Engineering aspects of immobilized lipases on esterification: A special emphasis of crowding,confinement and diffusion effects

Cross‐linked enzyme crystal (CLEC) and sol‐gel entrapped pseudomonas sp. lipase were investigated for the esterification of lauric acid with ethanol by considering the effects of reaction conditions on reaction rate. The activation energy for the reaction was estimated to be 1097.58 J/mol and 181.75 J/mol for sol‐gel and CLEC entrapped lipase respectively. CLEC lipase exhibited a marginal internal diffusion effect on reaction rate over sol‐gel lipases and found to be interesting. The overall reaction mechanism was found to conform to the Ping Pong Bi Bi mechanism. The higher efficiency of sol‐gel lipases over CLEC lipases in esterification reaction is mainly due to the combined effects of crowding, confinement and diffusional limitations.     

13C and 15N isotope effects for conversion of L-dihydroorotate to N-carbamyl-L-aspartate using dihydroorotase from hamster and Bacillus caldolyticus

In the pyrimidine biosynthetic pathway, N-carbamyl-L-aspartate (CA-asp) is converted to L-dihydroorotate (DHO) by dihydroorotase (DHOase). The mechanism of this important reaction was probed using primary and secondary 15N and 13C isotope effects on the ring opening of DHO using isotope ratio mass spectrometry (IRMS). The reaction was performed at three different temperatures (25, 37, and 45 degrees C for hamster DHOase; 37, 50, and 60 degrees C for Bacillus caldolyticus), and the product CA-asp was purified for analysis. The primary and secondary kinetic isotope effects for the ring opening of the DHO were determined from analysis of the N and C of the carbamyl group after hydrolysis. In addition, the beta-carboxyl of the residual aspartate was liberated enzymatically by transamination to oxaloacetate with aspartate aminotransferase and then decarboxylation with oxaloacetate decarboxylase. The 13C/12C ratio from the released CO2 was determined by IRMS, yielding a second primary isotope effect. The primary and secondary isotope effects for the reaction catalyzed by DHOase showed little variation between enzymes or temperatures, the primary 13C and 15N isotope effects being approximately 1% on average, while the secondary 13C isotope effect is negligible or very slightly normal (>1.0000). These data indicate that the chemistry is at least partially rate-limiting while the secondary isotope effects suggest that the transition state may have lost some bending and torsional modes leading to a slight lessening of bond stiffness at the carbonyl carbon of the amide of CA-asp. The equilibrium isotope effects for DHO --> CA-asp have also been measured (secondary 13K(eq) = 1.0028 +/- 0.0002, primary 13K(eq) = 1.0053 +/- 0.0003, primary 15K(eq) = 1.0027 +/- 0.0003). Using these equilibrium isotope effects, the kinetic isotope effects for the physiological reaction (CA-asp --> DHO) have been calculated. These values indicate that the carbon of the amide group is more stiffly bonded in DHO while the slightly lesser, but still normal, values of the primary kinetic isotope effect show that the chemistry remains at least partially rate-limiting for the physiological reaction. It appears that the ring opening and closing is the slow step of the reaction.     

Ultrasonic monitoring of glycerol settling during transesterification of soybean oil

A low-intensity ultrasonic measurement system was used to monitor the products of transesterification of soybean oil in methanol to FAME (biodiesel). The byproducts of the transesterification reaction are methyl esters, glycerol and other products. During the transesterification reaction, the glycerol, having a higher density than the methyl ester, settles at the bottom of the reaction vessel. The aim of this study was to measure the glycerol deposition rate during transesterification and to assess the reaction rate and end time. Soybean oil was converted into biodiesel at four temperature levels. The amount of catalyst (KOH) used in the transesterification reactions was determined by titration. The ultrasonic waveforms captured during the reaction were recorded and analyzed automatically. The ultrasonic system monitored the effects of reaction temperatures on the glycerol settling rate and the reaction end times. The ultrasonic measurement of glycerol settling would be a useful non-destructive method for evaluating the effects of parameters such as catalyst amount, mixing time and temperature on transesterification reactions.     

The action of soybean lipoxygenase-1 on 12-iodo-cis-9-octadecenoic acid: the importance of C11-H bond breaking

Previous work has demonstrated that the ferric form of soybean lipoxygenase-1 will catalyze an elimination reaction on 12-iodo-cis-9-octadecenoic acid (12-IODE) to produce 9, 11-octadecadienoic acid and iodide ion. Elimination is accompanied by irreversible inactivation of the enzyme on 1 out of 10 turnovers. In the present work, 11,11-dideuterio-12-IODE (D(2)-12-IODE) was synthesized and used to demonstrate that both the elimination reaction and inactivation of the enzyme exhibit very large kinetic isotope effects. The rates with the deuterated compound are so low that the isotope effects are difficult to quantify, but they appear to be comparable to the isotope effects previously observed for the normal reaction catalyzed by lipoxygenase and much larger than can be explained by zero-point energy considerations. ESR spectroscopy was used to demonstrate that 12-IODE can reduce ferric lipoxygenase to the ferrous form, and a large isotope effect on this process was observed with D(2)-12-IODE. It is proposed that the pathway leading to reduction and inactivation by 12-IODE is initiated by homolytic cleavage of the C(11)-H bond. Elimination could be initiated either by homolytic or by heterolytic cleavage of this bond. The results suggest that very large isotope effects may be a general feature of C-H bond cleavages catalyzed by this enzyme.     

NADH- and NADPH-dependent formation of superoxide anions by bovine heart submitochondrial particles and NADH–ubiquinone reductase preparation

1. Both NADH and NADPH supported the oxidation of adrenaline to adrenochrome in bovine heart submitochondrial particles. The reaction was completely inhibited in the presence of superoxide dismutase, suggesting that superoxide anions (O(2) (-)) are responsible for the oxidation. The optimal pH of the reaction with NADPH was at pH7.5, whereas that with NADH was at pH9.0. The reaction was inhibited by treatment of the preparation with p-hydroxymercuribenzoate and stimulated by treatment with rotenone. Antimycin A and cyanide stimulated the reaction to the same extent as rotenone. The NADPH-dependent reaction was inhibited by inorganic salts at high concentrations, whereas the NADH-dependent reaction was stimulated. 2. Production of O(2) (-) by NADH-ubiquinone reductase preparation (Complex I) with NADH or NADPH as an electron donor was assayed by measuring the formation of adrenochrome or the reduction of acetylated cytochrome c which does not react with the respiratory-chain components. p-Hydroxymercuribenzoate inhibited the reaction and rotenone stimulated the reaction. The effects of pH and inorganic salts at high concentrations on the NADH- and NADPH-dependent reactions of Complex I were essentially similar to those on the reactions of submitochondrial particles. 3. These findings suggest that a region between a mercurialsensitive site and the rotenone-sensitive site of the respiratory-chain NADH dehydrogenase is largely responsible for the NADH- and NADPH-dependent O(2) (-) production by the mitochondrial inner membranes.     

Effects of water activity on reaction rates and equilibrium positions in enzymatic esterifications

A technique of continuous water activity control was used to examine the effects of water activity on enzyme catalysis in organic media. Esterification catalyzed by Rhizopus arrhizus lipase was preferably carried out at a water activity of 0.33, which resulted in both maximal initial reaction rate and a high yield. When Pseudomonas lipase was used as catalyst it was beneficial to start the reaction at high water activity (giving the optimal reaction rate with this enzyme) and then shift to a lower water activity toward the end of the reaction to obtain a high yield. The apparent equilibrium constant of the reaction was influenced by the water activity of the organic solvent. (c) 1994 John Wiley & Sons, Inc.     

Stimulation of liver glycogen particle synthase D phosphatase activity by caffeine, AMP, and glucose 6-phosphate

Caffeine stimulates synthase and phosphorylase phosphatase activities by independent mechanisms. Both effects of caffeine are concentration-dependent with different apparent A_0.5 for each reaction. Stimulation of the synthase phosphatase reaction was independent of the initial phosphorylase a concentration, was immediate, and did not follow in sequence the depletion of phosphorylase a. Glucose 6-phosphate also was stimulatory to the synthase phosphatase reaction (A_0.5 = 0.14 mM) with little effect on phosphorylase phosphatase. In combination glucose 6-phosphate and caffeine effects were additive suggesting the existence of separate binding sites. The synthase phosphatase reaction also was stimulated by AMP (binding affinity 2.3 mm) but with no effect on phosphorylase phosphatase activity. Together caffeine and AMP effects were not additive suggesting that they share a common binding site or closely interrelated sites. The location of the AMP and caffeine site(s) has not yet been determined.     

Regeneration of NADH in a bioreactor using yeast cells immobilized in alginate fiber: I. Method and effect of reactor variables

Saccharomyces cerevisiae cells immobilized in a calcium alginate fiber reactor were used as a source of alcohol dehydrogenase for the NAD(+)-to-NADH reaction. The reaction was catalyzed by enzyme in cells on the surface of the fiber. Internal diffusional effects were present. The enzyme cell concentration was optimized by harvesting cells finally grown under anaerobic conditions. The results were expressed as an apparent reaction rate constant that was independent of NAD(+) and excess ethanol concentration, was slightly affected by flow rate above a minimum value, and increased with immobilized cell concentration in the fiber. The reaction was complete after 6 to 7 h under optimal conditions of 36 degrees C and 9.5 pH. The latter was 0.5 pH units above the free enzyme optimum, indicating that microenvironmental effects were in evidence. (c) 1993 John Wiley & Sons, Inc.     

Bull sperm acrosome reaction induced by gamma-aminobutyric acid (GABA) is mediated by GABAergic receptors type A

The effect of gamma-aminobutyric acid (GABA) on the bull sperm acrosome reaction was evaluated, and the interaction of progesterone, a physiologic inducer of the acrosome reaction, with the GABA receptor was explored. The acrosome reaction was stimulated by GABA in a dose-dependent manner. This effect was inhibited completely by bicuculline, a GABA A receptor antagonist, but GABA B and C receptor antagonists had no effect. Accordingly, muscimol, a GABA A receptor agonist, stimulated the acrosome reaction to the same extent as GABA, whereas baclofen (GABA B receptor agonist) and CACA (GABA C receptor agonist), had no effect. Preincubation with progesterone followed by the addition of GABA resulted in a significant increase in the percentage of acrosome reacted spermatozoa compared with progesterone or GABA alone. Taking into account that this increase was less than a simple addition of effects, it might be suggested that GABA and progesterone act through the same receptor and/or use the same mechanism of action. To test this hypothesis, the abilities of GABA and progesterone to induce acrosome reaction were tested in the presence of bicuculline, which suppressed both stimulatory effects. Given that the GABA A receptor is linked to the Cl(-) channel, we tested whether picrotoxin, a blocker of this channel, could modulate the effects of progesterone or GABA. Cl(-) channel blocker picrotoxin dramatically reduced the GABA and progesterone-initiated AR. In conclusion: GABA and progesterone stimulate the acrosome reaction in bull spermatozoa acting through a classical GABA A receptor. The mechanism of action requires the functional integrity of the Ca(2+) Cl(-) channel.     

Peroxidase-catalyzed N-demethylation reactions. Substrate deuterium isotope effects

Deuterium isotope effects on the kinetic parameters for the hydroperoxide-supported N-demethylation of N,N-dimethylaniline catalyzed by chloroperoxidase and horseradish peroxidase were determined using N,N-di-(trideuteromethyl)aniline. The isotope effect on the Vmax for the chloroperoxidase-catalyzed demethylation reaction supported by ethyl hydroperoxide was 1.42 +/- 0.31. The isotope effects on the Vmax for the horseradish peroxidase-catalyzed reaction supported by ethyl hydroperoxide and hydrogen peroxide were 1.99 +/- 0.39 and 4.09 +/- 0.27, respectively. Isotope effects ranging from 1.76 to 5.10 were observed on the Vmax/Km for the hydroperoxide substrate (i.e. the second order rate constant for the reaction of the hydroperoxide with the peroxidase to form compound I) in both enzyme systems when the N-methyl groups of N,N-dimethylaniline were deuterated. These results are not predicted by the simple ping-pong kinetic model for peroxidase-catalyzed N-demethylation reactions. The data are most simply explained by a mechanism involving the transfer of deuterium (or hydrogen) from N,N-dimethylaniline to the enzyme during catalysis. The deuterium must subsequently be displaced from the enzyme by the hydroperoxide, causing the observed isotope effects.     

Immunoregulative effects of homocarnosine and gamma-aminobuthyric acid

The effects of homocarnosine and GABA on antibody production (PFC reaction) and cellular immunity (delayed hypersensitivity reaction, DHR) were examined in vivo. In mice treated with these agents, PFC reaction to 2 X 10(7) SRBC was enhanced but that to 1 X 10(9) SRBC was suppressed; moreover, immunoreaction was reduced in immature mice (2-2.5 weeks old) but was increased in aged mice (30 weeks old or above). These agents had optimal doses on the PFC reaction in mice given 1 X 10(8) SRBC and DHR, and induced recovery of immunofunction suppressed by the administration of MMC.     

Deodorizing effects of tea catechins on amines and ammonia

Deodorizing effects of tea catechins on amines were examined under alkaline conditions to eliminate the neutralization reaction. They showed deodorizing activity on ethylamine, but none on dimethylamine or trimethylamine. Deodorizing activity on ethylamine was found to be in the order of (-)-epigallocatechin gallate > gallic acid > (-)-epigallocatechin (EGC) > (-)-epicatechin gallate > ethyl gallate > (+)-catechin = (-)-epicatechin. Further, reaction products of EGC with methylamine, ethylamine, and ammonia were detected by HPLC, indicating that a deodorizing reaction other than neutralization occurs. From structural analysis of the reaction product with the methylamine isolated as a peracetylated derivative, the product was presumed to be methylamine substituted EGC, in which the hydroxyl group of EGC at the 4' position is replaced by the methylamino group. The same replacement reaction took place in the case of ethylamine and ammonia.     

Effects of platelet activating factor on capacitation and acrosome reaction in mouse spermatozoa

Platelet activating factor (PAF) plays an important role in mammalian reproduction. The aim of this study was to investigate the effects of PAF on capacitation and acrosome reaction of mouse spermatozoa by chlortetracycline (CTC) fluorescence assay and coomassie blue staining. The percentage of capacitated mouse spermatozoa was increased (P < 0.05) by incubation with 50 ng/ml PAF for 20-120 min. The peak response occurred between 80 to 100 min of exposure to PAF. In contrast, the effects of PAF on acrosome reaction may be not receptor-mediated since lyso-PAF had the same effects. Ionophore A23187 stimulated an increase in acrosome-reacted spermatozoa of PAF-treated spermatozoa, but not of lyso-PAF-treated ones. These results suggest that PAF mainly acts on sperm capacitation.     

Structural Stability of Streptococcal Serum Opacity Factor

Streptococcal serum opacity factor (SOF) is a protein that clouds the plasma of multiple mammalian species by disrupting high density lipoprotein (HDL) structure. Intravenous infusion of low dose SOF (4 µg) into mice reduces their plasma cholesterol concentrations?~?40% in 3 h. Here we investigated the effects of pH, ionic strength, temperature, and denaturation with guanidinium chloride (GdmCl) on SOF stability and its reaction vs HDL. SOF stability was tested by pre-incubation of SOF at various temperatures, pH’s, and GdmCl concentrations and measuring the SOF reaction rate at pH 7.4 and 37?°C. SOF retained activity at temperatures up to 58?°C, at pH 4 to 10, and in 8.5 M GdmCl after being returned to standard buffer conditions. The effects of GdmCl, pH, and ionic strength on the SOF reaction rates were also measured. SOF was inactive at GdmCl?≥?1 M; SOF was most active at pH 5, near its isoelectric point and at an ionic strength of 3 (in NaCl). These data reveal that SOF is a stable protein and suggest that its activity is determined, in part, by the effects of pH and ionic strength on its overall charge relative to that of its reaction target, HDL.     

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.     

Effects of neutral salts on the conformational state of glycogen, interacting with iodine

The effects of salts on helix formation of linear (outer) chains of branched alpha-glucane, muscle glycogen, was judged upon by the state of polyiodine chromophore of the iodine reaction surrounded by a polysaccharide asymmetric helix and recorded by the circular dichroism method. It was demonstrated that apart from the known changes in the absorption of the iodine-glycogen complex the salts induce changes in the ellipticity by affecting the helix formation of the linear chains of the polysaccharide. The nature of these effects depends on the type and concentration of the salt used. Monovalent metal salts produce a more favourable effect on helix formation than the divalent metal salts. Among divalent metal salts transient metal salts produce a weaker effect. The action of neutral salts on helix formation is of dual nature: helix formation and helix destruction. The helix formation is largely impaired by high concentrations of LiCl (greater than 6 M) or CaCl2 (greater than 2M). It was shown that a certain role in the mechanism of neutral salt action on helix formation belongs both to electrostatic and lyotropic effects. The latter determine the effects of salts on helix formation at concentrations above the monomolar one, when the specific effect of a salt is especially well-pronounced. The salts which enhance the orderliness of H2O structure produce a favourable effect on helix formation of the linear polysaccharide chains and, consequently, on the iodine reaction. In terms of molar efficiency of their action on the iodine reaction the salts correspond to the Hofmeister lyotropic sequence.     

Different pH Optima in the Two Steps of an Indirect Fluorescent Antibody Reaction for Clostridium botulinum Type E

Quantitative measurements of the effects of ions on fluorescent antibody reactions have not been reported in the literature. Data in this report show the effects of ranges of H(+), phosphate, Mg(2+), and Ca(2+) molarities on antigen-antibody coupling during an indirect fluorescent antibody (IFA) reaction for Clostridium botulinum type E. These effects were quantified in two ways: (i) by microphotometric measurement of cell fluorescence intensity; and (ii) by visual estimation of cell fluorescence intensity on long glass strips treated with antibody in ion gradients. Optimal pH for the first part of the reaction (coupling of rabbit antibody to cells) was 7.25, and optimum in the second part (coupling of tracer globulin to the rabbit globulin) was pH 7.37. Running the reaction as little as 0.10 to 0.15 pH units off from the optima considerably reduced fluorescence intensity. Sodium phosphate buffer up to 0.1 M did not significantly affect either portion of the reaction. Ca(2+) and Mg(2+) showed no effect on the first part of the reaction. These results support the use of higher-strength phosphate buffer and indicate for the first time that IFA reactions may have two independent, narrow pH optima.     

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。     

Characterization of mitochondrial glutamate dehydrogenase from dark‐grown soybean seedlings

Purified preparations of NAD(H)‐glutamate dehydrogenase (GDH, EC 1.4.1.2.) were assayed to determine the effects of mono‐ and divalent cations, nucleotides and select carbon compounds on NAD(H)‐dependent GDH activity. The amination reaction was stimulated 2‐ to 17‐fold by divalent cations (Ca²⁺ > Cd²⁺ > Co²⁺ > Mg²⁺ > Mn²⁺ > Zn²⁺ between 1 and 1000 µ M ), but the reaction was unaffected by monovalent cations (Na ⁺ and K ⁺). The amination reaction was most responsive to changes in Ca²⁺ in a NADH‐dependent manner. The addition of EDTA or EGTA nullified the stimulatory effects of Ca²⁺. Calmodulin alone or in combination with calmodulin antagonists did not affect the amination reaction. Divalent cations (at 1 m M ) inhibited the rate of the deamination reaction by 15 to 25%, while monovalent cations had no effect. ATP inhibited the amination reaction by 10 to 60%, while ADP had little or no effect. ATP or ADP decreased the rate of the deamination reaction 23 to 60 or 20 to 38%, respectively. Many tricarboxylic acid cycle intermediates inhibited the amination reaction, 20 to 50% of the inhibition could be attributed to the chelating capacity of intermediates. Conversely, most of the carbon sources tested did not affect the deamination reaction, the only appreciable differences were increases in activity with sucrose (21%) and glucose (41%) and a decrease in activity with pyruvate (34%). Inhibitors of sulfhydryl groups were used to examine the importance of reduced thiol groups in the amination or deamination reactions. The amination was not dependent on reduced thiol groups, whereas the deamination reaction was dependent on reduced thiol groups.     

The mechanism of glycogen synthetase as determined by deuterium isotope effects and positional isotope exchange experiments

The reaction mechanism for glycogen synthetase from rabbit muscle was examined by alpha-secondary deuterium isotope effects and positional exchange experiments. Incubation of glycogen synthetase with [beta-18O2,alpha beta-18O]UDP-Glc did not result in any detectable positional isotope exchange from the beta-nonbridge position to the anomeric oxygen of the glucose moiety. Glucono-1,5-lactone was found to be a noncompetitive inhibitor versus UDP-Glc. The kinetic constants, K(is) and K(ii), were found to be 91 +/- 4 microM and 0.70 +/- 0.09 mM, respectively. Deoxynojirimycin was a nonlinear inhibitor at pH 7.5. The alpha-secondary deuterium isotope effects were measured with [1-2H]UDP-Glc by the direct comparison method. The isotope effects on Vmax and Vmax/K were found to be 1.23 +/- 0.04 and 1.09 +/- 0.06, respectively. The inhibitory effects by glucono-lactone and deoxynojirimycon plus the large alpha-secondary isotope effect on Vmax have been interpreted to show that an oxocarbonium ion is an intermediate in this reaction mechanism. The lack of a detectable positional isotope exchange reaction in the absence of glycogen suggests the formation of a rigid tight ion pair between UDP and the oxocarbonium ion intermediate.