Hydrogen‐Bonding‐Driven Enantioselective Resolution against the Kazlauskas Rule To Afford γ‐Amino Alcohols by Candida rugosa Lipase

Most lipases resolve secondary alcohols in accordance with the “Kazlauskas rule” to give the R enantiomers. In a similar manner to other lipases, Candida rugosa lipase (CRL) exhibits R enantioselectivity towards heptan‐2‐ol, although the enantiomeric ratio (E) is low (E=1.6). However, unexpected enantioselectivity (i.e., S enantioselectivity, E=58) of CRL towards 4‐(tert‐butoxycarbonylamino)butan‐2‐ol, which has a similar chain length to heptan‐2‐ol, has been observed. To develop a deeper understanding of the molecular basis for this unusual enantioselectivity, we have conducted a series of molecular modeling and substrate engineering experiments. The results of these computational and experimental analyses indicated that a hydrogen bond between the Ser450 residue and the nitrogen atom of the carbamate group is critical to stabilize the transition state of the S enantiomer.     

Utilization of Catalytic Properties of the Encapsulated Lipase with Calix[4]arene-Adorned Sporopollenin

The dihydrazide calix[4]arene was immobilized onto sporopollenin in order to encapsulate Candida rugosa lipase (CRL) via sol-gel entrapment. The kinetic resolution of the new encapsulated lipase was investigated for enantioselective hydrolysis of racemic naproxen methyl ester and 2-phenoxypropionic acid methyl ester. The results demonstrated that the activity and enantioselectivity of the lipase were improved when the lipase was encapsulated in the presence of calix[4]arene-immobilized sporopollenin. The encapsulated lipase showed an excellent rate of enantioselectivity against the (R/S)-naproxen methyl and (R/S)-2-phenoxypropionic acid methyl esters, with E = 350 and 295, respectively, compared to the free enzyme.     

Prediction of the Candida antarctica lipase A protein structure by comparative modeling and site-directed mutagenesis

A number of model structures of the CalA suggested by comparative modeling were tested by site-directed mutagenesis. Enzyme variants were created where amino acids predicted to play key roles for the lipase activity in the different models were replaced by an inert amino acid (alanine). The results from activity measurements of the overproduced and purified mutant enzymes indicate a structure where the active site consists of amino acid residues Ser184, His366, and Asp334 and in which there is no lid. This model can be used for future targeted modifications of the enzyme to obtain new substrate acceptance, better thermostability, and higher enantioselectivity.     

Effect of co‐solvent addition on the reaction kinetics of the lipase‐catalyzed resolution of ibuprofen ester

BACKGROUND: The addition of co‐solvent is not limited to enhancing the catalytic rate, it could also assist in situ racemization in the dynamic kinetic resolution of racemic compounds by increasing the reactivity of the base catalyst employed. In the current work, reaction media with the presence of DMSO were investigated in Candida rugosa lipase (EC 3.1.1.3)‐catalyzed hydrolysis of ibuprofen ester that focuses on the thermodynamic effect, reaction stability and implication for the kinetic parameters. RESULTS: The introduction of 2% DMSO increased the reaction rate, conversion, and enantioselectivity of the Candida rugosa lipase‐mediated resolution. However, the performance of the particular enzymatic reaction was reduced when a higher DMSO concentration was added. At lower reaction temperatures, the medium with 2% DMSO exhibited an increase in enantioselectivity, which was attributed to a higher activation energy difference between the fast‐ and slow‐reacting enantiomers compared with the water‐isooctane medium. Additionally, the presence of DMSO had a significant effect on the kinetic parameters, shown by a lower value of Michaelis constant compared with that of a normal reaction without DMSO, which resulted in a fast reaction rate. Finally, inhibition due to the uncompetitive substrate inhibitor was reduced, while the non‐competitive product inhibitor consequently increased. CONCLUSION: This work has demonstrated that only 2% of DMSO can be tolerated by the free Candida rugosa lipase in the resolution of ibuprofen ester. However, it is still able to give significant positive effects on the hydrolysis rate, kinetic parameters and enantioselectivity as well as reaction stability. © 2012 Society of Chemical Industry     

Variability within the Candida rugosa Upases family

Several fungi secrete lipase isozymes differing in biochemicalproperties and in some cases in substrate specificity. In theyeast Candida rugosa, a family of related genes encodes formultiple lipase proteins, highly homologous in sequence butpartially different in the regions interacting with the substratemolecule. Analysis of these substitutions performed on the basisof multiple alignments and using a 3-D model of the enzyme,allows identification of a restricted number of amino adds possiblyinvolved in substrate specificity of Candida lipases.     

Enantioselective Hydrolysis of d,l‐Menthyl Benzoate to L‐(–)‐Menthol by Recombinant Candida rugosa Lipase LIP1

The stereoselective synthesis of L ‐menthol is an attractive process in the flavor and fragrance industry. One promising way to obtain optically pure menthol is the enantioselective hydrolysis of menthol esters under enzymatic catalysis. We developed an effective and highly enantioselective method for the synthesis of L ‐(−)‐menthol (>99% EE) by hydrolyzing the key industrial starting compound, d, l ‐menthyl benzoate. The enzyme of choice was the lipase from Candida rugosa (CRL). While commercially available preparations of this lipase showed only minor selectivity (E=15), excellent enantiomeric purity (E>100) was achieved using the heterologously expressed isoenzyme LIP1.     

Dynamic enzymatic resolution of Naproxen methyl ester in a membrane bioreactor

A lipase‐catalyzed enantioselective continuous hydrolysis process under in situ racemization of substrate using sodium hydroxide as catalyst was developed for the production of (S)‐Naproxen from racemic Naproxen methyl ester in an aqueous–organic biphase system. Use of a tubular silicone rubber membrane in the stirred tank reactor to separate the chemical catalytic racemization and biocatalytic resolution processes, served to avoid the key problem associated with conventional dynamic resolution, viz the incompatibility of in situ chemical racemization with the presence of a biocatalyst. To overcome product inhibition and to facilitate product recovery from the aqueous–organic emulsion containing substrate and lipase, a hydrophilic porous semipermeable membrane was used in the stirred tank reactor. Greater than 60% conversion of the racemate with an enantiomeric excess of product (ee_p) greater than 96% was obtained. In addition, transformation of Candida rugosa lipase (CRL) isoenzymes was observed in the reaction process. © 2001 Society of Chemical Industry     

有机介质中脂肪酶促动力学拆发外消旋1-三甲基硅乙醇

The enantioselective esterification of racemic 1-trimethylsilylethanol with acids catalyzed by lipase in organic solvent was successfully performed. The influence of some factors on the reaction was investigated. Among the four lipases explored, Candlda rugosa lipase (CRL) showed the highest activity and enantioselectivity. Octanoic acid was the best acyl donor among the eleven acids studied and n-hexane was the most suitable medium for the reaction. The optimum shaking rate and temperature were found to be 150 r-rain-i and 20~(3 to 30~C, respectively.The enantiomeric excess of the remaining (S)-(-)-1-trimethylsilylethanol was 93% when substrate conversion was 53% upon incubation of the reaction mixture at 30~C, 150 r-rain-i for 12 h.     

Effects of Additives on the Activity and Enantioselectivity of Candida rugosa Lipase in a Biphasic Medium

The effects of Li⁺, Na⁺, K⁺, Mg²⁺ and Zn²⁺ ions on the activity and enantioselectivity of Candida rugosa lipase (CRL) were investigated in a biphasic medium composed of phosphate buffer solution (containing a metal ion within a 50–500 mM concentration range) and isooctane. The hydrolytic activities of CRL towards p‐nitrophenyl acetate were measured after incubation of the enzyme in the presence of metal ions for 24 h, and they were compared to that obtained after incubation in the absence of any metal ion. The CRL activity was stimulated by the chloride salts of Li⁺, K⁺ and Mg²⁺ for all concentrations considered and the highest enhancement was achieved by Li⁺ with a 1.24–1.75 fold increase observed. The effects of metal ions on the enantioselectivity of CRL were investigated by performing the hydrolysis of racemic Naproxen methyl ester in the same biphasic medium containing Li⁺, Na⁺, K⁺, Mg²⁺ and Zn²⁺ ions. The addition of metal ions increased the hydrolysis rate by ca. 1.31–1.45 fold relative to the control, whereas the enantiomeric excess of product increased slightly in the presence of the metal ions. The effect of Triton X‐100 on the activity and enantioselectivity of the CRL was also investigated by employing 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mM concentrations of it in phosphate buffer solution of the biphasic medium. High concentrations of Triton X‐100 stimulated the enzyme activity up to 1.66 fold after 24 h incubation. Triton X‐100 increased the hydrolysis rate almost independently of the concentration.     

Enhancement of enantioselectivity and reaction rate on the synthesis of (S)‐ketoprofen hydroxyalkyl ester in organic solvents via isopropanol‐dried immobilized lipase

Lipase‐catalyzed enantioselective esterification between (R,S)‐ketoprofen and alkanediol in organic solvents was developed to produce (S)‐ketoprofen hydroxyalkyl esters. The acyl acceptor of 1,6‐hexanediol for the resolution of (R,S)‐ketoprofen yielded only the enantioselectivity (the enantiomeric ratio of initial rate for (S)‐ketoprofen to that of (R)‐ketoprofen) V_S/V_R = 8, when crude Lipase MY originating from Candida rugosa was used. However, isopropanol‐dried immobilized lipases (IPA‐dried IM‐lipase) effectively enhanced the enantioselectivity to greater than 20 in the esterification of (R,S)‐ketoprofen when 1,4‐butanediol, 1,5‐pentanediol or 1,6‐hexanediol was employed. IPA‐dried IM‐lipase and isooctane were selected to use for optimally immobilized lipase and reaction medium, respectively. The IPA‐dried IM‐lipase exhibited the highest enantioselectivity, E = 26.7, to the (S)‐enantiomer with 1,5‐pentanediol and the best enzyme activity to the (S)‐enantiomer with 1,4‐butanediol. The finding indicates that the carbon chain length of the alkanediol strongly affected the enzyme activity and enantioselectivity of lipase‐catalyzed esterification. A maximum enantioselectivity of 37 at 27 °C was generated by IPA‐dried IM‐lipase for the enantioselective esterification of racemic ketoprofen with 1,4‐butanediol. IPA‐dried IM‐lipase can effectively increase the enantioselectivity of lipase. Copyright © 2005 Society of Chemical Industry     

Lipase-catalyzed esterification of glycerol and oleic acid

The enzymatic synthesis of glycerides from glycerol and oleic acid in organic solvent was studied, and the optimal conditions for glyceride synthesis by lipases were established. Of the commercially available lipases that were investigated, Candida rugosa lipase and porcine pancreas lipase resulted in the highest extent of esterification. Iso-octane and hexane were particularly useful organic solvents in glyceride synthesis. The water content in the reaction mixture was of primary importance. For C. rugosa lipase and porcine pancreas lipase, the optimal water contents were 5 and 1%, respectively. Candida rugosa lipase and porcine pancreas lipase manifested contrasting positional specificities in glyceride synthesis.     

Application of Immobilized Lipase from Candida rugosa to Synthesis of Cholesterol Oleate

Candida rugosa lipase (CRL) has been immobilized on two kinds of ion-exchange resins, Duolite A 568 and Amberlite IRC 50. These preparations were investigated as a tool for the production of cholesterol oleate in organic media. An increase in temperature up to 40°C increased the rate of reaction and improved the final ester yield. Under optimal conditions, the reaction yield was followed as a time function, for both lipase preparations with an initial water content of 20%. Then, it was observed that about 78% of the oleic acid was esterified after 10 h using CRL immobilized on Duolite, whereas 73% synthesis of cholesterol oleate was reached with CRL immobilized on Amberlite, for the same incubation time. Also, a difference in reaction yield was noticed for the preparations containing sorbitol. In fact, sorbitol treatment might improve the activity of immobilized lipase by preserving the watershell around the catalyst and by increasing the accessibility of the active site to the substrates. In this way, the reaction yield was enhanced, and an increase of 10% synthesis of cholesterol oleate was obtained in both cases. © 1997 SCI.     

High‐pressure enzymatic hydrolysis of oil

The hydrolysis of sunflower and soybean oil, catalyzed by two enzymes, non‐immobilized Candida rugosa and immobilized Candida antarctica lipase, was performed at atmospheric and high‐pressure. The results showed that at atmospheric pressure between 40 °C and 60 °C initial reaction rates were influenced by the temperature variation, as expected. Due to favorable physico‐chemical properties of dense gases as reaction media, hydrolysis of soybean oil was performed in non‐conventional solvents: in supercritical (SC) CO₂ and near‐critical propane. In SC CO₂ the activity of non‐immobilized Candida rugosa lipase decreased while the reaction rates of hydrolysis catalyzed by immobilized Candida antarctica lipase were 1.5‐fold higher than at atmospheric pressure. However, the reaction rates for the hydrolyses catalyzed by both lipases, were much higher in propane than at atmospheric pressure.     

Acylation of glucose catalysed by lipases in supercritical carbon dioxide

The acylation of glucose with lauric acid in a reaction catalysed by two Candida lipases and a Mucor miehei lipase in supercritical carbon dioxide (SCCO₂) was investigated. A linear dependence of the reaction rate on enzyme concentration was observed. Studies on the effect of temperature on enzyme activity showed that Candida antarctica lipase remains stable at temperatures as high as 70°C. Non-immobilised Candida rugosa lipase was found to have a temperature optimum at 60°C. The acylation reaction rate depended on the initial water activity of both substrates and enzyme; the optimum was 0·75 for Candida antarctica lipase, 0·53 for Candida rugosa lipase, and between 0·3 and 0·5 for Mucor miehei lipase. Candida rugosa lipase was most active at a molar ratio of sugar: acyl donor of 1: 3, while the optimum ratio was found to increase to 1: 6 when the reaction was catalysed by Candida antarctica and Mucor miehei lipases. © 1998 SCI     

High pressure enhances activity and selectivity of Candida rugosa lipase immobilized onto silica nanoparticles in organic solvent

High hydrostatic pressure has been increasingly utilized to improve functions of enzymes, and most of such studies are currently focused on free enzymes in aqueous solution or organic solvent. In this work, Candida rugosa lipase (CRL) was immobilized onto silica nanoparticles and its activity and enantioselectivity in organic solvent were evaluated at high pressures under different water activities. The application of high hydrostatic pressures (50–200 MPa) led to improved activities of immobilized CRL for transesterification of (R)-1-phenylpropan-2-ol with vinyl acetate by 4–6 folds. Additionally the immobilization of CRL resulted in a significant change of selectivities, shifting the enantiomeric excess from the (R)- towards (S)-1-phenylpropan-2-yl acetate product at atmospheric pressure. The application of high pressures led to either enantiomeric excess towards (R)-1-phenylpropan-2-yl or no enantiomeric selectivity, depending on the water activities in the organic solvent and the level of pressures. The interesting behaviour of immobilized CRL under high pressures offers new opportunities to modulate enzyme functions through combination of high pressures and enzyme immobilization.     

Enhancing Effect of Calix[4]arene Amide Derivatives on Lipase Performance in Enantioselective Hydrolysis of Racemic Arylpropionic Acid Methyl Esters

Calix[4]arene amide derivatives were employed as new additives within the sol-gel encapsulation of lipase from Candida rugosa (CRL) to improve its catalytic properties. Evaluation of catalytic activity of the encapsulated lipases was acheived by enantioselective hydrolysis of both racemates, Naproxen methyl ester and 2-phenoxypropionic acid methyl ester, in aqueous buffer solution/isooctane reaction system. Results show that enantioselectivity was improved by using calix[4]arene amide derivatives-based encapsulated lipases. The reaction of naproxen methyl ester resulted in 47.6% conversion (x) in 24 h with 88.9% enantiomeric excess of substrate (ee_s), analogous to an enantioselectivity (E) value of 297 (E = 137 for the encapsulated free enzyme). The conversion of 2-phenoxypropionic acid methyl ester, obtained was 48.4% with E value of 327, enantiomeric excess of substrate (ee_s) of 92% for the reaction time of 1 h (E = 211 for the encapsulated free enzyme).     

Lipase‐catalyzed synthesis of butyl esters by direct esterification in solvent‐free system

BACKGROUND: Reactions performed under solvent‐free conditions give processes that are environmentally friendly, since most solvents are polluting agents. In this work, the performance of Candida rugosa lipae (CRL) immobilized on styrene‐divinylbenzene (STY‐DVB) or controlled pore silica (CPS), and the commercial lipase Novozym 435, was evaluated for the synthesis of butyl esters in solvent–free systems (SFS). A 2² full factorial design was used to study the influence of the organic acid chain length and the biocatalyst concentration on the esterification performance. RESULTS: When CRL on STY‐DVB was used, the ester formation was influenced by both variables and their interaction. The reaction conversion was higher (63%) using 10% of immobilized system and lauric acid, corresponding to a productivity of 3.62 g L^?1 h^?1 For CRL on CPS, only the effect of biocatalyst concentration was significant, and the highest yield was attained using 20% of immobilized system and caprilic acid. In the case of Novozym 435, the highest yield (49%) was obtained using butyric acid as acyl donor at 15% of immobilized lipase. CONCLUSION: The results allowed better understanding of the influence of important parameters in this environmentally friendly process, which also has the process advantage of a higher volumetric productivity when compared with a solvent system. Copyright © 2007 Society of Chemical Industry     

Sustainable production of the emulsifier methyl oleate by Candida rugosa lipase nanoconjugates

Acid functionalization of multi-walled carbon nanotubes (F-MWCNTs) using a mixture of HNO₃ and H₂SO₄ (1:3, v:v) was used as support materials for the adsorption of Candida rugosa lipase (CRL) as nanoconjugates (CRL-MWCNTs) for producing methyl oleate. To evaluate the competency of the CRL-MWCNTs nanoconjugates, parameters viz. reaction time, surfactant as well as thermostability and reusability were investigated. The characterization of CRL-MWCNTs nanoconjugates using Fourier transform infrared spectroscopy, Field Scanning Electron Microscopy and Transmission Electron Microscopy revealed successful attachment of CRL onto the F-MWCNTs. Utilization of CRL-MWCNTs nanoconjugates resulted in a higher acid conversion in the synthesis of methyl oleate (79.85% at 11 h of reaction time) when compared with the free CRL i.e. an approximately 1.5-fold improvement over the free CRL. The highest percentage of esterification (83.62%) was observed following the use of nonionic surfactant when compared with the anionic and cationic ones. The CRL-MWCNTs nanoconjugates could be used up to 5 cycles, retaining 50% of its residual activity. Since the preparation of the CRL-MWCNTs nanoconjugates was facile and cheap while producing reasonable yield, the CRL-MWCNTs nanoconjugates developed here were found as promising biocatalysts for the production of methyl oleate.     

Immobilized lipase-catalysed esterification and transesterification reactions in non-aqueous media for the synthesis of tetrahydrofurfuryl butyrate: comparison and kinetic modeling

Immobilized lipase-catalyzed synthesis of tetrahydrofurfuryl butyrate is reported in this paper. Esterification and transesterification of tetrahydrofurfuryl alcohol (THFA) with butyric acid (BA) and transesterification with ethyl butyrate (EB) to prepare tetrahydrofurfuryl butyrate (THFB) were studied systematically including kinetic modeling. A series of immobilized lipases such as Novozym 435, Lipozyme IM 20, Pseudomonas species lipase on toyonite (PSL/Toyo), Candida rugosa lipase (CRL) on polypropylene, CRL on egg shells and CRL on celite were screened to establish that Novozym 435 was the best catalyst for both esterification and transesterification at 30°C. The effects of various parameters on reaction rates were studied in detail for both reactions with Novozym 435. The ping-pong bi-bi mechanism with inhibition by the substrate THFA fits the data for esterification whereas the ping-pong bi-bi mechanism with inhibition by both the reactants (THFA and EB) and both the products (THFB and ethanol) is valid for the transesterification reaction. The kinetic parameters deduced from these models were used to simulate the conversions, which are in good agreement with the experimental values. Since transesterification suffers inhibition by both the substrates and products, esterification is a better method compared to transesterification.     

Insights from molecular dynamics simulations into pH-dependent enantioselective hydrolysis of ibuprofen esters by Candida rugosa lipase

An interesting observation was found during our continued studieson the hydrolysis of ibuprofen esters by Candida rugosa lipase(CRL). An important role is played by pH in the stereospecifichydrolysis of these esters. The flap region of CRL plays a significantrole in the access of the substrate to the active site of theenzyme. At pH 5.6, 48% of the methyl ester and 5% of the butylester of ibuprofen were hydrolysed in 5.5 h, whereas at pH 7.2,9% of methyl ester and 45% of the butyl ester of ibuprofen washydrolysed in a identical reaction time using CRL. This leadus to assume that CRL prefers the methyl ester of ibuprofenas a substrate at an acidic pH and the butyl ester of ibuprofenat a neutral pH. Therefore, in order to understand the roleof pH in the substrate selection by CRL for the esters of ibuprofenwe used the crystallographic coordinates of the open form ofthe CRL (1CRL) for molecular dynamics (MD) simulations underacidic and neutral conditions for 2 ns using GROMACS. The finalstructures obtained after simulation in acidic and neutral conditionswere compared with the energy-minimized structure, and the root-mean-squaredeviations (r.m.s.ds) were calculated. The r.m.s.d. of the CRLflap at neutral pH was found to be greater than that of theCRL flap at acidic pH. The extent to which the flap opens atneutral pH allowed the bulkier substrate, the butyl ester ofibuprofen, to diffuse into the active site and provides thebest enzyme–substrate fit for this specific substrate.At acidic pH there is a decreased opening of the flap therebyaccommodating a more compact substrate, namely the methyl esterof ibuprofen. Thus, simulation experiments using MD providereasonable insight for the pH-dependent substrate selectivityof CRL in aqueous environments. Received March 27, 2003; revised October 27, 2003; accepted October 30, 2003