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     

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.     

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.     

Enantioselective synthesis ofN-acetyl-l-methionine with aminoacylase in organic solvent

We have developed an enzymatic procedure for the enantiospecific synthesis ofN-acetyl-l-methionine with aminoacylase in an organic solvent.N-Acetyl-l-methionine was most effectively synthesized with a yield of about 90% (on the basis of thel-methionine used) when the reaction mixture, composed of 100 mm sodium acetate, 20 MMdl-methionine and aminoacylase (1000 units) immobilized on celite in 1 ml ethyl acetate saturated with 32 l 140mm sodium phosphate buffer (pH 7.0) containing 0.1 mm CoCl₂, was incubated at 30°C for 24 h.N-Acetyl-l-methionine was isolated from the reaction mixture and the enantiomeric excess was 100%.d-Methionine was also isolated from the mixture with a yield of about 95% and 90% enantiomeric excess. The method is applicable to the synthesis of otherN-acetyl-l-amino acids.     

Production of l-phenylalanine from phenylpyruvate by Paracoccus denitrificans containing aminotransferase activity

Summary To establish an efficient production method for l-phenylalanine, the production of l-phenylalanine from phenylpyruvate by Paracoccus denitrificans pFPr-1 containing aminotransferase activity was investigated. By using intact cells, 0.74M l-phenylalanine was produced from 0.8M phenylpyruvate (conversion yield, 92.5%). Moreover, by using immobilized cells with -carrageenan, when the space velocity was 0.1 h^-1 at 30°C, 0.135 M l-phenylalanine was produced from 0.15 M phenylpyruvate (conversion yield, 90%). The half-life of the l-phenylalanine-forming activity of the column was estimated to be about 30 days at 30°C.     

Subtilisin-catalysed peptide synthesis and transesterification in organic solvents

Summary Subtilisin from Bacillus subtilis was modified with polyethylene glycol (PEG), or adsorbed either on celite or porous glass, or directly used as a suspended powder to catalyse peptide synthesis and transesterification reactions in organic solvents. The rather low yield of peptide synthesis probably resulted from the enzyme tendency to catalyse hydrolysis and transesterification side reactions. The kinetics of transesterification catalysed by PEG-subtilisin was consistent with a ping-pong mechanism modified by a hydrolytic branch. Initial rates of transesterification were found to be dependent on alcohol and organic base concentrations in the reaction mixture. The high affinity of benzyloxycarbonyl-l-serine-methyl ester for the enzyme indicated that a change in substrate specificity of subtilisin occurred in organic phase. The 50-fold increase in the rate of synthesis of benzyloxycarbonyl-l-serine-l-phenylalanine amide which was observed when PEG-subtilisin was used instead of immobilized or powdered enzyme, suggested that a higher flexibility of the polypeptide chain modified by the covalent attachment of a number of soluble PEG moieties occurred in organic solvents. This also resulted in a lower stability of PEG-subtilisin at high temperature.Offprint requests to: A. Puigserver     

A mutant leucine aminopeptidase from <Emphasis Type="Italic">Streptomyces cinnamoneus</Emphasis> with enhanced <Emphasis Type="SmallCaps">l</Emphasis>-aspartyl <Emphasis Type="SmallCaps">l</Emphasis>-amino acid methyl ester synthetic activity

l-Aspartyl l-amino acid methyl ester was synthesized using a mutant of a thermostable leucine aminopeptidase from Streptomyces cinnamoneus, D198 K SSAP, obtained in previously. A peptide of high-intensity sweetener, l-aspartyl-l-phenylalanine methyl ester, was selected as a model for demonstrating the synthesis of l-aspartyl l-amino acid methyl ester. The hydrolytic activities of D198 K SSAP toward l-aspartyl-l-phenylalanine and its methyl ester were, respectively, 74-fold and fourfold higher than those of wild type. Similarly, the initial rate of the enzyme for l-aspartyl-l-phenylalanine methyl ester synthesis was over fivefold higher than that of wild-type SSAP in 90% methanol (v/v) in a one-pot reaction. Furthermore, other l-aspartyl l-amino acid methyl esters were synthesized efficiently using D198 K SSAP. Results show that the substitution of Asp198 of SSAP with Lys is effective for synthesizing l-aspartyl l-amino acid methyl ester.     

Synthesis of aspartame precursor with an immobilized thermolysin in tert-amyl alcohol

N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the synthetic sweetner asparatame, was synthesized from N-(benzyloxycarbolyl)-L-aspartic acid (Z-Asp) and L-phenylalanine methyl ester (PheOMe) with an immobilized thermolysin in various organic solvents. We found that in tert-amyl alcohol containing a small amount of water the immobilized enzyme showed a high activity comparble to that in ethyl acetate with quite a high stability. The immobilized enzyme was fully stable up to 70 degrees C in tert-amyl alcohol in the absence of the subatrate, and up to 50 degrees C in the presence of the substrate. The high stability in the presence of the substrate was found due to the fact that the release of calcium ions, the stabilizing factor of thermolysin, is suppressed.The substrate concentration dependence of the initial synthetic rate with the immobilized enzyme was quite different from that with the free enzyme in the biphasic system, in contrast to that in ethyl acetate. Finally, Z-AspPheOMe was continuously synthesized in a column reactor using 200 mM PheOMe and 120 mM Z-Asp as the substrate for over 300 h at 45 degrees C and a space velocity of 1 h(-1) without any loss of acivity. (c) 1994 John Wiley & Sons, Inc.     

Purification,characterization and regulation of a monomeric l-phenylalanine dehydrogenase from the facultative methylotroph Nocardia sp. 239

In Nocardia sp. 239 d-phenylalanine is converted into l-phenylalanine by an inducible amino acid racemase. The further catabolism of this amino acid involves an NAD-dependent l-phenylalanine dehydrogenase. This enzyme was detected only in cells grown on l- or d-phenylalanine and in batch cultures highest activities were obtained at relatively low amino acid concentrations in the medium. The presence of additional carbon- or nitrogen sources invariably resulted in decreased enzyme levels. From experiments with phenylalanine-limited continuous cultures it appeared that the rate of synthesis of the enzyme increased with increasing growth rates. The regulation of phenylalanine dehydrogenase synthesis was studied in more detail during growth of the organism on mixtures of methanol and l-phenylalanine. Highest rates of l-phenylalanine dehydrogenase production were observed with increasing ratios of l-phenylalanine/methanol in the feed of chemostat cultures. Characteristic properties of the enzyme were investigated following its (partial) purification from l- and d-phenylalanine-grown cells. This resulted in the isolation of enzymes with identical properties. The native enzyme had a molecular weight of 42 000 and consisted of a single subunit; it showed activity with l-phenylalanine, phenylpyruvate, 4-hydroxyphenyl-pyruvate, indole-3-pyruvate and -ketoisocaproate, but not with imidazolepyruvate, d-phenylalanine and other l-amino acids tested. Maximum activities with phenylpyruvate (310 mol min^-1 mg^-1 of purified protein) were observed at pH 10 and 53°C. Sorbitol and glycerol stabilized the enzyme.Abbreviations RuMP ribulose monophosphate - HPS hexulose-6-phosphate synthase - HPT hexulose-6-phosphate isomerase - FPLC fast protein liquid chromatography     

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.     

Resolution of d,l-menthol by interesterification with triacetin using the free and immobilized lipase of Candida cylindracea

Summary Interesterification in isooctane with triacetin as an acyl donor was found to be a new and effective method of racemic resolution of d,l-menthol, when using the free and immobilized lipase of Candida cylindracea. No water was produced by this highly stereoselective type of reaction in contrast to ester synthesis with acetic acid as an acyl donor. Even with diacetin no possible back reaction occurred and the enzyme was easily separated from the reaction solution as opposed to ester hydrolysis in aqueous systems. Inhibition of interesterification was caused by increasing concentrations of the acyl donor triacetin by more than 10 mmol·l^-1 on the one hand, and especially by diacetin on the other hand. The reaction product menthyl acetate had no influence. By adding water the interesterification activity of the lipase was reduced significantly. An alteration of the acyl donor triacetin to longerchained triglycerides caused changes in higher specific activities but poor enantioselectivities of the products, as in the case of ester synthesis starting from longer-chained organic acids.Dedicated to Prof. Dr. Fritz Wagner on the occasion of his 60th birthday     

Hollow-Fibre Enzyme Reactor Integrated with Solvent Extraction for Synthesis of Aspartame Precursor

A new process for the simultaneous enzymic synthesis and purification of N-(benzyloxycarbonyl)- -aspartyl- -phenylalanine methyl ester (ZAPM), a precursor of aspartame, has been developed. The enzymic reaction between N-(benzyloxycarbonyl)- -aspartic acid (ZA) and -phenylalanine methyl ester (PM) was carried out in a biphasic hollow-fibre rector with an aqueous phase an a butyl acetate phase. The reaction took place in the aqueous phase and by maintaining the pH at 5, the product (ZAPM) was extracted into the organic phase. Product purity was greater than 90% and reasonable productivity could be achieved with this system.     

A new approach to preparative enzymatic synthesis

A new approach to preparative organic synthesis in aqueous–organic systems is suggested. It is based on the idea that the enzymatic process is carried out in a biphasic system “water–water-immiscible organic solvent.” Thereby the enzyme is localized in the aqueous phase—this eliminates the traditional problem of stabilizing the enzyme against inactivation by a nonaqueous solvent. Hence, in contrast to the commonly used combinations “water–water-miscible organic solvent,” in the suggested system the content of water may be infinitely low. This allows one to dramatically shift the equilibrium of the reactions forming water as a reaction product (synthesis of esters and amides, polymerization of amino acids, sugars and nucleotides, dehydration reactions, etc.) toward the products. The fact that the system consists of two phases provides another very important source for an equilibrium shift, i.e., free energies of the transfer of a reagent from one phase to the other. Equations are derived describing the dependence of the equilibrium constant in a biphasic system on the ratio of the volumes of the aqueous and nonaqueous phases and the partition coefficients of the reagents between the phases. The approach has been experimentally verified with the synthesis of N-acetyl-L -tryptophan ethyl ester from the respective alcohol and acid. Porous glass was impregnated with aqueous buffer solution of chymotrypsin and suspended in chloroform containing N-acetyl-L -tryptophan and ethanol. In water (no organic phase) the yield of the ester is about 0.01%, whereas in this biphasic system it is practically 100%. The idea is applicable to a great number of preparative enzymatic reactions.     

Isobutene production by Rhodotorula minuta

Summary Isobutene production by Rhodotorula minuta IFO 1102 was studied. It was confirmed that the gas species produced by this yeast was isobutene from the result of analysis with a gas chromatograph mass spectrometer. Oxygen supply was essential to the microbial production of isobutene. The optimum pH was found to be approximately pH 6.0 and optimum temperature 25°–27° C. Isobutene production rate was maximal when l-leucine and l-phenylalanine in the medium were being uptaken by the yeast.The results from an investigation of the role of l-leucine and l-phenylalanine suggested that l-leucine was the precursor of isobutene and l-phenylalanine the inducer for the enzyme concerned with isobutene production.     

l-Phenylalanine dehydrogenase from Brevibacterium sp. for production of l-phenylalanine by reductive amination of phenylpyruvate

Summary For production of l-phenylalanine the reductive amination of phenylpyruvate, catalyzed by phenylalanine-dehydrogenase was examined. To reach high levels and a sufficient stability of the inducible intracellular enzyme, growth conditions of Brevibacterium sp. are optimized. For continuous production of l-phenylalanine in an enzyme membrane reactor, the kinetic parameters of the partially purified enzyme are determined.In continuous production a space time yield of 37.4 g l-Phe l^-1 d^-1 can be reached.By means of the measured kinetic parameters and simultaneous calculation of the mass balances of all reaction components the behaviour of the reactor can be simulated. For certain conditions the multi-enzyme-system shows multiple steady-states.Abbreviations l-phe l-phenylalanine - phepy phenylpyruvate - PEG polyethylenglycol - pheDH l-phenylalanine dehydrogenase     

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.     

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.     

Enzymes in preparative organic synthesis: Coincidence of the pH optimum for catalyst effectiveness with the pH optimum for the catalyzed reaction equilibrium

The study concerned the pH profile of the apparent equilibrium constant for synthesis of N-benzoyl-L -phenylalanine ethyl ester from the respective acid and ethanol in the biphasic system chloroform + 5% (v/v) water. The substitution of water (as a reaction medium) for the biphasic aqueous–organic system shifted the pH profile toward neutral pH values. As a result the pH range thermodynamically conducive to synthesis of the final product in the biphasic system coincided with the pH optimum of the catalytic activity of the enzyme used (α-chymotrypsin). This approach should, in principle, be considered as general: first, per se it is independent of a catalyst (enzyme) nature; second, the biphasic method helps the shift ionic equilibria involving not only organic acids, but also bases. A physical mechanism of the ionic equilibrium shift is the same is both cases, namely, a preferable extraction from water into an organic phase of one generally nonionic (more hydrophobic) form of the reagent.     

Enantioselective ester hydrolase from Sphingobacterium sp. 238C5 useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis

A novel enzyme, β-phenylalanine ester hydrolase, useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis was characterized. The enzyme purified from the cell free-extract of Sphingobacterium sp. 238C5 well hydrolyzed β-phenylalanine esters (S)-stereospecifically. Besides β-phenylalanine esters, the enzyme catalyzed the hydrolysis of several α-amino acid esters with l-stereospecificity, while the deduced 369 amino acid sequence of the enzyme exhibited homology to alkaline d-stereospecific peptide hydrolases from Bacillus strains. Escherichia coli transformant expressing the β-phenylalanine ester hydrolase gene exhibited an about 8-fold increase in specific (S)-β-phenylalanine ethyl ester hydrolysis as compared with that of Sphingobacterium sp. 238C5. The E. coli transformant showed (S)-enantiomer specific esterase activity in the reaction with a low concentration (30 mM) of β-phenylalanine ethyl ester, while it showed both esterase and transpeptidase activity in the reaction with a high concentration (170 mM) of β-phenylalanine ethyl ester and produced β-phenylalanyl-β-phenylalanine ethyl ester. This transpeptidase activity was useful for β-phenylalanine β-peptide synthesis.     

Isolation of l-phenylalanine dehydrogenase from Rhodococcus sp. M4 and its application for the production of l-phenylalanine

Summary l-Phenylalanine dehydrogenase [l-phenylalanine: NAD⁺-oxidoreductase (deaminating)] of Rhodococcus sp. strain M4 was studied emphasizing its application for the production of l-phenylalanine. A high enzyme level (30,000 U·l^-1, 25–30 U·mg^-1 in the crude extract) could be reached during aerob degradation of l-phenylalanine (10 g·l^-1) under optimized growth coditions. A partial purification of the intracellular enzyme by liquid-liquid extraction, and DEAE-cellulose led to a specific activity of more than 1300 U·mg^-1. The continuous production of l-phenylalanine in an enzyme-membrane-reactor for 350h resulted in a space-time yield of 456 g·l^-1·d^-1 with a mean substrate conversion of 95%. Consumption of phenylalanine dehydrogenase was 1,500 U·kg Phe^-1.Abbreviations BSA bovine serum albumine - pheDH l-phenylalanine dehydrogenase - phepyr phenylpyruvate - OD optical density - FDH formate dehydrogenase