Enantioselective production of 2,2-dimethylcyclopropane carboxylic acid from 2,2-dimethylcyclopropane carbonitrile using the nitrile hydratase and amidase of Rhodococcus erythropolis ATCC 25544

The enantioselective production of (S)-2,2-dimethylcyclopropane carboxylic acid was investigated in 53 Rhodococcus and Pseudomonas related strains. Rhodococcus erythropolis ATCC 25544 was selected as it showed the highest enantioselectivity. The enantioselectivity was due to the amidase activity in a two-step reaction involving nitrile hydratase. The enantiomeric excess of the amidase was highest at pH 7.0 and decreased significantly above 20 °C. For the enantioselective production of (S)-2,2-dimethylcyclopropane carboxylic acid, the optimum reaction conditions of the cells were determined to be pH 7.0, 20 °C, and 10% (v/v) methanol and were the same as the optimum pH and temperature for the enantioselective conversion by the amidase. Under these conditions, the R. erythropolis ATCC 25544 cells, which harbored nitrile hydratase and amidase enzymes, produced 45 mM (S)-2,2-dimethylcyclopropane carboxylic acid from racemic 100 mM 2,2-dimethylcyclopropane carbonitrile with an 81.8% enantiomeric excess after 64 h.     

Modulation of Mucor miehei lipase properties via directed immobilization on different hetero-functional epoxy resins: Hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid

Purified lipase from Mucor miehei (MML) has been covalently immobilized on different epoxy resins (standard hydrophobic epoxy resins, epoxy-ethylenediamine, epoxy-iminodiacetic acid, epoxy-copper chelates) and adsorbed via interfacial activation on octadecyl-Sepabeads support (fully coated with very hydrophobic octadecyl groups). These immobilized enzyme preparations were used under slightly different conditions (temperature ranging from 4 to 25 °C and pH values from 5 to 7) in the hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid.

Different catalytic properties (activity, specificity, enantioselectivity) were found depending on the particular support used. For example, the epoxy-iminodiacetic acid-Sepabeads gave the most active preparation at pH 7 while, at pH 5, the ethylenediamine-Sepabeads was superior.

More interestingly, the enantiomeric ratio (E) also depends strongly on the immobilized preparation and the conditions employed. Thus, the octadecyl-MML preparation was the only immobilized enzyme derivative which exhibited enantioselectivity towards R isomer (with E values ranging from 5 at 4 °C and pH 7 to 1.2 at pH 5 and 25 °C).

The other immobilized preparations, in contrast, were S selective. Immobilization on iminodiacetic acid-Sepabeads afforded the catalyst with the highest enantioselectivity (E=59 under optimum conditions).     

Preparation of enantiomerically pure (S)-flurbiprofen by an esterase from Pseudomonas sp. KCTC 10122BP

Esterase PF1-K from Pseudomonas sp. KTCC 10122BP was overproduced by the fed-batch culture of Escherichia coli. The soluble expression of esterase PF1-K was achieved by shifting the culture temperature from 37 to 25 °C at the time of IPTG induction. The enzyme was partially purified to about 75% purity by a single-step hydrophobic interaction column chromatography. The purified enzyme exhibited a fairly high enantioselectivity towards the hydrolysis of rac-flurbiprofen ethyl ester. The enzymatic chiral resolution was further improved by optimizing the reaction conditions in terms of reaction rate and enantioselectivity. The optimal reaction conditions were found to be 40 °C, pH 10.5 and 600 mM of initial rac-flurbiprofen ethyl ester. After 90 min of batch reaction under the optimal conditions, 50% of the initial rac-flurbiprofen was hydrolyzed with an enantiomeric excess of 99%.     

Catalytic performance of a highly enantioselective (R)-ester hydrolase from a new isolate Acinetobacter sp. CGMCC 0789

A highly enantioselective (R)-ester hydrolase was partially purified from a newly isolated bacterium, Acinetobacter sp. CGMCC 0789, whose resting cells exhibited a highly enantioselective activity toward the acetate of (4R)-hydroxy-3-methyl-2-(2-propynyl)- cyclopent-2-enone (R-HMPC). The optimum pH and temperature of the partially purified enzyme were 8.0 and 60 °C, respectively. The enantioselectivity of the crude enzyme was increased by 1.2-fold from 16 to 20 when the reaction temperature was raised from 30 to 60 °C. The activity of the crude enzyme was enhanced by 4.1-fold and the enantioselectivity (E-value) was markedly enhanced by 4.3-fold from 16 to 68 upon addition of a cationic detergent, benzethonium chloride [(diisobutyl phenoxyethoxyethyl) dimethyl benzylammoniom chloride]. The hydrolysis of 52 mM (R,S)-HMPC acetate to (R)-HMPC was completed within 8 h, with optical purity of 91.4% ee_p and conversion of 49%.     

Resolution of 2-phenoxy-1-propanols through the enantioselective acylation mediated by Achromobacter sp. lipase as biocatalyst

Enantioselective acylation employing vinyl alkanoates as acyl donors was exploited for the resolution of 2-(substituted phenoxy)-1-propanols carrying different substituents on the benzene ring using Achromobacter sp. lipase. These primary alcohols with an oxygen atom at the stereocenter, were resolved with moderate to good enantioselectivity, based on the enantiomeric ratio E (up to 27), through acylation with vinyl butanoate in diisopropyl ether, after the examination of potential factors affecting the reaction such as organic solvents and acyl donors. Using this procedure, enantiomerically enriched (R)-2-(4-chlorophenoxy)-1-propanol was prepared in 97% e.e. and 33% yield in a gram-scale reaction.     

Isolation and characterization of a metagenome-derived and cold-active lipase with high stereospecificity for (R)-ibuprofen esters

We report on the isolation and biochemical characterization of a novel, cold-active and metagenome-derived lipase with a high stereo-selectivity for pharmaceutically important substrates. The respective gene was isolated from a cosmid library derived from oil contaminated soil and designated lipCE. The deduced aa sequence indicates that the protein belongs to the lipase family l.3, with high similarity to Pseudomonas fluorescens lipases containing a C-terminal secretion signal for ABC dependent transport together with possible motifs for Ca²⁺-binding sites. The overexpressed protein revealed a molecular weight of 53.2 kDa and was purified by refolding from inclusion bodies after expression in Escherichia coli. The optimum temperature of LipCE was determined to be 30 °C. However, the enzyme still displayed 28% residual activity at 0 °C and 16% at −5 °C. Calcium ions strongly increased activity and thermal stability of the protein. Further detailed biochemical characterization of the recombinant enzyme showed an optimum pH of 7 and that it retained activity in the presence of a range of metal ions and solvents. A detailed analysis of the enzyme's substrate spectrum with more than 34 different substrates indicated that the enzyme was able to hydrolyze a wide variety of substrates including the conversion of long chain fatty acid substrates with maximum activity for pNP-caprate (C₁₀). Furthermore LipCE was able to hydrolyze stereo-selectively ibuprofen-pNP ester with a high preference for the (R) enantiomer of >91% ee and it demonstrated selectivity for esters of primary alcohols, whereas esters of secondary or tertiary alcohols were nearly not converted.     

Lipase catalysed synthesis of sebacic and phthalic esters

The enzymatic synthesis and hydrolysis of alkyl sebacates and o-, m-, p-phthalates were studied. Biosyntheses were conducted through alcoholysis of dimethyl phthalates and dimethyl sebacate with 2-ethylhexanol and 3,5,5-trimethylhexanol in a solvent-free medium, using lipases from Candida antarctica (Novozym 435), Rhizomucor miehei (Lipozyme IM) and Porcine pancreas (PPL). It was found that the synthesis and hydrolysis of sebacic acid esters were characterised by a satisfactory rate, however, by low enantioselectivity. The yield of synthesis of di-3,5,5-trimethylhexyl sebacate catalysed by Novozym 435 at 50 °C was 84%, after 20 h of reaction. The degree of conversion, 62.9% after 350 h, was obtained for alcoholysis reaction of dimethyl m-phthalate with 3,5,5-trimethylhexanol. For the enzymes used, no activity was detected at all on both the synthesis and hydrolysis of di-2-ethylhexyl o-phthalate and di-3,5,5-trimethylhexyl o-phthalate.     

Determination of the thermodynamics of carbonic anhydrase acid-unfolding by titration calorimetry

The enthalpy of unfolding (Δ_uH) of carbonic anhydrase II was determined by titrating the protein with acid and measuring the heat using isothermal titration calorimetry (ITC) in the temperature range of 5 to 59 °C. By combining the ITC results with our previous findings by differential scanning calorimetry (DSC) in the temperature range of 39 to 72 °C, the Δ_uH dependence over a wide temperature range was obtained. The temperature dependence of the enthalpy displays significant curvature indicating that the heat capacity of unfolding (Δ_uC_p) is dependent on temperature. The T-derivative of Δ_uC_p was equal to 100 ± 30 J/(mol × K²), with the result that the Δ_uC_p is equal to 15.8 kJ/(mol × K) at 5 °C, 19.0 kJ/(mol × K) at 37 °C and 21.8 kJ/(mol × K) at 64 °C. The enthalpy of unfolding is zero at 17 °C. At lower temperatures, the Δ_uH becomes exothermic.

This method of determining protein unfolding thermodynamics using acid-ITC, significantly widens the accessible T-range, provides direct estimate of the thermodynamic parameters at physiological temperature, and gives further insight into the third T-derivative of the Gibbs free energy of unfolding.     

Resolution of 1,3-dioxolane derivatives using the lipase from an Acinetobacter junii SY-01

Acinetobacter junii SY-01 producing a lipase enantioselectively hydrolyzing 1,3-dioxolane derivatives was isolated from water sludge sample and the effect of solvent, acyl donor, vinyl acetate concentration, substrate concentration, operating temperature and immobilization on activity and enantioselectivity was studied for the resolution of 1,3-dioxolane derivatives through transesterification reaction using a lipase from the isolated strain. Best selectivity was obtained at lower substrate concentration (3–5 mM), higher vinyl acetate concentration (500–1000 mM) and lower temperature (30–40 °C) in the reaction mixture. Lipase immobilized onto Accurel MP-1000 (micro-porous polypropylene) gave the best results and the reactivity was about 29-fold higher than the free enzyme without the decrease of enantioselectivity. Resolution of 1,3-dioxolane derivatives was carried out in flask scale containing 100 ml solvents using the lipase immobilized onto Accurel MP-1000. In this reaction, the yield and enantiomeric excess of the remaining (2R, 4S)-alcohol were 31.2% and 98.2%, respectively. This result suggests that it can be used as an alternative method, compared to the present synthetic method, for the production of optically pure (2R, 4S)-itraconazole.     

Kinetic resolutions with novel, highly enantioselective fungal lipases produced by solid state fermentation

Thirty-eight filamentous fungi cultivated under solid state fermentation (SSF) conditions were screened for lipase activity and enantioselectivity in kinetic resolutions of racemic secondary alcohols (rac-1a–c) by acetylation with vinyl acetate performed in organic solvents. Many of the target fungi have not been studied previously for lipase/esterase activity and enantioselectivity. Without special enzyme isolation processes, the room temperature (25 °C) dried SSF cultures as such were tested in the enantiomer selective biotransformations. The majority of these SSF preparations proved to be effective as enantiomer selective biocatalysts exhibiting high but usual enantioselectivities according to the Kazlauskas rule. However, the SSF preparation of Mucor hiemalis origin acted as a selective anti-Kazlauskas catalyst. The best SSF products were successfully applied in preparative scale resolutions.     

Latent infection: a low temperature survival strategy in steinernematid nematodes

1. Entomopathogenic nematodes penetrate and kill Galleria mellonella within 48 h at optimal temperatures.

2. Low temperature induces infection latency, preventing host death until optimal conditions resume.

3. Infected Galleria survived 25 days at 5°C. On transfer to 25°C, 100% and 12.5% of Steinernema carpocapsae and Steinernema riobravis infected larvae died within 72 h.

4. Infective juvenile penetration decreased with decreasing temperature; declining from 49.7 and 49.3 nematodes/host at 25°C to 6.3 and 0.25 nematodes/host at 5°C for S. carpocapsae and S. riobravis, respectively.

5. Latent infection occurs, albeit infrequently, due to low host penetration at low temperature.

Determination of the maximum and minimum lethal temperatures (LT50) for Loxosceles intermedia Mello-Leitão, 1934 and L. laeta (Nicolet, 1849) (Araneae, Sicariidae)

In this study, the maximum and minimum lethal temperatures (LT₅₀) of L. intermedia and L. laeta were determined in two treatments: gradual heating (25–50°C) and cooling (25°C to −5°C), and 1 h at a constant temperature. In gradual temperatures change, L. intermedia mortality started at 40°C and the LT₅₀ was 42°C; for L. laeta, mortality began at 35°C and the LT₅₀ was 40°C. At low temperatures, mortality was registered only at −5°C for both species. In the constant temperature L. intermedia showed a maximum LT₅₀ at 35°C and L. laeta at 32°C; the minimum LT for both species was −7°C.     

Primary allenic alcohols of high optical purity via lipase catalyzed resolution

The lipase from Candida rugosa lipase (CRL) was used for the preparation of optically active primary allenic alcohols with axial chirality. The biocatalytic material used was the commercial CRL (C-CRL) and propan-2-ol-treated CRL (PT-CRL). The kinetic resolution of (±)-1 and (±)-3a–c in water using C-CRL resulted in either the absence of or low enantiomeric ratio, whereas PT-CRL increased the E-value. Under optimized conditions (temperature and medium used) (+)-S-3a and (−)-R-4 were isolated in excellent yields and high optical purity (o.p.). The resolution was carried out on multigram scale in water/n-hexane at 4 °C.     

Effects of Temperature on Stereochemistry of Enzymatic Reactions

A brief discussion of the theoretical basis for effects of temperature on stereoselectivity of enzyme catalysed reactions is presented. In theory, the stereoselectivity of an enzymatic reaction can either increase or decrease as the reaction temperature is raised. The secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus reduces 2-butanone to (R)-2-butanol at 37° C, with increased stereoselectivity at higher temperatures and in the presence of NADP analogues. In contrast, at 37°, 2-pentanone and 2-hexanone are reduced to (S)-2-pentanol and (S)-2-hexanol, respectively, but the stereoselectivity decreases at higher temperatures and in the presence of NADP analogues. Reduction of racemic 2-methylbutanal by the primary alcohol dehydrogenase from T. ethanolicus gives (S)-2-methyl-1-butanol with greater stereospecificity at 35° (51% e.e.) than at 15° (14% e.e.). Horse liver alcohol dehydrogenase shows a preference for oxidation of the (S)-enantiomers of acyclic secondary alcohols at 25°, with a decrease in stereospecificity at higher temperatures.     

A nicotinamide mononucleotide adenylyltransferase with unique adenylyl group donor specificity from a hyperthermophilic archaeon, Pyrococcus horikoshii OT-3

A gene encoding a nicotinamide mononucleotide (NMN) adenylyltransferase (NMNAT, EC 2.7.7.1) homologue was identified via genome sequencing in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3. The gene encoded a protein of 186 amino acids with a molecular weight of 21,391. The deduced amino acid sequence of the gene showed 59% identities to the NMNAT from Methanococcus jannaschii. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified to homogeneity. Characterization of the enzyme revealed that it is an extremely thermostable NMNAT; the activity was not lost after incubation at 80 °C for 30 min. The native molecular mass was estimated to be 77 kDa. The K_m values for ATP and NMN were calculated to be 0.056 and 0.061 mM, respectively. The optimum temperature of the reaction was estimated to be around 90 °C. The adenylyl group donor specificity was examined by high-performance liquid chromatography (HPLC). At 70 °C, ATP was a prominent donor. However, above 80 °C, a relatively small, but significant, NMNAT activity was detected when ATP was replaced by ADP or AMP in the reaction mixture. To date, an NMNAT that utilizes ADP or AMP as an adenylyl group donor has not been found. The present study provides interesting information in which a di- or mono-phosphate nucleotide can be utilized by adenylyltransferase at high temperature.     

Effect of nifedipine on core cooling in rats during tail cold water immersion

Male rats (450 g, n=11/group) were heated at an ambient temperature of 42°C until a rectal temperature of 42.8°C was attained. Rats, then received either saline (30°C)+tail ice water immersion (F+I) or saline (30°C)+tail ice water immersion+Nifedipine, a peripheral vasodilator, (F+I+N) to determine cooling rate effectiveness and survivability. The time to reach a rectal temperature of 42.8°C averaged 172 min in both groups resulting in similar heating rates (0.029°C/min). The cooling rates in group F+I and F+I+N were not significantly different from each other. We conclude that since Nifedipine did not improve cooling rates when combined with fluid+tail ice water immersion, its use as a cooling adjunct does not seem warranted.     

Morphology and germination characteristics of the cysts of Chattonella ovata (Raphidophyceae), a novel red tide flagellate in the Seto Inland Sea, Japan

To elucidate the mechanism of bloom outbreaks of Chattonella ovata (Raphidophyceae), we investigated the cysts of C. ovata and succeeded in finding them from the bottom sediments of Hiroshima Bay. The morphology of the cysts was mostly hemispherical in shape, with a diameter of ca. 30 μm and height of ca. 20 μm. The cysts were usually adhering to solid materials, such as diatom frustules, yellow-greenish in color and had several dark brown grains. The cyst wall was smooth and had no ornamentation. Because the morphological characteristic of the cysts was in general agreement with those of Chattonella antiqua and Chattonella marina, it was difficult to differentiate the cysts of these three species. Germination of the cysts of C. ovata was observed at temperatures from 17.5 to 30 °C, but not at 15 °C or below. The number of the germinated cysts increased with increasing temperature and the optimum temperature for germination was 30 °C. Although cysts of C. antiqua and C. marina germinated at temperatures from 15 to 30 °C, optimum temperature of germination was 22.5 °C. The lower limit and optimum temperatures for germination of C. ovata cysts was higher than for C. antiqua and C. marina. The role of cysts in the population dynamics of C. ovata is discussed.     

Metabolic characteristics of sea cucumber Apostichopus japonicus (Selenka) during aestivation

Metabolic characteristics of the sea cucumber Apostichopus japonicus (Selenka) during aestivation were studied in the laboratory. The effects of water temperature on oxygen consumption rate (OCR) and ammonia-N excretion rate (AER) in A. japonicus were determined by the Winkler and Hypobromite methods, respectively. Mature (large, 148.5 ± 15.4 g, medium 69.3 ± 6.9 g) and immature (small, 21.2 ± 4.7 g) individuals aestivated at water temperatures of 20 and 25 °C, respectively. The metabolic characteristics of mature individuals were different from immature individuals during this period. The OCR of mature sea cucumbers peaked at 20 °C, and then dropped significantly at higher temperatures, whereas the OCR of the immature animals continued to increase slightly, even beyond the aestivation temperature. The AER of mature individuals peaked at 20 °C, while that of the immature animals peaked at 25 °C. The relationships between dry weight (DW) and absolute oxygen consumption (R) and absolute ammonia-N excretion (N) could be described by the regression equation R or N = aW^b. With the exception of 15 °C, the O / N ratios (calculated in atomic equivalents) of large size sea cucumbers was close to 20 across the temperatures used in this study, indicating that their energy source was a combination of lipid and protein. On the other hand, apart from small individuals maintained at 10 °C, the O / N ratios of the medium and small sea cucumbers were close to 10, indicating that protein was their major energy source. The O / N ratios in all size groups remained unchanged after aestivation was initiated.     

Kinetic modeling of immobilized-lipase catalyzed transesterification of n-octanol with vinyl acetate in non-aqueous media

Organic esters are employed as solvents, fragrance, flavors, and precursors in a variety of industries. Particularly, aliphatic esters are greatly used in flavor industry, mainly as fixatives and modifiers, and aromatic esters in fragrance compositions. Esters are produced by a variety of methods among which esterification and transesterification with acid catalysts under reflux conditions are prominent. The use of biocatalysts provides an opportunity for carrying out reactions under milder conditions leading to better quality products suitable in fragrance and flavor industry. Transesterification of n-octanol with vinyl acetate was studied at 30 °C as a model reaction by employing different lipases as catalysts such as Psedomonas species lipase immobilized on diatomite, free Candida rugosa lipase. Novozym 435 (lipase B from Candida antarctica; immobilized on macro-porous polyacrylic resin beads) and Lipozyme IM 20 (Mucor miehei lipase immobilized on anionic resin). Novozym 435 was found to be the most active catalyst in heptane as a solvent. A conversion of 82% with 100% selectivity of n-octyl acetate was obtained at 30 °C in 90 min using equimolar quantities of the reactants with 0.833 g l⁻¹ of Novozym 435. Transesterification of other alcohols such as n-decanol, benzyl alcohol, cinnamyl alcohol, 2-ethyl-1-hexanol, 1-phenyl ethyl alcohol, and 2-phenyl ethyl alcohol was also studied with vinyl acetate. The analysis of the initial rate data and progress curve data showed that the reaction obeys the ternary complex bi–bi mechanism with inhibition by n-octanol. The experimental and theoretical values matched very well.

The order of transesterification reactivity of vinyl acetate with various alcohols in presence of Novozym 435 under otherwise identical conditions at 30 °C was found to be as follows:

n-octanol>n-decanol>benzylalcohol>cinnamylalcohol>2-ethyl-1-hexanol>2-phenylethylalcohol>1-phenylethylalcohol.