A novel approach for estimating the relationship between the kinetics and thermodynamics of glycoside hydrolases

A series of experiments were performed, in which p-nitrophenyl-β-D-cellobioside (PNPC) was hydrolyzed by 1, 4-β-D-glucan-cellobiohydrolase (CBHI: EC 3.2.1.91), and O-nitrophenyl-β-D-galactoside (ONPG) was hydrolyzed by β-galactosidase (EC 3.2.1.23) under different combinations of temperature and time period. The combined effects of temperature and time on p-nitrophenyl and O-nitrophenyl formation were characterized as the change of the instantaneous reaction velocity occurrence per temperature range termed as v(inst)· T(-1). This parameter was used as a stable index to evaluate the apparent activation energy (E(a)) based on the Arrhenius approach, instead of the reaction velocity constant, k. It was found that E(a) for PNPC hydrolysis by CBHI first decreased with temperature increase and then slightly increased at higher temperature, and its minimum value was obtained just at the maximum point of v(inst). In addition, E(a) for PNPC hydrolysis by dilute sulfuric acid was not a constant, but was continuously increased with temperature. The present studies demonstrated that E(a) obtained by Arrhenius approach for the hydrolysis reaction of β-hydrolases appears to be only an empirical kinetic parameter for the dependence of the reaction velocity on temperature and time, and has no meaning in the sense of thermodynamic energy.     

The kinetics and thermodynamics of reversible denaturation of crystalline soybean trypsin inhibitor

Crystalline soybean trypsin inhibitor protein undergoes denaturation on heating which is reversed on cooling. In the range of temperature of 35 to 50 degrees C. a solution of the protein consists of a mixture of native and denatured forms in equilibrium with each other. The equilibrium is only slowly established and its final value at any temperature is the same whether a heated, denatured solution of the protein is cooled to the given temperature or whether a fresh solution is raised to that temperature. The kinetics of reversible denaturation of the soybean protein as well as the reversal of denaturation is that of a reversible unimolecular reaction, each process consisting at a given temperature of the same two simultaneous reactions acting in opposite directions. The experimental data on the effect of temperature on the velocity and the equilibrium constants of the opposing reaction were utilized in evaluating the reaction energies and activation energies. The reaction energies for denaturation were found to be as follows:- Change in total heat of reaction DeltaH = 57,000 calories per mole Change in entropy of reaction DeltaS = 180 calories per degree per mole The heat of activation DeltaH(1) (double dagger) for denaturation = 55,000 The heat of activation DeltaH(2) (double dagger) for the reversal of denaturation = -1900 The entropy DeltaS(1) (double dagger) for denaturation = 95 The entropy DeltaS(2) (double dagger) for reversal of denaturation = -84     

Effects of pressure and temperature on enzymatic reactions in supercritical fluids

Supercritical fluids (SCFs) are receiving increasing attention as reaction media because they permit higher reaction rates compared with the conventional solvents. The ease of manipulating the physical properties of the SCFs enables easier control of the reaction conditions and easier solvent removal after the reaction. This review focuses on effects of pressure, temperature and the properties of the SCFs, on enzymatic reactions. Phase behavior, reaction rate and activation volume in SCFs are discussed. Within the ranges of pressure (10-40 MPa) and temperature (35-60 degrees C) that typically characterize the supercritical region, an increase in pressure and/or a decrease in temperature lead to a decrease in the enzyme turnover because the diffusion coefficients of the substrates migrating to the active sites of enzymes are affected.     

A model for optimal reaction norms: the case of the pregnant garter snake and her temperature-sensitive embryos

We present a model to test Osgood's ( 1978 ) proposition that viviparous snakes have optimal reaction norms for temperature-sensitive meristic traits, such as scale counts. Our model predicts that traits that are subject to temperature effects during development will evolve a flat or [Formula: see text]-shaped reaction norm (average scale count as a function of developmental temperature). We tested this prediction by maintaining 67 female garter snakes (Thamnophis elegans) at eight different constant temperatures (21 degrees -33 degrees C) during pregnancy and making a series of scale counts on their newborn offspring (n = 491). To insure that the experimental temperatures were ecologically relevant, we used automated radiotelemetry to record the body temperature of pregnant, free-ranging females. The resulting temperature data allowed us to test the prediction that the inflection points of reaction norms would correspond to the average temperature experienced by embryos in the field. In line with predictions of the Osgood model, reaction norms were flat or U-shaped. In the case of U-shaped reaction norms, the inflection point of the curves corresponded to the average temperature imposed on embryos by free-ranging females. In contrast to some past studies, none of the standard scale scores (ones commonly used in systematics) showed significant temperature effects in either sex. Reaction norms were flat. In contrast, incidences of various abnormalities showed U-shaped reaction norms. Temperature effects were more pronounced in males than in females. The results have implications for systematics and for the evolution of canalization and phenotypic plasticity.     

Optimization of ultrasound-accelerated synthesis of enzymatic octyl hydroxyphenylpropionate by response surface methodology

The ultrasound-accelerated enzymatic synthesis of octyl hydroxyphenylpropionate (OHPP) from p-hydroxyphenylpropionic acid (HPPA) and octanol was investigated in this study. A commercially available immobilized lipase from Candida antarctica, Novozym 435, was used as the biocatalyst. A three-level-three-factor Box-Behnken design experiment and response surface methodology were used to evaluate the effects of temperature, reaction time, and enzyme activity on percent yield of OHPP. The results indicated that temperature and enzyme activity significantly affected percent yield, whereas reaction time did not. A model for the synthesis of OHPP was established. Based on a ridge max analysis, the optimum conditions for OHPP synthesis were predicted to use a reaction temperature of 58.8°C, a reaction time of 14.6 h, and an enzyme activity of 410.5 PLU with a yield of 98.5%. A reaction was performed under these optimal conditions, and a yield of 97.5% ± 0.1% was obtained.     

温度、pH及CO2对白藜芦醇与过氧亚硝基相互作用的影响

白藜芦醇(Resveratrol,Res)是植物在遇到紫外线照射、真菌感染等不利条件下自然产生的抗毒素,存在于多种植物中,具有明显的消炎、抗癌、抗血栓等作用。本文利用紫外-可见光谱法研究了Res与过氧亚硝基阴离子(Peroxynitrite anion,ONOO-)的相互作用,提出了反应机理。研究了温度、pH及CO2对Res与ONOO-反应的影响,结果表明低温、偏碱性pH有利于反应的进行;CO2存在时Res仍能与ONOO-反应,但反应机理与前面不同。     

Optimization studies on acid hydrolysis of oil palm empty fruit bunch fiber for production of xylose

Oil palm empty fruit bunch fiber is a lignocellulosic waste from palm oil mills. It is a potential source of xylose which can be used as a raw material for production of xylitol, a high value product. The increasing interest on use of lignocellulosic waste for bioconversion to fuels and chemicals is justifiable as these materials are low cost, renewable and widespread sources of sugars. The objective of the present study was to determine the effect of H(2)SO(4) concentration, reaction temperature and reaction time for production of xylose. Batch reactions were carried out under various reaction temperature, reaction time and acid concentrations and Response Surface Methodology (RSM) was followed to optimize the hydrolysis process in order to obtain high xylose yield. The optimum reaction temperature, reaction time and acid concentration found were 119 degrees C, 60 min and 2%, respectively. Under these conditions xylose yield and selectivity were found to be 91.27% and 17.97 g/g, respectively.     

A network of hydrogen bonds in the reaction centers of Rhodobacter sphaeroides serves as a regulatory factor of the temperature dependence of the recombination rate constant of photooxidized bacteriochlorophyll and primary quinone acceptors

The dark recombination rate constant for the photooxidized bacteriochlorophyll (P) and reduced primary quinone acceptor (QA) in the photosynthetic reaction centers (RC) from purple bacterium Rhodobacter sphaeroides depends nonmonotonically on temperature. The time of this reaction is approximately 100 ms at 270-300 K and decreases as the temperature both increases and decreases beyond this temperature range. It is known that the dome-shaped dependence of the thermodynamic stability on temperature is an intrinsic feature of many proteins in solution. The experimental results on the nonmonotonous temperature dependence of P+ and QA- recombination rate constant are discussed in terms of general thermodynamic approaches. The dynamic properties of the network of hydrogen bonds that are involved in the relaxation processes accompanying the electron transport are considered as a regulatory factor of the efficiency of electron transfer.     

连续温度变化对β-1,3-葡聚糖酶酶解酵母β-葡聚糖的影响

为了探索反应温度对产物组分的影响,利用自制连续变化的温度梯度实验装置,研究了22 ℃~60 ℃ (±0.1 ℃) 区间内温度对一内切β-1,3-葡聚糖酶酶解酵母β-葡聚糖的影响,获得了酶解过程多点温度特性数据。分析表明：该酶酶解酵母β-葡聚糖的活化能为84.17 kJ/mol;以产物积累表示的最适酶解温度随时间延长呈指数下降;酶解产物组分受温度的影响,低温较高温获得的寡糖链长,高温区大于46 ℃可以获得以昆布二糖、昆布三糖为主的组分,而低温区小于30 ℃可以获得昆布五糖及更大分子量的产物。研究结果可为寡糖     

耐热碱性磷酸酶突变体耐热性变化机理研究

从耐热碱性磷酸酶（TAP）200多个随机突变体的克隆库中选出耐热性明显下降的4株突变体,进行全序列及表达产物的最高耐受温度和最适反应温度测定和酶分子高级结构的模拟,分析突变位点、高级结构和耐热性表现三者的关系,探讨引起耐热性变化的机理。结构模拟显示所有突变位点都仅能引起细微的、局部的结构变化,除T320→I外都未直接触及酶的活性中心;结构上的细微改变虽然对最适反应温度影响不明显,但却使最高耐受温度降低了10℃左右;T320→I靠近酶的活性中心,尽管未能引起结构的较大变化,但却使最高耐受温度和最适反应温度同时显著降低。可见,多数点突变对高级结构的影响都不剧烈,但对耐热性尤其是最高耐受温度的影响却比较明显,一般地,在非活性区的突变通常只能引起最高耐受温度的降低,靠近活性区的突变则能同时引起最适反应温度和最高耐受温度的降低。     

Preparation and characterization of poly(acrylic acid)-hydroxyethyl cellulose graft copolymer

Poly(acrylic acid) hydroxyethyl cellulose [poly(AA)-HEC] graft copolymer was prepared by polymerizing acrylic acid (AA) with hydroxyethyl cellulose (HEC) using potassium bromate/thiourea dioxide (KBrO(3)/TUD) as redox initiation system. The polymerization reaction was carried out under a variety of conditions including concentrations of AA, KBrO(3) and TUD, material to liquor ratio and polymerization temperature. The polymerization reaction was monitored by withdrawing samples from the reaction medium and measuring the total conversion. The rheological properties of the poly(AA)-HEC graft copolymer were investigated. The total conversion and rheological properties of the graft copolymer depended on the ratio of KBrO(3) to TUD and on acrylic acid concentration as well as temperature and material to liquor ratio. Optimum conditions of the graft copolymer preparation were 30mmol KBrO(3) and 30mmol TUD/100g HEC, 100% AA (based on weight of HEC), duration 2h at temperature 50°C using a material to liquor ratio of 1:10.     

THE DENATURATION OF EGG ALBUMIN BY ULTRA-VIOLET RADIATION

The coagulation of isoelectric egg albumin solutions, on exposure to ultraviolet radiation, involves three distinct processes, (1) the light denaturation of the albumin molecule, (2) a reaction between the light denatured molecule and water which may be similar to heat denaturation but occurs at a lower temperature, and (3), the flocculation of the denatured molecules to form a coagulum. The light denaturation is unimolecular, independent of temperature, and occurs over a wide pH range. The reaction between the light denatured molecule and water has a temperature coefficient of 10⁺ and occurs rapidly at 40°C., a temperature at which heat denaturation is inappreciable.     

Biophysical studies on subcellular particles V. Effects of temperature on the ferricyanide-Hill reaction, the light-induced pH shift and the light scattering response of isolated spinach chloroplasts

Effects of temperature on the ferricyanide-Hill reaction, thelight-induced pH shift (JpH) and the light scattering response(4LS) of isolated spinach chloroplasts were studied at temperaturesranging from 5 to 60°C. Activities which produce JpH and JLS under actinic illuminationdecreased with a rise in the reaction temperature above 30°Cand disappeared at 50°C. The ferricyanide-Hill reactionat pH 6.0 was stimulated by raising the reaction temperaturefrom 5 to 35°C, was slowed at higher temperatures and wasinactivated at 50°C. 4LS activity at temperatures below20°C depended on the activity of electron transport. With a rise in temperature of a suspension of chloroplasts whichexhibited JLS under actinic illumination, the scattering intensitydecreased. The decrease in scattering intensity was limited,at most, to an extent equal to ALS. The behavior of JLS whichdiffered from that of basal scattering is discussed in connectionwith a mechanism of JLS formation other than the shrinkage-swellingcycle. When activities were measured at 25°C with chloroplastswarmed transiently (2 min) at various temperatures (25–60°C),the dependence of JLS on the wanning temperature was identicalwith that on the reaction temperature, while the activitiesof both ferricyanide reduction and JpH formation were higherthan those measured at the corresponding reaction temperature.This suggested that the mechanism for 4LS formation could beirreversibly altered by a warming treatment and that it wasless stable against heat than the mechanisms of ferricyanidephotoreduction and 4pH formation. That is, transiently warmedchloroplasts showed a similar behavior to alkane-treated chloroplasts.The similarity is discussed in relation to the activity changethrough the possible and common cause of disordering of theorganized structures of lipophilic components in the lamellarmembranes. (Received June 5, 1971; )     

Gas phase enantioselective reduction catalyzed by immobilized ketoreductase: Effects of water activity and reaction temperature

The performance (activity, stability, enantioselectivity and productivity) of the commercial ketoreductase immobilized on non-porous glass supports was investigated as functions of the water activity and the reaction temperature in a continuous gas phase reactor. The enantioselective reduction of 2-butanone to (S)-2-butanol with the in situ regeneration of β-nicotinamide adenine dinucleotide phosphate by 2-propanol catalyzed by the immobilized ketoreductase was used as a model reaction. The activity, stability and enantioselectivity were strongly influenced by the water activity and the reaction temperature. The optimal water activity and reaction temperature were obtained at 0.8 and 313–323 K in terms of the productivity, respectively. Successfully, the enantioselectivity for the gas phase system attained the level identical to that for the aqueous phase system.     

Cephalexin synthesis by immobilised penicillin G acylase under non-isothermal conditions: reduction of diffusion limitation

The effect of thermodialysis on the enzymatic kinetic synthesis of the antibiotic cephalexin was investigated. As reference points, two existing models for an immobilised enzyme (Assemblase^®) and for the free enzyme were used. For Assemblase^®, it is known that diffusion limitation occurs and that therefore considerably more of the undesired side-product phenylglycine is formed.

The enzyme was immobilised on a membrane, and under isothermal conditions (293 K) the course of the reaction resembled that of the Assemblase^® enzyme. However, if a temperature gradient was applied across the membrane, with an average temperature of 293 K for the enzyme, than the course of the reaction changed. For large temperature gradients (30° and more), the course of the reaction resembled that of free enzyme. Thermodialysis enhances mass transfer across the membrane and therewith reduces diffusion limitations in the immobilised enzyme on the membrane.

The stability of the immobilised enzyme is such that the reactor can be re-used repeatedly. This, together with the positive effect of the temperature gradient on the course of the reaction, makes thermodialysis an interesting new technique that has potential to be applied on a larger scale if the membrane surface area per volume of reactor can be improved.     

Fmoc-Asn(Trt)-OH与Wang树脂的合成研究

研究了Fmoc-Asn(Trt)-OH与Wang树脂的酯化反应工艺。探讨了反应策略、溶剂体系、投料比、反应时间以及反应温度等条件对手工合成Fmoc-Asn(Trt)-Wang树脂反应的影响。并用微波辅助方法与常规方法进行了对比。实验结果表明了采用HBTU/HOBt/DIEA方法的连接效率最高。对反应的优化结果为:NMP/DCM体积比为1:7,体系、摩尔比为3:1,每次反应时间4 h,温度40℃。微波对本酯化反应影响不大。     

Acclimation to the growth temperature and the high-temperature effects on photosystem II and plasma membranes in a mesophilic cyanobacterium, Synechocystis sp. PCC6803.

High-temperature effects on Photosystem II and plasma membranes, temperature dependence of growth, and acclimation to the growth temperature were studied in a mesophilic cyanobacterium, Synechocystis sp. PCC6803. The following results were obtained. (1) Small but distinct temperature acclimation of the PSII reaction center activity was shown for the first time when the activity was measured at inhibitory high temperatures. However, the reaction center activity showed no apparent acclimation when it was measured at growth temperatures after heat stress. (2) Oxygen-evolving activity and the permeability of plasma membranes showed higher resistance to heat when PCC6803 cells were grown at higher temperatures. (3) Acclimation of photosynthesis to the growth temperature seemed to occur so as to maintain photosynthesis activity not at a maximum level but in a certain range at the growth temperatures. (4) Neither sensitivity to high-temperature-induced dissociation of phycobilisomes from the PSII reaction center complexes nor degradation of phycocyanin were altered by changes in the environmental temperature. (5) A close relationship between the viability of cells and the structural changes of plasma membranes (but not the inactivation of photosynthesis) was observed. The denaturation process of PSII complexes and the relationship between the temperature dependence of the growth of Synechocystis PCC6803 cells and that of the photosynthetic activity are also discussed.     

响应面法优化Zn-Mn-Co/HZSM-5催化乙醇脱水制乙烯

采用响应面法研究温度、乙醇浓度、质量空速对锌、锰、钴改性的HZSM-5催化乙醇脱水制备乙烯过程中乙烯收率的影响。结果表明反应温度对乙烯收率影响最大,并且各因素之间存在交互作用。用响应面方法确定乙醇脱水制备乙烯的最佳工艺条件是：温度261.3 ℃,乙醇浓度34.4％,质量空速1.18 h?1,在该条件下乙烯收率达到98.69％。     

Vibrationally enhanced hydrogen tunneling in the Escherichia coli thymidylate synthase catalyzed reaction

The enzyme thymidylate synthase (TS) catalyzes a complex reaction that involves forming and breaking at least six covalent bonds. The physical nature of the hydride transfer step in this complex reaction cascade has been studied by means of isotope effects and their temperature dependence. Competitive kinetic isotope effects (KIEs) on the second-order rate constant (V/K) were measured over a temperature range of 5-45 degrees C. The observed H/T ((T)V/K(H)) and D/T ((T)V/K(D)) KIEs were used to calculate the intrinsic KIEs throughout the temperature range. The Swain-Schaad relationships between the H/T and D/T V/K KIEs revealed that the hydride transfer step is the rate-determining step at the physiological temperature of Escherichia coli (20-30 degrees C) but is only partly rate-determining at elevated and reduced temperatures. H/D KIE on the first-order rate constant k(cat) ((D)k = 3.72) has been previously reported [Spencer et al. (1997) Biochemistry 36, 4212-4222]. Additionally, the Swain-Schaad relationships between that (D)k and the V/K KIEs reported here suggested that at 20 degrees C the hydride transfer step is the rate-determining step for both rate constants. Intrinsic KIEs were calculated here and were found to be virtually temperature independent (DeltaE(a) = 0 within experimental error). The isotope effects on the preexponential Arrhenius factors for the intrinsic KIEs were A(H)/A(T) = 6.8 +/- 2.8 and A(D)/A(T) = 1.9 +/- 0.25. Both effects are significantly above the semiclassical (no-tunneling) predicted values and indicate a contribution of quantum mechanical tunneling to this hydride transfer reaction. Tunneling correction to transition state theory would predict that these isotope effects on activation parameters result from no energy of activation for all isotopes. Yet, initial velocity measurements over the same temperature range indicate cofactor inhibition and result in significant activation energy on k(cat) (4.0 +/- 0.1 kcal/mol). Taken together, the temperature-independent KIEs, the large isotope effects on the preexponential Arrhenius factors, and a significant energy of activation all suggest vibrationally enhanced hydride tunneling in the TS-catalyzed reaction.     

Influence of the energy of H-bond protons on the electron transfer rate in photosynthetic reaction centers

We present here a theoretical interpretation of the temperature dependence of the rate of dark recombination between a primary quinone (Q_A) and a bacteriochlorophyll dimer in the reaction center of Rhodobacter sphaeroides. We were able to describe qualitatively the nonmonotonous character of this dependence using the energy of interaction between an excess electron and H-bond protons. We considered a molecular model of Q_A and two reaction center fragments that make H-bonds with Q_A: His(M219) and Asn(M259)-Ala(M260). We used the two-center approach with regard for electron-phonon interaction in order to calculate the characteristic time of electron tunneling during the recombination reaction. The energy of the phonon emitted/ absorbed during the electron tunneling was determined by the relative shift of donor and acceptor energy levels, the detuning of levels. The detuning was shown to depend on temperature nonmonotonously for H-bonds with double-well potential energy surface. The characteristic time (or the reaction rate) depended on temperature parametrically. The computed dependence was in qualitative agreement with the experimental one.