Synthesis of aspartame precursor with an immobilized thermolysin in mixed organic solvents

N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester, a precursor of the synthetic sweetener, aspartame, was synthesized from N-(benzyloxycarbonyl)-L-aspartic acid and L-phenylalanine methyl ester with an immobilized thermolysin (EC 3.4.24.4) in the mixed organic solvent system of tert-amyl alcohol and ethyl acetate. A mixed solvent consisting of tert-amyl alcohol and ethyl acetate at a ratio of 33:67 (v/v) was found to be the most suitable with respect to synthetic rate and stability of the immobilized enzyme. The reaction continued to proceed quite successfully in a column reactor at 40 degrees C and at a space velocity of 3.6 h(-1) with a yield of 99%, using 40 mM Z-Asp and 200 mM PheOMe dissolved in the mixed solvent as the substrate. (c) 1995 John Wiley & Sons, Inc.     

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.     

Synthesis of Aspartame Precursor Using Protease Suspended in Microaqueous Molten Amino Acids Mixture

Enzymatic synthesis of the aspartame precursor, N -(benzyloxycarbonyl)- l -aspartyl- l -phenylalanine methyl ester (Z-AspPheOMe) was performed with highly concentrated molten substrates. A mixture composed of molten N -(benzyloxycarbonyl)- l -aspartic acid (Z-Asp) and l -phenylalanine methyl ester (PheOMe) mixtures of 20 M could be prepared at 50°C. This Z-Asp/PheOMe mixture was applied to the enzymatic synthesis of Z-AspPheOMe using free thermolysin. Synthesis of Z-AspPheOMe was observed in the range of 100-150 &#119 l of NaOH solution (12.5 M) addition to a reaction mixture consisting of 1.0 mmol Z-Asp and 1.0 mmol PheOMe at 50°C. The enzymatic activity increased with increasing water addition, and reached a maximum at 100 &#119 l in addition to the reaction mixture of 1.0 mmol Z-Asp, 1.0 mmol PheOMe and 125 &#119 l of the NaOH solution. In this reaction system, the conversion at the reaction equilibrium was about 60%, the initial reaction rate calculated on the basis of the enzyme weight was 2.2 &#119 mol/g s, and the productivity calculated on the basis of the reaction mixture volume was 300 mol/m 3 h.     

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

We studied kinetics of thermolysin-catalyzed peptide synthesis in an aqueous/organic biphasic system theoretically and experimentally. As a model reaction producing a condensation product having no dissociating groups, we used the synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-phenylalanine methyl ester (Z-Phe2OMe) from N-(benzyloxycarbonyl)-L-phenylalanine (Z-Phe) and L-phenylalanine methyl ester (PheOMe). Usually, ethyl acetate was used as the organic solvent. First we studied the kinetics of the synthesis of Z-Phe2OMe in a buffer solution saturated with ethyl acetate. Then, factors that may affect the kinetics in the biphasic system were examined. The course of Z-Phe2OMe synthesis in the biphasic system was explained by the rate equations obtained, using the partitions of substrate and product and non-enzymatic decomposition of PheOMe. In the biphasic reaction system, the rate of synthesis was lower for a wide range of pH due to the unfavorable partition of PheOMe in the aqueous phase, but yields were higher than in the buffer solution. The effects of the organic solvents on the rate of synthesis could also be explained by variations in the partition coefficient of PheOMe. Finally, we gave a way to predict the aqueous-phase pH change caused by partitioning of the substrate. The significance of the pH change was shown in connection with the reaction using the immobilized enzyme in an organic solvent.     

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.     

Synthesis of aspartame precursor: alpha-L-aspartyl-L-phenylalanine methyl ester in ethyl acetate using thermolysin entrapped in polyurethane

Cross-linked polyurethane (PU) was prepared for entrapping thermolysin. Using the immobilized thermolysin (IT), Z-L-aspartic acid (ZA) was reacted with -Lphenylalanine methyl ester (L-PM) in water-saturated ethyl acetate to give only alpha-Z-L-aspartylL-phenylalanine methyl ester (alpha-ZAPM). Ninety-four percent conversion of alpha-ZAPM was obtained for 30 h of reaction at 40 degrees C when 46 mg of enzyme was entrapped. PU support prepared from polypropylene glycol (#2000) showed better properties than from polypropylene (#1000) and polyethylene (#1000). Addition of polyol could increase the gel fraction of PU. The IT PU-ll-G-3, prepared from 1/2 mole ratio of PPG (#2000)/glycerin, gave the highest gel fraction and best swelling, and 89.0% of residual activity was obtained after four times of reuse (72 h). The stability of immobilized thermolysin was good; the activity loss resulting from degradatin and leak of enzyme in each time of reuse were found only about 2%. The kinetics of immobilized thermolysin-catalyzed condensation reaction of ZA with L-PM in water-saturated ethyl acetate was found to be first order in L-PM and the Lineweaver-Burk plot of 1/V against 1/[ZA] yields a straight line, showing that the reaction involves consecutive reactions of ZA and L-PM with the immobilized enzyme and with the ZA-immobilized enzyme complex, with the second reaction being the rate determining step.     

Biotransformation of alkyl and aryl carbonates: enantioselective hydrolysis

Summary N-(Benzyloxycarbonyl)-l-asparty-l-phenylalanine methyl ester, the precursor of the synthetic sweetener aspartame, was continuously synthesized in an immobilized thermolysin plug-flow type reactor at 25° C with the substrates (N-benzyloxycarbonyl-l-aspartic acid and l-phenylalanine methyl ester) dissolved in ethyl acetate. The immobilized enzyme was quite stable in ethyl acetate containing 2.5% 0.01 M 2-(N-morpholino)ethanesulphonic acid-NaOH buffer, pH 6.0, and 20 mM CaCl₂ with or without the substrate at 25° C. By periodically washing the column, we could conduct a continuous reaction for over 500 h with an average yield of 95% and a space velocity of 1.85 h ^–1.Offprint requests to: K. Nakanishi     

Repeated batch and continuous syntheses of N-(benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester with immobilized thermolysin

Summary N-(Benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester was synthesized from N-(benzyloxycarbonyl)-l-phenylalanine and l-phenylalanine methyl ester in an aqueous solution (aqueous phasic reaction), in an aqueous/organic biphasic system (biphasic reaction), and in an organic solvent (organic phasic reaction) with immobilized thermolysin. In the aqueous phasic reaction with thermolysin immobilized on Amberlite XAD-7, the whole product was trapped inside the support; extraction with ethyl acetate was needed to recover the product, and the equilibrium yield was low (about 65%). With the biphasic and organic phasic reactions with ethyl acetate as an organic solvent, the yield was around 95%. Because of the high yield and feasibility of operation, repeated batch and continuous reactions were done in the biphasic and organic phasic systems, respectively. The half-lives of the activity for the immobilized enzyme used in the biphasic system at 40°C by repeated batch operation and in a plug flow reactor fed with substrate dissolved in ethyl acetate at 40°C and 30°C were estimated to be about 200 h (67 batches), 420 h, and 1100 h, respectively.     

Production of L-lysine by immobilized trypsin. Study of DL-lysine methyl ester resolution

The possibility of producing L-lysine from chemically synthesized DL-lysine has been investigated. Optical resolution of racemic DK-lysine may be achieved by using the stereospecific esterasic activity of trypsin on DL-lysine methyl ester, which gives L-lysine and unchanged D-lysine methyl ester. SL-lysine methyl ester spontaneous hydrolysis may be neglected when operating at pH 5.5 and 30 degrees C. Effect of pH and substrate concentration on hydrolysis rate has been investigated when using as a catalyst either soluble or immobilized trypsin. For this purpose, trypsin was coupled onto an amine porous silica, Spherosil, activated with glutaraldehyde. The optimal pH is 5.8 for soluble trypsin and 6.0 for immobilized trypsin. It was yet possible to lower the parent optimal pH of immobilized trypsin, and thus increase its activity at 5.5, by co-grafting onto Spherosil an aminosilane, for enzyme coupling via glutaraldehyde activation and a positively charged diethyl amino ethyl (DEAE) silane, for decreasing the pH of trypsin microenvironment.     

Biosensor for direct determination of organophosphate nerve agents. 1. Potentiometric enzyme electrode

A potentiometric enzyme electrode for the direct measurement of organophosphate (OP) nerve agents was developed. The basic element of this enzyme electrode was a pH electrode modified with an immobilized organophosphorus hydrolase (OPH) layer formed by cross-linking OPH with bovine serum albumin (BSA) and glutaradehyde. OPH catalyses the hydrolysis of organophosphorus pesticides to release protons, the concentration of which is proportional to the amount of hydrolysed substrate. The sensor signal and response time was optimized with respect to the buffer pH, ionic concentration of buffer, temperature, and units of OPH immobilized using paraoxon as substrate. The best sensitivity and response time were obtained using a sensor constructed with 500 IU of OPH and operating in pH 8.5, 1 mM HEPES buffer. Using these conditions, the biosensor was used to measure as low as 2 microM of paraoxon, ethyl parathion, methyl parathion and diazinon. The biosensor was completely stable for at least one month when stored in pH 8.5, 1 mM HEPES + 100 mM NaCl buffer at 4 degrees C.     

Enzymatic synthesis of the precursor of Leu-enkephalin in water-immiscible organic solvent systems.

The precursor of Leu-enkephalin, Z-L-TyrGlyGly-L-Phe-L-LeuOEt, was synthesized from amino acid derivatives with three proteinases without the protection of the side chain of L-Tyr. First, Z-GlyGlyOBut and Z-L-TyrGlyGlyOBut were synthesized in quite a high yield, 83% and 99%, in an aqueous/organic biphasic system by papain and alpha-chymotrypsin, respectively. Then, Z-L-Phe-L-LeuOEt was synthesized by thermolysin from Z-L-Phe and L-LeuOEt either in buffer or in a biphasic system; the yields were 95% and 100%, respectively. The synthesis of Z-L-TyrGlyGly-L-Phe-L-LeuOEt from Z-L-TyrGlyGly and L-Phe-L-LeuOEt was performed effectively by thermolysin immobilized on Amberlite XAD-7 in a buffer and in an aqueous/organic biphasic system, as well as in saturated ethyl acetate, while the yield was low in reactions by free thermolysin. In the reaction with the immobilized enzyme (IME) in saturated ethyl acetate, the maximum yield of the precursor of Leu-enkephalin was 68%. The reasons for effective synthesis with IME are: (1) higher concentration of L-Phe-L-LeuOEt inside support, which resulted in rising the rate of the synthesis reaction and protecting the competitive hydrolysis of Z-L-TyrGlyGly by thermolysin, (2) entrapment of the product inside the support where thermolysin could not act in the case of reaction in buffer, and (3) extraction of the product with the organic solvent in the case of reaction in a biphasic system or in saturated organic solvent.     

Inhibitory effects of alcohols on thermolysin activity as examined using a fluorescent substrate

Alcohols inhibit the thermolysin-catalyzed hydrolysis of N-[3-(2-furyl)acryloyl]-Gly-L-Leu-NH(2) and decrease the NaCl-induced activation of thermolysin in a concentration-dependent manner [K. Inouye et al. (1997) J. Biochem. 122, 358-364]. In this study, the inhibitory effects of alcohols on thermolysin activity were examined in detail using 10 different alcohols and a fluorescent substrate, (7-methoxycoumarin-4-yl) acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diamino-propionyl]-L-Ala-L-Arg-NH(2). The inhibition by all alcohols examined is completely reversible, and thermolysin activity is recovered by dilution. The inhibitor constants (K(i)) are in the range of 35-430 mM, and the order of the inhibitory effect is 1-pentanol, 1-propanol, 2-butanol, 2-methyl-1-propanol > 1-butanol > 2-propanol > ethanol, tert-amyl alcohol > tert-butyl alcohol > methanol. Linear and secondary alcohols whose mains chains consist of more than 3 carbons inhibit thermolysin effectively. Thermolysin activity is decreased by decreasing the dielectric constant, D, of the reaction medium containing the alcohol, and the decrease depending on the D value was almost the same manner for all alcohols except methanol, tert-butyl alcohol, and tert-amyl alcohol. Alcohols may inhibit thermolysin activity both by binding to the active site, most possibly to the S1' subsite, of thermolysin and by altering the electrostatic and hydrophobic environment around the thermolysin molecule.     

小分子封闭提高固定化嗜热菌蛋白酶的热稳定性

为了提高固定化嗜热菌蛋白酶的热稳定性,在制备共价固定化嗜热菌蛋白酶的基础上,通过选择氨基酸和醇类小分子来封闭载体表面未反应的活化基团,并考察了固定化酶的催化活性及热稳定性。结果发现：L-Trp和L-Val封闭修饰固定化酶时,在80℃的水浴中加热150 min后其剩余活力仍为93.4%和98.6%,其效果约为未经小分子封闭的固定化嗜热菌蛋白酶的2倍。所筛选的几种小分子物质中,叔戊醇、L-Trp、L-Val及L-Ala不仅能提高固定化嗜热菌蛋白酶的热稳定性,而且也可以提高固定化酶的相对活力,从而更有利于其在工业生产中的应用。     

Effects of subzero temperatures on the kinetics of protease catalyzed dipeptide synthesis in organic media

A depeptide synthesis was drastically influenced by the reaction temperature, in the range from -30 degrees to 25 degrees C. This article shows the kinetic reasons of this effect. alpha-Chymotrypsin was immobilized on celite and used in four different water-miscible solvents containing small amounts of water-miscible solvents containing small amounts of water. The reaction studied was the aminolysis of N-acetyl-L-phenylalanine ethyl ester (Ac-PheOEt) with L-alaninamide (Ala-NH(2)) and water for the acylenzyme complex, the nucleophile was favoured by low reaction temperatures. This effect (quantified as p-values) was observed in all four solvents, and it was greatest in acetonitrile and tetrahydrofuran. The esterase and amidase activities of the enzyme were studies using AcPheOEt and N-acetyl-L-phenylalanyl-L-ananinamide (AcPheAla-NH(2)) as substrates. The Michaelis-Menten parameters, K(m,app) and V(max), were determined for ester hydrolysis and dipeptide hydrolysis. Both K(m,app) and V(max) tended to increase with increasing temperature. Secondary hydrolysis was reduced at subzero temperatures because ester hydrolysis was favoured in relation to depeptide hydrolysis. Depeptide synthesis was thus favored by low temperatures in two ways: first, in the competition between the nucleophile and water for the acyl enzyme; and, second, in the competition between the ester substrate and the peptide substrate for the free enzyme. As a result, in acetonitrile containing 10% water, the maximal yield was 99% at -20%C compared with 84% at 25 degrees C. (c) 1995 John Wiley & Sons, Inc.     

Chymotrypsin substrate analogues inhibit endocytosis of insulin and insulin receptors in adipocytes

To explore the possible role of proteolytic step(s) in receptor-mediated endocytosis of insulin, the effects of inhibitors of various classes of proteases on the internalization process were studied in isolated rat adipocytes. Intracellular accumulation of receptor-bound 125I-insulin at 37 degrees C was quantitated after rapidly dissociating surface-bound insulin with an acidic buffer (pH 3.0). Of the 23 protease inhibitors tested, only chymotrypsin substrate analogues inhibited insulin internalization. Internalization was decreased 62-90% by five different chymotrypsin substrate analogues: N-acetyl-Tyr ethyl ester, N-acetyl-Phe ethyl ester, N-acetyl-Trp ethyl ester, benzoyl-Tyr ethyl ester, and benzoyl-Tyr amide. The effect of the substrate analogues in inhibiting insulin internalization was dose-dependent, reversible, and required the full structural complement of a chymotrypsin substrate analogue. Cell surface receptor number was unaltered at 12 degrees C. However, concomitant with their inhibition of insulin internalization at 37 degrees C, the chymotrypsin substrate analogues caused a marked increase (160-380%) in surface-bound insulin, indicating trapping of insulin-receptor complexes on the cell surface. Additionally, 1 mM N-acetyl-Tyr ethyl ester decreased overall insulin degradation by 15-20% and also prevented the chloroquine-mediated increase in intracellular insulin, further indicating that surface-bound insulin was prevented from reaching intracellular chloroquine-sensitive degradation sites. The internalization of insulin receptors that were photoaffinity labeled on the cell surface with B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin was also inhibited 70-90% by the five chymotrypsin substrate analogues, as determined by the effects of the analogues on the accumulation of trypsin-insensitive (intracellular) 440-kD intact labeled receptors. In summary, these results show that chymotrypsin substrate analogues efficiently inhibit the internalization of insulin and insulin receptors in adipocytes and implicate a possible role for endogenous chymotrypsin-like enzyme(s) or related substances in receptor-mediated endocytosis of insulin.     

Peptide synthesis with a proteinase from the extremely thermophilic organism Thermus Rt41A

A proteinase isolated from Thermus RT41a was immobilized to controlled pore glass beads and was used in the free and immobilized forms for peptide synthesis. The observed maximum yield was the same in both cases. a number of dipeptides were produced from amino acid esters and amides. The best acyl components, from those tested, were found to be Ac-Phe-OEt and Bz-Ala-OMe. Tur-NH(2), Trp-NH(2), Leu-pNA, and Val-pNA were all reactive nucleophiles.The kinetically controlled synthesis of Bz-ala-Tyr-NH(2) was optimized by studying the effect of pH, temperature, solvent concentration, ionic strength, and nucleophile and acyl donor concentration, ionic strength, and nucleophile and acyl donor concentration on the maximum yield. The initial conditions used were 25 mM Bz-ala-OMe, 25 mM Tyr-NH(2), 70 degrees C, pH 8.0, and 10% v/v dimethylformamide (DMF). The optimum conditions were 90% v/v DMF using 80 mM bz-Ala-OMe and 615 mM Tyr-NH(2) at 40 degrees C and pH 10. These conditions increased the maximum conversion from 0.75% to 26% (of the original ester concentration). In a number of other cosolvents, the best peptide yields were observed with acetonitrile and ethyl acetate. In 90% acetonitrile similar yields were observed to those in 90% DMF under optimized conditions except that the acyl donor and nucleophile concentrations could be reduced to 25 mM and 100mM, respectively. The effect of the blocking group on the nucleophile was also investigated; -betaNA and -pNA as blocking groups improved the yields markedly. The blocking and leaving groups of the acyldonor had no effect on the dipeptide yield. (c) 1994 John Wiley & Sons, Inc.     

Synthesis of ethyl isovalerate using Rhizomucor miehei lipase: optimization

Immobilized lipase from Rhizomucor miehei (Lipozyme IM-20) was used to catalyze the esterification reaction between isovaleric acid and ethanol to synthesize ethyl isovalerate in n-hexane. Response surface methodology based on five-level four-variable central composite rotatable design was employed to optimize four important reaction variables such as enzyme/substrate E/S ratio, substrate concentration, incubation time, and temperature affecting the synthesis of ethyl isovalerate. The optimum conditions predicted for achieving maximum ester yield (500 mM) are as follows: E/S ratio, 48.41 g/mol; substrate concentration, 1 M; reaction time, 60 h; temperature, 60 degrees C. The predicted value matched well with experimentally obtained value of 487 mM.     

Kinetics of inhibition of thermolysin-catalyzed peptide synthesis by alcohols in aqueous organic one-phase system

The kinetic patterns and parameters of 12 alcoholic organic solvents of different classes inhibiting thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-phenylalanine methyl ester (Z-Phe-Phe-OMe) in aqueous organic one-phase reaction system have been determined. All alcohols showed a linear mixed type inhibition. A kinetic model of inhibition is suggested. It was presumed that alcohols interact with substrate in the active site of thermolysin.     

A versatile esterase from Bacillus subtilis: cloning, expression, characterization, and its application in biocatalysis

An esterase from Bacillus subtilis DSM402 (BS2) was cloned and functionally expressed in E. coli. The enzyme is active up to 50 degrees C, and the V(max) (1449 mM/min) and K(M) values (119 mM) were determined using p-nitrophenyl acetate as substrate. BS2 belongs to the few hydrolases that can act on tertiary alcohols and was therefore used to resolve racemic acetates of selected tertiary alcohols, but also to selectively remove the tert-butyl ester protecting group from peptides. In addition, the enzyme shows promiscuous amidase activity.