Purification and biochemical properties of highly efficient alkaline phosphatase from Fenneropenaeus merguiensis brain

Accepted Manuscript Title: Purification and biochemical properties of highly efficient alkaline phosphatase from Fenneropenaeus merguiensis brain Author: Ahmad Homaei PII: DOI: Reference:

S1381-1177(15)00121-6 http://dx.doi.org/doi:10.1016/j.molcatb.2015.04.013 MOLCAB 3153

To appear in:

Journal of Molecular Catalysis B: Enzymatic

Received date: Revised date: Accepted date:

3-3-2015 28-4-2015 29-4-2015

Please cite this article as: A. Homaei, Purification and biochemical properties of highly efficient alkaline phosphatase from Fenneropenaeus merguiensis brain, Journal of Molecular Catalysis B: Enzymatic (2015), http://dx.doi.org/10.1016/j.molcatb.2015.04.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Highlights

The alkaline phosphatase showed the strongest affinity with p-NPP as the substrate.

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The structure of enzyme–substrate complex at the transition state was more ordered that the native enzyme.

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The pH profile of enzyme showed a broad range of activity

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The highest catalytic efficiency for hydrolysis of the substrate at pH 11 and 50 ◦C.

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Purification alkaline phosphatase from Fenneropenaeus merguiensis brain, opening new opportunities for biotechnology and medicine industry applications.

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Purification and biochemical properties of highly efficient alkaline phosphatase from Fenneropenaeus merguiensis brain

Department of Biochemistry, Faculty of Sciences, Hormozgan University, Bandar Abbas, Iran

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Ahmad Homaei a, *

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Corresponding authors: Tel: (98) 7617665054; Fax: (98) 7616670716; P.O. Box 3995.E-mail addresses:

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[email protected] (Homaei)

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Abstract Alkaline phosphatase was purified from Fenneropenaeus merguiensis from Persian Gulf to homogeneity level by using (NH4)2SO4 precipitation, DEAE-cellulose and DEAE-Sephadex

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anion-exchange chromatography and gel filteration chromatography. The molecular weight of the enzyme was 70 KD, measured by SDS-page. The alkaline phosphatase showed the strongest

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affinity with p-NPP as the substrate, and the highest catalytic efficiency for hydrolysis of the substrate at pH 11 and 50 ºC. Activation energy (Ea) for catalysis of alkaline phosphatase was 8.3

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kcal mol−1 K-1, while temperature quotient (Q10) was 3.25. It exhibited Michaelis–Menten

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Kinetics with kcat of 95 s−1 and Km of 0.3. µM, respectively. Thermodynamic parameters for soluble p-NPP hydrolysis were as follow: ∆H#=7.7 kcal mol-1 K-1, ∆G#= 14.7 kcal mol-1 K-1,

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∆S#=-23. 5 cal mol−1 K-1, ΔG#E-S=-0.7 kcal mol-1 K-1 and ΔG#E-T=-3.4 kcal mol-1 K-1. Thermodynamic parameters (∆H*, ∆G*, ∆S*) for irreversible inactivation of alkaline

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phosphatase at different temperatures (35–50 ◦C) were also determined.

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Keywords: Purification; Fenneropenaeus merguiensis alkaline phosphatase; Thermodynamic

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parameters; Catalytic efficiency; Persian Gulf.

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1. Introduction The growing knowledge and technique improvement about protein extraction and purification lead to the production of many enzymes at an analytical grade purity for research and

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biotechnological applications. Recent advances in biotechnology, particularly in protein engineering, have provided the basis for the efficient development of enzymes with improved

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properties. This has led to establishment of novel, tailor-made enzymes for completely new

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applications [1]. A marine enzyme may be a unique protein molecule not found in any terrestrial organism or it may be a known enzyme from a terrestrial source but with novel properties.

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Alkaline phosphatase (EC 3.1.3.1) is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process

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of removing the phosphate group is called dephosphorylation. As the name suggests, alkaline phosphatases are most effective in an alkaline environment. It is sometimes used synonymously

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as basic phosphatase [2]. Information about the reaction mechanism of ALPs comes mostly from studies on the E. coli enzyme, but it is believed to be generally the same for all Aps [3]. The

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catalytic mechanism involves the formation of a serine phosphate at the active site, which reacts

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with water at alkaline pH to release Pi from the enzyme [4, 5]. Alkaline phosphatases are widely distributed in nature and knowledge of the alkaline phosphatases has increased remarkably during the last few years [6]. ALPs in marine organisms play important roles in cell phosphate metabolism, which is related to the absorption of phosphate and calcium from seawater and the biomineralization process in marine organisms [7, 8]. Furthermore, alkaline phosphatase is an important enzyme in recycling phosphate in marine environments as well as within living cells. Its distribution is dependent on environmental factors such as phosphate availability, dominant

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habitants, water depth, salinity, temperature, and the internal ratio of nitrogen to phosphorus in higher organisms and microbial communities [8, 9]. Alkaline phosphatase is an enzyme of high research interest, having also a variety of industrial

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applications. This enzyme is routinely used as a labeling enzyme in electrochemical enzyme

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immunoassays and immunosensors and numerous approaches have been reported for determining herbicides, pesticides, and different biologically active organic compounds [10-12]. Additionally,

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prospects of using different alkaline phosphatases bearing zinc and magnesium ions in their catalytic and allosteric sites, respectively, in pharmaceutical and clinical analysis were

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demonstrated. Also their application for the determination of zinc in insulin to control injection quality and magnesium in human urine for the diagnosis and treatment of magnesium deficiency

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was shown [11]. Marine organism have been shown to be a valuable source for the isolation of novel APs with potential practical application [12]. Current report has novelty as it explained for

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the first time kinetics and thermodynamics of soluble p-NPP hydrolysis and irreversible

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inactivation of alkaline phosphatase from Fenneropenaeus merguiensis brain and compatibility with other enzymes to exploit its efficiency in a variety of medicine and biotechnology

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applications. Some properties and the substrate specificity of this AP make it advantageous as a tool for practical application in comparison with the commercially available APs. All the results obtained in this paper would provide a sound basis to the further exploration. 2. Materials and methods 2.1. Materials

p-NPP was from Sigma (St. Louis, MO, USA), DEAE-cellulose, DEAE-Sephadex and Sephadex G-200 provided by Pharmacia (Uppsala, Sweden). All other chemicals were reagent grade and purchased from Merck (Darmstadt, Germany).

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2.2. Specimen collection and cell extraction Fenneropenaeus merguiensis, caught from the Persian Gulf, State of Hormozgan, Iran, was immediately frozen and transported to our enzymology laboratory in Bandar Abbas. The frozen

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samples were then stored at -80 °C until used. 50 gr fresh shrimp brain was freed from the adhering meninges and blood and the grey matter

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was removed by gross dissection. Brain tissue was re-suspended in 10 ml of 50 mM Tris-HCl buffer, pH 7.4. The suspension was subjected to sonic disruption at 0 °C ,10 s on, 45 s off for 15

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min, and cell debris were discarded by centrifugation at 15,000 × g for 20 min. The supernatant

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Total proteins in the cell-free supernatant were precipitated by adding ammonium sulphate to 85% saturation and the resulting suspension was kept for an overnight. Subsequently, the

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precipitate was then collected using centrifuge at 12000 rpm for 10 min at 4 ºC . The protein was then dissolved in minimal amount of Tris-HCl buffer and dialyzed against the same buffer for 24

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h at 4 °C, changing dialysis buffer every 8 hours. 2.3. Enzyme purification procedure

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15 ml the dialyzed sample was subjected to anion-exchange chromatography, using a DEAEcellulose column, previously equilibrated with 50 mM Tris-HC1 buffer pH 7.4 at room

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temperature. The column was washed with the same buffer until no protein was detected in the eluent. Then it was eluted using a gradient of 0–1 M NaCl in the same buffer at a flow rate 1 ml/min and 2 ml fractions were collected. To determine the protein content of each fraction the absorbance at 280 nm was measured using UV–vis spectrophotometer. The active fractions from this and subsequent chromatographies were measured with p-NPP as a substrate at room temperature. Fractions with phosphatase activity were pooled and applied to a DEAE -Sephadex fast flow column, pre-equilibrated with 50 mM Tris-HC1 buffer at pH 7.4. The column was washed with the same buffer until no protein was detected in the eluent. Then it was eluted using

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a gradient of 0–1 M NaCl in the same buffer at a flow rate 1 ml/min. The enzyme from the previous step was placed on a column of Sephadex G-200 previously equilibrated with 50 mMTris-HCl buffer pH 7.4. A flow rate of 1 ml/min was maintained and fractions of 2 ml were

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collected. The enzyme appeared in the effluent, immediately after the void volume, and the active fractions from tubes were pooled and concentrated in an ultrafiltration chamber with a 30 kDa

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membrane cut-off and analyzed for proteins and enzyme activity. The concentrated enzyme was

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dialysed against 50 mM Tris-HC1 buffer pH 7.4.

2.4. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)

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SDS-PAGE was carried out using a 12.5% polyacrylamide gel by the method of Laemmli [13] and the gels were stained with Comassie Brilliant Blue R-250 [14]. 2.5. Measurement of enzyme activity and protein concentration

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Phosphatase activity was determined in Alkaline phosphatase buffer, containing 50 mM Tris-

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HCl, 100 mM NaCl and 2 mM MgCl2 at pH 11 and room temperature, using 2 mM p-NPP as substrate. To 100 µl of enzyme solution diluted in 500 µl of ALP buffer, 400 µl of 2 mM p-NPP

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was added and the reaction mixture was incubated at room temperature for 5 min. The hydrolysis of p-NPP to p-NP (a chromogenic product with absorbance at 405 nm) in ALP buffer was

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measured for 5 min. The reaction was stopped by adding 500 µl of 1 M NaOH and the amount of p-NP liberated was estimated by measuring its absorbance at 405 nm in a Perkin Elmer's LAMBDA 850 UV/Vis Spectrophotometer. One unit of phosphatase is defined as the amount of enzyme that hydrolyzes p-NPP to produce equivalent absorbance to 1µmol of p-NP/min. Protein concentration was estimated by the Bradford method [15] using bovine serum albumin as standard. 2.6. Characterization of Alkaline phosphatase

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Physiochemical, kinetic and thermodynamic properties of alkaline phosphatase from Fenneropenaeus merguiensis. were determined as mentioned below. 2.6.1. Determination of optimum temperature, activation energy and temperature quotient (Q10)

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The activity versus temperature profiles were graphed on the basis of the activity values measured at different temperatures in the range 20–90 °C, in alkaline phosphatase buffer of pH

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11.0, as described. Ea was calculated by using Arrhenius plot [16]; the Arrhenius plot was graphed utilizing the activity values in the temperature range of 30–50 °C for alkaline

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phosphatase. The effect of temperature on the rate of reaction was expressed in terms of

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temperature quotient (Q10), which is the factor by which the rate increases due to a raise in the temperature by 10 ◦C. Q10 was calculated by rearranging the equation given by Dixon and

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Webb[17]:

whereE=Ea= activation energy.

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 E  10  Q10  anti loge  2   RT 

2.6.2. Determination of optimum pH

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The activity versus pH profiles of alkaline phosphatase was graphed measuring the alkaline

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phosphatase activity at room temperature, as reported above, in a mixed buffer containing 50 mM acetate, phosphate and glycine in the range 2–12 pH values. 2.6.3. Effect of pH and temperature on enzyme stability To determine the thermal stability, alkaline phosphatase was incubated at 60 and 70oC in 50 mM in Na2CO3–NaHCO3 buffer pH 11.0, for different intervals of time, then cooled on ice and the residual activity determined under the assay conditions. The stability to pH was checked after incubation of the enzyme in 50 mM of mixed buffer (pH 3 and 12) for different intervals of time at room temperature, then pH value was adjusted to 11.0 and the residual activity determined

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according to the assay conditions. Control measurements were carried out measuring the activity of the same enzyme solution kept on ice for the thermal stability and in the buffer at pH 11.0 for the pH stability experiments.

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2.6.4. Effect of different concentration of bivalent chloride salts on the activity of alkaline phosphatase

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The relative activity of enzyme was measured in the presence of various chloride salts with final concentrations of 0- 5 mM under assay conditions.

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2.6.5. Determination of kinetic parameters

Catalytic activity of alkaline phosphatase was investigated at different p-NPP concentrations

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under assay conditions. Km, Vmax, Kcat and Kcat/Km values were determined using Lineweaver– Burk plots. The reciprocal of substrate concentration (1/S) was plotted against the reciprocal of

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reaction rate (1/V) according to the following equation:

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(1)

where [S] is the concentration of substrate, V and Vmax represented the initial and maximum rate

the rate is half of Vmax.

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of reactions, respectively. Km is the Michaelis–Menten constant, the substrate concentration when

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2.6.6. Calculation of thermodynamic parameters of p-NPP hydrolysis The rate constants of phosphatic reaction (kcat) and inactivation (kinact) were used to calculate the activation energy according to the Arrhenius equation [16]. (2)

Where k (s−1) is the rate constant at temperature T (K), A is a pre-exponential factor related to steric effects and the molecular collision frequency, R is the gas constant (8.314 J mol−1 K−1), and Ea the activation energy of the reaction. Hence, a plot of ln k as a function of 1/T gives a curve with slope of −Ea/R. The thermodynamic parameters of activation were determined as follows:

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(3) (4) (5)

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(6.6256×10−34J. s), and kcat(s−1) is the rate constant at temperature T(K).

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Where kB is the Boltzmann constant (1.3805×10−23J. K−1), h the Planck’s constant

2.6.7. Thermodynamics of enzyme stability

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Thermal inactivation of enzyme was determined by incubating the enzyme solution in 50 mM in Na2CO3–NaHCO3 buffer pH 11. various temperatures in the range 35-50 °C. Aliquots were

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withdrawn at different time intervals, cooled on ice for 30 min and assayed for phosphatase activity at room temperature. The data was fitted to first-order plot and inactivation rate constants

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(Kd) were determined as described previously [18-20]. The first-order rate constants for denaturation (kd)of the enzyme at different temperatures were determined from the slopes of

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semi-logarithmic plots according to Eq. (6) and activation energy for denaturation (Ea,d) was obtained from the slope (−Ea,d/R) of Arrhenius plot of lnkd versus 1/T. Free energy (∆G#d),

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enthalpy (∆H#d) and entropy (∆S#d) for the enzyme denaturation were calculated from Eqs. (7)–

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(9), respectively. The half-life of the enzyme was obtained from Eq.(10). lnV max  B  k d t d G d#   RT ln

(6)

kd h k bT

(7)

H d#  E a ,d  RT S d# 

(8)

H d#  G d# T

(9)

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t 1/2 

ln 2 kd

(10)

The free energy of substrate binding and transition state formation was calculated using the

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following derivations [21, 22]:  free energy of substrate binding ∆G*E-S = -RT ln Ka

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(11)

 free energy for transition state formation ∆G*E-T =-RT ln (Kcat/Km)

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(12)

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where Ka = 1/Km

All of the results are taken as the mean value obtained from at least three repeated experiments in

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a typical run to confirm reproducibility. 3. Results and discussion

3.1. Extraction and purification of the enzyme

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The supernatant obtained from cell free extract was concentrated by precipitation with ammonium sulfate. The resulting protein solution was then loaded into chromatography columns.

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Purified Fenneropenaeus merguiensis alkaline phosphatase with a specific activity of 421 U/ mg

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was obtained using chromatography on DEAE-cellulose, DEAE-Sephadex and Sephadex G-200, respectively. The overall purification procedure is summarized in Table 1. Fractions exhibiting phosphatase activity were pooled out and concentrated using Amicon 8050 ultrafiltration system equipped with a 30 kDa membrane cut-off. SDS–PAGE course showed electrophoretic purity grade for the enzyme. The molecular mass of Fenneropenaeus merguiensis alkaline phosphatase was about 70 kDa (Fig. 1). 3.2. Kinetic and thermodynamic parameters

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Phosphatase hydrolysis of p-NPP by alkaline phosphatase catalysis follows the Michaelis-Menten equation. As reported in Table 2, the values of kcat, Km, and kcat/Km measured from Lineweaver Burk plot, were 95 s-1, 0.3 µM and 316.6 s-1µM-1, respectively. Affinity for the substrate was

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increased in the Fenneropenaeus merguiensis alkaline phosphatase as compared to E. coli alkaline phosphatase and Vibrio alkaline phosphatase [3]. The Km of Fenneropenaeus

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merguiensis alkaline phosphatase is 70 and 266 times lower than that of the E. coli ALP and

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Vibrio ALP, respectively [3].

The profile of relative activity vs pH shows higher activity in basic pH ranges, values of relative

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activity higher than 70% in the pH range 8.0-12.0 were found (Fig. 2). The enzyme exhibited maximum activity at about pH 11 and a relative high residual activity at pH 12 (80% of the

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maximum). As known, at the pH values ≥10.0, the substrate-binding residues oriented toward the bound substrate become neutral and negatively charged, respectively [8, 23, 24]. In the case of

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some alkaline phosphatases can be inactive at some pH values. For example, activity of Physarum polycephalum can be quenched at pH 9.2 [25], that of Homo sapiensat pH 6–9 [26],

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and the phosphatasse of the Nilaparvata lugensbecomes inactive at about pH 9.5 [27].

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From Fig.3, where the effect of temperature on the activity of alkaline phosphatase is shown, it appears that maximum activity was obtained at 50 °C, a value similar of that found for marine bacterium Cobetia marina and Neurospora crassa [8, 28], but higher than that referred to bos Taurus alkaline phosphatase (25 °C) [29], Pyrococcus furiosus alkaline phosphatase (37 °C) [30] and Pinctada fucata alkaline phosphatase (45 °C) [7]. Enzyme retains a high activity in a wide temperature range from about 30-70 °C if compared with other alkaline phosphatases [8, 31, 32]. Moreover, it appears that about 35% of its maximum phosphatase activity is retained at low (25 °C) and very high (80 °C) temperatures.

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The Arrhenius plot, graphed utilizing the activity values of alkaline phosphatase in the temperature range 20–50 °C (inset of Fig.3), clearly suggested that beyond 50 °C the activity declined, indicating the inactivation of enzyme. Activation energy (Ea) of the p-NPP phosphatic

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reaction catalyzed by alkaline phosphatase was 8.3 kcal mol-1K-1. The temperature quotient (Q10) for alkaline phosphatase was 3.25. The two major effects of temperature observed on the activity

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of the enzyme were increase in the rate of the reaction at 30– 50 ◦C, as the enzyme gains kinetic

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energy and a decrease in the rate of activity due to increase in the rate of denaturation of the enzyme at temperatures greater than 50 ◦C.

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In Table 2, values of activation free energy (G#), activation enthalpy (H#) and activation entropy (S#) for the catalytic reaction are also reported. The negative value of activation entropy indicates

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that the structure of enzyme–substrate complex at the transition state was more ordered that the native enzyme [33].

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Enzymes are proteins, whose three dimensional structures are stabilized by weak forces, because of the weak nature of these forces they are disrupted at high temperatures. The time dependence

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of the alkaline phosphatase stability with temperature, measured after incubating the enzyme at

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60 and 70 °C, evidences a half life of more than 10 and of 5 min respectively (Fig. 4a). This enzyme appear to be distinct from other ALPs in thermostability [3, 8]. A relatively low thermostability can be useful for AP removing from the reaction mixture without additional purification in DNA techniques [12]. Furthermore, Fenneropenaeus merguiensis alkaline phosphatase can be a potential marker for specific plasma membrane purification, additionaly this enzyme can be used as a marker to differentiate testicular origin azoospermia or oligospermia from ejaculatory failure, as well as ejaculatory failure and excurrent duct blockages in an inexpensive way [8-12].

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Experimental measurements of stability of alkaline phosphatase at extreme pH values (Fig. 4b) indicate that, at pH alkaline values, the enzyme appears more stable with respect to acid values. In fact, after 60 min of incubation at pH 12, alkaline retained about 98% of activity, while, under

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the same conditions at pH=2, a residual activity 0% was observed. The rate-determining step of the alkaline phosphatase enzymes reaction is pH-dependent. At acidic pH, the hydrolysis of the

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covalent phosphoseryl intermediate is the slowest step, but at basic pH, the dissociation of the

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phosphate from the noncovalent enzyme–phosphate complex becomes rate-determining [3, 23, 24].

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Thermostability represents the capability of an enzyme molecule to resist against thermal unfolding in the absence of substrate, while thermophilicity is the ability of an enzyme to work at

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elevated temperature in the presence of substrate [22, 34].. Alkaline phosphatase was incubated at temperatures ranging from 35 to 50 ◦C and the effect of temperature on denaturation of the

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enzyme was showed in fig. 5, which was fitted with Eq. (6). The rate of denaturation of this enzyme increase rapidly with temperature: its half lifes (t1/2) decreased from 597 min at 35 °C to

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23 min at 50 °C (table 3). It is interesting to underline that at this last temperature (50°C) alkaline

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phosphatase shows maximum activity. It is probable that the structural features of the enzyme active site provide a barrier to prevent contact of denaturants, including chelators, with the active site. Metal ions released from the active site by heat led to decrease of enzymatic activity [8]. These may coincide with the loss of the magnesium ion from the active site because of increased mobility of the binding ligands [3]. Thermal inactivation of enzymes occurred in two steps [35, 36] as shown below: NUI

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where N is the native enzyme, U the unfolded inactive enzyme, which could be reversibly refolded upon cooling, and I is the inactivated enzyme formed, after prolonged exposure to heat, which cannot be recovered upon cooling. The thermal inactivation of enzymes is accompanied by

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the disruption of lots of non-covalent linkages including hydrophobic interactions with concomitant increase in the enthalpy of activation (∆H#) [37, 38]. The opening up of the enzyme

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structure is accompanied by a lot of increase in the disorder or entropy of activation (∆S#) [39,

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40]. Activation energy for irreversible inactivation ‘Ea(d)’of the alkaline phosphatase, determined by applying Arrhenius plot and Gibbs free energy (∆G#) for activation of thermal unfolding of

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enzyme, was 21.91 kcal mol−1 (Fig. 6). Increasing the temperature, a decrease in free energy was observed. The enthalpy of activation of thermal unfolding (∆H#) of the enzyme at 35 ◦C was 48.0

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kcal mol−1; this value was unchanged up to 50 ◦C. The entropy of activation (∆S#) for unfolding of transition state of the alkaline phosphatase was 85.0 cal mol−1K−1 and was slightly increased at 50 C (Table 3). The decrease of ∆G# at higher temperatures indicates a higher thermal instability of

the enzyme [22, 41].

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The free activation energy of substrate binding (∆G#E–S) and the free energy for the formation of

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activation complex (∆G#E–T) were -0.7 and -3.4 kcal mol−1, respectively. These values again confirm the great affinity of enzyme towards substrate and the following hydrolysis reaction. 3.3. Effect of bivalent metal ions on alkaline phosphatase activity In Table 4, the results relative to the effect of metal ions at different concentration on phosphatase activity are reported. Experimental data show that monovalent ions, such as Li+, Na+, K+ have little effect decreasing activity with concentration, probably owing to ionic strength effect. Calcium and magnesium ions strongly increase activity, with a factor of about 50% in the case of Ca2+ and until 100% for Mg2+. A great increase in enzymatic activity may be to the increased

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binding affinity for Mg2+ in alkaline phosphatases [42]. In contrast, ALPase from V.cholerae has been reported to be inhibited significantly with the addition of 2 mM Mg2+ [43]. This enzyme appears strongly inhibited by Hg2+, Cu2+ and Pb2+, In particular, Hg2+concentration

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lower than 1 mM is sufficient to totally inhibit alkaline phosphatase activity, while a value lower than 2 mM is requested in the case of Cu2+ and Pb2+. From the experimental data, the following

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scale of alkaline phosphatase inhibition from bivalent ions can be drawn up: Hg2+>Cu2+>Pb2+.

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These data clearly indicate that the sensitivity of the enzyme increases against the inhibitory effects ions of the first transition series that are elements in marine pollution. Such inhibition may

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be due to masking of binding sites on the enzyme by these metal ions [41]. The experimental results showed that all these metal ions also inhibited the activity of ALP from Fenneropenaeus

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merguiensis. Therefore, marine pollution might disrupt the production of Fenneropenaeus

4. Conclusion

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merguiensis.

A unique and hitherto not reported alkaline phosphatase was detected in Fenneropenaeus

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merguiensis brain. This enzyme was purified to homogeneity according to the following schematic procedure: i) (NH4)2SO4 precipitation; ii) DEAE-Cellulose and DEAE-Sephadex

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anion-exchange chromatography and iii) Sephadex G-200 gel filtration chromatography. Kinetic and thermodynamic parameters of the enzyme displays extremely high catalytic efficiency. Biotechnology, analysis and medicine applications appear very interesting, in particular on the possibility to generate some structural variations of Fenneropenaeus merguiensis alkaline phosphatase in order to improve its phosphatic applications. Acknowledgements Authors are grateful to the University of Hormozgan for the financial support to this research. References

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[6] R.L. Olsen, K. Overb, B. Myrnes, Comp. Biochem. Physiol. 99 (1991) 755-761.

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[7] R. Xiao, L.P. Xie, J.Y. Lin, C.H. Li, b, Q.X. Chen, H.M. Zhou, R.Q. Zhang,. J. Mol. Catal. BEnzymol. 17 (2002) 65–74.

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[13] U.K Laemmli, Nature (London). 227 (1970) 680–5.

[14] C.M Wilson, Methods Enzymol. 91(1983) 236-247. [15]. M. M Bradford. Anal. Biochem. 72 (1976)248–54.

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[16] S. Arrhenius, Z. Phys. Chem. 4 (1889) 226-228. [17] Dixon M, Webb EC. Enzyme kinetics. Enzymes, vol. 3. New York: Academic Press; 1979. pp. 47–206.

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[18] I.P. Street, J.B. Kempton, S.G. Withers, Biochemistry. 31 (1992) 9970–9978. [19] J. Ellis, C.R. Bagshaw, W.V. Shaw, Biochemistry. 34 (1995) 16852–16859.

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[20] M.N. Namchuk , S.G. Withers, Biochemistry, (1995) 16194–16202. [21] H. Eyring, A.E. Stearn, Chem. Rev. 24 (1939) 253–270. [22] A. Homaei, R. Etemadipour, Inter. J. Biol Mol. [23] P. Gettins, J.E. Coleman, J. Biol. Chem. 258 (1983) 408–416. [24] W.E. Hull, S.E. Halford, H. Gutfreund, B.D. Sykes, Biochemistry. 15 (1976) 1547–1561. [25] K. Furuhashi, Arch. Microbiol. 189 (2008) 151-156. [26] R.A. Stinson, J.R.A. Chan, Adv. Prot. Phosphatases. 4 (1987) 127-151. [27] Z. Wang, S. Liu, B. Yang, Z. Liu, Arch. Insect. Biochem. Physiol. 78 (2011) 30-45. [28] K.R. Bogo, D.C. Masui, F.A. Leone, J.A. Jorge, R.P. Furriel, Microbiol. 51(2006) 431-437. [29] P. Schrenkhammer, I.C. Rosnizeck, A. Duerkop, O.S. Wolfbeis, M. Schäferling, J. Biomol.

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Screen. 13 (2008) 9-16. [30] K. Minamihata, M. Goto, N. Kamiya, Biotechnol. Lett. 34 (2012):2055-2060. [31] H. Kobori, C.W. Sullivan, H. Shizuya, Proc. Natl. Acad. Sci. 81 (1984) 6691-6695. [32] M. Politino, J. Brown, J.J. Usher, Prep. Biochem. Biotechnol. 26 (1996) 171-181.

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[33] A. Homaei, A. Bahari, R. Sariri, E. Kamrani, R. Stevanato, S.M. Etezad, K. Khajeh, J. Photoch. Photobio B. 125 (2013) 131-136.

[34] Georis J, Esteves FL, Brasseur JL, Bougnet V, Devreese B, Giannotta F, Protein Sci 2000; 9: 466–75.

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[35] L. Plaza, T. Duvetter, S. Monfort, E. Clynen, L. Schoofs, A.M. Van Loey, M.E. Hendrickx, J. Agric. Food. Chem. 55 (2007) 9259–9265.

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[36] U. Jakob, R. Kriwacki, V.N. Uversky, Chem. Rev. 114 (2014) 6779–6805.

[37] V. Sant’Anna, M. Utpott, F. Cladera-Olivera, A. Brandelli, J. Agric. Food. Chem. 58 (2010) 3147–3152.

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[38] U. Jakob, R. Kriwacki, V.N. Uversky, Chem. Rev. 114 (2014) 6779–6805. [39] H. Gouzi, C. Depagne, T. Coradin, J. Agric. Food Chem. 60 (2012) 500–506.

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[40] H. N. Bhatti, M. Asgher, A. Abbas, R. Nawaz, M.A. Sheikh, J. Agric. Food Chem. 54 (2006) 4617–4623. [41] A. Homaei, Int. J. Biol. Macromol. 75 (2015) 373–377

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[42] J.E. Murphy, X. Xu, R.E. Kantrowitz, J. Biol. Chem. 268 (1993) 21497-21500.

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[43] N.K. Roy, R.K. Ghosh, J. Das, J. Bacteriol. 150 (1982) 1033-1039.

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Table 1 Purification procedures of alkaline phosphatase from Fenneropenaeus merguiensis brain. Total protein (mg)

Total activity (U)

Specific activity (U/mg)

Purification fold

Yield (%)

Cell extract

1700

13250

7.8

1

100

(NH4)2So4

1121

10083

9

1.2

76

DEAE-cellulose

427

9347

22

2.8

70

DEAE-Sephadex

57

7398

129.8

16.6

56

Sephadex G-200

11.2

4716

421

54

36

Ac ce

pt

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M

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Steps of purification

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Table 2 The kinetic and thermodynamic parameters for p-NPP hydrolysis catalyzed by Fenneropenaeus merguiensis alkaline phosphatase. Conditions were 50 mM Tris-HCl, 100 mM NaCl and 2 mM MgCl2 at pH 11, using 2 mM p-NPP as substrate. Each number is the average of at least three independent experiments. Kinetics parameters

Energy and entropy parameters

Vmax

kcat

kcat/Km

Ea

ΔH#

ΔG#

ΔS#

ΔG#E-S

ΔG#E-T

(µM)

(µM.min-1)

(s-1)

(s-1 µM-1)

(kcal mol-1)

(kcal mol-1)

(kcal mol-1)

(cal molK-1)

(kcal mol-1)

(kcal mol-1)

0.3

73

95

316.6

8.3±0.1

7.7±0.5

14.7±0.3

-23.5±0.1

-0.7±0.1

-3.4±0.2

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M

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Km

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Table 3 Thermodynamic parameters for irreversible inactivation of alkaline phosphatase from Fenneropenaeus merguiensis. Conditions were 50 mM Tris-HCl, 100 mM NaCl and 2 mM MgCl2 at pH 11, using 2 mM p-NPP as substrate. Each number is the average of at least three independent experiments. kd (min-1)×10-3 1.16±0.1 3.03±0.1 12±0.8 30±0.6

t1/2 (min-1) 597±1.3 228±1.6 57±1.1 23±0.94

ΔH# (kcal mol-1) 48.09±0.8 48.08±0.6 48.07±0.5 48.06±0.3

ΔG# (kcal mol-1) 21.91±0.2 21.88±0.1 21.37±0.3 22.6±0.1

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M

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Ea,d=48.7 kcal mol-1, pH 11.0

ΔS# (cal molK-1) 85.0±0.8 83.7±0.5 83.9±0.3 78.8±0.7

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Temperature (K) 308 313 318 323

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Table 4 Effect of various chloride metal ions and their concentrations on Fenneropenaeus merguiensis alkaline phosphatase. The enzyme activity at zero concentration of each ion was taken as 100% and

the other concentrations were measured with respect to zero concentration.

100 99±1.3 96±1.1 90±0.8 95±1.5

Mg2+

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1 2 3 4 5

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100 100 99±0.8 95±0.5 91±1.1

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K+

1 2 3 4 5

Relative activity (%) Ca2+ 114±0.3 125±1.6 96±0.8 90±1.1 83±1.4 2+ Hg 5±0.2 0 0 0

1 2 3 4 5

123±1.1 156±1.7 134±1.4 109±0.8 93±0.5

1 2 3 4 5

Pb2+

36±1.7 0 0 0

Cu2+ 19±0.7 0 0 0

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1 2 3 4 5

Na+

100 97±0.5 92±0.2 87±1.3 83±1.6

M

1 2 3 4 5

Li+

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1 2 3 4 5

[Me ] (mM)

pt

1 2 3 4 5

2+

Relative activity (%)

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2+

[Me ] (mM)

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LEGENDS

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Fig. 1. SDS-PAGE analysis of samples obtained by the different purification steps. Proteins were detected by coomassie brilliant blue. Lane 1 (30 µg), crude extract; lane 2 (20 µg), DEAESephadex pool; lane 3 (10 µg), DEAE-cellulose pool; lane 4 (3 µg), G-200 Sephadex pool. Lane M is the molecular mass marker.

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Fig. 2. Effect of pH on the alkaline phosphatase activity. 100% relative activity refers to the percentage of the initial reaction rate obtained by the enzyme at the pH value of maximum activity.

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Fig. 3. Effect of temperature, in the range 20–90 °C, on the enzyme activity. The activity at optimal temperature was taken as 100. In the inset, the relative Arrhenius plot (Ln Activity versus 1/T) is reported.

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Fig. 4. a) Irreversible thermoinactivation at 60 ºC (filled triangles), 70 ºC (filled squares). The activity of the same enzyme solution, kept on ice, was considered as the control (100%). b) pH stability of alkaline phosphatase at pH 3.0 (filled triangles) and 12.0 (filled squares) in mixed buffer. Control data were obtained measuring the activity of the same stock of enzyme solution kept for the same times at room temperature.

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Fig. 5. First-order plot for thermal denaturation of alkaline phosphatase. The enzyme solution was incubated at various temperature in the range 35- 50 °C in 50 mM Tris-Hcl pH 11.

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Fig. 6. Arrhenius plot of the irreversible thermal inactivation/denaturation of alkaline phosphatase.

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ip t cr us an M ed pt Ac ce Figure 1

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Figure 2

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Figure 3

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Figure 4

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M

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Ac ce

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M

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Figure 5

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ip t cr us an M ed pt Ac ce

Figure 6

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Ac ce p

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*Graphical Abstract (for review)

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