Synthesis of dipeptide precursors with an immobilized thermolysin in ethyl acetate

N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the aspartame, and N-(benzyloxycarbonyl)-L-phenylalanyl-Lphenylalanine methyl ester (Z-PhePheOMe) were synthesized from the respective amino acid derivatives with an immobilized thermolysin (EC 3.4.24.4) in ethyl acetate. Various factors affecting the synthesis of these dipeptide precursors were clarified. The initial synthetic rate was the highest at the water content of 3.5% for both reactions. The substrate concentration dependencies of the initial synthetic rate of Z-AspkPheOMe and Z-PhePheOMe with the immobilized enzyme in ethyl acetate were different from those in an aqueous buffer solution saturated with ethyl acetate but similar to those in the aqueous/organic biphasic system using the free enzyme. Particularly, the initial synthetic rate of Z-AspPhOMe increased in order higher than first order with respect to the concentration of L-phenylalanine methyl ester (PheOMe), whereas it decreased sharply with the concentration of N-(benzyloxycarbonyl)-L-aspartic acid (Z-Asp). Such kinetic behavior could be explained by regarding the inside of the immobilized enzyme as being a biphasic mode composed from the organic phase and aqueous phase where the enzymatic reaction takes place. The reaction in the aqueous/organic biphasic system using the free enzyme could be simulated by taking into consideration the partition of the substrate and the initial rate of synthesis in the aqueous buffer saturated with ethyl acetate. Based on this analysis, the rate of reaction with the immobilized enzyme in ethyl acetate could also be predicted. Z-AsPheOMe and Z-PhePheOMe were synthesized by the fed-batch method where the acid component of the substrate was intermittently added during the course of reaction and by the batch method. In the synthesis of Z-AspPheOMe, the synthetic rate and maximum yield of reaction as well as the stability of the immobilized enzyme were higher in the fed-batch reaction than those in the batch reaction. In the synthesis of Z-PhePheOMe, the results obtained by both methods were similar. (c) 1994 John Wiley & Sons, Inc.     

Comparative study of the enzymatic synthesis of cephalexin at high substrate concentration in aqueous and organic media using statistical model

Synthesis of cephalexin with immobilized penicillin acylase at high substrates concentration at an acyl donor to nucleophile molar ratio of 3 was comparatively evaluated in aqueous and ethylene glycol media using a statistical model. Variables under study were temperature, pH and enzyme to substrate ratio and their effects were evaluated on cephalexin yield, ratio of initial rates of cephalexin synthesis to phenylglycine methyl ester hydrolysis, volumetric and specific productivity of cephalexin synthesis, that were used as response parameters. Results obtained in both reaction media were modeled using surface of response methodology and optimal operation conditions were determined in terms of an objective function based on the above parameters. At very high substrates concentrations the use of organic co-solvents was not required to attain high yields and actually almost stoichiometric yields were obtained in a fully aqueous media with the advantages of higher productivities than in an organic co-solvent media and compliance with the principles of green chemistry.     

Anhydrous tert-pentanol as a novel media for the efficient enzymatic synthesis of amoxicillin

The efficient enzymatic synthesis of amoxicillin using anhydrous tert-pentanol as a novel media has been demonstrated for the first time. p-OH-Phenylglycine methyl ester (HPGM) was selected as the activated acyl donor due to its good solubility in organic solvents. The screening results of 21 organic solvents showed that solvents with either strong polarity or poor substrate solubility were unfavorable. Remarkable catalytic activity of the immobilized penicillin acylase (IPA) from Escherichia coli was retained in tert-pentanol, and high yield could be obtained. Effects of various parameters such as acyl donor, water content or cosolvents of tert-pentanol, substrate concentration, temperature, etc., on the enzymatic synthesis of amoxicillin in tert-pentanol were investigated systematically. The best reaction medium, the optimal temperature, initial concentration of 6-APA and HPGM and concentration of enzyme were tert-pentanol, 15 °C, 100, 200 mM and 20 IU/mL, respectively. Under the optimal conditions, the yield of amoxicillin was as high as 88% after a reaction time of 20 h.     

Strategies for enzymatic esterification in organic solvents: comparison of microaqueous, biphasic, and micellar systems.

The kinetics of butyl butyrate synthesis by a lipase from Mucor miehei in different types of organic media were investigated. The three systems studied were a microaqueous medium containing enzyme in suspension in hexane, a water-hexane two-phase system, and reverse micelles. The synthesis of butyl butyrate was possible in all cases because of a favorable partition of the ester into the organic solvent. A sufficient stirring rate was necessary to achieve good reaction rates in the case of the liquid-liquid biphasic medium. The effect of water content was different according to the type of system used. The dependence of reaction rate and of conversion yield on enzyme and substrate concentrations was also investigated. From an applied point of view, the best performances were obtained with either microaqueous or liquid-liquid two-phase systems. The use of reverse micelles can be advocated only in particular conditions, such as low enzyme concentration, compatible with the specific constraints it involves.     

Novozym 435-catalyzed regioselective benzoylation of 1-β-d-arabinofuranosylcytosine in a co-solvent mixture of C4MIm·PF6 and pyridine

Regioselective acylation of 1-β-d-arabinofuranosylcytosine (ara-C), using vinyl benzoate (VB) as acyl donor and Novozym 435 as catalyst, was carried out in various reaction media including pure organic solvents, organic solvent mixtures, and ionic liquid (IL)-containing systems. Although the reaction was highly regioselective in all the media assayed, remarkable enhancement of substrate conversion was achieved with a co-solvent mixture of 1-butyl-3-methylimidazolium hexafluorophosphate (C₄MIm·PF₆) and pyridine as the reaction medium, compared with other media tested. Additionally, the results demonstrated that the anions of ILs had a significant effect on the initial rate and substrate conversion. To better understand the reaction performed in IL-containing system, several variables were examined. The optimum molar ratio of VB to ara-C, initial water activity, temperature and shaking rate were 25:1, 0.11, 40°C and 250rpm, respectively. Under these optimum reaction conditions, the initial rate, substrate conversion, and regioselectivity were 0.49mMmin^?1, 99.4 and 99%, respectively. The product of the lipase-catalyzed reaction was characterized by ¹³C NMR and was shown to be 5′-O-benzoyl ara-C.     

The role of hydration in enzyme activity and stability: 1. Water adsorption by alcohol dehydrogenase in a continuous gas phase reactor

The enzymatic synthesis of the tripeptide derivative Z-Gly-Trp-Met-OEt is reported. This tripeptide is a fragment of the cholecystokinin C-terminal octapeptide CCK-8. Studies on the alpha-chymotrypsin catalyzed coupling reaction between Z-Gly-Trp-R(1) and Met-R(2) have focused on low water content media, using deposited enzyme on inert supports such as Celite and polyamide. The effect of additives (polar organic solvents), the acyl-donor ester structure, the C-alpha protecting group of the nucleophile, enzyme loading, and substrate concentration were tested. The best reaction medium found was acetonitrile containing buffer (0.5%, v/v) and triethylamine (0.5%, v/v) using the enzyme deposited on Celite as catalyst (8 mg of alpha-chymotrypsin/g of Celite). A reaction yield of 81% was obtained with Z-Gly-Trp-OCam as acyl donor, at an initial concentration of 80 mM. The tripeptide synthesis was scaled up to the production of 2 g of pure tripeptide with an overall yield of 71%, including reaction and purification steps. (c) 1996 John Wiley & Sons, Inc.     

The lipase-catalyzed hydrolysis of lutein diesters in non-aqueous media is favored at extremely low water activities

The enzymatic hydrolysis of a mixture of lutein diesters from Marigold flower (Tagetes erecta) was performed both in organic solvents and supercritical CO(2) (SC-CO(2)) using two commercial lipases: lipase B from Candida antarctica (Novozym 435) and the lipase from Mucor miehei (Lipozyme RM IM). Both lipases showed an unexpected dependence of initial reaction rate with the initial water activity (a(wi)) in hexane, with the highest rates of hydrolysis taking place at the lowest a(wi) of the biocatalyst particles. The same result was observed using isooctane, toluene, or SC-CO(2). It is proposed that an increase in a(wi) generates a hydrophilic microenvironment that prevents efficient partitioning of the highly hydrophobic lutein diesters to the enzyme. The critical role of water in this system has not been reported for other hydrolytic reactions in low water media. Calculations of water available for hydrolysis from isotherm analysis, Karl-Fischer titration, and substrate conversion at a(wi) = 0.13, indicate that the extent of reaction is not limited by the amount of available water. Accordingly, the enzyme that holds the largest amount of water after prehydration at the same a(wi) (0.13) will yield the greatest substrate conversion and concentration of the free lutein product. The highest conversion occurred in SC-CO(2), which opens up new opportunities to develop a combined extraction-reaction process for the environmentally benign synthesis of lutein, an important nutraceutical compound.     

Steady-state kinetics of immobilized enzymes at very low substrate concentrations studied using the asarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase

A method for determining initial velocities of enzymatic reactions at very low substrate concentrations is presented. It is based on teh continuous perfusiion of substrate-containing media through the enzyme, previously deposited as a thin layer on a solid support. An analytical rationalization of the dependence of the enzymatic activity upon the substrate supply and the flow rate was developed (substrate supply (μmol/min) = flow rate (ml/min) × inflowing substrate concentration (μmol/ml). This paper shows that a straight line should be expected from a double-reciprocal plot of the velocity of the enzymatic reaction and flow rate. The reciprocal of the ordinate at the origin is the strict initial velocity for a given, constant, and very low substrate concentration, since substrate consumption and product accumulation tend to zero. Results obtained with two different sarcoplasmic reticulum (Ca²⁺ + Mg²⁺)-ATPase preparations agree with the theoretical predictions. The method enabled the use of ATP concentrations in the range of 10^?8 M: it required neither an ATP-regerating system nor the dilution of the enzyme protein, and it presented no limitations for the reaction time. Both ATPase preparations showed two apparent K_m values for the substrate in the submicromolar and micromolar ranges: 0.25–12.0 μM for the purified ATPase, and 0.17–1.65 μM for the microsomal ATPase.     

Production of Ethyl Butyrate by Candida rugosa Lipase Immobilized in Polyurethane

The enzymatic production of ethyl butyrate was studied: the lipase of Candida rugosa (E.C. 3.1.1.3.) was immobilized in a polyurethane matrix and subsequently introduced in an organic medium containing the substrates in appropriate concentrations. The large majority of experiments was carried out in n-hexane. Two further solvents were tested, namely n-heptane and n-dodecane. The partition coefficients matrix/solvent were estimated for the various solvent systems. The initial esterification rate, the molar yield ester/acid and the degree of conversion were found to be solvent independent when the reaction media were designed so that similar concentrations were created in the microenvironment. Initial rate experiments indicated that in n-hexane the threshold of inhibitory substrate concentrations lies (i) between 0.40 M and 0.50 M for butyric acid, according to the purity of the enzyme preparation and (ii) at 0.30 M for ethanol. Batch operational stability tests indicate that no enzyme deactivation occurs after 20 consecutive batches.     

Importance of solute partitioning in biphasic oxidation of benzyl alcohol by free and immobilized whole cells of pichia pastoris

Using free and immobilized whole cells of Pichia pastoris, the biocatalytic oxidation of benzyl alcohol was investigated in different two-phase systems. This reaction was strongly influenced by both the substrate and product inhibitions, and the production rate of benzaldehyde in the aqueous system became maximum at the initial substrate concentration of ca. 29 g/L with the aldehyde formation less than 4 to 5 g/L even after a longer reaction period. The reaction rates in the two-liquid phase systems were predominantly determined by the partitioning behaviors of the substrate and product between the two phases rather than by enzyme deactivation by the organic solvents. In the two-liquid phase systems, consequently, the organic solvent acted as a reservior to reduce these inhibitory effects, and it was essential to select the organic solvent providing the optimal partitioning of the substrate into the aqueous phase as well as the preferential extraction of the product into the organic phase. The whole cells immobilized in a mixed matrix composed of silicone polymer [>50% (v/v)] and Ca alginate gel (<50%) worked well in the xylene and decane media, providing comparable activities with the free cells. The production rate of aldehyde was also influenced by the solute partitioning into the hydrophilic alginate phase where the cells existed. (c) 1994 John Wiley & Sons, Inc.     

非水相脂肪酶催化合成L-抗坏血酸棕榈酸酯的研究Ⅰ

对催化合成Ｌ－捩民血酸棕榈酸本反应的脂肪酶（ＮＯＶＯ４３５、ＭＭＬ、ＬＩＰＯＬＡＳＥ、ＰＰＩ）和反应介质进行比较,得出最佳酶为ＮＯＶＯ４３５,最佳介质为椒戊醇,同时对影响合成Ｌ－抗坏血酸棕榈酸酯反应的初速度的因素（转速、温度、水分含量、酶氏物浓度）进行了探讨,确定了最适反应１条件,转速为２００ｔ／ｍｉｎ,温度为５５℃,水分含量为０,酶浓度为１２．５％。     

Study of vitamin ester synthesis by lipase-catalyzed transesterification in organic media

Immobilized lipase from Candida antarctica (Novozym 435) was used in organic media to catalyze esterifications of vitamins (ascorbic acid and retinol) from hydroxy acid. We described the synthesis of retinyl L-lactate by transesterification between retinol and L-methyl lactate with yield reaching 90% and the synthesis of ascorbyl L-lactate by transesterification between ascorbic acid and L-methyl lactate with yield reaching 80%. The kinetic study of the esterification of vitamins with L-methyl lactate in organic media has been carried out and agrees with ping-pong-ordered Bi-Bi when the initial vitamin concentration is low. When initial vitamin concentration is high, the kinetic is similar to a hybrid ping-pong-ordered Bi Bi or hybrid ping-pong-random Bi Bi mechanism. However, with high initial substrate concentration, change of the kinetic by other phenomena, such as interaction of substrates with molecular sieves, adsorption of the methanol formed, and decreases of substrate diffusion, could be considered. It is obvious that in these conditions, classical enzymology (i.e., Michaelian enzymology) cannot be used for the interpretation of results.     

Predicting enzyme behavior in nonconventional media: correlating nitrilase function with solvent properties

The insolubility of nitrile substrates in aqueous reaction mixture decreases the enzymatic reaction rate. We studied the interaction of fourteen water miscible organic solvents with immobilized nitrile hydrolyzing biocatalyst. Correlation of nitrilase function with physico-chemical properties of the solvents has allowed us to predict the enzyme behavior in such non-conventional media. Addition of organic solvent up to a critical concentration leads to an enhancement in reaction rate, however, any further increase beyond the critical concentration in the latter leads to the decrease in catalytic efficiency of the enzyme, probably due to protein denaturation. The solvent dielectric constant (epsilon) showed a linear correlation with the critical concentration of the solvent used and the extent of nitrile hydrolysis. Unlike alcohols, the reaction rate in case of aprotic solvents could be linearly correlated to solvent log P. Further, kinetic analysis confirmed that the affinity of the enzyme for its substrate (K (m)) was highly dependent upon the aprotic solvent used. Finally, the prospect of solvent engineering also permitted the control of enzyme enantioselectivity by regulating enantiomer traffic at the active site.     

Influence of solvent and water activity on kinetically controlled peptide synthesis.

alpha-Chymotrypsin deposited on Celite was used to catalyse peptide synthesis reactions between N-protected amino acid esters and leucine amide in organic media with low water content. The influence of the solvent and the thermodynamic water activity on the reaction kinetics was studied. The substrate specificity in the reactions was shown to be a combination of the substrate specificity of the enzyme in aqueous media and the influence of the solvents. The magnitude of the solvent effects differed greatly depending on the substrates used. In hydrophobic solvents high reaction rates were observed and the competing hydrolysis of the ester substrate occurred to only a minor extent. Reactions occurred at water activities as low as 0.11, but the rate constants increased with increasing water activity and were about two orders of magnitude higher at the highest water activity tested (0.97).     

Protease-catalyzed tripeptide (RGD) synthesis

The tripeptide Bz-Arg-Gly-Asp(-OMe)-OH was synthesized by enzymatic method. Bz-Arg-Gly-OEt was synthesized by trypsin in ethanol containing 0.1 M Tris/HCl buffer (pH 8.0), and then H-Asp(-OMe)(2) was incorporated into the Bz-Arg-Gly-OEt using chymopapain in 0.25M CHES/NaOH buffer (pH = 9.0, EDTA 10 mM). The yield of Bz-Arg-Gly-OEt and Bz-Arg-Gly-Asp(-OMe)-OH were 80% and 70% using 1M Bz-Arg-OEt and 0.5M Bz-Arg-Gly-OEt, respectively. For Bz-Arg-Gly-OEt synthesis reaction at high concentrations of the substrates, the buffer content in ethanol was a key factor to determine the optimal reaction condition. In Bz-Arg-Gly-Asp(-OMe)-OH synthesis reaction, the yield was low in organic solvent due to various side products such as Bz-Arg-OH, Bz-Arg-Gly-OH, and Bz-Arg-Gly-Asp(-OMe)-Asp(-OMe)-OH, suggesting that chymopapain has a very broad substrate specificity of the S(1) site. The Bz-Arg-Gly-Asp(-OMe)-OH synthesis rate and its yield were dramatically elevated and the side reactions were reduced using only the CHES/NaOH buffer (pH = 9.0, EDTA 10 mM) as a reaction media. The final product Bz-Arg-Gly-Asp(-OMe)-OH was identified to be formed via C-terminal hydrolysis of Bz-Arg-Gly-Asp(-OMe)(2) after the nucleophile, H-Asp(-OMe)(2), was added.     

Environmentally Friendly Approach to the Synthesis of 3-[Benzylideneamino]-2-methylquinazolin-4(3H)-one Derivatives and Calculation of Their Toxicity

Application of deep eutectic solvents in synthesis of different heterocyclic compounds was proven very efficient. These solvents are a new generation of green solvents showing excellent potential for different purposes, where they are used as environmentally acceptable substitute for toxic and volatile organic solvents. This research describes their application in the synthesis of series of quinazolinone Schiff bases in combination with microwave, ultrasound-assisted and mechanochemical methods. First, a model reaction was performed in 20 different deep eutectic solvents to find the best solvent and then reaction conditions (solvent, temperature and reaction time) were optimized for each method. Afterwards, 40 different quinazolinone derivatives were synthesized in choline chloride/malonic acid (1 : 1) DES by each method and compared by their yields. Here we show that deep eutectic solvents can be very efficient in the synthesis of quinazolinone derivatives as an excellent substitution for volatile organic solvents. With green chemistry approach in mind, we have also performed a calculation on compounds’ toxicity and solubility, showing that most of them possess toxic and mutagenic properties with low water solubility.     

Continuous production of (R)-phenylacetylcarbinol in an enzyme-membrane reactor using a potent mutant of pyruvate decarboxylase from Zymomonas mobilis.

The optimization of a continuous enzymatic reaction yielding (R)-phenylacetylcarbinol (PAC), an intermediate of the L-ephedrine synthesis, is presented. We compare the suitability of three pyruvate decarboxylases (PDC), PDC from Saccharomyces cerevisiae, PDC from Zymomonas mobilis, and a potent mutant of the latter, PDCW392M, with respect to their application in the biotransformation using acetaldehyde and benzaldehyde as substrates. Among these, the mutant enzyme was the most active and most stable one. The reaction conditions of the carboligation reaction were investigated by determining initial rate velocities with varying substrate concentrations of both aldehydes. From the resulting data a kinetic model was inferred which fits the experimental data with sufficient reliability to deduce the optimal concentrations of both substrates for the enzymatic process. The results demonstrate that the carboligation is most efficiently performed using a continuous reaction system and feeding both aldehydes in equimolar concentration. Initial studies using a continuously operated enzyme-membrane reactor gave (R)-PAC with a space-time yield of 81 g L(-1). d(-1) using a substrate concentration of 50 mM of both aldehydes. The yield was easily increased by cascadation of enzyme-membrane reactors. The new strategy allows the synthesis of (R)-PAC from cheap substrates in an aqueous reaction system. It thereby overcomes the limitation of by-product formation that severely limits the current fermentative process.     

NMR for direct determination of K(m) and V(max) of enzyme reactions based on the Lambert W function-analysis of progress curves

(1)H NMR spectroscopy was used to follow the cleavage of sucrose by invertase. The parameters of the enzyme's kinetics, K(m) and V(max), were directly determined from progress curves at only one concentration of the substrate. For comparison with the classical Michaelis-Menten analysis, the reaction progress was also monitored at various initial concentrations of 3.5 to 41.8mM. Using the Lambert W function the parameters K(m) and V(max) were fitted to obtain the experimental progress curve and resulted in K(m)=28mM and V(max)=13μM/s. The result is almost identical to an initial rate analysis that, however, costs much more time and experimental effort. The effect of product inhibition was also investigated. Furthermore, we analyzed a much more complex reaction, the conversion of farnesyl diphosphate into (+)-germacrene D by the enzyme germacrene D synthase, yielding K(m)=379μM and k(cat)=0.04s(-1). The reaction involves an amphiphilic substrate forming micelles and a water insoluble product; using proper controls, the conversion can well be analyzed by the progress curve approach using the Lambert W function.     

Kinetics and equilibrium of enzymatic synthesis of peptides in aqueous/organic biphasic systems. Thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester

We studied kinetics and the equilibrium relationship for the thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-Asp-PheOMe) from N-(benzyloxycarbonyl)-L-aspartic acid (Z-Asp) and L-phenylalanine methyl ester (PheOMe) in an aqueous-organic biphasic system. This is a model reaction giving a condensation product with dissociating groups. The kinetics for the synthesis of Z-Asp-PheOMe in aqueous solution saturated with ethyl acetate was expressed by a rate equation for the rapid-equilibrium random bireactant mechanism, and the reverse hydrolysis reaction was zero-order with respect to Z-Asp-PheOMe concentration. The courses of synthesis of Z-Asp-PheOMe in the biphasic system were well explained, by the rate equations obtained for the aqueous solution and by the partition of substrate and condensation product between the both phases. The rate of synthesis in the biphasic system was much lower than in aqueous solution due to the unfavorable partition of PheOMe in the aqueous phase. The equation for the equilibrium yield of Z-Asp-PheOMe in the biphasic system was derived assuming that only the non-ionized forms of the substrate and condensation product exist in the organic phase. It was found theoretically and experimentally that the yield of Z-Asp-PheOMe is maximum at the aqueous-phase pH of around 5, lower than for synthesis in aqueous solution. The effect of the organic solvent on the rate and equilibrium for the synthesis of Z-Asp-PheOMe could be explained by the variation in the partition coefficient. The effect of the partitioning of substrate on the aqueous-phase pH change was also shown.     

Enzymatic synthesis of butyl hydroxycinnamates and their inhibitory effects on LDL-oxidation

The potential of the Aspergillus niger type A feruloyl esterase (AnFaeA) for the synthesis of various phenolic acid esters was examined using a ternary-organic reaction system consisting of a mixture of n-hexane, 1- or 2-butanol and water. Reaction parameters including the type of methyl hydroxycinnamate, the composition of the reaction media, the temperature, and the substrate concentration were investigated to evaluate their effect on initial rate and conversion to butyl esters of sinapic acids. Optimisation of the reaction parameters lead to 78% and 9% yield for the synthesis of 1-butyl and 2-butyl sinapate, respectively. For the first time, a feruloyl esterase was introduced in the reaction system as cross-linked enzyme aggregates (CLEAs), after optimisation of the immobilisation procedure, allowing the recycling and reuse of the biocatalyst. The inhibition of copper-induced LDL oxidation by hydroxycinnamic acids and their corresponding butyl esters was investigated in vitro. Kinetic analysis of the antioxidation process demonstrates that sinapate derivatives are effective antioxidants indicating that esterification increases the free acid's antioxidant activity especially on dimethoxylated compounds such as sinapic acid compared to methoxy-hydroxy-compounds such as ferulic acid.