Thermal Inactivation Of Enzyme Research Articles

A thermostable and inhibitor resistant β-glucosidase from Rasamsonia emersonii for efficient hydrolysis of lignocellulosics biomass.

The present study reports a highly thermostable β-glucosidase (GH3) from Rasamsonia emersonii that was heterologously expressed in Pichia pastoris. Extracellular β-glucosidase was purified to homogeneity using single step affinity chromatography with molecular weight of ~ 110kDa. Intriguingly, the purified enzyme displayed high tolerance to inhibitors mainly acetic acid, formic acid, ferulic acid, vanillin and 5-hydroxymethyl furfural at concentrations exceeding those present in acid steam pretreated rice straw slurry used for hydrolysis and subsequent fermentation in 2G ethanol plants. Characteristics of purified β-glucosidase revealed the optimal activity at 80°C, pH5.0 and displayed high thermostability over broad range of temperature 50-70°C with maximum half-life of ~ 60h at 50°C, pH 5.0. The putative transglycosylation activity of β-glucosidase was appreciably enhanced in the presence of methanol as an acceptor. Using the transglycosylation ability of β-glucosidase, the generated low cost mixed glucose disaccharides resulted in the increased induction of R. emersonii cellulase under submerged fermentation. Scaling up the recombinant protein production at fermenter level using temporal feeding approach resulted in maximal β-glucosidase titres of 134,660units/L. Furthermore, a developed custom made enzyme cocktail consisting of cellulase from R. emersonii mutant M36 supplemented with recombinant β-glucosidase resulted in significantly enhanced hydrolysis of pretreated rice straw slurry from IOCL industries (India). Our results suggest multi-faceted β-glucosidase from R. emersonii can overcome obstacles mainly high cost associated enzyme production, inhibitors that impair the sugar yields and thermal inactivation of enzyme.

Nonthermal inactivation effects on oxidative enzymes in grape juice influenced by moderate electric fields: Effect of constant exposure electrical treatments combined with temperature

Inactivation of polyphenol oxidase (PPO) and peroxidase (POD) enzymes in fresh grape juice was studied by subjecting the juice to moderate electric field (MEF) treatments of 82 and 87 V/cm field strengths. We used an electro-processing device that was built to minimize ohmic heating and enhance cooling during application of electric fields. Kinetic studies indicate that the MEF treatments increase the inactivation of both enzymes at 65 and 75 °C, compared to the corresponding control treatments without electric fields. The increase in the inactivation becomes statistically significant ( P ≤ 0.05) in the MEF treatment at 75 °C. Molecular motion simulations of PPO and POD enzymes correlate with the experimental results. The study results further indicate the efficacy of MEF treatments on the nonthermal inactivation of enzymes, which are held at or near their thermal inactivation threshold temperatures (≤80 °C for PPO and POD). • Moderate electric field (MEF) treatments inactivate enzymes (PPO, POD) in grapes. • The efficacy of MEFs increases with increasing field strength and temperature. • The thermal impact on grape juice can be reduced by applying MEF treatments. • Enzyme's thermal inactivation threshold temperature is useful in MEF studies. • Thermostability and electric field sensitivity of enzymes affect the inactivation.

Enzymatic hydrolysis of soy and chickpea protein with Alcalase and Flavourzyme and formation of hydrogen bond mediated insoluble aggregates

Food applications involving plant proteins require modification of their functionality to mimic the unique properties of animal proteins. Enzymatic hydrolysis is commonly used to alter the functionality of plant proteins, particularly to improve their solubility near the isoelectric point. Current methodological approaches mostly indicate improved solubility upon hydrolysis. However, published methods include the removal of insoluble material before analysis, and calculations are based on only the solubilized material as a percentage of the filtered protein. This approach artificially increases solubility estimation and gives an incorrect assessment of the efficacy of hydrolysis. By using the total amount of protein, this study aims to determine the effect of two microbial proteases, Flavourzyme and Alcalase, on the solubility and structural and thermal properties of soy and chickpea proteins. Protein isolates were first extracted from soy and chickpea flour and hydrolyzed from 0 to 3 h. Then, their degree of hydrolysis and solubility at a range of pHs were determined using the o-phthaldialdehyde (OPA) and Lowry methods, respectively. Proteins’ electrophoretic mobility, protein-protein interactions, thermal properties, and protein secondary structures were also determined. Solubility decreased over time though the solubility of the hydrolysate improved near the isoelectric point. Soy Flavourzyme hydrolysates remained the most soluble and chickpea Flavourzyme hydrolysates showed the least solubility. Thermal data suggested that Alcalase reduced the protein denaturation temperature, leading to a loss of solubility upon thermal enzyme inactivation. The loss of solubility of hydrolysates was strongly associated with hydrogen bonding, which may result from the formation of polar peptide termini. These results challenge commonly accepted beliefs that hydrolysis inevitably improves solubility of plant proteins. Instead, it is shown that hydrolysis causes structural changes that result in aggregation, thus potentially limiting the application of enzymatic hydrolysis without the addition of further processing methods.

Production, Biochemical Characterization, and Kinetic/Thermodynamic Study of Inulinase from Aspergillus terreus URM4658.

Inulinases are enzymes involved in the hydrolysis of inulin, which can be used in the food industry to produce high-fructose syrups and fructo-oligosaccharides. For this purpose, different Aspergillus strains and substrates were tested for inulinase production by solid-state fermentation, among which Aspergillus terreus URM4658 grown on wheat bran showed the highest activity (15.08 U mL−1). The inulinase produced by this strain exhibited optimum activity at 60 °C and pH 4.0. A detailed kinetic/thermodynamic study was performed on the inulin hydrolysis reaction and enzyme thermal inactivation. Inulinase was shown to have a high affinity for substrate evidenced by very-low Michaelis constant values (0.78–2.02 mM), which together with a low activation energy (19.59 kJ mol−1), indicates good enzyme catalytic potential. Moreover, its long half-life (t1/2 = 519.86 min) and very high D-value (1726.94 min) at 60 °C suggested great thermostability, which was confirmed by the thermodynamic parameters of its thermal denaturation, namely the activation energy of thermal denaturation (E*d = 182.18 kJ mol−1) and Gibbs free energy (106.18 ≤ ΔG*d ≤ 111.56 kJ mol−1). These results indicate that A. terreus URM4658 inulinase is a promising and efficient biocatalyst, which could be fruitfully exploited in long-term industrial applications.

Impact of Enzymatic Hydrolysis and Heat Inactivation on the Physicochemical Properties of Milk Protein Hydrolysates.

This study determined the physicochemical properties (apparent viscosity (ηapp), turbidity (A550nm), particle size and molecular mass distribution) of hydrolysates generated from whey protein concentrate (WPC), milk protein concentrate (MPC) and sodium caseinate (NaCN), following incubation with Debitrase HYW20™ and Prolyve™ at 50 °C, pH 7.0 for 1 and 4 h, before and after heat inactivation (80 °C for 10 min). The degree of hydrolysis (DH) increased with incubation time, giving values of 6.56%, 8.17% and 9.48%, following 1 h hydrolysis of WPC, MPC and NaCN with Debitrase HYW20™, and 12.04%, 15.74% and 17.78%, respectively, following 4 h incubation. These DHs were significantly higher compared to those obtained following 4 h incubation with Prolyve™. Hydrolysis with Debitrase HYW20™ gave >40% of peptides with molecular masses < 1 kDa for all substrates, which was higher than the value obtained following hydrolysis with Prolyve™. The effect of hydrolysis on the physicochemical properties was substrate dependent, since ηapp decreased in WPC and NaCN hydrolysates, particle size decreased for all the substrates, with aggregate formation for MPC, and turbidity decreased in WPC and MPC hydrolysates, while it increased in NaCN hydrolysates. The physical properties of the hydrolysates were influenced by the enzyme thermal inactivation step in a DH-dependent manner, with no significant effect on turbidity and viscosity for hydrolysates at higher DHs.

Inactivation Kinetics and Thermodynamics Parameters of Polyphenol Oxidase and Peroxidase Activities in an Extract from of Violet Eggplant (Solanum melongena L.)

Enzymatic browning is associated with the action of polyphenol oxidases (PPO) and peroxidases (POD). The products of these enzymes cause undesirable changes of color and flavor of processed eggplant products. The present study aimed to evaluate kinetic properties and thermodynamics parameters of PPO and POD activities for controlling this undesirable process in extract from of violet eggplant. The effect of heat treatment on polyphenol oxidase and peroxidase activities in violet eggplant were studied over a range of 30 to 80 °C. T1/2-values of enzymatic activities are between 6.15 ± 0.03 and 13,27 ± 0,04 min at 80 °C, they decreased with increasing temperature, indicating a difference thermostability of each enzyme. D- and k-values decreased and increased, respectively, with increasing temperature, indicating faster of these enzymes inactivation at higher temperatures. Results suggested that polyphenol oxydase and peroxidase were relatively thermostable enzymes with a Z-value which from 50.25 and from 88.33 °C and Ea of 41.21 and of 27.78 kJmol-1. Thermodynamic parameters were also calculated. The Gibbs free energy ΔG values range from 43.24 ± 0,03 to 91.45 ± 0,01 kJ/mol. These kinetic data can be used to predict prevention of browning in the violet eggplant (Solanum melongena L.) by thermal inactivation of enzymes.

Inhibition kinetic and thermal inactivation of horseradish peroxidase in the presence of Zn2+ ion

Peroxidase as a heme-containing metalloprotein, is an important enzyme for food industries. Hence, the current study is aimed to evaluate the inhibitory properties of zinc ion in terms of activity and thermal stability of horseradish peroxidase (HRP) in the range of 40-70°C for 1-45min. The results showed that zinc ion potently inhibited HRP activity by noncompetitive mechanism. The thermal inactivation of enzyme followed first-order reaction kinetics in the presence of ZnCl2 up to 300μM. However, higher concentrations of Zn2+ -induced irregularities in HRP thermo inactivation pattern from first-order kinetics. According to the kinetic parameters, K and Ea , the rate of reactivation of HRP may be dependent on the amount of zinc ion, inactivation time, and heating duration. PRACTICAL APPLICATIONS: Thermal inactivation kinetics of HRP was investigated in the presence of Zn2+ . The results showed that thermal inactivation of HRP followed first-order kinetics for up to 300μM of Zn2+ . HRP inactivation was consistent with biphasic models for higher concentrations of Zn2+ at 60°C. The transition free energy of inactivation confirmed that zinc ion increased the enzyme unfolding transition states stability as denatured structure of HRP is more favorable than native one in this condition. Moreover, reactivation potential of enzyme decreased by increasing the zinc ion concentration.

Kinetic and Thermodynamic Analysis of Two Carboxymethylcellulases from Macrotermes subhyalinus Little Soldier

Optimization of thermal processes relies on adequate degradation kinetic models to warrant food safety and quality. The knowledge on thermal inactivation of enzymes is necessary to allow their proper utilization in food industry and technology applications, enabling the reduction of heating times and optimization of heating temperatures. In this work, the kinetic of thermal inactivation was studied for the previously characterized carboxylmethylcellulases Ab-CX1 and Ab-CX2 from Macrotermes subhyalinus little soldier. Samples of carboxymethylcellulases were treated at different time-temperature combinations in the range of 5-60 min at 50-65°C and the kinetic and thermodynamic parameters for carboxymethylcellulases were calculated. Results showed that inactivation followed a first-order reaction with k-values between 0.0103 ± 0.0003 to 0.1217 ± 0.0005 and 0.0149 ± 0.0007 to 0.0416 ± 0.0003 min-1 for Ab-CX1 and Ab-CX2, respectively. At high temperatures, Ab-CX2 was less resistant, with a significant decrease in residual activity compared to Ab-CX1. The D- and k-values decreased and increased, respectively, with increasing temperature, indicating faster inactivation of carboxymethylcellulases. Activation energy (Ea) and Z-values were estimated to 76.74 ± 1.98 kJ.mol-1 and 24.21 ± 1.92 °C for Ab-CX1, 62.80 ± 2.05 kJ.mol-1 and 33.33 ± 2.78 °C for Ab-CX2. Thermodynamic parameters (ΔH#, ΔS# and ΔG#) were also calculated. The high value obtained for the variation in enthalpy of activation indicates that a high amount of energy is required to initiate denaturation, probably due to the molecular conformation of carboxymethylcellulases. All results suggest that both carboxymethylcellulases are relatively resistant to long heat treatments up to 50°C.

Effect of protein load on stability of immobilized enzymes.

Different lipases have been immobilized on octyl agarose beads at 1mg/g and at maximum loading, via physical interfacial activation versus the octyl layer on the support. The stability of the preparations was analyzed. Most biocatalysts had the expected result: the apparent stability increased using the highly loaded preparations, due to the diffusional limitations that reduced the initial observed activity. However, lipase B from Candida antarctica (CALB) was significantly more stable using the lowly loaded preparation than the maximum loaded one. This negative effect of the enzyme crowding on enzyme stability was found in inactivations at pH 5, 7 or 9, but not in inactivations in the presence of organic solvents. The immobilization using ethanol to reduce the immobilization rate had no effect on the stability of the lowly loaded preparation, while the highly loaded enzyme biocatalysts increased their stabilities, becoming very similar to that of the lowly loaded preparation. Results suggested that CALB molecules immobilized on octyl agarose may be closely packed together due to the high immobilization rate and this produced some negative interactions between immobilized enzyme molecules during enzyme thermal inactivation. Slowing-down the immobilization rate may be a solution for this unexpected problem.

Thermal inactivation of enzymes and pathogens in biosamples for MS analysis.

Protein denaturation is the common basis for enzyme inactivation and inactivation of pathogens, necessary for preservation and safe handling of biosamples for downstream analysis. While heat-stabilization technology has been used in proteomic and peptidomic research since its introduction in 2009, the advantages of using the technique for simultaneous pathogen inactivation have only recently been addressed. The time required for enzyme inactivation by heat (≈1 min) is short compared with chemical treatments, and inactivation is irreversible in contrast to freezing. Heat stabilization thus facilitates mass spectrometric studies of biomolecules with a fast conversion rate, and expands the chemical space of potential biomarkers to include more short-lived entities, such as phosphorylated proteins, in tissue samples as well as whole-blood (dried blood sample) samples.

Waste heat and water recovery opportunities in California tomato paste processing

Water and energy efficiency are important for the vitality of the food processing industry as demand for these limited resources continues to increase. Tomato processing, which is dominated by paste production, is a major industry in California – where the majority of tomatoes are processed in the United States. Paste processing generates large amounts of condensate as moisture is removed from the fruit. Recovery of the waste heat in this condensate and reuse of the water may provide avenues to decrease net energy and water use at processing facilities. However, new processing methods are needed to create demand for the condensate waste heat. In this study, the potential to recover condensate waste heat and apply it to the tomato enzyme thermal inactivation processing step (the hot break) is assessed as a novel application. A modeling framework is established to predict heat transfer to tomatoes during the hot break. Heat recovery and reuse of the condensate water are related to energy and monetary savings gained through decreased use of steam, groundwater pumping, cooling towers, and wastewater processing. This analysis is informed by water and energy usage data from relevant unit operations at a commercial paste production facility. The case study indicates potential facility seasonal energy and monetary savings of 7.3 GWh and $166,000, respectively, with most savings gained through reduced natural gas use. The sensitivity of heat recovery to various process variables associated with heat exchanger design and processing conditions is presented to identify factors that affect waste heat recovery.

Purification and Characterization of Glucose-6-Phosphate Dehydrogenase from Pigeon Pea (Cajanus cajan) Seeds

Glucose-6-phosphate dehydrogenase has been purified from pigeon pea (Cajanus cajan) seeds and subjected to characterization. The enzyme was purified 123.69 fold with a yield of 21.37% by ammonium sulphate fractionation, PEG-4000 precipitation, CM cellulose column chromatography and DEAE cellulose column chromatography. The catalytically active enzyme is a dimer of 113 KDa with a subunit molecular weight of 55 KDa. Thermal inactivation of enzyme follows first order kinetics at 30&#176C and 40&#176C with half life of 6 and 1.5 min respectively. Km value for glucose-6-phosphate and NADP+ was found to be 2.68 mM and 0.75 mM respectively whereas Vmax value was found to be 0.11 U/mL and 0.13 U/mL respectively. The enzyme shows more affinity towards NADP+ than glucose-6-phosphate. The pKa value was found to be 10.41 indicating that the amino acid residue at active site might be lysine. The enzyme exhibited maximum catalytic activity at pH 8.2. The enzyme was found to be highly thermosensitive with gradual loss of activity above 30&#176C temperature.

Fungal Beta-glucosidases: a bottleneck in industrial use of lignocellulosic materials.

Profitable biomass conversion processes are highly dependent on the use of efficient enzymes for lignocellulose degradation. Among the cellulose degrading enzymes, beta-glucosidases are essential for efficient hydrolysis of cellulosic biomass as they relieve the inhibition of the cellobiohydrolases and endoglucanases by reducing cellobiose accumulation. In this review, we discuss the important role beta-glucosidases play in complex biomass hydrolysis and how they create a bottleneck in industrial use of lignocellulosic materials. An efficient beta-glucosidase facilitates hydrolysis at specified process conditions, and key points to consider in this respect are hydrolysis rate, inhibitors, and stability. Product inhibition impairing yields, thermal inactivation of enzymes, and the high cost of enzyme production are the main obstacles to commercial cellulose hydrolysis. Therefore, this sets the stage in the search for better alternatives to the currently available enzyme preparations either by improving known or screening for new beta-glucosidases.

Mathematical Modeling and Analysis of the Kinetics of Thermal Inactivation of Enzyme

A theoretical model of Illeova et al. (2003) thermal inactivation of urease is discussed. Analytical expressions pertaining to the molar concentrations of the native and denatured enzyme are obtained in terms of second-order reaction rate constant. Simple and closed form of theoretical expression pertains to the temperature are also derived. In this paper, homotopy analysis method (HAM) is used to obtain approximate solutions for a nonlinear ordinary differential equation. The obtained approximate result in comparison with the numerical ones is found to be in satisfactory agreement.

γ-Glutamyl transferase inactivation in milk and cream: A comparative kinetic study

The degree and rate of inactivation of γ-glutamyl transferase in raw cow's milk containing two levels of fat (skimmed [0%] and whole [3.5%], and cream [45%]) were measured at different time–temperature combinations in order to evaluate the suitability of this enzyme as a marker for the pasteurization of milk and milk products. A detailed comparative kinetic study of γ-glutamyl transferase inactivation was carried in the temperature range of 60 to 85 °C. Kinetic studies showed that the thermal inactivation of enzyme followed first-order kinetics. The influence of temperature on the inactivation rate constant was quantified using the Arrhenius equation and was characterized by activation energy values of 214.8 ± 4.6, 190.2 ± 3.7 and 174.5 ± 3.5 kJ/mol in skimmed milk, whole milk and cream, respectively. The kinetic data regarding inactivation of milk enzymes can be valuable in terms of implementing food safety management systems, including traceability of the heat treatment step as a critical control point for milk and dairy processes. Heat stability profiles of bovine milk γ-GT were examined with the object of being able to monitor heat treatments slightly or even more severely than typical pasteurization conditions by measurements of residual enzyme activity after heating. Assay procedure was limited to simple colorimetric method that would be most likely to form the basis of a quick, simple and inexpensive test. The importance of this work is related to the possibility of establishing potential markers to evaluate the destruction of the target microorganism associated with each heating process, taking into consideration the safety and quality aspects.

Bacteriophage enzymes for the prevention and treatment of bacterial infections: Stability and stabilization of the enzyme lysing Streptococcus pyogenes cells

The effect of various compounds on the activity and stability of a phage-associated enzyme lysing cells of streptococci of groups A and C (PlyC) was investigated. Substantial inhibition of the enzyme activity was revealed at an increased ionic strength (in the presence of NaCl) and upon the addition of carbohydrates (mono-, di-, and polysaccharides), i.e., agents stabilizing many enzymes. It was established that the enzyme activity was substantially reduced in the presence of positively charged polyelectrolytes and surfactants, whereas incubation with micelle-forming substances and negatively charged polyelectrolytes led to PlyC activation and stabilization. It was shown that, in the micellar polyelectrolyte composition M16, the enzyme retained its activity for 2 months; while in a buffer solution under the same conditions (pH 6.3, room temperature), ture), it practically completely lost its activity in 2 days. Characteristics of the enzyme thermal inactivation were found, in particular, its half-inactivation time at various temperatures; these allowed us to estimate its behavior at any temperature and to recommend conditions for its storage and use.

A generic model for glucose production from various cellulose sources by a commercial cellulase complex

The kinetics of cellulose hydrolysis by commercially available Cellubrix were described mathematically, with Avicel and wheat straw as substrates. It was demonstrated that hydrolysis could be described by three reactions: direct glucose formation and indirect glucose formation via cellobiose. Hydrolysis did not involve any soluble oligomers apart from low amounts of cellobiose. Phenomena included in the mathematical model were substrate limitation, adsorption of enzyme onto substrate, glucose inhibition, temperature dependency of reaction rates, and thermal enzyme inactivation. In addition, substrate heterogeneity was described by a recalcitrance constant. Model parameters refer to both enzyme characteristics and substrate-specific characteristics.Quantitative model development was carried out on the basis of Avicel hydrolysis. In order to describe wheat straw hydrolysis, wheat straw specific parameter values were measured. Updating the pertinent parameters for wheat straw yielded a satisfactory description of wheat straw hydrolysis, thus underlining the generic potential of the model.

THE THERMAL STABILITY OF ASPERGILLUS ORYZAE ALPHA‐AMYLASE IN PRESENCE OF SUGARS AND POLYOLS

ABSTRACT The aim of this study was to test whether it is possible to estimate the heat stability of Aspergillus oryzae alpha‐amylase (α‐amylase) based on the amount of hydroxyl (OH) groups provided in a buffer solution. The thermal stability of the enzyme in a presence of different sugars (sucrose and trehalose) and polyols (mannitol, sorbitol, lactitol and glycerol) was investigated in the temperature range of 62–68C on a kinetic basis. It was investigated if the protective effect of additional substances was correlated to the number of hydroxyl groups (nOH) provided by each of them (per volume unit of the enzyme solution nOH). All additives showed a protective effect on the enzyme's heat stability, which was strongly dependent on the added compound concentration. Among all stabilizing compounds investigated, sucrose exhibited the largest protective effect. The decimal reduction time of α‐amylase activity increased by 33.9 times when 420 mg/mL of sucrose was added to the environment. When the same concentration of trehalose was used, the D‐value increased by 6.4 times compared to the value in the buffer system. The nOH provided in the enzyme solution could not be related to the D‐values for the enzyme thermal inactivation, meaning that the enzyme heat stability was not dependent on the nOH.

Thermal inactivation of polyphenoloxidase in pineapple puree

Prevention of browning in pineapple puree by thermal inactivation of enzyme, Polyphenoloxisase (PPO), was examined between 40 and 90 °C and in relation to exposure time. The amount of inactivation was measured as a function of time and temperature under isothermal conditions. Reaction rate constant and activation energy ( E a) as well as Decimal reduction time ( D) and z-value of thermal inactivation, were determined. The rate of inactivation varied with temperatures and follows a logarithmic law. Kinetic studies showed that the thermal inactivation (40–90 °C) of the PPO followed first-order kinetics.

Production of whey protein hydrolysates with reduced allergenicity in a stable membrane reactor

In this paper, a process for the stable production of low allergenicity hydrolysates is presented. Whey protein was hydrolysed at 50 °C and pH 8.5 using a bacterial protease in a continuous stirred tank membrane reactor including a polyethersulfone plate and frame ultrafiltration module with a molecular weight cut-off of 3 kDa. The reactor was maintained in operation for 16 h. Conversion reached a steady value around 80% after 10 h of operation, while a steady permeate flow was achieved after 13 h. A slight, first order enzyme thermal inactivation was detected. A hydrolysate with an average peptide chain length around 4 amino acids was obtained. The antigenic whey protein in the product was reduced 99.97%, which suggests that it can be incorporated as nitrogen source in infant formula and enteral nutrition.