Thermal stability enhancement of Candida rugosa lipase using ionic liquids

The thermal stability of Candida rugosa (C. rugosa) lipase was investigated and compared in n-hexane, benzene, dibutyl-ether as well as [bmim]PF₆ and [omim]PF₆ ionic liquids and the effect of solvent polarity and water activity were evaluated. Deactivation of the enzyme followed a series-type kinetic model. First order deactivation rate constants and the ratios of specific activities were determined and the kinetics of deactivation were studied. Among the organic solvents, the best stability was observed in n-hexane with a half-life of 6.5 h at water activity of 0.51. In ionic liquids, however, even longer half lives were obtained, and the enzyme was stable in these solvents at 50°C. The highest half-life times were obtained in [bmim]PF₆ (12.3 h) and [omim]PF₆ (10.6 h). A direct correlation was found between solvent polarity and thermal stability since the higher the polarity of the solvent, the lower was the stability decrease at 50°C comparing to that at 30°C.     

Carboxypeptidase Y-Catalyzed Reaction in Systems Containing Organic Solvent

The effect of organic solvents on carboxypeptidase Y (a serine carboxypeptidase from yeast)-catalyzed hydrolysis of amino acid ester and peptide synthesis from N-acyl amino acid ester and amino acid amide was investigated.

The K_m value of ester hydrolysis increased with an increase in the solvent content. Dioxane was the most effective and dimethyl sulfoxide (DMSO) the least, whilst K_cat showed a tendency to increase slightly in N, N-dimethylformamide (DMF) and DMSO. For dioxane and acetonitrile (MeCN) a maximum was observed.

In peptide formation from Fua-Phe-OEt and Gly-NH₂, dioxane and MeCN supported high product yield at molar fractions smaller than ca. 0.05 but the yield decreased significantly at higher fractions, although a relatively constant selectivity (ratio of the peptide bond formed to the ester consumed) was maintained. DMSO gave rather low peptide yields and selectivity even at lower molar fractions. DMF showed an intermediate tendency.

An apparent saturation parameter of the amine component was evaluated and the dissociation constant of a complex between acyl-enzyme and amino acid amide (K_n), as well as the rate constant of aminolysis exerted by the amino acid amide bound correctly on the enzyme (K_n), was calculated by initial rate analysis of peptide formation. In contrast to K_m values, K_n decreased with increasing concentrations of organic cosolvent. while a suppressive effect was observed (except for DMSO) on the K_n parameter.

Effects of the solvent practically immiscible in water was also studied by use of the enzyme physically “immobilized” on glass beads.     

The preparation and properties of the membranal DPNH dehydrogenase from Escherichia coli

1. The membrane bound DPNH oxidase of Escherichia coli can reduce the artificial electron acceptors: ferricyanide, dichlorophenolindophenol (DCIP) and menadione. All three are reduced by the flavoprotein of DPNH oxidase, but at different sites of the enzyme.

2. Freeze-drying of the bacterial membranes causes a selective detachment of DPNH dehydrogenase (DPNH: (acceptor) oxidoreductase, EC 1.6.99.3) from the membranes. This solubilization is accompanied by a decrease of K_m(K₃Fe(CN)₆) from 2.0 to 0.25 mM, while no change is detected in K_m(DPNH). This enzyme is not the DPNH diaphorase found in the bacteria.

3. DPNH dehydrogenase of E. coli is a metalloflavoprotein, containing non-heme iron, labile sulfide, FMN and FAD.

4. Reduction of the enzyme with DPNH in the absence of electron acceptor (ferricyanide or DCIP) causes a rapid and irreversible change to a less active state, Form II. Form II is characterized by a higher K_m(DPNH) and slower v_max., while the K_m(K₃Fe(CN)₆) remains unchanged.

5. The transformation of the enzyme to Form II is accompanied by the reduction of the non-heme iron component. The role of non-heme iron in the enzymic reaction is discussed.     

Stability of free and immobilised peroxidase in aqueous–organic solvents mixtures

The activity and stability of horseradish (Amoracia rusticana) peroxidase (HRP) free in solution and immobilised onto silica microparticles was studied in the presence of organic co-solvents.

The effect of several hydrophilic organic solvents, namely dimethyl sulfoxide, dimethylformamide, dioxan, acetonitrile and tetrahydrofuran, in the activity and stability of free HRP was studied. From the solvents tested, DMSO led to the highest activities and stabilities. After 2 h of incubation at 35°C, the remaining activity of the enzyme in the presence of 30% of each solvent was less than 30%, with exception of DMSO for which the enzyme remained fully active.

In order to increase stability, HRP was covalently immobilised onto silica microparticles. The half-life of the enzyme in buffer at 50°C increased from 2 to 52 h when the enzyme was immobilised. The stability of both free and immobilised HRP was also studied at 50°C in aqueous mixtures of 3.5, 20, 35 and 50% (v/v) DMSO. Free HRP stability was not affected by the presence of 3.5 and 20% DMSO, but higher contents lead to a more pronounced deactivation. Immobilised HRP stability increased with DMSO content up to 20%, decreasing for higher contents. The enzyme half-life increased more than 300% when changing from buffer to 20% DMSO.

The deactivation of free HRP was modelled using the simple exponential decay, and the deactivation of immobilised HRP was described by a two-step inactivation model.     

Cortisol metabolism in vitro—III. Inhibition of microsomal 6β—hydroxylase and cytosolic 4-ene-reductase

The in vitro metabolism of cortisol in human liver fractions is highly complex and variable. Cytosolic metabolism proceeds predominantly via A-ring reduction (to give 3,5β-tetrahydrocortisol; 3,5β-THF), while microsomal incubations generate upto 7 metabolites, including 6β-hydroxycortisol (6β-OHF), and 6β-hydroxycortisone (6β-OHE), products of the cytochrome P450 (CYP) 3A subfamily. The aim of the present study was, therefore, to examine two of the main enzymes involved in cortisol metabolism, namely, microsomal 6β-hydroxylase and cytosolic 4-ene-reductase. In particular, we wished to assess the substrate specificity of these enzymes and identify compounds with inhibitory potential. Incubations for 30 min containing [³H]cortisol, potential inhibitors, microsomal or cytosolic protein (3 mg), and co-factors were followed by radiometric HPLC analysis. The K_m value for 6β-OHF and 6β-OHE formation was 15.2 ± 2.1 μM (mean ± SD; n = 4) and the V_max value 6.43 ± 0.45 pmol/min/mg microsomal protein. The most potent inhibitor of cortisol 6β-hydroxylase was ketoconazole (K_i = 0.9 ± 0.4 μM; N = 4), followed by gestodene (K_i = 5.6 ± 0.6 μM) and cyclosporine (K_i = 6.8 ± 1.4 μM). Both betamethasone and dexamethasone produced some inhibition (K_i = 31.3 and 54.5 μ, respectively). However, substrates for CYP2C (tolbutamide), CYP2D (quinidine), and CYP1A (theophylline) were essentially non-inhibitory. The K_m value for cortisol 4-ene-reductase was 26.5 ± 11.2 μM (n = 4) and the V_max value 107.7 ± 46.0 pmol/min/mg cytosolic protein. The most potent inhibitors were androstendione (K_i = 17.8 ± 3.3 μM) and gestodene (K_i = 23.8 ± 3.8 μM). Although both compounds have identical A-rings to cortisol, and undergo reduction, inhibition was non-competitive.     

A nitrite reductase with cytochrome oxidase activity from Micrococcus denitrificans

The formation of nitrite reductase and cytochrome c in Micrococcus denitrificans was repressed by O₂. The purified nitrite reductase utilized reduced forms of cytochrome c, phenazine methosulphate, benzyl viologen and methyl viologen, respectively, as electron donors. The enzyme was inhibited by KCN, NaN₃ and NH₂OH each at 1 mM, whereas CO and bathocuproin, diethyl dithiocarbamate, o-phenanthroline and ,'-dipyridyl at 1 mM concentrations were relatively ineffective. The purified enzyme contains cytochromes, probably of the c and a₂ types, in one complex. A K_m of 46 μM for NO₂⁻ and a pH optimum of 6.7 were recorded for the enzyme. The molecular weight of the enzyme was estimated to be around 130000, and its anodic mobility was 6.8·10⁻⁶ cm²·sec⁻¹·V⁻¹ at pH 4.55.

The most highly purified nitrite reductase still exhibited cytochrome c oxidase activity with a K_m of 27 μM for O₂. This activity was also inhibited by KCN, NaN₃ and NH₂OH and by NO₂⁻.

A constitutive cytochrome oxidase associated with membranes was also isolated from cells grown anaerobically with NO₂⁻. It was inhibited by smaller amounts of KCN, NaN₃ and NH₂OH than the cytochrome oxidase activity of the nitrite reductase enzyme and also differed in having a pH optimum of about 8 and a K_m for O₂ of less than 0.1 μM. Spectroscopically, cytochromes b and c were found to be associated with the constitutive oxidase in the particulate preparation. Its activity was also inhibited by NO₂⁻.

The physiological role of the cytochrome oxidase activity associated with the purified nitrite reductase is likely to be of secondary importance for the following reasons: (a) it accounts for less than 10% of total cytochrome c oxidase activity of cell extracts; (b) the constitutive cytochrome c oxidase has a smaller K_m for O₂ and would therefore be expected to function more efficiently especially at low concentrations of O₂.     

Enantioselective oxidation of secondary alcohols by quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni

Purified and reconstituted quinohaemoprotein alcohol dehydrogenase (QH-EDH) from Comamonas testosteroni is shown to oxidize secondary alcohols enantioselectively. The products formed during the oxidation of secondary alcohols were positively identified as the corresponding ketones. In the oxidation of chiral secondary n-alkyl alcohols a preference of the enzyme for the S(+)alcohols was found. The apparent kinetic parameters (K_m and K_max) for a range of n-alkyl alcohols depend on the length of the alcohol chain and the location of the hydroxyl function in the chain. The enzyme is stable up to a temperature of 37 °C. Above this temperature the activity is irreversibly lost. The pH optimum of the enzyme in the conversion of secondary alcohols is 7.7.     

Kinetic analysis of duodenal and testicular cytochrome P450c17 in the rat

In the adult rat, the duodenal tissue of both sexes can convert progesterone to 17-hydroxyprogesterone, androstenedione and testosterone. The transition from C₂₁ to C₁₉ steroids is apparently controlled by the same cytochrome P450c17 expressed in the testis, which catalyzes both 17-hydroxylation and C-17,20 bond scission at a single bifunctional active site. The kinetic parameters of this enzyme were measured at the steady state for both reactions using [1,2-³H]progesterone and [1,2-³H]17-hydroxyprogesterone as substrates. In the testis and male and female duodena, the K_m values for progesterone 17-hydroxylation were 14.2, 23.8 and 23.2 nM, whereas the V_max values were 105, 3.5 and 3.1 pmol/mg protein/min, respectively. With respect to C-17,20 lyase activity, the K_m values for exogenous 17-hydroxyprogesterone were 525, 675 and 637 nM, whereas the V_max values were 283, 7.8 and 7.8 pmol/mg protein/min, respectively. However, when the K_m values were calculated with respect to intermediate 17-hydroxyprogesterone formed from progesterone, they were similar to the K_m values for 17-hydroxylase, being 15, 31.4 and 24.8 nM, whereas the V_max values were 26.3, 2 and 1.8 pmol/mg protein/min, respectively. The similarity of K_m values is due to the fact that the relative androgen formation efficiency (bond scission events/total 17-hydroxylation events ratio) was remarkably constant in both testicular and duodenal incubates, irrespective of progesterone concentration. Efficiency values were 2-fold higher in duodenal tissue (0.54) than in testis (0.25). Estradiol-17β inhibited 17-hydroxylation but not bond scission on intermediate 17-hydroxyprogesterone, because it did not affect the efficiency value. Rat duodenal P450c17 has the same substrate affinity, a lower specific activity and a higher androgen formation efficiency than testicular P450c17.     

A xylose-tolerant beta-xylosidase from Paecilomyces thermophila: characterization and its co-action with the endogenous xylanase

An extracellular β-xylosidase from the thermophilic fungus Paecilomyces thermophila J18 was purified 31.9-fold to homogeneity with a recovery yield of 2.27% from the cell-free culture supernatant. It appeared as a single protein band on SDS–PAGE with a molecular mass of approx 53.5 kDa. The molecular mass of β-xylosidase was 51.8 kDa determined by Superdex 75 gel filtration. The enzyme was a glycoprotein with a carbohydrate content of 61.5%. It exhibited an optimal activity at 55 °C and pH 6.5, respectively. The enzyme was stable in the range of pH 6.0–9.0 and at 55 °C. The purified enzyme hydrolyzed xylobiose and higher xylooligosaccharides but was inactive against xylan substrates. It released xylose from xylooligosaccharides with a degree of polymerization ranging between 2 and 5. The rate of xylose released from xylooligosaccharides by the purified enzyme increased with increasing chain length. It had a K_m of 4.3 mM for p-nitrophenol-β-d-xylopyranoside and was competitively inhibited by xylose with a K_i value of 139 mM. Release of reducing sugars from xylans by a purified xylanase produced by the same organism increased markedly in the presence of β-xylosidase. During 24-hour hydrolysis, the amounts of reducing sugar released in the presence of added β-xylosidase were about 1.5–1.73 times that of the reaction employing the xylanase alone. This is the first report on the purification and characterization of a β-xylosidase from Paecilomyces thermophila.     

Structure and function of psychrophilic alanine racemase

We describe the structure and function of psychrophilic alanine racemases from Bacillus psychrosaccharolyticus and Pseudomonas fluorescens. These enzymes showed high catalytic activities even at 0°C and were extremely labile at temperatures over 35°C. The enzymes were also found to be less resistant to organic solvents than alanine racemases from thermophilic and mesophilic bacteria, both in vivo and in vitro. Both enzymes have a dimeric structure and contain 2 mol of pyridoxal 5′-phosphate (PLP) per mol as a coenzyme. The enzyme from B. psychrosaccharolyticus was found to have a markedly large K_m value (5.0 μM) for PLP in comparison with other reported alanine racemases, and was stable at temperatures up to 50°C in the presence of excess amounts of PLP. The dissociation of PLP from the P. fluorescens enzyme may trigger the unfolding of the secondary structure. The enzyme from B. psychrosaccharolyticus has a distinguishing hydrophilic region around residue no. 150 in its deduced amino acid sequence, whereas the corresponding regions of other Bacillus alanine racemases are hydrophobic. The position of this region in the three dimensional structure of this enzyme was predicted to be in a surface loop surrounding the active site. This hydrophilic region may interact with solvent, reduce the compactness of the active site, and destabilize the enzyme.     

Multi-competitive enzymatic reactions in organic media: Application to the determination of lipase alcohol specificity

Multicompetitive reactions catalyzed by lipases in organic media were used for the determination of lipase specificity towards alcohols. The competitive factors (, defined as the ratio of the kinetic powers, k_cat/K_m, for two substrates in competition for the enzyme active site) were estimated in a one-step experiment and a scale of specificity was easily deduced. The specificity towards the alcohol chain length and degree of substitution of seventeen commercial lipase preparations was investigated. The results show that, like fatty acids, alcohols greatly influence the reaction rates of lipase catalyzed reactions in organic solvents. Five groups of alcohol specificity are proposed after using the statistical method of principal component analysis.     

Enhanced activity and stability of immobilized lipases by treatment with polar solvents prior to lyophilization

Lipases from Candida rugosa, Mucor javanicus and Rhizopus oryzae were respectively adsorbed on Amberlite XAD-7 followed by incubation in 2-propanol and then lyophilization. The activities of the immobilized enzymes were 1.6–3.4 times higher than those of the immobilized enzymes without incubation in the organic solvent before lyophilization for esterification of lauric acid (0.1 M) and 1-propanol (0.1 M) in isooctane at 37 °C. The immobilized C. rugosa lipase (Sigma) without the incubation did not show any activity but displayed considerable activity (19.8 μmol h⁻¹ mg⁻¹) after the incubation before lyophilization. Besides 2-propanol, acetone, 1-propanol and ethyl acetate were also found to be good solvents for treating M. javanicus lipase immobilized on Amberlite XAD-7 and acetone was the best among them. When incubated in isooctane at 25 °C for 120 h, the immobilized M. javanicus lipase prepared by incubation in acetone for 1 h before lyophilization retained 70% of its initial activity while the immobilized enzyme without the solvent treatment kept only 50% of its initial activity.     

Temperature and the regulation of activity of some mitochondrial enzyme systems in ecto- and endo-thermic animals

1. 1.|No obvious correlation was found between the temperature dependence of the apparent K_m values for either mitochondrial succinoxidase or NADH oxidase systems prepared from rat or rainbow trout liver tissue and the previously observed pattern of susceptibility of these same enzymes to thermal inactivation.

2. 2.|Apparent activation energies (E_A) have been calculated from the data and, although these are equivocal when considered in the above context, it is concluded that the possibility is not excluded that entropy may be a more important variable than enthalpy in the action of these enzymes in ectothermic animals.

Studies on adenine nucleotide exchange in mitochondria from the muscle tissue of Ascaris lumbricoides (Nematoda)

Adenine nucleotide exchange between the intra- and extramitochondrial compartments of mitochondria isolated from the muscle tissue of Ascaris lumbricoides was investigated. The exchange was specific for ATP and ADP, AMP, adenosine and non-adenine nucleotides were not exchanged at significant rates. All combinations of counter exchange were found to be possible between intra- and extramitochondrial ATP and ADP. Adenine nucleotide exchange in Ascaris muscle mitochondria was inhibited by atractyloside; was strongly temperature dependent; activated by potassium and magnesium and only slightly activated by calcium. The K_m for adenine nucleotide exchange in Ascaris mitochondria was 4·1 and 2·85 μm for ATP and ADP respectively. The properties of adenine nucleotide exchange in Ascaris muscle mitochondria are thus similar in general features to the adenine nucleotide translocase system of mammalian mitochondria.     

Kinetic evidence for the involvement of a common enzyme in the microsomal reduction of retinal and androstenedione in rat liver

Androst-4-ene-3,17-dione (androstenedione) was found to be a potent competitive inhibitor of the NADH-supported reduction of retinal in rat hepatic microsomes (K_i 42 μM, K_m/K_i ratio 1.1). Similarly, the NADH-mediated reduction of androstenedione was inhibited in mixed fashion by retinal (K_i 12 μM, K_m/K_i ratio 0.34). In subsequent experiments the cofactor NADH exhibited an identical K_m (8 μM) in the microsomal reductions of both substrates. Acidic pH markedly stimulated the microsomal reduction of androstenedione to testosterone and was also found to enhance retinal reduction to retinol, although the latter reaction exhibited a district pH optimum between 6.0 and 6.5. These results suggest that a common enzyme may participate in the reduction of both substrates but at least one other enzyme probably participates in hepatic microsomal testosterone production.     

Oxidative phosphorylation and effects of aerobic conditions on Rhodopseudomonas viridis

Particles prepared from photosynthetically grown Rhodopseudomonas viridis, which has previously been described as an obligate photo-organotroph, catalyse oxidative phosphorylation. The P:O ratio is 0.3 with succinate as substrate and 0.45 with NADH. The reaction is inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone and oligomycin. Antimycin A or 2-heptyl-4-hydroxyquinoline-N-oxide inhibit succinate or NADH oxidation as well as oxidative phosphorylation.

After repeated transfers of cultures from agar plates, kept aerobic in the dark, cells of R. viridis were capable of limited exponential aerobic growth. In such cells no bacteriochlorophyll and very little carotenoid was detected; there was also a loss of the characteristic membrane structure found in photosynthetic cells. The K_m for oxygen of these cells was lowered from 0.35 (±0.09) to 0.007 (±0.002) mM and an additional cytochrome-like pigment was observed, with an absorption maximum at 560 nm at 77 °K. There was also an increased amount of a CO-binding pigment in aerobically grown cells.     

Immobilization on Eupergit C of cyclodextrin glucosyltransferase (CGTase) and properties of the immobilized biocatalyst

The extreme thermophilic cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. was covalently attached to Eupergit C. Different immobilization parameters (incubation time, ionic strength, pH, ratio enzyme/support, etc.) were optimized. The maximum yield of bound protein was around 80% (8.1 mg/g support), although the recovery of β-cyclodextrin cyclization activity was not higher than 11%. The catalytic efficiency was lower than 15%. Results were compared with previous studies on covalent immobilization of CGTase.

The enzymatic properties of immobilized CGTase were investigated and compared with those of the soluble enzyme. Soluble and immobilized CGTases showed similar optimum temperature (80–85 °C) and pH (5.5) values, but the pH profile of the immobilized CGTase was broader at higher pH values. The thermoinactivation of the CGTase coupled to Eupergit C was slower than the observed with the native enzyme. The half-life of the immobilized enzyme at 95 °C was five times higher than that of the soluble enzyme. The immobilized CGTase maintained 40% of its initial activity after 10 cycles of 24 h each. After immobilization, the selectivity of CGTase (determined by the ratio CDs/oligosaccharides) was notably shifted towards oligosaccharide production.     

Purification and properties of a Mg-dependent ATPase from chloroplasts of Euglena gracilis

Isolated chloroplasts of Euglena gracilis contain highly active ATPases. The ATPase activity in the chloroplasts is usually much higher than the maximal rate of photophosphorylation obtainable and is due to a number of different enzymes.

One of these ATPases, Mg²⁺-dependent and with a low pH optimum, has been solubilized with the aid of detergents and purified approx. 20-fold. The optimal activity of the enzyme is at pH 5.5, and the optimal Mg²⁺ concentration is 5·10⁻³ M. The enzyme hydrolyzes dATP more rapidly than ATP, although the K_m for both substrates is the same. Other nucleotide triphosphates are hydrolyzed very slowly while ADP or pyrophosphate are not hydrolyzed at all. Oligomycin, carbonylcyanide m-chlorophenylhydrazone, and ADP inhibit the enzyme, the latter competitively.

The level of the enzyme is highest in chloroplasts from rapidly dividing cells, and reaches a minimum in chloroplasts from maturing cultures which have attained their maximal photosynthetic activity. It is postulated that the enzyme has a role in controlling chloroplast development.     

Purification and characterization of an enantioselective carbonyl reductase from Candida viswanathii MTCC 5158

A highly enantioselective carbonyl reductase produced by a new yeast strain Candida viswanathii MTCC 5158, which was isolated using an acetophenone enriched medium, has been purified and characterized. The enzyme has been purified to near homogeneity using ammonium sulfate precipitation, ion exchange and gel filtration chromatography. The molecular properties of the carbonyl reductase suggested the native enzyme to be tetrameric, with an apparent molecular weight of 120 kDa, the monomer being about 29 kDa. Acetyl aryl ketones were found to be the preferred substrates for the enzyme and the best reaction was the enantioselective reduction of acetophenone. The enzyme yielded (S)-alcohol in preference to (R)-alcohol and utilized NADH, but not NADPH as the cofactor. The purified enzyme exhibited maximum enzyme activity at pH 7.0 and 60 °C. The enzyme retained about 80% of its activity after 7 h incubation at 25 °C in sodium phosphate buffer (50 mM, pH 7.0). The addition of reducing agents like dithiothreitol and β-mercaptoethanol enhanced the enzyme activity while organic solvents, detergents and chaotropic agents had deleterious effect on enzyme activity. Metal chelating agents like hydroxyquinoline and o-phenanthroline have significant effect on enzyme activity suggesting that the carbonyl reductase required the presence of a tightly bound metal ion for activity or stability. The maximum reaction rate (V_max) and apparent Michaelis–Menten constant (K_m) for acetophenone and NADH were 59.21 μmol/(min mg) protein and 0.153 mM and 82.64 μmol/(min mg) protein and 0.157 mM at a concentration range of 0.2–2 mM acetophenone (NADH fixed at 0.5 mM) and 0.1–0.5 mM NADH (acetophenone fixed at 2 mM), respectively.     

Cloning and characterization of a fish microsomal epoxide hydrolase of Danio rerio and application to kinetic resolution of racemic styrene oxide

A potent bacterial strain, Pseudomonas aeruginosa, has been isolated from the soil which produces extracellular lipase that can carry out the excellent stereospecific hydrolysis of trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM)] to give [(−)-MPGM], an intermediate required in the synthesis of cardiovascular drug, diltiazem. As a preliminary experiment for enzymatic resolution, we characterized the fractionated enzyme. The enzyme had a pH and temperature optima of 8.0 and 60 °C, respectively. The enzyme showed high degree of thermostability. Also, the enzyme was found to be stable in alkaline condition and in organic solvents. The activity of the enzyme increased by the addition of magnesium ions. The small-scale hydrolysis of (±)-MPGM (250 mg) with partially purified enzyme (21,000 U) gave (−)-MPGM with good isolated yield (44%) and excellent enantiomeric excess (99.9%) in a very short time (12 h).