The kinetics of enzyme catalyzed synthesis of sterol ester

The production of sterol ester by transesterification of β‐sitosterol with fish oil (TAG) catalyzed by Thermomyces lanuginosus immobilized lipase enzyme with varied reaction parameters such as temperature, substrate molar ratio, concentration of enzyme to deduce the enzyme kinetics for the reaction was investigated. For this transesterification reaction, the kinetic model was derived by using Ping Pong Bi Bi Mechanism. The K_m value from the first plot containing fish oil as substrate was 1.31 ± 0.05, while K_m from the second plot containing β‐sitosterol as substrate was 1.01 ± 0.04; identical V_max (0.213 ± 0.06) values were obtained by keeping one of the substrate concentration constant and varying the other. Practical applications : Deduction of reaction kinetics is an important criterion to ascertain the viability of any chemical process. Enzymatic processes need special attention to set model reaction parameters which could help in optimization or design of the actual process. In the present study we have derived the enzyme kinetics for the production sterol ester, an important nutraceutical, and calculated its K_m and V_max values along with the Arhenius activation energy to establish the viability of the reaction.     

Lipase‐catalysed esterification of oleic acid and ethanol in a continuous packed bed reactor,using supercritical CO2 as solvent: approximation of system kinetics

This work investigated the immobilised lipase kinetics of esterification of oleic acid and ethanol. The reaction was conducted under supercritical conditions (13 × 10⁶ Pa and 40 °C) using carbon dioxide as solvent in a continuous packed bed (plug flow) reactor. Biocatalyst Lypozyme^TM IM60, which is lipase from Rhizomucor miehei (EC.3.1.1.3), immobilised on Duolite (anionic exchange resin) was used as biocatalyst. Kinetically, with regard to oleic acid, the reaction was successfully modelled by the Michaelis–Menten mechanism. The reaction rate constants K_m and V_max were evaluated. Furthermore, it was found to undergo competitive inhibition by ethanol, and the inhibition constant K_i was evaluated. © 2000 Society of Chemical Industry     

Effect of pressure on the extractive biocatalysis of ethanol

The effect of pressure on the esterification reaction of ethanol with water-immiscible organic acids, catalysed by a lipase from Mucor miehei (pH 4.5; 30°C), was studied through analysis of the kinetics and equilibrium parameters. An increase of the ethanol distribution between the aqueous and organic phases was observed by the addition of lipase and the increase of the pressure in the system. Furthermore, the enzyme showed high specificity for the acid substrate, esterifying preferentially long chain fatty acids (C₈-C₁₈). In the studies described oleic acid was used as substrate for the esterification reaction. A kinetic study with the free enzyme, showed that pressure affected the extraction system, increasing the maximum reaction rate (> V_max), the affinity (< K_m) and the specificity (> V_max/K_m = k_sp) of the enzyme to the substrate, probably due to the effect of pressure on the electrostatic interactions in biological systems. The enzyme operational stability, at 30°C, improved significantly with the increase of pressure, having lower values for the deactivation constant (k) (8.3 × 10^?3 h^?1) and higher values for the half-life times (t_1/2) (77 h) in comparison with those obtained under atmospheric pressure conditions (k = 2.3 × 10^?2h^?1; t_1/2 = 30 h).     

Factors affecting catalytic performance in yadh-catalyzed reductions in non-conventional media

The effects of parameters including water content, the amount of celite and the co-factor regeneration system on reduction of benzaldehyde to benzyl alcohol by yeast alcohol dehydrogenase (YADH) immobilized on celite in non-conventional media were investigated. Kinetic parameters (apparent) such as V_max, K_m, and k_car for different organic solvents used (butyl acetate, hexane, heptane and i-octane) were determined. K_m values for benzaldehyde increased as the solvent was varied in the following manner: butyl acetate < hexane < i-octane < heptane. The highest K_m value (20.26 mmol/L) was noted in heptane and the lowest (3.24 mmol/L) in butyl acetate. The V_max value in heptane was more than an order of magnitude higher than that in butyl acetate. Catalytic efficiencies (as expressed by k_car/K_m) ranged from 0.703 × 10^?4 L/mol · s for hexane to 0.171 × 10^?4 L/mol · s for butyl acetate. In general, linear relationships were observed between k_car/K_m and the following solvent physico-chemical constants: dielectric constant logarithm of the saturated molar solubility of water in the organic solvents, log S_w/o, logarithm of partition coefficient of organic solvent in water/octanol two-phase system, log P, Hildebrand solubility parameter, δ, and solvatochromism of pyridinium-N-phenoxide betaine dye, ET(30).     

Catalysis by Dihydrofolate Reductase from the Psychropiezophile Moritella profunda

The influence of temperature and pH on the stability and catalytic activity of dihydrofolate reductase (MpDHFR) from the cold‐adapted deep‐sea bacterium Moritella profunda was studied. The thermal melting temperature was found to be ～38 °C and was not affected by pH, while activity measurements demonstrated that its stability was maximal at pH 7 and was reduced dramatically below pH 6 or above pH 8. The steady‐state rate constant (k_cat) was maximal at neutral pH and higher temperatures, while the Michaelis constants (K_M) for both substrate and cofactor were optimal at lower temperatures and at elevated or reduced pH. For both temperature and pH, any change in k_cat was therefore offset by a similar change in K_M. Both the activation enthalpy and entropy of the MpDHFR‐catalysed reaction were lower than those of DHFR from E. coli leading overall to a very small difference in activation free energy and therefore similar steady‐state rate constants at the same temperature. The chemical step of the reaction is not rate limiting at pH 7, but becomes progressively more rate limiting as the pH increases. These results demonstrate adaptation of MpDHFR to its environment and show compromises between enthalpic and entropic contributions to the reaction, and between k_cat and K_M.     

Kinetic studies of Mucor miehei lipase in phosphatidylcholine microemulsions

Ethanol—oleic acid esterification by a free and microencapsulated lipase from Mucor miehei, using dodecane as solvent and phosphatidylcholine as surfactant, was studied. The initial esterification rate was influenced by the water content in the biphasic system. Kinetic studies with free and microencapsulated enzyme showed that the microencapsulation led to an increase of the kinetic parameters (V_max and K_m), probably due to an increase of the interfacial area. The reaction rate was also affected by the shaking rate, the temperature and the pH. The optimal temperature and pH achieved were, respectively, 40°C and 4.5 using free enzyme, and 50°C and 6 using microencapsulated enzyme.     

Enzyme-Catalyzed Synthesis of Monoacylglycerols Citrate: Kinetics and Thermodynamics

The kinetics and thermodynamics of the esterification of citric acid (CA) and monoglycerides (MGs) for citrate esters of monoacylglycerols catalyzed by Novozym 435 in tert-butyl alcohol system was studied in this work. The relationship between initial reaction rate and temperature was established, based on the Arrhenius law. A linear relationship was established between the initial reaction rate and enzyme load up to 3 g/L, which demonstrated that the influence of external mass transfer limitations on the reaction could be eliminated. The reaction kinetics agreed with the Ping-Pong Bi–Bi mechanism with CA inhibition characterized by V _max, K _B, K _A, and K _iA , values of 0.7092 mmol/(min g), 0.0553, 0.0136 and 0.1948 mol/L, respectively. The model was used to simulate the reaction process. The values calculated from the kinetic mode agreed well with the experimental rate data under the different MG and CA concentrations.     

Synthesis and Characterization of Pyridine Compounds for Amperometric Measurements of Leukocyte Esterase

We introduce a new class of substrates (compounds I – III ) for leukocyte esterase (LE) that react with LE yielding anodic current in direct proportion to LE activity. The kinetic constants K_m and k_cat for the enzymatic reactions were determined by amperometry at a glassy carbon electrode. The binding affinity of I – III for LE was two orders of magnitude better than that of existing optical LE substrates. The specificity constant k_cat/K_m was equal to 2.7, 3.8, and 5.8×10⁵ m ^?1 s^?1 for compounds containing the pyridine ( I ), methoxypyridine ( II ), and (methoxycarbonyl)pyridine ( III ), respectively, thus showing an increase in catalytic efficiency in this order. Compound III had the lowest octanol/water partition coefficient (log p=0.33) along with the highest topological surface area (tPSA=222 Ų) and the best aqueous solubility (4.0 mg mL^?1). The average enzymatic activity of LE released from a single leukocyte was equal to 4.5 nU when measured with compound III .     

Studies of L-phenylalanine production by immobilised aminoacylase in stabilized calcium alginate beads

Aminoacylase I (EC 3.5. 1.14) was immobilised by entrapment in uncoated calcium alginate beads and calcium alginate beads coated with chitosan, polyethyleneimine and polyethyleneimine-glutaraldehyde for the production of L-phenylalanine by the hydrolysis of a racemic mixture of N-acetyl-DL-phenylalanine. The operational stability, thermal stability, effects of pH and temperature and kinetic constants, K_m and V_max values of free and immobilised enzymes were studied. Scanning electron micrographs revealed differences in the structures of the surfaces of coated and uncoated beads. Chitosan-coated calcium alginate beads was found to be the best among the immobilised systems studied. The activity and the optimum temperature of immobilised aminoacylase were higher than those of the free enzyme. In addition, the beads showed stable activity under operational conditions. The immobilised aminoacylase lost about 20% of its initial activity after ten cycles of reuse. Polyethyleneimine and polyethyleneimine-glutaraldehyde treatments were also found to enhance the operational stability of the enzyme but its activity was greatly reduced.     

Kinetic properties of the periplasmic hydrogenase from Desulfovibrio desulfuricans NCIMB 8372 and use in photosensitized H2-production

The periplasmic hydrogenase (hydrogen: ferricytochrome-c₃ oxido-reductase EC 1.12.2.1) from Desulfovibrio desulfuricans (NCIMB 8372) was isolated and the catalytic properties of the enzyme determined with different electron mediators. In the direction of H₂-oxidation with the physiological electron mediator cytochrome c₃, the value of the apparent second order rate constant (k_cat/K_m) is close to that expected for a diffusion-controlled reaction. In H₂-production however, the value of k_cat/K_m with cytochrome c₃ is almost 200 times lower. When methyl viologen is used as mediator, the value of k_cat/K_m is the same in both directions and is intermediate to the values obtained with cytochrome c₃. The close to optimum value of k_cat/K_m in H₂-oxidation with cytochrome c₃ as electron acceptor is discussed with respect to the biological role of this and related hydrogenases. H₂-production occurred with the hydrogenase in a system where the mediator was reduced with either of three photosensitizers (proflavin, 5-deazariboflavin, Zn-tetraphenyl porphyrin sulphonate) upon illumination. The efficiency of different mediators is discussed.     

Immobilization of pepsin on an acrylamide/2‐hydroxyethyl methacrylate copolymer and its use in casein hydrolysis

Pepsin was immobilized through covalent bonding on a copolymer of acrylamide and 2‐hydroxyethyl methacrylate via the individual and simultaneous activation of both groups. The extent of enzyme coupling upon the activation of both the amino and hydroxyl groups of the copolymer resulted in a synergistic effect. However, the order of activation of the support was critical. The covalently bound enzyme retained more than 50% of its activity even after six cycles. The storage stability of the covalently bound enzyme was 60% after storage for 1 month, whereas the free enzyme lost all of its activity within 10 days of storage at 35°C. The Michaelis constant (K_m) and maximum reaction velocity (V_max) were 1.1 × 10^?6 and 0.87 for the free enzyme and 1.2 × 10^?6 and 0.98 for the covalently bound enzyme when the enzyme concentration was kept constant and the substrate concentration was varied. Similarly, K_m and V_max were 6.73 × 10^?11 and 0.47 for the free enzyme and 7.59 × 10^?11 and 0.545 for the covalently bound enzyme when the substrate concentration was kept constant and the enzyme concentration was varied; this indicated no conformational change during coupling, but the reaction was concentration‐dependent. The hydrolysis of casein was carried out with a fixed‐bed reactor (17 cm × 1 cm). Maximum hydrolysis (90%) was obtained at a 2 cm³/min flow rate at 35°C with a 1 mM casein solution. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 96: 1544–1549, 2005     

Enhancement of enzymatic catalysis of Bacillus amyloliquefaciens α-amylase by nonionic surfactant micelles

Some enzymes are considerably more stable when formulated with nonionic surfactants than when formulated with anionic surfactants. The effect of a nonionic surfatant, polyoxyethylene mono-N-dodecyl ether (Brij 35; number of units of ethylene oxide moieties, 23), on the kinetic behavior of hydrolysis of amylopectin with Bacillus amyloliquefaciens α-amylase (BAA) was studied at a temperature of 25°C and a pH of 7.0. The hydrolytic rate was accelerated by the addition of the nonionic surfactant above its critical micelle concentration. Lineweaver-Burk plots for the enzymatic hydrolysis in the absence and presence of the nonionic surfactant at 0.5 to 2.5% (wt/vol) had linear relationships, and the kinetic parameters, K _m and k _cat were obtained. The value of k _cat was increased with an increased concentration of Brij 35, whereas the K _m value was approximately constant. Therefore, the increase in k _cat contributed to the acceleration of the apparent hydrolytic rate. The interaction of amylopectin with the surfactant was examined by a surface tension measurement, and the result confirmed the corresponding binding between the substrate and the surfactant. A fluorescence analysis due to tryptophan in BAA suggested that BAA bound to the nonionic micelles. The increase in k _cat suggested that hydrolytic catalysis at the micellar pseudophase was more efficient than that at the aqueous pseudophase. The enhancement of the catalytic rate contributed to the effective removal of food stains containing starch when BAA was added with Brij 35 in a laundry detergent washing test.     

Enzymatic Synthesis of Thioesters in Non-conventional Solvents

The feasibility of enzymatic thioesterification between oleic acid and butanethiol in n-hexane, with the immobilised lipase (Lipozyme) from Mucor miehei, has been demonstrated. The immobilised enzyme quantity (100 mg), temperature (40°C), pH range (6–9) and water content (10%) were studied and their optimum values were determined. A preliminary kinetic study indicated a low butanethiol affinity for the enzyme (K_m = 1·85 mol dm⁻³). Even when butanethiol was used without solvent, no substrate inhibition was observed. The possibility of carrying out this reaction in a natural solvent, supercritical carbon dioxide (SCCO₂), was successfully verified. After 8 h reaction, a conversion yield of oleic acid of about 33% was obtained. © 1997 SCI.     

Entrap-immobilization of invertase on composite gel fiber of cellulose acetate and zirconium alkoxide by sol–gel process

We prepared a composite gel fiber by the gel formation of cellulose acetate and zirconium tetra-n-butoxide. Gel fiber is stable in common solvents, phosphate solution, and electrolyte solution. Invertase was entrap-immobilized on the gel fiber. The immobilization was easily performed under the mild conditions. The apparent Michaelis constant (K_m) and maximum reaction velocity (V_max) were estimated from Eadie–Hofstee plot for immobilized invertase. The K_m of immobilized invertase was larger than that of native invertase, while the opposite tendency was observed for the V_max. The activity for the immobilized invertase became higher with increasing fiber diameter. It indicates that the hydrolysis of sucrose occurs in the neighborhood of the fiber surface. The thermal stability of the immobilized invertase was higher than those of its native counterpart. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 81: 2084–2088, 2001     

Highly efficient hydrolysis of phosphodiester by a copper(II)-chelated chitosan magnetic nanocarrier

A novel heterogeneous magnetic nanocatalyst was designed by conjugating copper(II) ions with chitosan-bound magnetic nanocarrier. Its hydrolytic activity for phosphodiesters was evaluated by investigating the hydrolysis of secondary ester bis-(nitrophenyl) phosphate (BNPP) in aqueous media. The maximum hydrolysis rate of BNPP was observed around the pK_a of nanocatalyst which was found to be 12. The kinetic studies revealed that the binding affinity of BNPP to the copper(II)-chelated chitosan magnetic nanocarrier was low as compared to other catalyst systems. However, the rate constants (k_cat) and maximum reaction rate (V_max) were found to be higher with lower activation energy for the developed nanocatalyst. Moreover, the developed nanocatalyst was magnetically recoverable and also had good reusability.     

Acyl-peptide releasing enzyme from Sulfolobus tokodaii (ST0779) as a novel promiscuous biocatalyst for aldol addition

This work, for the first time, demonstrated the catalytic promiscuity of an acyl-peptide releasing enzyme from Sulfolobus tokodaii (ST0779) for aldol addition reaction, which shows accelerated activity at elevated temperature. The turnover number k_cat (s^− 1) of this thermostable enzyme at 55 °C is 7.78-fold higher than that of porcine pancreatic lipase (PPL) at its optimum temperature of 37 °C, which is one of the best reported enzymes for promiscuous catalysis of aldol reaction; and the molecular catalytic efficiency k_cat/K_m (M^− 1 s^− 1) adds up to 140 times higher than PPL.     

A comparison of properties of inulinases of Fusarium oxysporum immobilised on various supports

Extracellular inulinase was obtained after growing Fusarium oxysporum for 9 days on a medium containing an aqueous extract of Cichorium intybus roots. After partial purification, inulinase was immobilised by cross-linking it with water insoluble protein supports prepared from flour of soybean and mungbean, and egg white. Inulinase was also immobilised by adsorbing it on DEAE-cellulose. Inulinase immobilised on soybean and mungbean supports had a higher optimum temperature of 45°C as compared to 37°C of the free enzymes. Inulinase adsorbed on DEAE-cellulose was thermally the most stable whereas the inulinase cross-linked on soybean support was the least stable. Inulinase immobilised on DEAE-cellulose and on supports obtained from mungbean and soybean had a pH optima of 5.5, 6.5 and 6.0, respectively. The K_m values of various immobilised inulinases with inulin varied from 0.25 to 0.44 mmol dm^?3.     

Biochemical Characterization of Purified Esterase from Soybean (Glycine max) Seed

Alkaline esterase (carboxylic‐ester hydrolases; EC 3.1.1.1) extracted from germinated soybean seeds (Glycine max) was purified approximately 3.6 times by chromatography in a DEAE‐cellulose anion exchange column and filtration in Sephadex G100 gel. The molecular mass of the enzyme was estimated at 45 kDa by gel electrophoresis (SDS‐PAGE). The purified enzyme showed a specific activity of 5.6 U mg^?1 using p‐nitrophenyl butyrate as substrate. The esterase showed optimal activity at 47 °C in moderately alkaline pH, low stability in temperatures higher than 50 °C, and high stability at pH values between 6 and 9.5. The Ca²⁺ and Co²⁺ ions proved to have a positive effect on enzyme activity; however, Hg²⁺ completely inhibited esterase activity. Using p‐nitrophenyl butyrate as substrate, the enzyme showed a K_m of 0.39 mM, V_max of 31.5 mM mg^?1 min^?1 and k_cat 7.60 × 10⁶ s^?1. Regarding substrate affinity, the enzyme showed greater activity for substrates containing short‐chain fatty acids, especially p‐nitrophenyl acetate. Such characteristics give the enzyme great potential for application in the production of low molecular weight esters, in the food industry, and in chemical products. This enzyme is another new member of the family of lipases and esterases from vegetable seeds with high activity and stability in alkaline pH.     

Immobilization of invertase onto dimer acid‐co‐alkyl polyamine

Invertase was immobilized onto the dimer acid‐co‐alkyl polyamine after activation with 1,2‐diamine ethane and 1,3‐diamine propane. The effects of pH, temperature, substrate concentration, and storage stability on free and immobilized invertase were investigated. Kinetic parameters were calculated as 18.2 mM for K_m and 6.43 × 10^?5 mol dm^?3 min^?1 for V_max of free enzyme and in the range of 23.8–35.3 mM for K_m and 7.97–11.71 × 10^?5 mol dm^?3 min^?1 for V_max of immobilized enzyme. After storage at 4°C for 1 month, the enzyme activities were 21.0 and 60.0–70.0% of the initial activity for free and immobilized enzyme, respectively. The optimum pH values for free and immobilized enzymes were determined as 4.5. The optimum temperatures for free and immobilized enzymes were 45 and 50°C, respectively. After using immobilized enzyme in 3 days for 43 times, it showed 76–80% of its original activity. As a result of immobilization, thermal and storage stabilities were increased. The aim of this study was to increase the storage stability and reuse number of the immobilized enzyme and also to compare this immobilization method with others with respect to storage stability and reuse number. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 1526–1530, 2004     

Invertase immobilised on montmorillonite: reusability enhancement and reduction in leaching

Invertase was immobilised on microporous montmorillonite K-10 via adsorption and covalent binding. The immobilised enzymes were tested for sucrose hydrolysis activity in a batch reactor. K_m for immobilised systems was greater than free enzyme. The immobilised forms could be reused for 15 continuous cycles without any loss in activity. After 25 cycles, 85% initial activity was retained. A study on leaching of enzymes showed that 100% enzyme was retained even after 15 cycles of reuse. Leaching increased with reaction temperature. Covalent binding resisted leaching even at temperatures of 70 °C.