Energy Of Deactivation Research Articles

Modifying Thermostability and Reusability of Hyperthermophilic Mannanase by Immobilization on Glutaraldehyde Cross-Linked Chitosan Beads

In the current study, the purified β-mannanase (Man/Cel5B) from Thermotoga maritima was immobilized on glutaraldehyde cross-linked chitosan beads. The immobilization of Man/Cel5B on chitosan beads was confirmed by Fourier-transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis. After immobilization, the protein loading efficiency and immobilization yield were found to be 73.3% and 71.8%, respectively. The optimum pH for both free and immobilized enzymes was found to be pH 5.5. However, the optimum temperature of immobilized Man/Cel5B increased by 10 °C, from 85 °C (free Man/Cel5B) to 95 °C (Immobilized). The half-life of free and immobilized enzymes was found to be 7 h and 9 h, respectively, at 85 °C owing to the higher thermostability of immobilized Man/Cel5B. The increase in thermostability was also demonstrated by an increase in the energy of deactivation (209 kJmol−1) for immobilized enzyme compared to its native form (92 kJmol−1), at 85 °C. Furthermore, the immobilized Man/Cel5B displayed good operational stability as it retained 54% of its original activity after 15 repeated catalytic reactions concerning its free form.

Single step immobilization of CMCase within agarose gel matrix: Kinetics and thermodynamic studies

In the current study, CMCase from Bacillus licheniformis KIBGE-IB2 was immobilized within the matrix of agarose gel through entrapment technique. Maximum immobilization yield (%) of the enzyme was obtained when 2.0 % agarose was used. The activation energy (Ea) of the enzyme increased from 16.38 to 44.08 kJ mol−1 after immobilization. Thermodynamic parameters such as activation energy of deactivation (ΔGd), enthalpy (ΔHd) and entropy (ΔSd) of deactivation, deactivation rate constant (Kd), half-life (t1/2), D-value and z-value were calculated for native/free and immobilized CMCase. The maximum reaction rate (Vmax) of the native enzyme was found to be 8319.47 U ml−1 min−1, which reduced to 7218.1 U ml−1 min−1after immobilization process. However, the Michaelis-Menten constant (Km) value of the enzyme increased from 1.236 to 2.769 mg ml−1 min−1 after immobilization. Immobilized enzyme within agarose gel matrix support can be reuse up to eight reaction cycles. Broad stability profile and improved catalytic properties of the immobilized CMCase indicated that this enzyme can be a plausible candidate to be used in various industrial processes.

Honeywell introduces next- generation catalyst to address ‘smell in cabin’ events

In the current study, CMCase from Bacillus licheniformis KIBGE-IB2 was immobilized within the matrix of agarose gel through entrapment technique. Maximum immobilization yield (%) of the enzyme was obtained when 2.0 % agarose was used. The activation energy (Ea) of the enzyme increased from 16.38 to 44.08 kJ mol−1 after immobilization. Thermodynamic parameters such as activation energy of deactivation (ΔGd), enthalpy (ΔHd) and entropy (ΔSd) of deactivation, deactivation rate constant (Kd), half-life (t1/2), D-value and z-value were calculated for native/free and immobilized CMCase. The maximum reaction rate (Vmax) of the native enzyme was found to be 8319.47 U ml−1 min−1, which reduced to 7218.1 U ml−1 min−1after immobilization process. However, the Michaelis-Menten constant (Km) value of the enzyme increased from 1.236 to 2.769 mg ml−1 min−1 after immobilization. Immobilized enzyme within agarose gel matrix support can be reuse up to eight reaction cycles. Broad stability profile and improved catalytic properties of the immobilized CMCase indicated that this enzyme can be a plausible candidate to be used in various industrial processes.

Catalytic oxidation and reaction kinetics of low concentration benzene over CuxMnyOz/SiO2

Reducing reaction temperature of catalytic combustion is the fundamental way to improve adaptability of catalysts to complex volatile organic compounds (VOCs) and off-design conditions. Using benzene which is the most difficult to be oxidized in VOCs as the catalytic target and by means of multiple characterization methods, silicon dioxide supported copper-manganese catalysts were optimized through changing the load of active material, the ratio of bimetal and calcination condition, the conclusions as following: Cu3Mn9/SiO2 with calcination temperature of 300 °C, molar ratio for Cu/Mn of 3:9 and total load mass for active substance of 11% demonstrates the best catalytic performance. And the benzene which is lower than 2000 mg/m3 in air can be completely oxidized at 265 °C. The change of activation energy and mechanism of deactivation in the process of catalyst optimization were obtained by catalytic reaction kinetic analysis. After the optimization, reaction activation energy decreased from 65.08 kJ/mol to 56.82 kJ/mol, and complete conversion temperature of the C6H6 decreased by nearly 20 °C. Additionally, the fundamental reason for deactivation of the catalyst is the structure changes at high temperature, and surface oxides agglomerate, which greatly reduces the oxygen content of the catalyst. With the increase of reaction temperature, the mass transfer capacity of O2 on the surface of catalyst increase less than that of C6H6, leading to carbon deposition, and the joint action results in the decrease of catalyst activity.

Improvement of catalytic, thermodynamics and antifungal activity of constitutive Trichoderma longibrachiatum KT693225 exochitinase by covalent coupling to oxidized polysaccharides

Our study full filled in two main goals preparation of constitutive exochitinase with low cost, utilizing non-chitin containing agricultural wastes, and improving the thermodynamics of purified Trichoderma longibrachiatum KT693225 exochitinase by covalent coupling to sodium periodate activated agar. Central composite design (CCD) was used to improve the chemical modification of Trichoderma longibrachiatum KT693225 exochitinase. Optimum temperature for conjugated exochitinase 60 °C was higher than native form 40 °C. Covalent coupling to oxidized agar caused 4.32, 2.75 and 2.44-fold increase in half-life values at 50, 55 and 60 °C, respectively. Also, conjugated exochitinase showed higher D-values (decimal reduction time) 1790.49 compared to 733.08 min for native form at 60 °C. Moreover, conjugated form had lower deactivation constant rate (kd) 0.39 × 10−3 min−1at 60 °C than native form 1.7 × 10−3 min−1. Native exochitinase exhibited higher activation energy (Ea) 3.39 Kcal·mol−1 and lower energy for denaturation (Ed) 6.88 Kcal·mol−1 compared to 3.21 and 13.05 Kcal·mol−1, respectively for conjugated form. The values of thermodynamic parameters for inactivation of native and conjugated exochitinase indicated that conjugation significantly decreased entropy (ΔS°) and increased enthalpy (ΔH°) and free energy (ΔG°) of deactivation. Conjugated exochitinase exhibited higher antifungal effect against Alternaria alternata, Fusarium oxysporium and Aspergillus niger than native form.

Investigation of deactivation thermodynamics of lipase immobilized on polymeric carrier.

In the present work, we have investigated biochemical thermo-kinetic stability of lipases immobilized on a biocompatible polymeric material. Immobilization of lipase Candida rugosa (CRL) was carried out on biocompatible blend of poly vinyl alcohol (PVA) and chitosan (CHY) support via entrapment and glutardehyde (Glu) cross-linking method to produce PVA:CHY:CRL and PVA:CHY:Glu:CRL as robust biocatalyst. These immobilized lipases were characterized by various physico-biochemical characterization techniques. Later on, thermal and solvent stability of polymer immobilized lipase was determined in term of half-life time (t 0.5), D values, enthalpy (ΔH°), entropy (ΔS°), and free energy (ΔG°) of deactivation at different temperatures and in various solvents. The thermodynamic deactivation stability trend was found as: cross-linked lipase CRL > entrapped lipase CRL > free lipase CRL. Moreover, kinetic parameters, such as K m, V max, and catalytic efficiency, were also determined to understand the kinetic features. The polymer immobilized enzyme was reused to investigate the economic viability of the developed biocatalyst.

Chemical modification with phthalic anhydride and chitosan: Viable options for the stabilization of raw starch digesting amylase from Aspergillus carbonarius

The raw starch digesting type of amylase (RSDA) presents greater opportunities for process efficiency at cheaper cost and shorter time compared to regular amylases. Chemical modification is a simple and rapid method toward their stabilization for a wider application. RSDA from Aspergillus carbonarius was modified with either phthalic anhydride (PA) or chitosan. Activity retention was 87.3% for PA-modified and 80.9% for chitosan-modified RSDA. Optimum pH shifted from 5 to 7 after PA-modification. Optimum temperature changed from 30°C (native) to 30–40°C and 60°C for PA-modified and chitosan-modified, respectively. Activation energy (kJmol−1) for hydrolysis was 13.5, 12.7, and 10.2 while the activation energy for thermal denaturation was 32.8, 80.3, 81.9 for free, PA-modified and chitosan-modified, respectively. The specificity constants (Vmax/Km) were 73.2 for PA-modified, 63.1 for chitosan-modified and 77.1 for native RSDA. The half-life (h) of the RSDA at 80°C was increased from 6.1 to 25.7 for the PA-modified and 138.6 for the chitosan derivative. Modification also led to increase in D value, activation enthalpy and Gibbs free energy of enzyme deactivation. Fluorescence spectra showed that center of spectral mass decreased for the PA-modified RSDA but increased for chitosan modified RSDA.

Improvement of recovered activity and stability of the Aspergillus oryzae β-galactosidase immobilized on duolite A568 by combination of immobilization methods

The immobilization and stabilization of Aspergillus oryzae ?-galactosidase on Duolite??A568 was achieved using a combination of physical adsorption, incubation step in buffer at pH 9.0 and cross-linking with glutaraldehyde and in this sequence promoted a 44% increase in enzymatic activity as compared with the biocatalyst obtained after a two-step immobilization process (adsorption and cross-linking). The stability of the biocatalyst obtained by three-step immobilization process (adsorption, incubation in buffer at pH 9.0 and cross-linking) was higher than that obtained by two-steps (adsorption and cross-linking) and for free enzyme in relation to pH, storage and reusability. The immobilized biocatalyst was characterized with respect to thermal stability in the range 55-65 ?C. The kinetics of thermal deactivation was well described by the first-order model, which resulted in the immobilized biocatalyst activation energy of thermal deactivation of 71.03 kcal/mol and 5.48 h half-life at 55.0 ?C.

Immobilization of β-galactosidase from Lactobacillus plantarum HF571129 on ZnO nanoparticles: characterization and lactose hydrolysis.

β-Galactosidase from Lactobacillus plantarum HF571129 was immobilized on zinc oxide nanoparticles (ZnO NPs) using adsorption and cross-linking technique. Immobilized β-galactosidase showed broad-spectrum pH optima at pH 5-7.5 and temperature 50-60°C. Fourier transform infrared spectroscopy (FTIR) and field emission scanning electron microscopy (FESEM) showed that β-galactosidase successfully immobilized onto supports. Due to the limited diffusion of high molecular weight substrate, K m of immobilized enzyme slightly increased from 6.64 to 10.22mM, while V max increased from 147.5 to 192.4µmolmin(-1)mg(-1) as compared to the soluble enzyme. The cross-linked adsorbed enzyme retained 90% activity after 1-month storage, while the native enzyme showed only 74% activity under similar incubation conditions. The cross-linked β-galactosidase showed activity until the seventh cycle and maintained 88.02% activity even after the third cycle. The activation energy of thermal deactivation from immobilized biocatalyst was 24.33kcal/mol with a half-life of 130.78min at 35°C. The rate of lactose hydrolysis for batch and packed bed was found to be 0.023 and 0.04min(-1).

Thermogravimetric Analysis of Activated Carbons, Ordered Mesoporous Carbide-Derived Carbons, and Their Deactivation Kinetics of Catalytic Methane Decomposition

This study presents the deactivation kinetics of methane decomposition for the activated carbons Fluka-05105 and Fluka-05120, ordered mesoporous carbon (CMK-3), and ordered mesoporous carbide-derived carbon (DUT-19). The experimental and thermodynamically predicted carbon deposition, the average and total hydrogen production, and the effect of flow rate on carbon formation rate of these catalysts were investigated. Results indicate that the experimental conditions chosen were within the reaction control regime. Catalytic activity was calculated via two different definitions present in literature: one in terms of carbon deposition rate and the other in terms of carbon mass deposited. Deactivation kinetics were obtained by fitting the experimental data by nonlinear regression analysis. Differences between the two methods in determining activity resulted in significant changes in the estimation of deactivation kinetics. The activity calculated based on the rate method results in the best fit of experimentally collected data. A deactivation order and methane concentration dependency of approximately 1.0 and 0.5 were determined for all the catalysts tested (Fluka-05105, Fluka-05120, CMK-3, and DUT-19). The activation energy of deactivation (Ed) was determined to be 192, 154, 166, and 181 kJ/mol for Fluka-05120, Fluka-05105, CMK-3, and DUT-19, respectively. DUT-19 was the best performing catalyst in terms of carbon formation rate, total carbon production, hydrogen production rate, average hydrogen production, and total hydrogen production.

Three phase partitioning of β-galactosidase produced by an indigenous Lactobacillus acidophilus isolate

Three Phase Partitioning (TPP) is reported here for the first time for β-galactosidase purification in single step and two step processes. The most favourable ammonium sulphate concentration (30% w/v), crude to t-butanol ratio (1:0.5) and temperature (25°C) resulted in 78% recovery with 7.5-fold purity of β-galactosidase in the single step process. Enhanced fold purity (21 times) with an enzyme recovery of 72% was achieved at the aforementioned temperature (25°C) and crude to t-butanol ratio (1:0.5) with 15–60% ammonium sulphate saturation in the second step. Characterisation showed that this purified enzyme exhibited a double pH optimum (3.0 and 7.0) that was more pronounced when lactose was used as the substrate. The enzyme was further characterised with respect to its optimum temperature, energy of enzyme activation and deactivation, kinetic constants (Vmax and Km) and pK1 and pK2 values and effect of monovalent and divalent cations.

β-Galactosidase of Aspergillus oryzae immobilized in an ion exchange resin combining the ionic-binding and crosslinking methods: Kinetics and stability during the hydrolysis of lactose

The strong dependence of the stability of the immobilized enzyme on the temperature. ► The use of ionic exchange resins for immobilization is used food industry. ► Glutaraldehyde activation of the carriers is one technique to immobilize enzymes. ► β-galactosidase e immobilized on the ionic exchange resin followed by crosslinking. ► The hydrolysis kinetics of lactose by β-galactosidase immobilized. ► The influence of temperature in the enzymatic activity of immobilized enzyme. In this work, the hydrolysis kinetics of lactose by Aspergillus oryzae β-galactosidase was studied using the ionic exchange resin Duolite A568 as a carrier. The enzyme was immobilized using a β-galactosidase concentration of 16 g/L in pH 4.5 acetate buffer and an immobilization time of 12 h at 25 ± 0.5 °C. Next, the immobilized β-galactosidase was crosslinked using glutaraldehyde concentration of 3.5 g/L for 1.5 h. The influence of lactose concentration was studied for a range of 5–140 g/L, and the Michaelis–Menten model was fitted well to the experimental results with V m and K m values of 0.71 U and 35.30 mM, respectively. The influence of the product galactose as an inhibitor on the hydrolysis reaction was studied. The model that was best fitted to the experimental results was the competitive inhibition by galactose with V m , K m and K i values of 0.77 U, 35.30 mM and 27.44 mM, respectively. The influence of temperature on the enzymatic activity of the immobilized enzyme was studied in the range of 10–80 °C, in which the temperature of the maximum activity was 60 °C, with an activation energy of 5.32 kcal/mol of lactose, using an initial concentration of lactose of 50 g/L in a pH 4.5 sodium acetate buffer solution. The thermal stability of the immobilized biocatalyst was determined to be in the range 55–65 °C. The first-order model described well the kinetics of thermal deactivation for all the temperatures studied. The activation energy of thermal deactivation from immobilized biocatalyst was 66.48 kcal/mol with a half-life of 8.9 h at 55 °C.

On the hyperthermostability of lipolytic enzymes from Thermus aquaticus YT‐1: exploring their application to polymer degradation

AbstractBACKGROUND: The investigation of enzymes from extremophilic microorganisms has generated great scientific interest in recent decades owing to their ability to operate in harsh conditions. Since lipolytic enzymes are one of the three groups most commercially demanded, the study of novel thermostable lipolytic enzymes is of much interest.RESULTS: Lipolytic enzymes produced by Thermus aquaticus YT1 have been investigated. Very interesting properties against thermal inactivation, with a half‐life time of 1 h at 95 °C were found. The use of compounds such as polyols is a strategy used to further improve the thermostability of these enzymes, and the modelling of the deactivation process with a series‐type equation, allows one to quantitatively ascertain the free energy of deactivation. Finally, the application to dibutyl adipate hydrolysis was investigated, as an indicator of the potential usefulness of the enzymes in polymer biodegradation.CONCLUSION: The crude lipolytic enzymes possess outstanding properties of thermostability (high lipolytic activity after 12 days at 80 °C, and a half‐life time of 1 h at 95 °C), and they can be significantly improved by adding polyols (addition of glycerol entailed activity losses of just 3% after 1 h at 95 °C). These enzymes are able to catalyze ester cleavage, showing a zero‐order kinetics. Copyright © 2011 Society of Chemical Industry

Optimal temperature control in a batch bioreactor with parallel deactivation of enzyme

A biotransformation process with parallel deactivation of biocatalyst running in a batch reactor has been analyzed. A non-linear deactivation model suggested by Do and Weiland has been taken into account. Based on variational calculus analytical computations aimed at finding an optimal selection of temperature conditions that ensure obtaining maximum conversion or minimum duration time necessary for its attaining have been carried out. Solutions for a stationary process and that carried out at the active upper and lower temperature limitations have been presented. Furthermore, an analysis of the course of the process under isothermal conditions as those indispensable for the appropriate choice of the optimal conditions has been presented. Based on an example of hydrogen peroxide decomposition process in the presence of the native catalase of yeast Saccharomyces cerevisae (CSC) the obtained solution has been described. In addition an influence of the quotient of energy of deactivation and reaction processes, initial and final biocatalyst activity and conversion on the course of the optimal temperature profiles have been considered. It has been proved that a decrease in the activation energy quotient and the final biocatalyst activity as well as an increase in conversion results in an increase of the overall process duration time. Also a comparison of the course of the stationary temperature profiles at different ranges of substrate concentration and the effect of the latter on the analyzed objective function has been made. It has been revealed that in case of processes which run at any range of the substrate concentrations, in order to achieve the shortest duration time of their course, it is appropriate to conduct them at possibly the lowest concentration range.

Effects of temperature and shear force on infectivity of the baculovirus Autographa californica M nucleopolyhedrovirus

Virus stability and infectivity during stressful conditions was assessed to establish guidelines for future virus filtration experiments and to contribute to the body of knowledge on a widely used virus. A recombinant baculovirus of Autographa californica M nucleopolyhedrovirus (Ac MNPV), vHSGFP, was incubated at 15–65 °C. A 2-log decrease in virus infectivity occurred after virus incubation above 45 °C. The activation energy of virus deactivation was circa 108 kJ/mol. Dynamic light scattering revealed an increase in apparent virus particle size from 150 ± 19 to 249 ± 13 nm at 55 °C. Protein and DNA concentrations in solution correlated well with virus aggregation as temperature was increased. Infectivity of vHSGFP stored for 5 months at 4 °C or exposed to shear stress from stirring (100 rpm, 1.02 × 10 −5 psi) and pumping (50–250 ml/min, 1.45 × 10 −5 to 7.25 × 10 −5 psi) did not change with time. Unlike temperature variations, cold storage and shear stress appeared to have little impact on infectivity.

Hidrólise da lactose pela β-galactosidase de Kluyveromyces fragilis: caracterização da enzima

The β-galactosidase enzyme from Kluyveromyces fragilis was characterized in the soluble form using lactose 5% w/v found in skimmed powdered milk as substrate. Enzyme diluted 50 times hydrolyzed the lactose in batch reactor of 50 mL capacity. Enzyme activity and its activation energy were determined as a function of temperature and pH. Temperature ranged from 20 to 55oC and pH from 5.5 to 8.0. Activation energy was 9.50 kcal/mol. The energy of deactivation was 33.74 kcal/mol. Although the enzyme presented a high specific activity at 45oC and pH 6.5 (3.312 U/mg protein), values indicate that the best use of the enzymatic activity occur at 40oC or below, with half-life higher than 12 hours. The activation energy increased proportionally to pH increase. Therefore, the activation energy depends on pH and varies according to the origin of the enzyme.

Study of the effects of temperature and pH on lactic acid production from fresh cassava roots in tofu liquid waste by Streptococcus bovis

We report a study of the effects of temperature and pH on the kinetics of lactic acid production from fresh cassava roots (FCR) by Streptococcus bovis in new media: tofu liquid waste (TLW); TLW with 2 wt% concentrated maguro waste (TLW + CMW2); and in a standard trypto-soya broth (TSB) compared with a standard medium (glucose in TSB). The results showed that 39 °C and pH 5.5 were optimal for fermentation properties (lactic acid concentration, productivity and specific growth rate) in all media, including the standard. The energy of activation ( E a) and the energy of deactivation ( E d) of lactic acid fermentation in all media were calculated using the Arrhenius relation. The E a and E d values increased in the order FCR in TLW < FCR in TLW + CMW2 < glucose in TSB < FCR in TSB, while values of productivity and specific growth rate decreased in the same order. The effects of pH on productivity and specific growth rate were evaluated by Michaelis–Menten and Monod-type equations. For pH, the inhibition is competitive for both productivity and specific growth rate. By controlling the pH at 5.80, the inhibition for both productivity and specific growth rate were minimized in all media.

Polyphenoloxidase deactivation kinetics during ohmic heating of grape juice

The heating method affects the temperature distribution inside a food, and directly modifies the time–temperature relationship for enzyme deactivation. Fresh grape juice was ohmically heated at different voltage gradients (20, 30, and 40 V/cm) from 20 °C to temperatures of 60, 70, 80 or 90 °C and the change in the activity of polyphenoloxidase enzyme (PPO) was measured. The critical deactivation temperatures were found to be 60 °C or lower for 40 V/cm, and 70 °C for 20 and 30 V/cm. Various kinetic models for the deactivation of PPO by ohmic heating at 30 V/cm were fitted to the experimental data. The simplest kinetic model involving one step first-order deactivation was better than more complex models. The activation energy of the PPO deactivation for the temperature range of 70–90 °C was found to be 83.5 kJ/mol.

Deactivation Kinetics of Metallocene‐Catalyzed Ethene Polymerization at High Temperature and Elevated Pressure

AbstractSummary: The kinetics of ethene polymerization with metallocene/[Me2PhNH]+[B(C6F5)4]−/AliBu3 (ternary systems) and metallocene/methylaluminoxan (MAO) systems respectively has been investigated at 100–140 °C and 7 MPa. Overall, eight different unbridged and ansa‐metallocenes were tested. The effect of ligand structure, cocatalyst and catalyst concentration on the thermostability and the activation energy of deactivation were studied. Deactivation with respect to the catalyst concentration followed a first order reaction. The half‐life as well as the activation energy of the ternary systems depended strongly on ligand structure while the ligand structure of MAO‐activated metallocenes merely influenced the half‐life. The half‐life associated with MAO activation is approximately twice as high as that in ternary activation. Based on these results, conclusions about the deactivation reactions have been drawn.Influence of catalyst concentration on deactivation.imageInfluence of catalyst concentration on deactivation.

Temperature Response of Mesophyll Conductance. Implications for the Determination of Rubisco Enzyme Kinetics and for Limitations to Photosynthesis in Vivo

CO(2) transfer conductance from the intercellular airspaces of the leaf into the chloroplast, defined as mesophyll conductance (g(m)), is finite. Therefore, it will limit photosynthesis when CO(2) is not saturating, as in C3 leaves in the present atmosphere. Little is known about the processes that determine the magnitude of g(m). The process dominating g(m) is uncertain, though carbonic anhydrase, aquaporins, and the diffusivity of CO(2) in water have all been suggested. The response of g(m) to temperature (10 degrees C-40 degrees C) in mature leaves of tobacco (Nicotiana tabacum L. cv W38) was determined using measurements of leaf carbon dioxide and water vapor exchange, coupled with modulated chlorophyll fluorescence. These measurements revealed a temperature coefficient (Q(10)) of approximately 2.2 for g(m), suggesting control by a protein-facilitated process because the Q(10) for diffusion of CO(2) in water is about 1.25. Further, g(m) values are maximal at 35 degrees C to 37.5 degrees C, again suggesting a protein-facilitated process, but with a lower energy of deactivation than Rubisco. Using the temperature response of g(m) to calculate CO(2) at Rubisco, the kinetic parameters of Rubisco were calculated in vivo from 10 degrees C to 40 degrees C. Using these parameters, we determined the limitation imposed on photosynthesis by g(m). Despite an exponential rise with temperature, g(m) does not keep pace with increased capacity for CO(2) uptake at the site of Rubisco. The fraction of the total limitations to CO(2) uptake within the leaf attributable to g(m) rose from 0.10 at 10 degrees C to 0.22 at 40 degrees C. This shows that transfer of CO(2) from the intercellular air space to Rubisco is a very substantial limitation on photosynthesis, especially at high temperature.