Immobilization of Bacillus subtilis α-amylase on zirconia-coated alkylamine glass with glutaraldehyde

Bacillus subtilis α-amylase (EC 3.2.1.1) has been immobilized on zirconia-coated alkylamine glass by using the process of glutaraldehyde coupling. The immobilized enzyme preparation exhibited 52% of the initial enzyme activity and a conjugation yield of 28 mg/g support. The K_m value of the immobilized α-amylase was decreased by immobilization while V_max was unaltered. E_a of the enzyme was decreased upon conjugation. The soluble enzyme was optimally active at pH 5.6 while the immobilized enzyme exhibited optimal activity in the pH range 5.4–6.2. The alkylamine-immobilized enzyme has also been characterized through its isoelectric point. The industrial importance of this work is discussed.     

Purification and characterization of methylglyoxal reductase from Hansenula mrakii

Methylglyoxal reductase was purified from Hansenula mrakii IFO 0895 to a homogenous state on polyacrylamide gel electrophoresis. The enzyme consisted of a single polypeptide chain with a molecular weight of 34,000. The enzyme was specific to methylglyoxal (K_m = 1.92 mM) and NADPH (K_m = 40.8 μM). The activity of the enzyme was inhibited by p-chloromercuribenzoate and HgCl₂. NADP also inhibited the activity of the enzyme, and the K_i value was calculated to be 0.25 mM.     

Purification and chemical modifications of porphobilinogen oxygenase

Porphobilinogen oxygenase from wheat germ was purified and was found to be a cationic protein containing 8 mol of nonheme iron and 8–10 mol of labile sulfide per mole of enzyme (M_r, 100,000). The enzyme isolated from either wheat germ or rat liver microsomes was found to exist in multiple molecular weight forms. When succinylated, only one molecular weight form of 25,000 was obtained and it retained full activity. It had lost all of the sigmoidal kinetics characteristic of the native enzyme. While the native enzyme had an n = 3.5, the succinylated enzyme showed Michaelian kinetics. A K_m of approximately 1.70 mm was determined for the succinylated wheat germ enzyme, and a K_m of approximately 2.5 mm was found for the succinylated microsomal enzyme. Acetylation of the enzyme afforded an active acetylated enzyme which showed allosteric kinetics and multiple molecular weight forms. The products formed by the succinylated enzyme were the same as those formed by the native enzyme.     

Purification and Characterization of NAD Malic Enzyme from Leaves of Eleusine coracana and Panicum dichotomiflorum

NAD malic enzyme (EC 1.1.1.39), which is involved in C₄ photosynthesis, was purified to electrophoretic homogeneity from leaves of Eleusine coracana and to near homogeneity from leaves of Panicum dichotomiflorum. The enzyme from each C₄ species was found to have only one type of subunit by SDS polyacrylamide gel electrophoresis. The M_r of subunits of the enzme from E. coracana and P. dichotommiflorum was 63 and 61 kilodaltons, respectively. The native Mr of the enzyme from each species was determined by gel filtration to be about 500 kilodaltons, indicating that the NAD malic enzyme from C₄ species is an octamer of identical subunits. The purified NAD malic enzyme from each C₄ species showed similar kinetic properties with respect to concentrations of malate and NAD; each had a requirement for Mn²⁺ and activation by fructose- 1,6-bisphosphate (FBP) or CoA. A cooperativity with respect to Mn²⁺ was apparent with both enzymes. The activator (FBP) did not change the Hill value but greatly decreased K_0.5 (the concentration giving half-maximal activity) for Mn²⁺. The enzyme from E. coracana showed a very low level of activity when NADP was used as substrate, but this activity was also stimulated by FBP. Significant differences between the enzymes from E. coracana and P. dichotomiflorum were observed in their responses to the activators and their immunochemical properties. The enzyme from E. coracana was largely dependent on the activators FBP or CoA, regardless of concentration of Mn²⁺. In contrast, the enzyme from P. dichotomiflorum showed significant activity in the absence of the activator, especially at high concentrations of Mn²⁺. Both immunodiffusion and immunoprecipitation, using antiserum raised against the purified NAD malic enzyme from E. coracana, revealed partial antigenic differences between the enzymes from E. coracana and P. dichotomiflorum. The activity of the NAD malic enzyme from Amaranthus edulis, a typical NAD malic enzyme type C₄ dicot, was not inhibited by the antiserum raised against the NAD malic enzyme from E. coracana.     

GTP Cyclohydrolase I: Purification, Characterization, and Effects of Inhibition on Nitric Oxide Synthase in Nocardia Species

GTP cyclohydrolase I (GTPCH) catalyzes the first step in pteridine biosynthesis in Nocardia sp. strain NRRL 5646. This enzyme is important in the biosynthesis of tetrahydrobiopterin (BH₄), a reducing cofactor required for nitric oxide synthase (NOS) and other enzyme systems in this organism. GTPCH was purified more than 5,000-fold to apparent homogeneity by a combination of ammonium sulfate fractionation, GTP-agarose, DEAE Sepharose, and Ultragel AcA 34 chromatography. The purified enzyme gave a single band for a protein estimated to be 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme was estimated to be 253 kDa by gel filtration, indicating that the active enzyme is a homo-octamer. The enzyme follows Michaelis-Menten kinetics, with a K_m for GTP of 6.5 μM. Nocardia GTPCH possessed a unique N-terminal amino acid sequence. The pH and temperature optima for the enzyme were 7.8 and 56°C, respectively. The enzyme was heat stable and slightly activated by potassium ion but was inhibited by calcium, copper, zinc, and mercury, but not magnesium. BH₄ inhibited enzyme activity by 25% at a concentration of 100 μM. 2,4-Diamino-6-hydroxypyrimidine (DAHP) appeared to competitively inhibit the enzyme, with a K_i of 0.23 mM. With Nocardia cultures, DAHP decreased medium levels of NO₂⁻ plus NO₃⁻. Results suggest that in Nocardia cells, NOS synthesis of nitric oxide is indirectly decreased by reducing the biosynthesis of an essential reducing cofactor, BH₄.     

Properties of Phosphoglycolate Phosphatase from Chlamydomonas reinhardtii and Anacystis nidulans

The levels of activity of 2-phosphoglycolate phosphatase in the green algae, Chlamydomonas reinhardtii and Chlorella vulgaris, were in the range of 37 to 60 micromoles per milligram chlorophyll per hour and in the blue-green algae, Anacystis nidulans and Anabaena variabilis were 204 to 310 micromoles per milligram chlorophyll per hour. The activity in each species was similar regardless of whether the algae were grown with air or 5% CO₂ in air. The enzyme purified 530-fold from Chlamydomonas was stable, had a broad pH optimum between 6 and 8.5, and was specific for the hydrolysis of P-glycolate with a K_m of 23 micromolar. The enzyme purified 18-fold from Anacystis was labile, had a sharp pH optimum at 6.3, and was also specific for P-glycolate with a K_m of 94 micromolar. The molecular weight of the enzyme from Chlamydomonas was estimated to be 92,000 by gel filtration.

The phosphatase from both sources required a divalent cation for activity. The Chlamydomonas enzyme was most effectively activated by Co²⁺, but was also activated by Mg²⁺ (K_a = 30 micromolar), Mn²⁺, and Zn²⁺. The Anacystis enzyme was most effectively activated by Mg²⁺ (K_a = 140 micromolar), and was also activated by Co²⁺ and Mn²⁺, but not by Zn²⁺. Anions were also required for maximum activity of the enzyme from both sources. The Chlamydomonas enzyme was activated about 2- to 3-fold by chloride (K_a = 140 micromolar), bromide, nitrate, bicarbonate (K_a = 600 micromolar) and formate. The Anacystis enzyme was activated over 10-fold by chloride (K_a = 870 micromolar), bromide, iodide, and nitrate, but was not activated by bicarbonate or formate.

The properties of the algal enzymes were similar to those previously reported for higher plants. The levels and kinetic properties of the enzyme seemed sufficient to account for the flux through the glycolate pathway that occurs in these algae. The phosphatase was not associated with the ribulose 1,5-bisphosphate carboxylase/oxygenase responsible for P-glycolate formation in the carboxysomes of Anacystis.

     

Liver protein phosphatases: studies of the presumptive native forms of phosphorylase phosphatase activity in liver extracts and their dissociation to a catalytic subunit of Mr 35,000.

Several aspects of the properties of phosphorylase phosphatase in crude rat liver extracts were investigated. Treatment of tissue extracts with either trypsin, ethanol, or urea was found to dissociate phosphorylase phosphatase activity to a form of M_r 35,000. The M_r 35,000 enzyme form was derived from three native enzyme forms. The major cytosolic form of phosphorylase phosphatase had a molecular weight of 260,000 as determined by gel filtration and was dissociated to a M_r 35,000 form by treatment with either ethanol or urea. Treatment of the M_r 260,000 form with trypsin led to its conversion to M_r 225,000 and a M_r 35,000 form. A minor cytosolic form of M_r 200,000 was also present. This minor activity was latent until activated by trypsin treatment and was converted to a M_r 35,000 form by such treatment. The third form was found to chromatograph as a form of molecular weight greater than 500,000 on gel filtration and, like the M_r 200,000 form, was only detected after activation with trypsin. Subsequent to this treatment, it too behaved as a M_r 35,000 enzyme. Although a single major enzyme form was present in the cytosol, multiple molecular weight forms could be generated in crude extracts simply by the use of vigorous mechanical homogenization procedures. This suggested that artifactual forms of enzyme may readily be produced, possibly by proteolytic cleavage of the native enzyme.     

Purification and properties of adenosine 3′,5′-monophosphate phosphodiesterase from baker's yeast

Cyclic AMP phosphodiesterase from Saccharomyces cerevisiae was purified about 20,000-fold to homogeneity. The purified enzyme had a molecular weight of about 60,000 as estimated by gel filtration.The enzyme activity was optimal at pH 8.5–9.0 and was not stimulated by imidazole. Among cyclic 3′,5′-nucleotides, cyclic AMP was the most active substrate for the purified enzyme (K_m = 0.25 mM), but it was inhibitory at concentrations above 4 mm. N⁶,O^2′-dibutyryl cyclic AMP was not hydrolyzed at all.Unlike other cyclic AMP phosphodiesterases from various sources, the purified yeast enzyme did not require divalent metal ions for maximal activity and was rather inhibited in various degrees by added metal ions. The enzyme was not very sensitive to thiol inhibitors.The purified yeast enzyme was strongly inhibited by theophylline and slightly by caffeine. In contrast to the enzyme from S. carlsbergensis, the enzyme from S. cerevisiae was not inhibited at all by ATP or PP_i.The enzyme activity was not released into the growth medium, and the intracellular distribution studies indicated that the enzyme was located mainly in the cytosol fraction.     

Purification, Characterization, and Mechanism of a Flavin Mononucleotide-Dependent 2-Nitropropane Dioxygenase from Neurospora crassa

A nitroalkane-oxidizing enzyme was purified to homogeneity from Neurospora crassa. The enzyme is composed of two subunits; the molecular weight of each subunit is approximately 40,000. The enzyme catalyzes the oxidation of nitroalkanes to produce the corresponding carbonyl compounds. It acts on 2-nitropropane better than on nitroethane and 1-nitropropane, and anionic forms of nitroalkanes are much better substrates than are neutral forms. The enzyme does not act on aromatic compounds. When the enzyme reaction was conducted in an ¹⁸O₂ atmosphere with the anionic form of 2-nitropropane as the substrate, acetone (with a molecular mass of 60 Da) was produced. This indicates that the oxygen atom of acetone was derived from molecular oxygen, not from water; hence, the enzyme is an oxygenase. The reaction stoichiometry was 2CH₃CH(NO₂)-CH₃ + O₂→2CH₃COCH₃ + 2HNO₂, which is identical to that of the reaction of 2-nitropropane dioxygenase from Hansenula mrakii. The reaction of the Neurospora enzyme was inhibited by superoxide anion scavengers in the same manner as that of the Hansenula enzyme. Both of these enzymes are flavoenzymes; however, the Neurospora enzyme contains flavin mononucleotide as a prosthetic group, whereas the Hansenula enzyme contains flavin adenine dinucleotide.     

The kinetics of rat liver citrate synthase in situ.

The kinetics of citrate synthase in situ in toluene-treated rat liver mitochondria were studied. The V_max, K_m, and kinetic pattern for oxaloacetate were the same as those for the pure rat liver citrate synthase. The K_m, for acetyl-CoA for the in situ enzyme was increased compared to pure enzyme (8.5 to 77 μm), and the sensitivity of the in situ enzyme to inhibition by ATP, NADPH, or tricarballylate was decreased. The change seen with ATP was not due to problems of small molecule diffusion.     

Exchange Properties of the Activator CO(2) of Spinach Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

The exchange properties of the activator CO₂ of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase were characterized both in vitro with the purified enzyme, and in situ within isolated chloroplasts. Carboxyarabinitol-1,5-bisphosphate, a proposed reaction intermediate analog for the carboxylase activity of the enzyme, was used to trap the activator CO₂ on the enzyme both in vitro and in situ. Modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in intact chloroplasts during a light/dark cycle was associated with a similar modulation in carboxyarabinitol-1,5-bisphosphate-trapped CO₂. The exchange kinetics of the activator CO₂ were monitored by activation of the enzyme to steady state in the presence of ¹²CO₂, followed by addition of ¹⁴CO₂ and determination of the amount of labeled CO₂ trapped on the enzyme by carboxyarabinitol-1,5-bisphosphate. Rate constants (K_obs) for exchange with both the purified enzyme (0.45 min⁻¹) and in illuminated chloroplasts (0.18 min⁻¹) were comparable to the observed rate constants for enzyme activation under the two conditions. A similar exchange of the activator CO₂ was not observed in chloroplasts in the dark. Kinetic analysis of the exchange properties of the purified enzyme were consistent with an equilibrium between active and inactive forms of the enzyme during steady state activation.     

Isolation and characterization of the iron-containing superoxide dismutase of Methanobacterium bryantii

Methanobacterium bryantii contains a single electrophoretically discernible superoxide dismutase, which constitutes 0.4% of the extractable protein. This enzyme has been purified to electrophoretic and ultracentrifugal homogeneity. It appears to be a tetramer. The subunits were tenaciously, but noncovalently bonded and were of identical size. The molecular weight of the enzyme was found to be 91,000 ± 2000. The specific activity of this enzyme was identical to that previously noted for the corresponding enzyme from Escherichia coli. The enzyme contained 2.7 atoms of Fe, 1.7 atoms of Zn, and less than 0.2 atoms Mn per tetramer. Its amino acid composition placed this enzyme with the other Mn- and Fe-containing superoxide dismutases. The M. bryantii enzyme was also similar to previously described Fe-containing superoxide dismutases in its optical and electron paramagnetic resonance spectra and in its susceptibility to inactivation by H₂O₂. The M. bryantii enzyme was ininhibited by N₃^?, but was less sensitive towards this inhibitor than other iron-containing superoxide dismutases.     

Role of chloride ion as an allosteric activator of angiotensin-converting enzyme

The nature of chloride ion as an activator of angiotensin-converting enzyme was studied by a series of kinetic experiments with hog plasma enzyme preparation. The enzyme required the presence of chloride ion for its full catalytic activity, but its requirement of monovalent anion was not absolute. The K_A value for the enzymechloride binding was estimated to be about 150 mm in all cases regardless of the peptide substrates employed. In the presence of chloride ion, the activity of the enzyme was increased, but its optimum pH was shifted gradually to the alkaline region up to pH 8.2 depending on the concentration of chloride ion. In addition, in the presence of chloride ion, the apparent K_m values were reduced markedly while the V_max values were not much altered; for example, for the hydrolysis of angiotensin I decapeptide, the K_m value decreased by a factor of 50 while only an 18% increase in V_max was observed when the enzyme was saturated with chloride ion. The result suggests that chloride ion acts as a conformational modifier inducing the affinity of synergistic binding of substrate.     

Pig liver glyceraldehyde-3-P dehydrogenase: purification, crystallization, and characterization.

Glyceraldehyde 3-P dehydrogenase was purified approximately 250-fold from pig liver and crystallized. The purification procedure consisted of treating liver homogenates with zinc chloride, followed by ammonium sulfate fractionation and ion exchange chromatography. The enzyme was monodisperse in the ultracentrifuge with a sedimentation coefficient of s_20,w = 7.85 S. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a single subunit band with an approximate molecular weight of 38,000. High-speed sedimentation equilibrium gave a molecular weight of 1.5 × 10⁵. Incubation of the enzyme with ATP at 0 °C caused a loss of its dehydrogenase activity; some of the lost activity was regained upon warming to room temperature. Sucrose density gradient studies of the ATP-treated enzyme revealed a decrease in its sedimentation coefficient from 7.8 to 3.85 S. In the forward reaction direction, the K_m for glyceraldehyde 3-P was 240 μm and the K_m for NAD was 12 μm. In the backward reaction direction, the K_m for NADH was 23 μm and the K_i for NAD was 850 μm. Pig liver glyceraldehyde-3-P dehydrogenase resembles the rabbit muscle enzyme in that it apparently contains 2 to 3 mol of tightly bound NAD. However, it differs strongly from that enzyme in its rate and extent of inactivation by ATP at 0 °C and by urea; the pig liver enzyme, like the yeast enzyme, dissociates much more slowly and much less completely than the rabbit muscle enzyme under comparable conditions.     

Cytochrome a1 of Nitrosomonas europaea resembles aa3-type cytochrome c oxidase in many respects

Cytochrome c oxidase of Nitrosomonas europaea has been called cytochrome a₁ by Erickson et al. (Erickson, R.H., Hooper, A.B. and Terry, K.R. (1972) Biochim. Biophys. Acta 283, 155–166) because the reduced form of their preparation had the α peak at 595 nm. In the present studies, the enzyme was purified to an electrophoretically almost homogeneous state and some of its properties were studied. The enzyme much resembled cytochrome aa₃-type oxidase although its reduced form showed the α peak at 597 nm. (1) The absorption spectra of the CO compound of the reduced enzyme and CN⁻ compounds of the oxidized and reduced enzyme were similar to those of the respective compounds of cytochrome aa₃, as well as the absorption spectrum of the intact enzyme resembled that of the cytochrome. (2) The enzyme possessed two molecules of haem a and 1–2 atoms of copper in the molecule. (3) The enzyme molecule was composed of two kinds of subunits of M_r 50000 and 33000, respectively, as are other bacterial cytochromes aa₃. Although the enzyme resembled other bacterial cytochromes aa₃ in many properties, it differed greatly in two properties; its CO compound was easily dissociated into the oxidized enzyme and CO in air, and 50% inhibition of its activity by CN⁻ required approx. 100 μM of the reagent. The enzyme oxidized 0.57, 1.6 and 1.8 mol horse, Candida krusei and N. europaea ferrocytochromes c per s per mol haem a, respectively, in 10 mM phosphate buffer, pH 6.0. The turnover numbers with eukaryotic ferrocytochromes c were increased to 32 and 14, respectively, by addition of cardiolipin (14 μ · ml⁻¹).     

Subcellular localization,metal ion requirement and kinetic properties of arginase from the gill tissue of the bivalve Semele solida

Arginase activity (3.1 ± 0.5 units/g (wet wt) of tissue) was found associated to the cytosolic fraction of the gill cells of the bivalve Semele solida. The enzyme, with a molecular weight of 120,000 ± 3000, was partially purified, and some of the enzymic properties were were examined. The activation of the enzyme by Mn²⁺ followed hyperbolic kinetics with a K_Mn value of 0.10 ± 0.02 μM. In addition to Mn²⁺, the metal ion requirement of the enzyme was satisfied by Ni²⁺, Cd²⁺ and Co²⁺; Zn²⁺ was inhibitory to ail the Values of K_m for arginine and K_i for lysine inhibition, were the same, regardless of the metal ion used to activate the enzyme; K_m values were 20 mM at pH 7.5 and 12 mM at the optimum pH of 9.5. Competitive inhibition was caused by ornithine, lysine and proline, whereas branched chain amino acids were non competitive inhibitors of the enzyme.     

Biomphalaria glabrata: lysozyme activities in the hemolymph, digestive gland, and headfoot of the intermediate host of Schistosoma mansoni.

Lysozyme (mucopeptide N-acetylmuramylhydrolase EC 3.2.1.17) activity has been found in the hemolymph, digestive gland, and headfoot extracts of Biomphalaria glabrata, the intermediate host of Schistosoma mansoni. Partial purification of the bacteriolytic enzyme was attained by gel chromatography on Sephacryl S-200 and active lytic fractions were concentrated by Amicon filtration. The properties of the lytic enzymes from the three tissue extracts were identical. Enzyme activity was determined by the rate of lysis of cell wall suspension of Micrococcus lysodeikticus. Lysis of the cell walls was accompanied by a release of reducing sugar groups and N-acetylhexosamines. The enzyme was stable to heating at 100 C for 2 min and had an optimum activity at pH 4.5 to 5.0 in 0.066 M glycylglycine buffer. Low concentrations (5 mM) of NaCl, KCl, and LiCl increased the activity of the enzyme, whereas high concentrations (25 mM) of the same ions caused about 50% inhibition of the enzyme activity. MgCl₂ and CaCl₂ also inhibited the enzyme activity. Addition of 1 mM EDTA or EGTA resulted in about a twofold increase in enzyme activity. Double reciprocal plots of enzyme velocities and substrate concentrations yielded an apparent Michaelis-Menten constant (K_m) of 0.05 ± 0.01 mg/ml of M. lysodeikticus.     

Purification and properties of mannitol dehydrogenase from Agaricus bisporus sporocarps

Mannitol dehydrogenase (mannitol: NADP⁺ 2-oxidoreductase: EC 1.1.1.138) was isolated from Agaricus bisporus by fractionation with protamine sulphate and (NH₄)₂SO₄, followed by chromatography on DEAE-Sephadex, then by affinity and gel chromatography. The products of enzyme reaction were identified by GLC and TLC. K_m, optimum pH, MW and pI of the enzyme as well as the influence of temperature, ions and inhibitors on enzymic activity were determined. In the sugar reducing reaction, the enzyme was specific for fructose but, in the reverse direction, some structurally related polyols could substitute for mannitol. The enzyme was very sensitive to alterations in the NADP⁺/NADPH ratio. The results are discussed in relation to the possible role of mannitol dehydrogenase in fungal metabolism.     

Solubilization and partial purification of dihydroxyacetone-phosphate acyltransferase from guinea pig liver

Dihydroxyacetone-phosphate:acyl coenzyme A acyltransferase (EC 2.3.1.42) was solubilized and partially purified from guinea pig liver crude peroxisomal fraction. The peroxisomal membrane was isolated after osmotic shock treatment and the bound dihydroxyacetone-phosphate acyltransferase was solubilized by treatment with a mixture of KCl-sodium cholate. The solubilized enzyme was partially purified by ammonium sulfate fractionation followed by Sepharose 6B gel filtration. The enzyme was purified 1200-fold relative to the guinea pig liver homogenate and 80- to 100-fold from the crude peroxisomal fraction, with an overall yield of 25–30% from peroxisomes. The partially purified enzyme was stimulated two- to fourfold by Asolectin (a soybean phospholipid preparation), and also by individual classes of phospholipid such as phosphatidylcholine and phosphatidylglycerol. The kinetic properties of the enzyme showed that in the absence of Asolectin there was a discontinuity in the reciprocal plot indicating two different apparent K_m values (0.1 and 0.5 mm) for dihydroxyacetone phosphate. The V_max was 333 nmol/min/mg protein. In the presence of Asolectin the reciprocal plot was linear, with a K_m = 0.1 mm and no change in V_max. The enzyme catalyzed both an exchange of acyl groups between dihydroxyacetone phosphate and palmitoyl dihydroxyacetone phosphate in the presence of CoA and the formation of palmitoyl [³H]coenzyme A from palmitoyl dihydroxyacetone phosphate and [³H]coenzyme A, indicating that the reaction is reversible. The partially purified enzyme preparation had negligible glycerol-3-phosphate acyltransferase (EC 2.3.1.15) activity.     

Purification and characterization of an aldehyde reductase from Candida magnoliae

An NADPH-dependent aldehyde reductase was purified to homogeneity from Candida magnoliae AKU4643 through four steps, including Blue-Sepharose affinity chromatography. The relative molecular mass of the enzyme was estimated to be 33,000 on high performance gel-permeation chromatography and 35,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The substrate specificity of the enzyme was broad and resembled those of other aldo–keto reductases. The partial amino acid sequences of the enzyme showed that it belongs to the aldo–keto reductase superfamily. The enzyme catalyzed the stereoselective reduction of ethyl 4-chloro-3-oxobutanoate to the corresponding (R)-alcohol, with a 100% enantiomeric excess. The enzyme was inhibited by 1 mM quercetin, CuSO₄, ZnSO₄ and HgCl₂. The thermostability of the enzyme was inferior to that of the (S)-CHBE-producing enzyme from the same strain.