Synthesis of propyl gallate by mycelium-bound tannase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> in organic solvent

Aspergillus niger with mycelium-bound tannase activity was employed to investigate the synthesis of propyl gallate from gallic acid and 1-propanol in organic solvents. The effects of various organic solvents (log P: −1.0 to 6.6) on the enzymatic reactions showed that benzene (log P: 2.0) was the most suitable solvent. The water content and protonation state of mycelium-bound enzyme both had significant effects on the activity of tannase. The maximum molar conversion (65%) was achieved with 7.3% (v/v) 1-propanol and 5.56 mM gallic acid at stirring speeds of 200 rev/min, 40 °C in presence of anhydrous sodium sulfate and PEG-10,000. Enzyme specificity for the alcohol portion (C₁–C₈) of the ester showed that the optimum synthesis was observed with alcohols ranging from C₃ to C₅.     

Lipase in aqueous‐polar organic solvents: Activity,structure, and stability

Studying alterations in biophysical and biochemical behavior of enzymes in the presence of organic solvents and the underlying cause(s) has important implications in biotechnology. We investigated the effects of aqueous solutions of polar organic solvents on ester hydrolytic activity, structure and stability of a lipase. Relative activity of the lipase monotonically decreased with increasing concentration of acetone, acetonitrile, and DMF but increased at lower concentrations (upto ～20% v/v) of dimethylsulfoxide, isopropanol, and methanol. None of the organic solvents caused any appreciable structural change as evident from circular dichorism and NMR studies, thus do not support any significant role of enzyme denaturation in activity change. Change in 2D [15N, 1H]‐HSQC chemical shifts suggested that all the organic solvents preferentially localize to a hydrophobic patch in the active‐site vicinity and no chemical shift perturbation was observed for residues present in protein's core. This suggests that activity alteration might be directly linked to change in active site environment only. All organic solvents decreased the apparent binding of substrate to the enzyme (increased K_m); however significantly enhanced the k_cat. Melting temperature (T_m) of lipase, measured by circular dichroism and differential scanning calorimetry, altered in all solvents, albeit to a variable extent. Interestingly, although the effect of all organic solvents on various properties on lipase is qualitatively similar, our study suggest that magnitudes of effects do not appear to follow bulk solvent properties like polarity and the solvent effects are apparently dictated by specific and local interactions of solvent molecule(s) with the protein.     

Imidazolium chloride‐based ionic liquid‐assisted improvement of lipase activity in organic solvents

The activity of a lipase from a newly isolated Pseudomonas sp. was investigated in the presence of organic solvents and imidazolium chloride‐based ionic liquids (IL) such as BMIM[Cl] and HMIM[Cl]. The lipase activity in the presence of IL was higher compared to that in common organic solvents such as methanol and 2‐propanol. A possible explanation for the enzyme activation might be the structural changes induced in the protein in organic systems. Since IL quench the intensity of fluorescence emission, it was not possible to investigate the major factor that influences the enzyme behavior in these new organic salts. Furthermore, the enzyme exhibited excellent activity in buffer mixtures containing both organic solvent and IL. The stability of the lipase at 50°C was considerably increased in the presence of 20% BMIM[Cl] compared with the untreated lipase in aqueous medium. The light scattering method clearly showed that prevention of aggregation could be the reason for thermal stabilization at 50°C in reactions containing IL. Kinetic analysis of the enzyme in the presence of different concentrations of IL showed that the K_m value increased from 0.45 mM in aqueous buffer to 2.4 mM in 50% v/v BMIM[Cl]/buffer. The increase in K_m indicates that IL can significantly reduce the binding affinity of the substrate to the enzyme. Also, a linear correlation was observed between the BMIM[Cl] concentration and V_max of the enzyme. As the concentration of BMIM[Cl] increased from 10 to 50% v/v, the V_max value increased from 1.8 to 46 μM/min.     

Synthesis of a highly fluorescent N,N‐dimethyl benzylamine–palladium(II) curcuminate complex and its application for determination of trace amounts of water in organic solvents

In this work a new highly fluorescent N,N‐dimethyl benzylamine–palladium(II) yu complex was synthesized by the reaction of [Pd₂{(C,N–C₆H₄CH₂N(CH₃)₂}₂(μ‐OAc)]₂] with curcumin. The structure of the synthesized complex was characterized using Fourier transform infra‐red (FT‐IR) spectroscopy, ¹H nuclear magnetic resonance spectroscopy, and elemental analysis. Fluorescence quantum yield (Φ_F) values of the synthesized complex in dimethyl sulfoxide (DMSO), acetonitrile, ethanol, and methanol were 0.160, 0.104, 0.068, and 0.061, respectively. The fluorescence signal of the complex in the organic solvents was very sensitive to the water content of the organic solvent. The quenching effect of water was used to determine trace amounts of water in the heteroatom‐containing organic solvents (ethanol, methanol, acetonitrile) and redox‐active solvents (DMSO). The linear ranges for determination of water (v/v %) in ethanol, DMSO and acetonitrile were found to be 0.03–14.5, 0.08–13.8, and 0.07–18.8, respectively. Two linear ranges were found for determination of water (v/v %) in methanol (0.1–1.2 and 4.7–25.0). Detection limit (DL) values were calculated to be 0.001, 0.05, 0.004, and 0.01 (v/v %) in ethanol, methanol, acetonitrile, and DMSO, respectively. The proposed method overcomes the problems of the standard Karl Fischer method for determination of water in DMSO. In addition, it gave the best DL value for determination of water in ethanol compared with all published papers to date.     

Influence of Water Miscible Organic Solvents on α-chymotrypsin in Solution and Immobilized on Eupergit CM

The effects of the water-miscible organic solvents (methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, N,N′-dimethylformamide and tetrahydrofuran) on the stability and catalytic activity of α-chymotrypsin (CT) immobilized on Eupergit CM were studied. Enhanced stabilities and activities were observed both as a consequence of immobilization and the presence of organic solvent, which in combination provide long term (at least 24 h) retention of activity, and up to 50-fold increase in 50% (v/v) methanol in buffer. Low quantities (20%, v/v) of acetonitrile not only prevented CT inactivation by autolysis at 20°C but also induced a significant increase in the activity of both free (six-fold) and immobilized (two-fold) CT.Linus Olofsson and Pernilla Söderberg authors have contributed equally to the work.     

Tolerance of β-diketone hydrolases as representatives of the crotonase superfamily towards organic solvents

Crotonase superfamily enzymes catalyze a wide variety of reactions, including hydrolytic C–C bond cleavage in symmetrical β‐diketones by 6‐oxo camphor hydrolase (OCH) from Rhodococcus sp. The organic solvent tolerance and temperature stability of OCH and its structurally related ortholog Anabaena β‐diketone hydrolase have been investigated. Both enzymes showed excellent tolerance toward organic solvents; for instance, even in the presence of 80% (v/v) THF or dioxane, OCH was still active. In most solvent mixtures, except methanol, the stereospecificity was conserved (>99% e.e. of product), hence neither the type of solvent nor its concentration appeared to have an effect on the stereoselectivity of the enzyme. Attempts to correlate the observed activities with log P, functional solvent group or denaturing capacity (DC) of the solvent were only successful in the case of DC for water miscible solvents. This study represents the first investigation of organic solvent stability for members of the crotonase superfamily. Biotechnol. Bioeng. 2011;108: 2815–2822. © 2011 Wiley Periodicals, Inc.     

Effects of imidazolium room temperature ionic liquids on the fluorescent properties of norfloxacin

The effects of 12 imidazolium room temperature ionic liquids (RTILs), including [C_nmim]BF₄, [C_nmim]PF₆, and [C_nmim]Br (n = 4, 6, 8, 10), on the fluorescent properties of norfloxacin were examined. The fluorescence intensity of norfloxacin at 0.1 mg/L in methanol significantly increased with the addition of [C_nmim]BF₄ and [C_nmim]PF₆ into the solvent at 0.1–15.0%. The sensitizing effect may result from the higher viscosity of the RTILs–methanol mixture solvent than that of the methanol itself. However, the quenching effect on fluorescence of norfloxacin was observed in [C_nmim]Br–methanol solvent. The fluorescence intensities of norfloxacin decreased with an increase in the alkyl chain length of the alkyl substituents of the imidazolium ring of RTILs. The main interaction between the RTILs and norfloxacin is not by hydrogen bonding. The fact, that some RTILs can significantly sensitize fluorescence of norfloxacin, indicates that RTILs could be a group of promising solvents for development of sensitive spectrofluorimetric methods for determination of norfloxacin at ultra‐trace levels in environmental samples. Copyright © 2011 John Wiley & Sons, Ltd.     

Stability and structure of Penicillium chrysogenum lipase in the presence of organic solvents

Abstract

The present work describes the enzymatic properties of Penicillium chrysogenum lipase and its behavior in the presence of organic solvents. The temperature and pH optima of the purified lipase was found to be 55?°C and pH 8.0 respectively. The lipase displayed remarkable stability in both polar and non-polar solvents upto 50% (v/v) concentrations for 72?h. A structural perspective of the purified lipase in different organic solvents was gained by using circular dichroism and intrinsic fluorescence spectroscopy. The native lipase consisted of a predominant α-helix structure which was maintained in both polar and non-polar solvents with the exception of ethyl butyrate where the activity was decreased and the structure was disrupted. The quenching of fluorescence intensity in the presence of organic solvents indicated the transformation of the lipase microenviroment P. chrysogenum lipase offers an interesting system for understanding the solvent stability mechanisms which could be used for rationale designing of engineered lipase biocatalysts for application in organic synthesis in non-aqueous media.     

Isolation of new toluene-tolerant marine strains of bacteria and characterization of their solvent-tolerance properties

Aims:  To isolate and characterize new marine bacteria capable of tolerating high concentrations of organic solvents, and to understand the toxic effects of these chemicals on marine bacteria. Methods and Results:  Five marine bacteria able to tolerate 0·1% (v/v) toluene were isolated and characterized on the basis of their growth and survival rates in the presence of different organic solvents. The toluene-tolerant marine bacteria identified in this study could not grow in the presence of 0·1% (v/v) of several organic solvents with a log P_ow higher than that of the toluene (which in theory should be less toxic than toluene). The mechanisms underlying solvent tolerance were explored. Conclusions:  Isolates of four different genera were identified as toluene-tolerant. Toxicity of a second phase of an organic solvent toward these isolates could not be predicted on the basis of the solvents’ log P_ow. Significance and Impact of the Study:  To improve the biodegradation rate of some water-insoluble compounds, double-phase bioreactors can be used. This type of bioreactor will require strains able to grow in a salt-containing environment and able to tolerate a second phase of an organic solvent.     

Activity,stability and enantioselectivity of lipase-coated microcrystals of inorganic salts in organic solvents

Lipase-coated microcrystals of inorganic salts were prepared by dissolving enzymes in buffers and then mixing with 3 volumes of saturated salt solutions followed by drop-wise addition into polar precipitating organic solvents. The Mucor javanicus lipase-coated microcrystals did not show any activity for esterification of lauric acid with 1-propanol in isooctane when NaCl and Na₂SO₄ were used as the salts but showed much higher activity than the enzyme powder when KCl (10.0 times) and K₂SO₄ (5.8 times) were used as the salts and precipitated in 1-propanol. Acetonitrile was found to be the best precipitating solvent for preparing M. javanicus lipase-coated microcrystals, with enzyme activities 26.2 and 22.4 times higher than that of the enzyme powder when KCl and K₂SO₄ were used as precipitating salts, respectively. The presence of water in the precipitating solvents markedly decreased the enzyme activity. The M. javanicus lipase-coated microcrystals prepared using K₂SO₄ as the salt and acetonitrile as the precipitating solvent was as active at 80°C as at 40°C. No significant improvement in enantioselectivity of Candida rugosa lipase-coated microcrystals was observed for transesterification of 1-phenylethanol with vinyl acetate in hexane when the microcrystals were prepared by dissolving the enzymes in salt solutions containing 25% (v/v) of acetone or 2-propanol before precipitating in polar solvents.     

Effects <Emphasis Type="Italic">per se</Emphasis> of Organic Solvents in the Cerebral Acetylcholinesterase of Rats

Acetylcholinesterase (AChE) was studied in different rat brain regions (cerebellum, hypothalamus, striatum, hippocampus and cortex) in the presence of different organic solvents normally used in the in vitro assay. The organic solvents used were acetone (C₃H₆O), acetonitrile (C₂H₃N), ethyl alcohol (C₂H₆O), isopropyl alcohol (C₃H₈O), methyl alcohol (CH₄O), tert-butyl alcohol (C₄H₁₀O) and dimethyl sulfoxide (DMSO, C₂H₆OS) ranging from 0.6 to 10%. Ethyl and methyl alcohol presented no effect on AChE activity at any of the concentrations and brain structures tested. In the hippocampus, isopropyl alcohol did not demonstrate a significant inhibitory effect, even at high concentrations. Tert-butyl alcohol presented an interesting result, increased AChE activity (P < .05) in the hypothalamus (1.8%), cortex (1.8 and 2.5) and striatum (1.2, 1.8 and 2.5%) and decreased activity at a concentration of 10% in the cortex (P < .05) and striatum (P < .01). Acetone and acetonitrile presented similar results, both significantly inhibiting AChE in all structures (5%, P < .05 and 10%, P < .01). DMSO exhibited a highly inhibitory effect at practically all concentrations tested (P < .01). In conclusion, for testing new compounds on AChE activity in vitro, methyl and ethyl alcohol may be the best organic solvent choice.     

Effects of Polar Organic Solvents on the Biocatalysis of Chlorophyllase in a Biphasic Organic System

The effects of polarity of various organic solvents, including acetone, ethanol, and propanol, used in a biphasic organic system, on the hydrolytic activity of a partially purified chlorophyllase from Phaeodactylum tricornutum were investigated. The different concentrations of each polar organic solvent, from 0 to 40%, were added to a mixture (45:55, v/v) of hexane and a buffer solution of Tris–HCl (20 mm, pH 7.5). The most appropriate concentrations of acetone, ethanol, and propanol for the hydrolytic activity of chlorophyllase were 12.5, 5.0, and 2.5%, respectively. The results indicated that the optimum reaction time for the chlorophyllase activity in the biphasic system decreased from 7.0 h to 3.0, 5.0, and 5.0 h, respectively, upon the addition of an appropriate amount of acetone, ethanol, or propanol. The V_max and K_m as well as the inhibitory effect of phytol on the chlorophyllase activity in the biphasic organic system containing a polar organic solvent were also investigated.     

Simple method for clinical determination of 13 carotenoids in human plasma using an isocratic high-performance liquid chromatographic method

We report a reversed-phase high-performance liquid chromatography method which resolves 13 identified carotenoids and nine unknown carotenoids from human plasma. A Nucleosil C₁₈ column and a Vydac C₁₈ column in series are used with an isocratic solvent system of acetonitrile–methanol containing 50 mM acetate ammonium–dichloromethane–water (70:15:10:5, v/v/v/v) as mobile phase at a flow-rate of 2 ml/min. The intra-day (4.5–8.3%) and inter-day (1.3–12.7%) coefficients of variation are suitable for routine clinical determinations.     

Improvement in lipase-catalyzed methanolysis of triacylglycerols for biodiesel production using a solvent engineering method

A solvent engineering strategy was applied to the lipase-catalyzed methanolysis of triacylglycerols for biodiesel production. The effect of different pure organic solvents and co-solvent mixtures on the methanolysis was compared. The substrate conversions in the co-solvent mixtures were all higher than those of the corresponding pure organic solvents. Further study showed that addition of co-solvent decreased the values of |log P_interface − log P_substrate| and thus led to a faster reaction. The more the values of |log P_interface − log P_substrate| decreased, the faster the reaction proceeded and the higher the conversion attained. Different co-solvent ratio was further investigated. The co-solvent mixture of 25% t-pentanol:75% isooctane (v/v) was optimal, with which both the negative effects caused by excessive methanol and by-product glycerol could be eliminated. There was no obvious loss in lipase activity even after being repeatedly used for 60 cycles (720 h) with this co-solvent mixture as reaction medium. Other lipases and lipase combinations can also catalyze methanolysis in this co-solvent mixture. Furthermore, other vegetable oils were also explored for biodiesel production in this co-solvent mixture and it had been found that this co-solvent mixture media has extensive applicability.     

Utilization of tannery solid waste for protease production by Synergistes sp. in solid‐state fermentation and partial protease characterization

Synergistes sp. DQ560074 produced a protease in submerged fermentation (SmF) at 400–420 U/mL and in solid‐state fermentation (SSF) at 745–755 U/g. The protease, which belongs to the aspartic protease class, was active over a wide range of pH (5–7) and at high temperatures (25–45°C). The protease is stable and active in various polar protic solvents (50% v/v) like ethanol, isopropanol, n–butanol, in polar aprotic solvents (50% v/v) like acetonitrile, and in non‐polar solvents (50% v/v) such as ethylacetate and toluene, but not in hydrophilic organic solvents (methyl alcohol and acetone). As far as we know, this is the first contribution to the production of a mesophilic protease with solvent stability in SSF using a proteinaceous solid waste.     

Cloning and characterization of a cyclohexanone monooxygenase gene from Arthrobacter sp. L661

Cyclohexanone monooxygenase (CHMO), a type of Baeyer-Villiger oxidation, catalyzes the oxidation of cyclohexanone into ɛ-caprolactone, which has been utilized as a building block in organic synthesis. A bacterium that is capable of growth on cyclohexanone as a sole carbon source was recently isolated and was identified as Arthrobacter sp. L661. The strain is believed to harbor a CHMO gene (chnB), considering the high degradablity of cyclohexanone. In order to characterize the CHMO, a chnB gene was cloned from Arthrobacter sp. L661. The deduced amino acids of the chnB gene evidenced the highest degree of homology (90% identity) with the CHMO of Arthrobacter sp. BP2 (accession no. AY123972). The CHMO of L661 was shown to be functionally expressed in Escherichia coli cells, purified via affinity chromatography, and characterized. The specific activity of the purified enzyme was 24.75 μmol/min/mg protein. The optimum pH was 7.0 and the enzyme maintained over 70% of its activity for up to 24 h in a pH range of 6.0 to 8.0 at 4°C. The CHMO of L661 readily oxidized cyclobutanone and cyclopentanone whereas less activity was detected with those of Arthrobacter sp. BP2, Rhodococcus sp. Phi1, and Rhodococcus sp. Phi2, thereby suggesting that the CHMO of L661 evidenced the different substrate specificities compared with other CHMOs. These results can provide us with useful information for the development of biocatalysts applicable to commercial organic syntheses, especially because only a few CHMO genes have been identified thus far.     

Examination of the solvent perturbation technique as a method to identify enzyme catalytic groups

The present study was undertaken for the purpose of evaluating the solvent perturbation technique as a method to identify enzyme catalytic residues. For establishment of expected directions and sizes of pKa perturbations for different types of acids in different classes of solvents, a study of the pKa of a series of acids in mixed solvent systems was carried out. Consistent with previous findings, the presence of organic solvents (25% v/v) increased the pKa values of neutral acids while it decreased or did not change the pKa values of cationic acids. The size of the perturbation observed was dependent on the nature of the organic solvent and on the polarity of the neutral form of the acid. The solvent perturbation studies were then extended to the catalytic aspartate residue of yeast hexokinase. The pKa of this residue was determined from the MgATP V/K profile measured in the presence and absence of organic solvents (25% v/v). While dimethylformamide and methanol induced small but perhaps significant increases in the observed pKa, dimethyl sulfoxide and propylene glycol did not. The pKa values, from the MgATP V/K profiles measured in the presence of fully saturating glucose, were not significantly increased by the organic solvents. The pKi vs. pH profile for the competitive inhibitor lyxose was also measured in the presence and absence of organic solvents. While methanol (25% v/v), dimethylformamide (25% v/v), and dioxane (17.5% v/v) induced a large increase in the pKa, propylene glycol and dimethyl sulfoxide (25% v/v) did not. The results from this investigation indicate that the solvent perturbation technique should not be relied upon indiscriminately.     

Toxicity assessment of common organic solvents using a biosensor based on algal photosynthetic activity measurement

The acute toxicities of common organic solvents (e.g., methanol, ethanol, isopropanol, acetone, acetonitrile, and dimethylformamide) were evaluated using a biosensor based on microalgal photosynthesis measurement. The biosensor was air-tight, with no headspace, preventing volatile organic toxicants from escaping into the environment as well as partitioning from the aqueous phase into the headspace until equilibrium was reached. Both the incubating and exposure times were set at 10 min. It was observed that only 2 h was needed to obtain complete dose-related inhibition of photosynthetic activity. The results showed that all the tested organic solvents inhibited algal photosynthesis with EC₅₀ ranging between 589 and 2,570 mM. The inhibition of these solvents was in the order: isopropanol > acetone > acetonitrile > ethanol > dimethylformamide > methanol. The quantitative structure-activity relationship (QSAR) between toxicity data and partition coefficient of the examined compounds could be modeled as follows: ${\text{log}}_{{10}} {\text{EC}}_{{50}} \;{\left( {\mu {\text{M}}} \right)} = - 0.6428\;{\text{log}}\;P + 5.76\;{\left( {{\text{R}}^{2} \approx 0.88} \right)}The acute toxicities of common organic solvents (e.g., methanol, ethanol, isopropanol, acetone, acetonitrile, and dimethylformamide) were evaluated using a biosensor based on microalgal photosynthesis measurement. The biosensor was air-tight, with no headspace, preventing volatile organic toxicants from escaping into the environment as well as partitioning from the aqueous phase into the headspace until equilibrium was reached. Both the incubating and exposure times were set at 10 min. It was observed that only 2 h was needed to obtain complete dose-related inhibition of photosynthetic activity. The results showed that all the tested organic solvents inhibited algal photosynthesis with EC₅₀ ranging between 589 and 2,570 mM. The inhibition of these solvents was in the order: isopropanol > acetone > acetonitrile > ethanol > dimethylformamide > methanol. The quantitative structure-activity relationship (QSAR) between toxicity data and partition coefficient of the examined compounds could be modeled as follows: \textlog₁₀ \textEC₅₀   ( m\textM ) = - 0.6428  \textlog  P + 5.76  ( \textR² ? 0.88 ){\text{log}}_{{10}} {\text{EC}}_{{50}} \;{\left( {\mu {\text{M}}} \right)} = - 0.6428\;{\text{log}}\;P + 5.76\;{\left( {{\text{R}}^{2} \approx 0.88} \right)}. This indicates that the photosynthetic activity of the microalga Pseudokirchneriella subcapitata is highly dependent on the hydrophobicity of these commonly used organic solvents.     

Lipase-catalyzed acylation of lily polysaccharide in ionic liquid-containing systems

A comparative study was made of immobilized Burkholderia cepacia lipase (PSL-C)-catalyzed acylation of lily polysaccharide (LP) with vinyl acetate in organic solvents, ionic liquids (ILs) and IL-containing systems. The degree of substitution (DS) of the modified LP was used to evaluate the extent of acylation and thus enzymatic activity. In this manner, an eco-friendly solvent, 2-methyltetrahydrofuran (MeTHF), was found to be the most suitable organic reaction medium. However, compared to MeTHF, enhanced enzyme activity was observed when 1-butyl-3-methylimidazolium tetrafluorobrate ([C₄MIm][BF₄]) was used as the solvent. To further enhance the DS of the modified LP product, co-solvent mixtures of [C₄MIm][BF₄] and MeTHF were investigated. Among the various MeTHF–[C₄MIm][BF₄] systems examined, 20% (v/v) MeTHF–[C₄MIm][BF₄] produced the highest DS. In this reaction medium, the optimal water activity, reaction temperature and time were 0.33, 55 °C and 18 h, respectively, producing a product DS as high as 0.67. The PSL-C enzyme exhibited a much higher stability in the IL-containing system. Additionally, PSL-C-catalyzed acylation of LP was highly regioselective, causing acylation of only C6OH.     

Burkholderia cepacia lipase is a promising biocatalyst for biofuel production

Lipases resistant to inhibition and denaturation by methanol are valuable tools for biotechnological applications, in particular for biofuel production. Microbial lipases have attracted a great deal of interest because of their stability at high concentrations of organic solvents. Burkholderia cepacia lipase (BCL) is tested here for robustness towards methanol in terms of conformational stability and catalytic activity in transesterification assays. This lipase turns out to be even more tolerant than the homologous and better characterized enzyme from Burkholderia glumae. BCL unfolding transition, as monitored by far‐UV circular dichroism (CD) and intrinsic fluorescence, displays a T_m above 60°C in the presence of 50% methanol. The protein unfolds at low pH, and the organic solvent affects the nature of the denatured state under acidic conditions. The protein performs well in transesterification assays upon prolonged incubations at high methanol concentrations. BCL is highly tolerant to methanol and displays particularly high conformational stability under conditions employed for transesterification reactions. These features depict BCL as a promising enzyme for biofuel industry.