Catalytic and thermodynamic characterization of protease from Halobacterium sp. SP1(1)

Alkaline proteases are one of the most important group of enzymes that are indispensable in a number of different industrial sectors. In this work, the effect of copper ions (Cu2+) was investigated for improving the thermostability and hydrolytic performance of Bacillus clausii GMBE 42 alkaline protease at different temperatures (45–65°C). Maximal residual activity was observed in the presence of 5 mM CuCl2. The enzyme was thermoinactivated according to first‐order kinetics. A stabilization effect caused by copper ions was the result of a decrease in both autolysis and thermoinactivation rates. Thermodynamic analysis of the thermoinactivation process showed that Ea,i, ΔGi, and ΔHi values of the enzyme were higher in the presence of copper ions, but there was no measurable change in ΔSi values. These results show the thermostabilizing potential of copper ions on the enzyme. Lower Km values and higher kcat and kcat/Km values were obtained in the presence of copper ions, which is an indication of the nonessential activation of the enzyme by copper ions. Thermodynamic analysis of casein hydrolysis showed that in presence of copper ions Ea, ΔG≠, ΔH≠, ΔnormalGnormalE−normalS≠, and ΔnormalGnormalE−normalT≠ values of enzyme were lower, but there was no change in ΔS≠ values. This is so far the first study that investigates the effect of cations on the basic catalytic and thermodynamic properties of an alkaline serine protease, which may be used to remove protein wastes from various industries such as food and leather processing.

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“…Similar observations were also reported by Akolkar et al. for the protease of Halobacterium sp. SP1(1).…”

Section: Resultssupporting

confidence: 91%

“…The second order rate constant, k cat /K m value, on the other hand, is the measure of the catalytic performance of enzyme. The higher k cat /K m values indicated that copper ions contributed to the more frequent collision of casein molecules with the conformationally active enzyme molecules . Capiralla et al.…”

Section: Resultsmentioning

confidence: 99%

The influence of copper on alkaline protease stability toward autolysis and thermal inactivation

Ozturk

Kazan

Denizci

et al. 2012

Engineering in Life Sciences

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“…Thermodynamic studies of β-glucosidase were carried out using Arrhenius plot and activation energy (E a ), change in enthalpy (∆H) and change in entropy (∆S) were calculated to be 52.17 KJ/mol, 49.90 KJ/mol and -71.69 J/mol.K respectively (Figures 8 and 9). Low values of enthalpy and negative values of entropy indicate the formation of a more efficient and ordered transition state complex between enzyme and substrate (Akolkar and Desai, 2010). Kvesitadze et al (1990) reported ∆H and ∆S of 125 KJ/mol and 65 J/mol.K respectively for β-glucosidase of Aspergillus wentii.…”

Section: Enzyme Characterizationmentioning

confidence: 99%

Metabolic engineering and thermodynamic characterization of an extracellular -glucosidase produced by Aspergillus niger

Zahoor¹,

Javed²,

Aftab³

et al. 2011

Afr. J. Biotechnol.

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The production of an extracellular β-glucosidase by Aspergillus niger NRRL 599 was optimized using submerged fermentation technique. Effect of different media, different carbon sources, initial pH of the fermentation medium, temperature, incubation period and inoculum size on the production of β-glucosidase enzyme was investigated. A. niger NRRL 599 produced maximum extracellular β-glucosidase (4.48 U/mg) in Eggins and Pugh medium with 1% wheat bran (w/v) at pH 5.5 inoculated with 4% conidial suspension after 96 h of incubation at 30°C. Purified β-glucosidase gave K m and V max values of 3.11 mM and 20.83 U/mg respectively for pNPG hydrolysis. The enzyme was optimally active at pH 4.8 and at temperature of 60°C. Thermodynamic parameters, E a , ∆H and ∆S were found to be 52.17 KJ/mol, 49.90 J/mol.K and -71.69 KJ/mol, respectively. The pKa 1 and pKa 2 of ionizable groups of active site residues involved in V max were calculated to be 4.1 and 6.0 respectively.

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“…Activation enthalpy (Δ H *), Gibbs activation free energy (Δ G *) and activation entropy (Δ S *) were calculated by rearranging Eyring's absolute rate equation based on transition state theory (Eyring & Stearn, ):

k_{cat} = ((), k_{b} T true/ h) \exp ((), prefix- Δ H^{*} true/ R T) \exp ((), Δ S^{*} true/ R),

where k b is Boltzmann's constant (1.38 × 10 −23 J K −1 ), h is Planck's constant (6.626 × 10 −34 J s), Δ H* = E a − RT , Δ G* = − RT ln ( k cat h / k b T ) and Δ S* = (Δ H* − Δ G* )/ T . The free energy of the substrate and enzyme combination,

Δ G_{normalE - normalS}^{*}

, was calculated with the following derivation (Akolkar & Desai, ):

Δ G_{normalE - normalS}^{*} = prefix- R T \ln K_{a},

where K a = 1/ K m .…”

Section: Methodsmentioning

confidence: 99%

Effect of arsenate contamination on free, immobilized and soil alkaline phosphatases: activity, kinetics and thermodynamics

Wang

Tan

et al. 2016

European J Soil Science

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Summary Soil alkaline phosphatase plays a vital role in phosphorus cycling in the soil ecosystem. Arsenic, an ecotoxic element, affects the normal function of phosphatase in the soil environment. To understand the effects of arsenic on phosphatase, the activity, kinetic and thermodynamic characteristics were investigated systematically following the addition of arsenate to free soluble, clay‐immobilized alkaline phosphatase (ALP) and soil ALP. The results showed that the ALP activity was strongly inhibited by arsenate. The Michaelis constant Km increased linearly with increasing arsenate concentration, indicating a decrease in affinity of the enzyme for the substrate. The inhibition constant Ki increased after the ALP was immobilized on montmorillonite and goethite. Thermodynamic properties such as activation energy, activation enthalpy, Gibbs activation free energy and activation entropy increased when arsenate was added, which suggests that more energy is required to form the activated molecule and the enzyme–substrate complex. The reaction was less spontaneous and the enzyme–substrate complex was less ordered. Immobilization of the ALP on minerals reduced the variation in kinetic and thermodynamic properties in the presence of arsenate, indicating that immobilization provided protection from arsenate stress. Arsenate toxicity to soil ALP was affected markedly by soil texture. The results suggest that the type of arsenate inhibition on free soluble ALP was competitive inhibition, whereas for immobilized and soil ALPs, the inhibition was competitive or mixed inhibition depending on the mineral or soil property. Highlights Alkaline phosphatase (ALP) activity was inhibited by arsenate. The kinetic and thermodynamic properties of ALP were changed in the presence of arsenate. Arsenate inhibition on free, mineral‐immobilized ALP and soil ALP was different. ALP immobilization on minerals reduced arsenate toxicity to ALP.

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Catalytic and thermodynamic characterization of protease from Halobacterium sp. SP1(1)

Cited by 35 publications

References 24 publications

The influence of copper on alkaline protease stability toward autolysis and thermal inactivation

The influence of copper on alkaline protease stability toward autolysis and thermal inactivation

Metabolic engineering and thermodynamic characterization of an extracellular -glucosidase produced by Aspergillus niger

Effect of arsenate contamination on free, immobilized and soil alkaline phosphatases: activity, kinetics and thermodynamics

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