Thermal Inactivation Rate Research Articles

Kinetics of the thermal inactivation and the refolding of bacterial luciferases in Bacillus subtilis and in Escherichia coli differ

Here we present a study of the thermal inactivation and the refolding of the proteins in Gram positive Bacillus subtilis. To enable use of bacterial luciferases as the models for protein thermal inactivation and refolding in B. subtilis cells, we developed a variety of bright luminescent B. subtilis strains which express luxAB genes encoding luciferases of differing thermolability. The kinetics of the thermal inactivation and the refolding of luciferases from Photorhabdus luminescens and Photobacterium leiognathi were compared in Gram negative and Gram positive bacteria. In B. subtilis cells, these luciferases are substantially more thermostable than in Escherichia coli. Thermal inactivation of the thermostable luciferase P. luminescens in B. subtilis at 48.5°С behaves as a first-order reaction. In E.coli, the first order rate constant (Kt) of the thermal inactivation of luciferase in E. coli exceeds that observed in B. subtilis cells 2.9 times. Incubation time dependence curves for the thermal inactivation of the thermolabile luciferase of P. leiognathi luciferase in the cells of E. coli and B. subtilis may be described by first and third order kinetics, respectively. Here we shown that the levels and the rates of refolding of thermally inactivated luciferases in B. subtilis cells are substantially lower that that observed in E. coli. In dnaK-negative strains of B. subtilis, both the rates of thermal inactivation and the efficiency of refolding are similar to that observed in wild-type strains. These experiments point that the role that DnaKJE plays in thermostability of luciferases may be limited to bacterial species resembling E. coli.

Desiccation and Thermal Resistance of Escherichia coli O121 in Wheat Flour

Non-O157 Shiga toxin-producing Escherichia coli infections have recently been associated with wheat flour on two separate accounts in the United States and Canada. However, there is little information regarding the thermal resistance and longevity of non-O157 Shiga toxin-producing Escherichia coli during storage in low-moisture environments. The objectives of this study were to determine the thermal inactivation kinetics of E. coli O121 in wheat flour and to compare the thermal inactivation rates with those of other pathogens. Wheat flour, inoculated with E. coli O121, was equilibrated at 25°C to a water activity of 0.45 in a humidity-controlled conditioning chamber. Inoculated samples were treated isothermally at 70, 75, and 80°C, and posttreatment population survivor ratios were determined by plate counting. D- and z-values calculated with a log-linear model, were compared with those obtained in other studies. At 70, 75, and 80°C, the D-values for E. coli O121 were 18.16 ± 0.96, 6.47 ± 0.50, and 4.58 ± 0.40 min, respectively, and the z-value was 14.57 ± 2.21°C. Overall, E. coli O121 was observed to be slightly less thermally resistant than what has been previously reported for Salmonella Enteritidis PT30 in wheat flour as measured under the same conditions with the same methods.

Thermal stability of myosin and protective effect of F-actin on myosin affect the thermal inactivation of calcium-ATPase in unstable kuruma prawn myofibrils

The thermal stability of myosin and the protective effect of F-actin on myosin in kuruma prawn myofibrils were investigated from the thermal inactivation rates at 25 °C of Ca-ATPase in myosin and myofibrils at various concentrations of KCl. The thermal inactivation rate constant (kD) of myofibrillar Ca-ATPase increased with increasing KCl concentration; however, the thermal inactivation followed a biphasic first-order reaction regardless of the KCl concentration: a relatively fast inactivation rate in the earlier phase (kDe) followed by a slower inactivation rate in the later phase (kDl) of the heat treatment. The thermal inactivation of myosin at various concentrations of KCl or sorbitol also followed a biphasic first-order reaction, and the differences between the kDe and kDl of myosin were always about twofold. kDe and kDl of myosin at 0.1 M KCl were decreased to 1/6 and 1/13, respectively, by the binding action of F-actin. These results suggest that the thermal stability of myosin and the protective effect of F-actin affect the stability and thermal inactivation of kuruma prawn myofibrils at physiological ionic strength.

Thermal inactivation of Pseudomonas aeruginosa 1244 in salted Sardinella fimbriata meat homogenate

This study established the thermal inactivation parameters, D and z values of Pseudomonas aeruginosa 1244 in a laboratory-prepared salted herring meat homogenate suspending medium. The food matrix was determined to have a pH equal to 5.85 and aw of 0.73. The moisture and NaCl contents were 38.25% and 13.81%, respectively. The test microorganism exhibited first-order inactivation behavior (R2 ≥ 0.90) at all heating temperatures. Thermal inactivation rates decreased with increasing temperatures. The D values established were 70.94 s (60°C), 60.46 s (75°C), 31.11 s (80°C) and 22.14 s (90°C).The thermal resistance parameter z value of 57.10°C was determined. The results provide information on the behavior of the test organism in heated salted herring meat, and could provide a basis for challenge studies involving other microbial species and strains in similar food matrices. The thermal inactivation kinetic parameters may be used in the establishment or validation or both of process schedules for salted herring meats, including mechanical dehydration and sun drying protocols.

Evaluation of Enterococcus faecium NRRL B-2354 as a surrogate for Salmonella during cocoa powder thermal processing

Salmonella is capable of surviving in a low moisture environment for long periods. Once adapted to the xeric conditions, the thermal resistance of Salmonella is enhanced. Cocoa powder is a low water activity (aw) food (LawF) that is an essential component in a wide variety of desserts and ready-to-eat (RTE) foods and drinks. The aim of this study was to evaluate the desiccation and thermal resistance of Salmonella in cocoa powder, as well as to examine the suitability of Enterococcus faecium NRRL B-2354 as a surrogate for Salmonella during cocoa powder thermal processing. Natural unsweetened cocoa powder was inoculated with a 3-strain Salmonella cocktail or E. faecium and was equilibrated to aw 0.30 and 0.45 at room temperature, then subjected to isothermal treatments at 70–80 °C or 12-month storage at RT (room temperature, 22.0 ± 0.5 °C, aw 0.30). At 70 and 80 °C, D-values of both Salmonella and E. faecium increased with decreasing aw. D-values of Salmonella at aw 0.30 cocoa powder were 46.2 ± 4.7, 20.5 ± 1.7, and 11.5 ± 0.9 min at 70, 75 and 80 °C, respectively. Higher heat resistance of E. faecium in aw 0.30 cocoa powder was observed with D-values of 59.9 ± 5.0, 28.9 ± 1.8, and 16.1 ± 1.4 min at 70, 75, and 80 °C, respectively. However, E. faecium demonstrated less heat resistance than Salmonella when aw was increased to 0.45. D-values for Salmonella at aw 0.45 were 31.6–7.0 min at 70–80 °C compared to 25.8–4.7 min for E. faecium. During 12 months of storage at RT, surviving E. faecium population in aw 0.30 cocoa powder was higher than that of the Salmonella cocktail; the population decreased by 1.39 and 3.75 logs, respectively. These findings indicate that the suitability of E. faecium as a surrogate organism for Salmonella is influenced by aw of cocoa powder. The aw correlates with thermal inactivation rates in both Salmonella and E. faecium, and should be considered as a significant contributor to the thermal resistance of Salmonella in cocoa powder.

Improve Salinivibrio zinc-metalloprotease function in less polar organic solvents by increasing surface hydrophobicity

Organic solvents tend to strip water from protein and thereby disrupt non-covalent forces and decrease enzyme activity and stability. In the present study, we have replaced the surface charge residues in Salinivibrio zinc-metalloprotease (SVP) with hydrophobic ones (E12V, D22I, D24A and D310I) in order to study the effects of surface hydrophobicity with hydrophobic strength of organic solvents. Compared to SVP, D24A exhibited an increase in kcat and catalytic efficiency and a reduction in thermal inactivation rate in aqueous solvent. Structural studies indicated that the replacement of surface charge residues with hydrophobic residues would not induce conformational changes. C50 value (the value of solvent concentration where 50% of enzyme activity remains), ki (irreversible thermoinactivation rate), and kinetic parameters of E12V, D22I, and D24A were higher in isopropanol and n-propanol. D24A is found to be the most efficient mutant for its remarkable decrease in ki value in the presence of isopropanol and n-propanol and a reduction in ki value in the presence of dimethylformamide (DMF) and methanol. C50 value in this variant was increased about 1.2% in DMF, 2% in methanol and isopropanol and 2.5% in n-propanol. Results revealed that, there was a correlation between surface hydrophobicity of SVP and hydrophobic strength of organic solvents.

The Influence of the Apical Domain of Groel Chaperone on the Kinetics and Thermodynamics of Zebrafish Dihydrofolate Reductase Under Thermal Stress

In the present study, we have attempted to characterize the chaperoning ability of the apical domain (residue 191–345) of bacterial chaperone GroEL (miniGroEL) in isolation. In native form, miniGroEL constitutes a hydrophobic pocket where partially folded substrate protein binds. In the present study various aspects of the thermal aggregation prevention of dihydrofolate reductase from zebrafish (zDHFR) were studied in the presence of miniGroEL. The thermal denaturation of zDHFR is irreversible, thus it is a very good model system for chaperone mediated aggregation prevention studies. The thermal aggregation of zDHFR was considerably prevented (42%) in the presence of miniGroEL. This miniature form of GroEL was able to delay the rate of thermal inactivation of zDHFR as monitored through Arrhenius plots. Furthermore, the reactivation yield of thermally inactivated zDHFR was significantly enhanced (35%) in the presence of miniGroEL. Through these findings we were able to ascertain the chaperoning ability of the miniature form of GroEL which exhibited all attributes of aggregation prevention, which could in turn be used as a very good in vitro and in vivo chaperone system akin to small heat shock proteins. It further delayed the thermal aggregation of zDHFR which could prove useful during in vivo over-expression of miniGroEL where the partially aggregating protein could be further acted upon by ATP-dependent chaperoning machineries. Since, it is non-homologous to known sHSPs, studying its chaperoning mechanism can provide novel mechanistic insights.

Effects of ultrasonication variables on the activity and properties of alpha amylase preparation.

Recently, ultrasound was demonstrated to increase enzyme activity in food industry. In most studies, an enzyme-substrate mixture was ultrasonicated. Very little is reported on the ultrasonication of enzyme preparation; the effects of ultrasonication variables on the enzyme activity and properties remained unclear. In this study, an α-amylase preparation was ultrasonicated under different conditions. At the ultrasonic frequency, power, temperature, and time of 20 kHz, 25 W/mL, 30°C, and 75 s, respectively, the secondary structure of the α-amylase was changed and that improved the catalytic activity by 47% over the control. The optimal temperature and pH of the α-amylase preparation did not change under the ultrasonic treatment. In addition, ultrasonic treatment increased kinetic parameters of the α-amylase including maximal velocity Vmax , Michaelis constant Km , turnover number Kcat , and catalytic specificity constant Kcat /Km . Nevertheless, the ultrasonicated α-amylase had lower thermal inactivation rate constant and half-life value than the nonultrasonicated enzyme. Ultrasonic treatment can be considered as a novel solution for improvement in enzyme activity. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:702-710, 2018.

Immobilized Cerrena sp. laccase: preparation, thermal inactivation, and operational stability in malachite green decolorization

Laccases are polyphenol oxidases with widespread applications in various industries. In the present study, the laccase from Cerrena sp. HYB07 was immobilized with four methods, namely entrapment in alginate, covalently binding to chitosan as well as formation of cross-linked enzyme aggregates (CLEAs) and magnetic CLEAs (M-CLEAs). The activity recovery rates of the immobilized laccases ranged from 29% to 68%. Immobilization elevated the reaction temperature optimum and reduced substrate specificity, but not necessarily the turnover rate. pH stability of immobilized laccases was improved compared with that of the free laccase, especially at acidic pH values. Thermal inactivation of all laccases followed a simple first-order exponential decay model, and immobilized laccases displayed higher thermostability, as manifested by lower thermal inactivation rate constants and longer enzyme half-life time. Operational stability of the immobilized laccase was demonstrated by decolorization of the triphenylmethane dye malachite green (MG) at 60 °C. MG decolorization with free laccase was accompanied by a shift of the absorption peak and accumulation of a stable, colored intermediate tetradesmethyl MG, probably due to lower thermostability of the free laccase and premature termination of the degradation pathway. In contrast, complete decolorization of MG was achieved with laccase CLEAs at 60 °C.

Kinetics and thermodynamics of the thermal inactivation and chaperone assisted folding of zebrafish dihydrofolate reductase

The maintenance of thermal stability is a major issue in protein engineering as many proteins tend to form inactive aggregates at higher temperatures. Zebrafish DHFR, an essential protein for the survival of cells, shows irreversible thermal unfolding transition. The protein exhibits complete unfolding and loss of activity at 50 °C as monitored by UV-Visible, fluorescence and far UV-CD spectroscopy. The heat induced inactivation of zDHFR follows first–order kinetics and Arrhenius law. The variation in the value of inactivation rate constant, k with increasing temperatures depicts faster inactivation at elevated temperatures. We have attempted to study the chaperoning ability of a shorter variant of GroEL (minichaperone) and compared it with that of conventional GroEL-GroES chaperone system. Both the chaperone system prevented the aggregation and assisted in refolding of zDHFR. The rate of thermal inactivation was significantly retarded in the presence of chaperones which indicate that it enhances the thermal stability of the enzyme. As minichaperone is less complex, and does not require high energy co-factors like ATP, for its function as compared to conventional GroEL-GroES system, it can act as a very good in vitro as well as in vivo chaperone model for monitoring assisted protein folding phenomenon.

Recombinant production of Aspergillus Flavus uricase and investigation of its thermal stability in the presence of raffinose and lactose.

Aspergillus flavus uricase (Rasburicase) with a molecular mass of 135kDa is currently used for the treatment of gout and hyperuricemia occurring in tumor lysis syndrome. To characterize the effects of raffinose and lactose osmolytes on the uricase structure, its coding sequence was cloned, expressed in E. coli BL21, and purified by Ni-NTA agarose affinity chromatography. Thermal inactivation studies at 40°C showed that nearly 15% of UOX activity was preserved, while the presence of raffinose and lactose reduced its activity to 35 and 45% of its original activity, respectively. Investigation of UOX thermal stability at 40°C in the course of time showed that the enzyme relatively lost almost 60% of its original activity after 40min, whereas more than 50% of UOX activity is preserved in the presence of lactose. Estimation of thermal inactivation rate constant, k in, showed that the UOX k in and UOX k in in the presence of raffinose was unchanged (0.018min-1), whereas for the presence of lactose, it was 0.015min-1. Half-life and T m analysis showed that UOX half-life is almost 38min and addition of raffinose did not change the half-life, whereas the presence of lactose had remarkable impact on UOX half-life (46min). The presence of raffinose increased UOX T m to a lesser extent, whereas lactose notably enhanced the T m from 27 to 37°C. Overall, our findings show that lactose has protective effects on UOX stability, while for raffinose, it is relatively compromised.

Modeling the Effect of Temperature and Water Activity on the Thermal Resistance of Salmonella Enteritidis PT 30 in Wheat Flour

Salmonella continues to be a problem associated with low-moisture foods, particularly given enhanced thermal resistance at lower water activity (aw). However, there is a scarcity of thermal inactivation models accounting for the effect of aw. The objective of this study was to test multiple secondary models for the effect of product (wheat flour) aw on Salmonella enterica Enteritidis phage type 30 thermal resistance. A full-factorial experimental design included three temperatures (75, 80, and 85°C) and four aw values (~0.30, 0.45, 0.60, and 0.70). Prior to isothermal treatment, sample aw was achieved by equilibrating samples in a humidity-controlled conditioning chamber. Two primary models (log linear and Weibull type) and three secondary models (second-order response surface, modified Bigelow type, and combined effects) were evaluated using the corrected Akaike information criterion and root mean squared errors. Statistical analyses of the primary models favored the log-linear model. Incorporating the three secondary models into the log-linear primary model yielded root mean squared errors of 2.1, 0.78, and 0.96 log CFU/g and corrected Akaike information criterion values of 460, -145, and -19 for the response surface, modified Bigelow, and combined-effects models, respectively. The modified Bigelow-type model, which exponentially scaled both temperature and aw effects on thermal inactivation rates, predicted Salmonella lethality significantly better (P < 0.05) than did the other secondary models examined. Overall, aw is a critical factor affecting thermal inactivation of Salmonella in low-moisture products and should be appropriately included in thermal inactivation models for these types of systems.

Low temperature MS2 (ATCC15597-B1) virus inactivation using a hot bubble column evaporator (HBCE)

In the treatment of household wastewater viruses are hard to eliminate. A new technique is described which tackles this major problem. The MS2 (ATCC15597-B1) virus was used as a surrogate to estimate the inactivation rates for enteric viruses by a hot (150°C) air bubble column evaporator (HBCE) system Its surface charging properties obtained by dynamic light scattering, have been studied in a range of aqueous salt solutions and secondary treated synthetic sewage water. A combination of MS2 virus surface charge properties with thermal inactivation rates, and an improved double layer plaque assay technique, allows an assessment of the efficiency of the HBCE process for virus removal in water. The system is a new energy efficient treatment for water reuse applications.

Use of power ultrasound to enhance the thermal inactivation of Clostridium perfringens spores in beef slurry

Clostridium perfringens is a pathogen of concern in pasteurised foods. The main objective of this study was to use power ultrasound to enhance the thermal inactivation of C. perfringens spores in beef slurry. The effect of simultaneous ultrasound and heat (TS, thermosonication) on the spore inactivation in beef slurry was first investigated. At 75°C, a 60min TS process (24kHz, 0.33W/g) resulted in a less than 1.5 log reduction for both C. perfringens NZRM 898 and NZRM 2621 spores. Then, the thermal inactivation first order kinetic parameters of C. perfringens spores in beef slurry were estimated for the two strains. The D105°C- and z-values were 2.5min and 10.6°C for NZRM 898 and 1.8min and 10.9°C for NZRM 2621. After, the effect of a spore heat shock followed by ultrasound on its thermal inactivation in beef slurry was investigated. This heat shock+ultrasound pretreatment was able to double the spore thermal inactivation rate in beef slurry. For example at 95°C D-value of 20.2min decreased to 9.8min, demonstrating that spore exposure to heat shock followed by ultrasonication enhanced its thermal inactivation.

Characteristics of Immobilized Urease on Grafted Alginate Bead Systems

This study evaluated the biological importance of immobilized urease enzyme over the free urease. The support material used for urease immobilization was alginate. Generally, the immobilization of urease in alginate gel showed a marked increase in Km and Vmax. However, the immobilized urease showed higher thermal stability than that of free enzyme. The rate of thermal inactivation of the immobilized enzyme decreased due to entrapment in gel matrix. Also, the activity of the immobilized urease was more stable in retention than that of the free enzyme during the storage in solution, although the activity of the immobilized enzyme was lower in comparison with the free enzyme. A stable immobilized system and long storage life are convenient for applications that would not be feasible with a soluble enzyme system. These results highlighted the technical and biochemical benefits of immobilized urease over the free enzyme.

Factors affecting the infectivity of tissues from pigs with classical swine fever: Thermal inactivation rates and oral infectious dose

Outbreaks of classical swine fever are often associated with ingestion of pig meat or products derived from infected pigs. Assessment of the disease risks associated with material of porcine origin requires knowledge on the likely amount of virus in the original material, how long the virus may remain viable within the resulting product and how much of that product would need to be ingested to result in infection. Using material from pigs infected with CSFV, we determined the viable virus concentrations in tissues that comprise the majority of pork products. Decimal reduction values (D values), the time required to reduce the viable virus load by 90% (or 1log10), were determined at temperatures of relevance for chilling, cooking, composting and ambient storage. The rate of CSFV inactivation varied in different tissues. At lower temperatures, virus remained viable for substantially longer in muscle and serum compared to lymphoid and fat tissues. To enable estimation of the temperature dependence of inactivation, the temperature change required to change the D values by 90% (Z values) were determined as 13°C, 14°C, 12°C and 10°C for lymph node, fat, muscle and serum, respectively. The amount of virus required to infect 50% of pigs by ingestion was determined by feeding groups of animals with moderately and highly virulent CSFV. Interestingly, the virulent virus did not initiate infection at a lower dose than the moderately virulent strain. Although higher than for intranasal inoculation, the amount of virus required for infection via ingestion is present in only a few grams of tissue from infected animals.

English

Thermal inactivation of a new recombinant Phlebia radiata manganese peroxidase (rPr-MnP3) in the presence and absence of additives (CaCl2 and EDTA) is described for the first time. The influence of temperature and melting points (Tm) on the stability of rPr-MnP3 and its mutant (E40H/E44H) were determined. There was no significant inactivation at 25 &ndash; 40&deg;C. However, we observed rapid inactivation of rPr-MnP3 at 50&deg;C and above. Addition of CaCl2 to the enzyme mixture resulted in a marked increase in the half-life (533 min) of the wild-type enzyme compared to E40H/E44H with the half-life of 92 min. Ethylenediaminetetraacetic acid (EDTA) increased the rate of rPr-MnP3 thermal inactivation as shown by the decay constant (kd) of 0.070 &plusmn; 0.007 min-1 and half-life of 10 min. The decay constant (kd) 0.029 &plusmn; 0.002 min-1 and half-life of 24 min were obtained for the control (untreated sample). Calcium ion had protective effect on the inactivation of the wild-type enzyme but not for mutant. The mutant (E40H/E44H) was observed to be more stable with a higher melting point of 58&deg;C than the wild-type (Tm =54&deg;C).The inactivation effect of EDTA on the E40H/E44H was lower than that of the wild-type. Calcium ions were found to be important structural elements responsible for the enzyme stability. Our findings showed that rPr-MnP3 is a highly stable enzyme and may be of significant industrial applications.&nbsp;&nbsp;&nbsp; Key words: Peroxidase, Phlebia radiata, thermal stability, thermal inactivation, reactivation, melting point, additives.

Gelatin and starch as stabilizers of the coupled enzyme system of luminous bacteria NADH:FMN–oxidoreductase–luciferase

We have studied the effects of a gel-like environment on the characteristics of enzyme preparations based on the coupled enzyme system of luminous bacteria, NADH:FMN-oxidoreductase-luciferase, to design a stable immobilizing reagent for bioluminescent analysis. Natural polymers, gelatin and starch, were used to create a viscous, structured microenvironment. The stability of the coupled enzyme system to such physical and chemical environmental factors as temperature, pH, and ionic strength in gelatin and starch-containing media was examined. It was shown that both gelatin and starch have a stabilizing effect on the enzymes of luminous bacteria under specific conditions. In particular, the enzymes' activity is increased twofold in the presence of 1 and 5% of gelatin at 20 °C and 25 °C, respectively (temperatures lower than the gel point). Also, the acceptable pH range of the coupled enzyme system expands into the alkaline region and becomes 6.8-8.1. Stabilization at low ionic strength (0.01-0.06 mol L(-1)) is observed. At the same time, microenvironments based on either gelatin or starch do not change the enzymes' thermal inactivation rate constants in the temperature range from 25 to 43 °C. Finally, gelatin and starch are suitable for development of a reagent for immobilization of enzymes which would be stable and resistant to physical and chemical environmental conditions.

Cloning of a 1‐pyrroline‐5‐carboxylate dehydrogenase from Taiwanofungus camphorata (583.1)

A cDNA (1890 bp) encoding a putative 1‐pyrroline‐5‐carboxylate dehydrogenase (P5CD) was cloned from Taiwanofungus camphorata. The DNA sequence encodes a protein of 547 amino acid residues with calculated molecular mass of 59.4 kDa. The deduced amino acid sequence is conserved among the reported P5CDs. To characterize the T. camphorata P5CD, the coding region was subcloned into an expression vector pET‐20b(+) and transformed into E. coli Rosetta (DE3). The recombinant His6‐tagged TcP5CD was expressed and purified by Ni2+‐nitrilotriacetic acid Sepharose. The purified enzyme showed a major band of ~59.4 kDa by 12 % SDS‐PAGE. The Michaelis constant (KM) value for 3,5‐dimethoxybenzaldehyde with NAD+ as cofactor was 0.56 mM. The enzyme’s half‐life of deactivation at 45oC was 4.4 min, and its thermal inactivation rate constant kd was 1.23 x 10‐2 min‐1. The enzyme was most active at pH 7.0. The enzyme’s preferred substrate is veratraldehyde. It can also use other aldehyde as substrates including acetaldehyde and propionaldehyde.Grant Funding Source: National Science Council of the Republic of China under grant NSC95‐2313‐B‐019‐007 to C‐T. L.

Thermal inactivation of a recombinant lipoxygenase fromPseudomonas aeruginosaBBE in the absence and presence of additives

Bacterial lipoxygenase (EC 1.13.11.12, LOX) is an important enzyme used as a brightener and strengthening agent during breadmaking. In this study, thermal inactivation of a recombinant LOX of Pseudomonas aeruginosa BBE was characterized by kinetic and thermodynamic analysis in the absence and presence of additives. As the heating temperature increased from 25 to 55 °C, the thermal inactivation rate (k) values for LOX without the additives ranged from 0.0407 to 0.2627 min(-1), while the half-life (t1/2) values were between 17.08 and 3.25 min. The activation energy (ΔE) values were increased with rise in heating temperatures from 13.26 to 108.9 kJ mol(-1) . Separate tests at 45 °C in the presence of additives (polyols, sugars and ions) at specific concentrations showed that xylitol (1 mol L(-1)) was the most effective stabilizer for recombinant LOX and increased the t1/2 value by 297%. Recombinant LOX was sensitive to heat treatment, and addition of polyols, sugars and ions could enhance its thermal stability. Our findings may provide useful information for stabilizing emerging bacterial LOXs.