A study on the enzyme catalysed enantioselective hydrolysis of methyl 2-methyl-4-oxopentanoate,a precursor of chiral γ-butyrolactones

Porcine pancreas lipase (PPL) resolution of the α-methyl group of racemic methyl 2-methyl-4-oxopentanoate, a valuable synthetic precursor of fragrances and marine natural products, was enhanced by salt modulation of the enzymatic hydrolysis. For the enantioselective hydrolysis of the title ester, PPL was selected from a series of esterases and lipases, and its enantioselectivity was evaluated by changing the reaction medium parameters. The use of 1.6?mol L^–1 sodium sulfate in phosphate buffer (pH 7.2) improved the enantioselectivity allowing the formation of methyl (2R)-(+)-2-methyl-4-oxopentanoate and (2S)-(–)-2-methyl-4-oxopentanoic acid with an enantiomeric excess of >99% and 71%, respectively. The study showed that a modulation of PPL enantioselectivity could be achieved by using kosmotropic salts in the reaction media. The present method consists of a practical and low-cost option to improve enzymatic kinetic resolution reactions.     

Resolution of 2-octanol via immobilized Pseudomonas sp. lipase in organic medium

Abstract

Pseudomonas sp. lipase (PSL) immobilization was performed using three different protocols. Lipase immobilized on Diaion HP20 (HP20-PSL) exhibited the highest catalytic activity and stability in the kinetic resolution of racemic 2-octanol. The reaction rate of HP20-PSL was approximately 20 times higher than that of free PSL and the residual activities of HP20-PSL and free PSL were respectively 84% and 19% after incubation in the reaction medium for 72 h. A study of the effect of different reaction parameters on HP20-PSL-catalyzed resolution of (R,S)-2octanol showed that the optimal water content of the immobilized matrix and the optimal molar ratio of vinyl acetate to 2-octanol were 60 ± 5% and 2.5:1, respectively. Under the optimized reaction conditions, (S)-2-octanol of high optically purity (enantiomeric excess > 99%) could be recovered at 53% conversion rate, and HP20-PSL could be reused for ten cycles without significant decrease in its activity and enantioselectivity.     

Improved production of (R)-2-octanol via asymmetric reduction of 2-octanone with Oenococcus oeni CECT4730 in a biphasic system

Abstract

Oenococcus oeni CECT4730, which catalyses the asymmetric reduction of 2-octanone to (R)-2-octanol with high enantioselectivity, was further studied to exploit its potential for production of (R)-2-octanol in an aqueous/organic solvent biphasic system. Variables such as the volume ratio of aqueous to organic phase (V_a/V_o), buffer pH, reaction temperature, shaking speed, co-substrates and the ratio of biocatalyst to substrate were examined with respect to the molar conversion, the initial reaction rate and the product enantiomeric excess (e.e.). Under the optimized conditions (V_a/V_o=1:1 (v/v), buffer pH=8.0, reaction temperature=30°C, shaking speed=150 rev/min, ratio of glucose to biomass=5.4:l (w/w), ratio of biocatalyst to substrate=0.51:l (g/mol)), the highest space time yield of (R)-2-octanol, 24 mmol L^?1 per h, and >98% product e.e. were obtained at a substrate concentration close to 1.0 mol L^?1 after 24 h reduction.     

复合诱变选育高产脂肪酶黑曲霉菌株及其固定化酶性质

通过硫酸二乙酯(DES)和微波复合诱变,获得遗传性状稳定的高产脂肪酶黑曲霉突变菌株CM2,酶活达174.93 U/mL.对菌株CM2培养条件的优化,以橄榄油和(NH_4)_2SO_4为最佳碳、氮源,在28℃、pH 7.5的条件下,发酵CM2菌株68 h,脂肪酶活为180.52 U/mL.大孔树脂固定化脂肪酶在35～55℃和pH 7.5～9.5之间有很好的稳定性.游离酶和固定化酶的表观失活活化能分别为52.6842 kJ/mol和30.8391 kJ/mol,固定化酶对温度的敏感度降低,耐受性增强.在微水相中脂肪酶催化2-辛醇和乙酸乙烯酯不对称酯交换反应中,(S)-乙酸辛酯的对映选择性高(游离酶e.e.s 85.7%;固定化酶e.e.s 87.7%),显示了该固定化酶在2-辛醇的手性拆分方面具有良好的应用前景.     

Enantioselective synthesis of phenylacetamides in the presence of high organic cosolvent concentrations catalyzed by stabilized penicillin G acylase. Effect of the acyl donor

Immobilization of penicillin G acylase on glyoxyl agarose and its further hydrophilization by physicochemical modification with ionic polymers has made it possible to perform the direct condensation between (+/-)-2-hydroxy-2-phenylethylamine [(+/-)-1] and different acyl donors in the presence of high concentrations of organic cosolvent (up to 90%) in the reaction medium. Using 50 mM phenyl acetic acid and these drastic reaction conditions, too harsh for any other PGA preparation, we have achieved an almost quantitative transformation (more than 99%) of 10 mM (+/-)-1 into the corresponding amide. Remarkably, the enantioselectivity of the enzyme immobilized on the amine was strongly dependent on the acyl donor employed. Thus, using phenylacetic acid (2), the enantioselectivity was almost negligible (1.3 favoring the S isomer), whereas using S-mandelic acid [(S)-4], the E factor reached a value of 21 (also favoring the S isomer). By using R-mandelic acid [(R)-4], we observed a different enantioselectivity (E was 3.6 favoring the R). At 4 degrees C, the E value reached a value higher than 100 when (S)-4 was used as the acyl donor. The reaction performed under these conditions allowed us to produce (2S,2'S)-N-2'-hydroxy-2'-phenyl)-2-hydroxyphenylacetamide [(2S,2'S)-7] with a diasteromeric excess higher than 98%.     

The stereoselectivity and catalytic properties of Xanthobacter autotrophicus 2-[(R)-2-Hydroxypropylthio]ethanesulfonate dehydrogenase are controlled by interactions between C-terminal arginine residues and the sulfonate of coenzyme M

2-[(R)-2-Hydroxypropylthio]ethanesulfonate (R-HPC) dehydrogenase (DH) catalyzes the reversible oxidation of R-HPC to 2-(2-ketopropylthio)ethanesulfonate (2-KPC) in a key reaction in the bacterial conversion of chiral epoxides to beta-keto acids. R-HPCDH is highly specific for the R-enantiomer of HPC, while a separate enzyme, S-HPCDH, catalyzes the oxidation of the corresponding S-enantiomer. In the present study, the features of substrate and enzyme imparting stereospecificity have been investigated for R-HPCDH. S-HPC was a substrate for R-HPCDH with a K(m) identical to that for R-HPC but with a k(cat) 600 times lower. Achiral 2-propanol and short-chain (R)- and (S)-2-alkanols were substrates for R-HPCDH. For (R)-alkanols, as the carbon chain length increased, K(m) decreased, with the K(m) for (R)-2-octanol being 1700 times lower than for 2-propanol. At the same time, k(cat) changed very little and was at least 90% lower than k(cat) for R-HPC and at least 22 times higher than k(cat) for S-HPC. (S)-2-Butanol and (S)-2-pentanol were substrates for R-HPCDH. The K(m) for (S)-2-butanol was identical to that for (R)-2-butanol, while the K(m) for (S)-2-pentanol was 7.5 times higher than for (R)-2-pentanol. Longer chain (S)-2-alkanols were sufficiently poor substrates for R-HPCDH that kinetic parameters could not be determined. Mutagenesis of C-terminal arginine residues of R-HPCDH revealed that R152 and R196 are essential for effective catalysis with the natural substrates R-HPC and 2-KPC but not for catalysis with 2-alkanols or ketones as substrates. Short-chain alkylsulfonates and coenzyme M (2-mercaptoethanesulfonate) were found to modify the kinetic parameters for 2-butanone reduction by R-HPCDH in a saturable fashion, with the general effect of increasing k(cat), decreasing K(m), and increasing the enantioselectivity of 2-butanone reduction to a theoretical value of 100% (S)-2-butanol. The modulating effects of ethanesulfonate and propanesulfonate provided thermodynamic binding constants close to K(m) for the natural substrates R-HPC and 2-KPC. The effects of alkylsulfonates on modulating the enantioselectivity and kinetic properties of R-HPCDH were abolished in R152A and R196A mutants but not in mutants of other C-terminal arginine residues. Collectively, the results suggest that interactions between the sulfonate of CoM and specific arginine residues are key to the enantioselectivity and catalytic efficiency of R-HPCDH. A model is proposed wherein sulfonate-arginine interactions within an alkylsulfonate binding pocket control the catalytic properties of R-HPCDH.     

Resolution of 4-amino-cyclopentanecarboxylic acid methyl esters using hydrolytic enzymes.

A number of esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3) of microbial and mammalian origin were screened for the ability to resolve racemic 4-amino-cyclopentanecarboxylic acid methyl ester derivatives as potential intermediates in the production of carbocyclic nucleosides. Surprisingly, functionalization of the remote amino group had a profound effect on both the rate and enantioselectivity of hydrolysis of the methyl ester. 4-(Benzoylamino)-2-cyclopentenecarboxylic acid, methyl ester (V) with pig liver esterase gave the highest enantioselectivity. The residual ester, which was of the correct absolute stereochemistry [(+) 1S, 4R] for carbocyclic nucleoside synthesis, could be obtained in high optical purity. Optimization of pH, solvent type, and concentration improved the enantioselectivity of the process by a further twofold.     

Diastereoselective synthesis of (R)-(alkyl)-beta-D-galactopyranoside by using beta-galactosidase (Aspergillus oryzae) in low-water media

A beta-galactosidase (from Aspergillus oryzae) preparation viz. EPRP (enzyme precipitated and rinsed with propanol), obtained by the removal of bulk water by precipitation with n-propanol, showed higher biological activity than the lyophilized powder. FT-IR study confirmed that EPRP had retained the alpha-helical content of the native structure better than the lyophilized form. Use of this formulation of beta-galactosidase under low water conditions (temperature 55 degrees C, reaction time of 4 h) gave enantioselectivity, E > 1000 for the stereoselective synthesis of (R)-(1-phenylethyl)-beta-D-galactopyranoside, starting from racemic 1-phenylethanol and D-galactose. For racemic 2-octanol also, EPRP worked better. Under similar conditions, (R)-(2-octyl)-beta-D-galactopyranoside was formed with an enantioselectivity, E = 38.     

Substrate specificity and stereoselectivity of horse liver alcohol dehydrogenase. Kinetic evaluation of binding and activation parameters controlling the catalytic cycles of unbranched, acyclic secondary alcohols and ketones as substrates of the native and active-site-specific Co(II)-substituted enzyme

1. The steady-state parameters kcat and Km and the rate constants of hydride transfer for the substrates isopropanol/acetone; (S)-2-butanol, (R)-2-butanol/2-butanone; (S)-2-pentanol, (R)-2-pentanol/2-pentanone; 3-pentanol/3-pentanone; (S)-2-octanol and (R)-2-octanol have been determined for the native Zn(II)-containing horse-liver alcohol dehydrogenase (LADH) and the specific active-site-substituted Co(II)LADH. 2. A combined evaluation of steady-state kinetic data and rate constants obtained from stopped-flow measurements, allowed the determination of all rate constants of the following ordered bi-bi mechanism: E in equilibrium E.NAD in equilibrium E.NAD.R1R2 CHOH in equilibrium E.NADH.R1R2CO in equilibrium E.NADH in equilibrium E. 3. On the basis of the different substrate specificities of LADH and yeast alcohol dehydrogenase (YADH), a procedure has been developed to evaluate the enantiomeric product composition of ketone reductions. 2-Butanone and 2-pentanone reductions revealed (S)-2-butanol (86%) and (S)-2-pentanol (95%) as the major products. 4. The observed enantioselectivity implies the existence of two productive ternary complexes; E.NADH.(pro-S) 2-butanone and E.NADH.(pro-R) 2-butanone. All rate constants describing the kinetic pathways of the system (S)-2-butanol, (R)-2-butanol/2-butanone have been determined. These data have been used to estimate the expected enantiomer product composition of 2-butanone reductions using apparent kcat/Km values for the two different ternary-complex configurations of 2-butanone. Additionally, these data have been used for computer simulations of the corresponding reaction cycles. Calculated, simulated and experimental data were found to be in good agreement. Thus, the system (S)-2-butanol, (R)-2-butanol/2-butanone is the first example of a LADH-catalyzed reaction for which the stereochemical course could be described in terms of rate constants of the underlying mechanism. 5. The effects of Co(II) substitution on the different steps of the kinetic pathway have been investigated. The free energy of activation is higher for alcohol oxidation and lower for ketone reduction when catalyzed by Co(II)LADH in comparison to Zn(II)LADH. However, the free energies of binding are affected by metal substitution in such a way that the enantioselectivity of ketone reduction is not significantly changed by the substitution of Co(II) for Zn(II). 6. Evaluation of the data shows that substrate specificity and stereoselectivity result from combination of the free energies of binding and activation, with differences in binding energies as the dominating factors. In this regard, the interactions of substrate molecules with the protein moiety are dominant over the interactions with the catalytic metal ion.     

Improving lipase enantioselectivity in organic solvents by forming substrate salts with chiral agents

We recently demonstrated (J Am Chem Soc 121:3334-3340, 1999) that enzymatic enantioselectivity in organic solvents can be markedly enhanced by temporarily enlarging the substrate via salt formation. In the present study, this approach was expanded by finding that, in addition to its size, the stereochemistry of the counterion can greatly affect the enantioselectivity enhancement. For example, the enantioselectivity [E = (k(cat)/K(M))(S)/(k(cat)/K(M))(R)] of crystalline Pseudomonas cepacia lipase in the propanolysis of phenylalanine methyl ester (PheOMe) in anhydrous acetonitrile was found to be 5.8 +/- 0.6; the E value doubled when PheOMe's salt with S mandelic acid was used as a substrate instead of the free ester, and rose sevenfold with R mandelic acid as a Bronsted-Lowry acid. Similar effects were observed with other bulky, but not petite, counterions. The greatest enantioselectivity enhancement was afforded by 10-camphorsulfonic acid: the E value increased to 18 +/- 2 for a salt with its R enantiomer and jumped to 53 +/- 4 for the S. These effects, also observed in other organic solvents, were explained by means of structure-based molecular modeling of the lipase-bound transition states of the substrate enantiomers and their diastereomeric salts.     

Enzymatic and chromatographic chiral discrimination of racemic (thio)glycidyl esters: Part I. A chemoenzymatic approach to both enantiomers of thioglycidyl esters

Both hitherto unknown (+)-(R)- and (?)-(S)-thioglycidyl esters, (R)-( 2 ) and (S)-( 2 ), have been synthesized with different high enantiomeric excesses (ee) by two routes from the corresponding rac-glycidyl esters rac-( 1 ). The first includes a porcine pancreatic lipase (PPL)-mediated kinetic resolution of these esters followed by sulfuration with practically complete inversion to the (+)-(R)-enantiomer (+)-(R)-( 2 ) (36–86% ee). (?)-(S)-Thioglycidyl esters (?)-(S)-( 2 ) are obtained by the reverse reaction sequence (43–80% ee). In the latter case the hydrolysis rate is lower than that of analogous glycidyl esters. Moreover, the dependence of enantiomeric excess on the size of the acyl-group is of the opposite tendency. Therefore, in both cases suitable selection of the acid residue gives rise to maximum enantioselectivity. The irreversible lipase-catalyzed acylation of rac-glycidol and rac-thioglycidol, however, was found to be a less suitable alternative. The enantiomeric excess of recovered homochiral esters was determined by chiral chromatography using modified cellulose stationary phases (OB, OD). © 1993 Wiley-Liss, Inc.     

Resolution of 2-octanol by SBA-15 immobilized Pseudomonas sp. lipase

Lipase from Pseudomonas sp. (PSL) was immobilized on SBA-15 (a highly ordered hexagonal array mesoporous silica molecular sieve) through physical adsorption and the immobilized PSL was used in resolution of (R,S)-2-octanol with vinyl acetate as acyl donor. Enhanced activity and enantioselectivity were observed for the immobilized PSL compared with those of the free one. The effects of reaction conditions, such as solvents, temperature, water activity and substrate ratio were investigated. Under the optimum conditions, the residual (S)-2-octanol was recovered with 99% enantiomeric excess at 52% conversion. The results also indicated that the immobilized PSL maintained 90% of its initial activity even after reusing it five times.     

Improvement of catalytic activity of Candida rugosa lipase in the presence of calix[4]arene bearing iminodicarboxylic/phosphonic acid complexes modified iron oxide nanoparticles

In the present study, iron oxide magnetite nanoparticles, prepared through a co-precipitation method, were coated with phosphonic acid or iminodicarboxylic acid derivatives of calix[4]arene to modulate their surfaces with different acidic groups. Candida rugosa lipase was then directly immobilized onto the modified nanoparticles through sol–gel encapsulation. The catalytic activities and enantioselectivities of the two encapsulated lipases in the hydrolysis reaction of (R/S)-naproxen methyl ester and (R/S)-2-phenoxypropionic acid methyl ester were assessed. The results showed that the activity and enantioselectivity of the lipase were improved when the lipase was encapsulated in the presence of calixarene-based additives; the encapsulated lipase with the phosphonic acid derivative of calix[4]arene had an excellent rate of enantioselectivity against the (R/S)-naproxen methyl and (R/S)-2-phenoxypropionic acid methyl esters, with E = 350 and 246, respectively, compared to the free enzyme. The encapsulated lipases (Fe-Calix-N(COOH)) and (Fe-Calix–P) showed good loading ability and little loss of enzyme activity, and the stability of the catalyst was very good; they only lost 6–11% of the enzyme’s activity after five batches.     

Microwave-assisted enzymatic resolution of (R,S)-2-octanol in ionic liquid

Combination use of microwave irradiation (MW) as heating mode and ionic liquid (IL) as reaction medium in enzymatic resolution of (R,S)-2-octanol with vinyl acetate as the acyl donor through transesterification by Novozym 435 was investigated. A synergistic effect of MW and IL [EMIM][NTf2], which was screened as the best reaction medium for this reaction, on improving enzyme activity and enantioselectivity was observed. The activity and enantioselectivity of Novozym 435 in [EMIM][NTf2] under MW were much higher than that in solvent free system under conventional heating, in solvent free system under MW, and in [EMIM][NTf2] under conventional heating, respectively. A systematic screening and optimization of the reaction parameters in [EMIM][NTf2] under MW were performed. Under the optimum conditions, 50% yield of (S)-2-octanol with 99% enantiomeric excess was obtained in 6 h. Furthermore, increased thermal stability and reusability of Novozym 435 under the combination use of MW and IL condition were also observed.     

Enantioselective synthesis of ibuprofen esters in AOT/isooctane microemulsions by Candida cylindracea lipase

The enantioselective esterification of racemic ibuprofen, catalyzed by a Candida cylindracea lipase, was studied in a water-in-oil microemulsion (AOT/isooctane). By using n-propanol as the alcohol, an optimal W(0) ([H(2)O]/[AOT] ratio) of 12 was found for the synthesis of n-propyl-ibuprofenate at room temperature. The lipase showed high preference for the S(+)-enantiomer of ibuprofen, which was esterified to the corresponding S(+)-ibuprofen ester. The R(-)-ibuprofen remained unesterified in the microemulsion. The calculated enantioselectivity value (E) for S-ibuprofen ester was greater than 150 (conversion 0.32). The enzyme activities of n-alcohols with different chain lengths (3-12) were compared, and it appeared that short- (propanol and butanol) and long-chained (decanol and dodecanol) alcohols were better substrates than the intermediate ones (pentanol, hexanol, and octanol). However, unlike secondary and tertiary alcohols, all of the tested primary alcohols were substrates for the lipase. The reversible reaction (i.e., the hydrolysis of racemic ibuprofen ester in the microemulsion) was also carried out enantioselectively by the enzyme. Only the S form of the ester was hydrolyzed to the corresponding S-ibuprofen. The reaction yield was, however, only about 4% after 10 days of reaction. The corresponding yield for the esterification of ibuprofen was about 35% (10 days). The high enantioselectivity displayed by the lipase in the microemulsion system was seen neither in a similar esterification reaction in a pure organic solvent system (isooctane) nor in the hydrolysis reaction in an aqueous system (buffer). The E value for S-ibuprofen ester in the isooctane system was 3.0 (conversion 0.41), and only 1.3 for S-ibuprofen in the hydrolysis reaction (conversion 0.32). The differences in enantioselectivity for the lipase in various systems are likely due to interfacial phenomena. In the microemulsion system, the water in which the enzyme is dissolved is separated from the solvent by a layer of surfactant molecules, thus creating an interface with a relatively large area. Such interfaces are not present in the pure organic solvent systems (no surfactant) nor in aqueous systems. (c) 1993 John Wiley & Sons, Inc.     

Improvement of the enantioselectivity and activity of lipase from Pseudomonas sp. via adsorption on a hydrophobic support: kinetic resolution of 2-octanol

Pseudomonas sp. lipase (PSL) was successfully immobilized on a novel hydrophobic polymer support through physical adsorption and the immobilized PSL was used for resolution of (R,S)-2-octanol with vinyl acetate as acyl donor. Enhanced activity and enantioselectivity were observed from the immobilized PSL compared with free PSL. The effects of reaction conditions such as temperature, water activity, substrate molar ratio and the amount of immobilized lipase were investigated. Under optimum conditions, the residual (S)-2-octanol was recovered with 99.5% enantiomeric excess at 52.9% conversion. The results also indicated that the immobilized PSL could maintain 94% of its initial activity even after reusing it five times.     

Efficient kinetic resolution of (R,S)-1-trimethylsilylethanol via lipase-mediated enantioselective acylation in ionic liquids

Efficient enantioselective acylation of (R,S)-1-trimethylsilylethanol {(R,S)-1-TMSE} with vinyl acetate catalyzed by immobilized lipase from Candida antarctica B (i.e., Novozym 435) was successfully conducted in ionic liquids (ILs). A remarkable enhancement in the initial rate and the enantioselectivity of the acylation was observed by using ILs as the reaction media when compared to the organic solvents tested. Also, the activity, enantioselectivity, and thermostability of Novozym 435 increased with increasing hydrophobicity of ILs. Of the six ILs examined, the IL C4MIm.PF6 gave the fastest initial rate and the highest enantioselectivity, and was consequently chosen as the favorable medium for the reaction. The optimal molar ratio of vinyl acetate to (R,S)-1-TMSE, water activity, and reaction temperature range were 4:1, 0.75, and 40 -50 degrees C, respectively, under which the initial rate and the enantioselectivity (E value) were 27.6 mM/h and 149, respectively. After a reaction time of 6 h, the ee of the remaining (S)-1-TMSE reached 97.1% at the substrate conversion of 50.7%. Additionally, Novozym 435 was effectively recycled and reused in C4MIm.PF6 for five consecutive runs without substantial lose in activity and enantioselectivity. The preparative scale kinetic resolution of (R,S)-1-TMSE in C4MIm.PF6 is shown to be very promising and useful for the industrial production of enantiopure (S)-1-TMSE.     

Different properties of the lipases contained in porcine pancreatic lipase extracts as enantioselective biocatalysts

The porcine pancreatic lipase (PPL) extracts contain a mixture of several lipases. Their fractioning was performed by sequential adsorption via interfacial activation on supports with different hydrophobicity. A protein of 25 KDa was preferentially adsorbed on octyl-Sepharose, another protein of 33 kDa was mainly adsorbed on octadecyl-Sepabeads support, and the PPL was mainly adsorbed on the support bearing phenyl groups. The different immobilized preparations showed different properties and different response due to change in the experimental conditions. Thus, in the hydrolysis of (+/-)-2-hydroxy-4-phenylbutyric acid ethyl ester [(+/-)-1] to produce the corresponding acid [2], the octyl-25KDa preparation showed the best enantioselectivity (E) value (E = 7) at pH 5 and 25 degrees C, whereas the phenyl-PPL was the most enantioselective (E = 10) at pH 5, 4 degrees C, and 10% dioxane. Using different preparations at different pHs it was possible to resolve (+/-)-2-O-butyryl-2-phenylacetic acid [(+/-)-3] with a high E value (E > 100); for example, with octadecyl-33 KDa enzyme at pH 8.     

微环境对脂肪酶催化拆分外消旋2－辛醇的影响

微环境对脂肪酶催化拆分外消旋２－辛醇的影响　　　　　　　杨红,曹淑桂,韩四平,黄仲丽,杨同书（吉林大学酶工程国家重点实验室,长春１３００２３）手性２－辛醇不仅是制备液晶材料不可缺少的重要手性原料,也是合成具有光学活性的医药和农药的重要手性中间体．本文...     

A two-step enzymatic resolution process for large-scale production of (S)- and (R)-ethyl-3-hydroxybutyrate

An efficient two-step enzymatic process for production of (R)- and (S)-ethyl-3-hydroxybutyrate (HEB), two important chiral intermediates for the pharmaceutical market, was developed and scaled-up to a multikilogram scale. Both enantiomers were obtained at 99% chemical purity and over 96% enantiomeric excess, with a total process yield of 73%. The first reaction involved a solvent-free acetylation of racemic HEB with vinylacetate for the production of (S)-HEB. In the second reaction, (R)-enriched ethyl-3-acetoxybutyrate (AEB) was subjected to alcoholysis with ethanol to derive optically pure (R)-HEB. Immobilized Candida antarctica lipase B (CALB) was employed in both stages, with high productivity and selectivity. The type of butyric acid ester influenced the enantioselectivity of the enzyme. Thus, extending the ester alkyl chain from ethyl to octyl resulted in a decrease in enantiomeric excess, whereas using bulky groups such as benzyl or t-butyl, improved the enantioselectivity of the enzyme. A stirred reactor was found unsuitable for large-scale production due to attrition of the enzyme particles and, therefore, a batchwise loop reactor system was used for bench-scale production. The immobilized enzyme was confined to a column and the reactants were circulated through the enzyme bed until the targeted conversion was reached. The desired products were separated from the reaction mixture in each of the two stages by fractional distillation. The main features of the process are the exclusion of solvent (thus ensuring high process throughput), and the use of the same enzyme for both the acetylation and the alcoholysis steps. Kilogram quantities of (S)-HEB and (R)-HEB were effectively prepared using this unit, which can be easily scaled-up to produce industrial quantities.