuphos®

406

phytase18, 45 kDa for the QuantumTM phytase19,20 and 67 kDa for the rye phytase14.

407

The individual phytate dephosphorylation pathways are indicated in bold22,23,30. Data

408

given are mean values of 5 independent experiments. Different letters indicate a ACS Paragon Plus Environment

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21

409

significant difference of data in a column at P < 0.05.



Page 22 of 22


Activity of Escherichia coli, Aspergillus niger, and Rye Phytase toward

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