Biocatalytic synthesis of poly(ε‐caprolactone) using modified lipase in ionic liquid media

Ionic liquids have been used as exceptional nonaqueous reaction media for enzymatic transformation. The ring‐opening polymerization of ε‐caprolactone catalyzed by Novozyme‐435 lipase was successfully conducted in 1‐butyl‐3‐methylimidazolium hexafluorophosphate ([Bmim]PF₆) ionic liquid. ¹H‐NMR and MALDI‐TOF analyses of poly(ε‐caprolactone) (PCL) formed by Novozyme‐435 lipase‐catalyzed reaction revealed an asymmetric telechelic α‐hydroxy‐ω‐carboxylic acid end group. The effects of enzyme concentration, temperature, reaction time, and water activities on monomer conversion and M_n were systematically evaluated. Through the optimization of reaction conditions, PCL was produced in 85% monomer conversion, with an M_n of 5942, in [Bmim]PF₆ at 60°C for 48 h. DSC results demonstrated that high‐molecular‐weight PCL exhibited an excellent thermal property. SEM results showed that PCL had a clear spherulites structure, which could provide a large surface area for cell adhesion. These results showed that [Bmim]PF₆ ionic liquid was suitable for the biocatalytic synthesis of PCL using Novozyme‐435 lipase, and could be used as alternative environmentally friendly media to replace the traditional organic solvents.     

Hydrolysis of crambe oil by enzymatic catalysis: An evaluation of the operational conditions

Abstract

In this work, the enzymatic hydrolysis of the crambe oil by using a commercial immobilized lipase Lipozyme RM IM was evaluated. The effect of the operational conditions, such as temperature, water/oil molar ratio, enzyme/substrate mass ratio and stirring speed were assessed based on the experimental designs. The experiments were performed in a closed and batch system with controlled temperature and stirring speed. In addition, the kinetics of the process was studied in the best operational conditions, wherein the experimental data were obtained and described by a mathematical model. The influence of the operational conditions was assessed based on the measured values of the free fatty acids (FFA) produced by the enzymatic hydrolysis. In 4?h of reaction, a yield of 42.6% was observed and the most significant operational conditions were the enzyme/substrate mass ratio and stirring speed. By the kinetic investigation, an initial reaction rate of 3.5?×?10⁴?mol?mL^?1?h^?1 and a maximum yield of 74% were observed after 40?h of reaction (in the equilibrium condition). The mathematical model was not only able to adequately describe the experimental data of FFA concentrations profiles but also showed predictive capacity to independents assays in different operational conditions. Therefore, based on the simulation analysis of the enzymatic hydrolysis of the crambe oil, the model can be useful for process optimization and phenomenological studies.     

Lipase‐Catalyzed Kinetic Resolution of (±)‐1‐(2‐Furyl) Ethanol in Nonaqueous Media

S‐1‐(2‐Furyl) ethanol serves as an important chiral building block for the preparation of various natural products, fine chemicals, and is widely used in the chemical and pharmaceutical industries. In this work, lipase‐catalyzed kinetic resolution of (R/S)‐1‐(2‐furyl) ethanol using different acyl donors was investigated. Vinyl esters are good acyl donors vis‐à‐vis alkyl esters for kinetic resolution. Among them, vinyl acetate was found to be the best acyl donor. Different immobilized lipases such as Rhizomucor miehei lipase, Thermomyces lanuginosus lipase, and Candida antarctica lipase B were evaluated for this reaction, among which C. antarctica lipase B, immobilized on acrylic resin (Novozym 435), was found to be the best catalyst in n‐heptane as solvent. The effect of various parameters was studied in a systematic manner. Maximum conversion of 47% and enantiomeric excess of the substrate (ee_s) of 89% were obtained in 2 h using 5 mg of enzyme loading with an equimolar ratio of alcohol to vinyl acetate at 60°C at a speed of 300 rpm in a batch reactor. From the analysis of progress curve and initial rate data, it was concluded that the reaction followed the ordered bi–bi mechanism with dead‐end ester inhibition. Kinetic parameters were obtained by using nonlinear regression. This process is more economical, green, and easily scalable than the chemical processes. Chirality 26:286–292, 2014. © 2014 Wiley Periodicals, Inc.     

Characterization of cross‐linked immobilized arylesterase from Gluconobacter oxydans 621H with activity toward cephalosporin C and 7‐aminocephalosporanic acid

Cross‐linked enzyme aggregates (CLEAs) were prepared from several precipitant agents using glutaraldehyde as a cross‐linking agent with and without BSA, finally choosing a 40% saturation of ammonium sulfate and 25 mM of glutaraldehyde. The CLEAs obtained under optimum conditions were biochemically characterized. The immobilized enzyme showed higher thermal activity and a broader range of pH and organic solvent tolerance than the free enzyme. Arylesterase from Gluconobacter oxydans showed activity toward cephalosporin C and 7‐aminocephalosporanic acid. The CLEAs had a Kcat/K_M of 0.9 M^?1/S^?1 for 7‐ACA (7‐aminocephalosporanic acid) and 0.1 M^?1/S^?1 for CPC (cephalosporin c), whereas free enzyme did not show a typical Michaelis–Menten kinetics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:36–42, 2016     

Lipase-catalyzed simultaneous biosynthesis of biodiesel and glycerol carbonate from corn oil in dimethyl carbonate

Biodiesel [fatty acid methyl esters (FAMEs)] and glycerol carbonate were synthesized from corn oil and dimethyl carbonate (DMC) via transesterification using lipase (Novozyme 435) in solvent-free reaction in which excess DMC was used as the substrate and reaction medium. Glycerol carbonate was also simultaneously formed from DMC and glycerol. Conversions of FAMEs and glycerol carbonate were examined in batch reactions. The FAMEs and glycerol carbonate reached 94 and 62.5% from oil and DMC (molar ratio of 1:10) with 0.2% (v/v) water and 10% (w/w) Novozyme 435 (based on oil weight) at 60°C. When Novozyme 435 was washed with acetone after each reaction, more than 80% activity still remained after seven recycling.     

A QM/MM study of the reaction mechanism of (R)‐hydroxynitrile lyases from Arabidopsis thaliana (AtHNL)

Hydroxynitrile lyases (HNLs) catalyze the conversion of chiral cyanohydrins to hydrocyanic acid (HCN) and aldehyde or ketone. Hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) is the first R‐selective HNL enzyme containing an α/β‐hydrolases fold. In this article, the catalytic mechanism of AtHNL was theoretically studied by using QM/MM approach based on the recently obtained crystal structure in 2012. Two computational models were constructed, and two possible reaction pathways were considered. In Path A, the calculation results indicate that the proton transfer from the hydroxyl group of cyanohydrin occurs firstly, and then the cleavage of C1‐C2 bond and the rotation of the generated cyanide ion (CN^?) follow, afterwards, CN^? abstracts a proton from His236 via Ser81. The C1‐C2 bond cleavage and the protonation of CN^? correspond to comparable free energy barriers (12.1 vs. 12.2 kcal mol^?1), suggesting that both of the two processes contribute a lot to rate‐limiting. In Path B, the deprotonation of the hydroxyl group of cyanohydrin and the cleavage of C1‐C2 bond take place in a concerted manner, which corresponds to the highest free energy barrier of 13.2 kcal mol^?1. The free energy barriers of Path A and B are very similar and basically agree well with the experimental value of HbHNL, a similar enzyme of AtHNL. Therefore, both of the two pathways are possible. In the reaction, the catalytic triad (His236, Ser81, and Asp208) acts as the general acid/base, and the generated CN^? is stabilized by the hydroxyl group of Ser81 and the main‐chain NH‐groups of Ala13 and Phe82. Proteins 2015; 83:66–77. © 2014 Wiley Periodicals, Inc.     

Enzymatic synthesis of palm-based ascorbyl esters

The synthesis of palm-based ascorbyl esters through transesterification of ascorbic acid and palm oil in tert-amyl alcohol catalyzed by immobilized lipase is described. Highest conversion (70–75%) was determined after 16 h reaction at 40 °C using lipase (Novozyme 435 from Candida antartica) with an ascorbic acid to palm oil mole ratio of 1:8. The purified product was further characterized by ¹³C NMR and GC–MS and the mixture of ascorbyl monoesters obtained were identified as ascorbyl monooleate (61%), ascorbyl monopalmitate (30%) and ascorbyl monostearate (9%). The antioxidant activity of palm-based ascorbyl esters was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) test. The results showed that pure palm-based ascorbyl esters have an antioxidant activity with an IC₅₀ value of 0.1 mg/mL.     

Improved enantioselective hydrolysis of racemic ethyl-2,2-dimethylcyclopropanecarboxylate catalyzed by modified Novozyme 435

S-(+)-2,2-dimethylcyclopropanecarboxylic acid (S-(+)-DMCPA) is a key chiral intermediate for the synthesis of Cilastatin. The enzymatic preparation of S-(+)-DMCPA has attracted much attention. In order to improve the activity and stability of Novozyme 435 for enzymatic preparation of S-(+)-DMCPA from 2,2-dimethylcyclopropane carboxylate (DMCPE), the glutaraldehyde modification for Novozyme 435 was investigated and the glutaraldehydemodified Novozyme 435 was used as biocatalyst for the synthesis of S-(+)-DMCPA. The results showed that the modified Novozyme 435 had a better reusing merit than unmodified enzyme. The maximum specific activity was obtained by modification Novozyme 435 with 1.5% glutaraldehyde solution under the conditions of shaking at 200 rpm and 30°C for 45 min. The optimal enzymatic hydrolysis conditions for glutaraldehyde-modified Novozyme 435 were also confirmed. The optimized hydrolytic reaction mixture contained 10 mL potassium phosphate buffer (1.0 mol/L, pH 7.6), 90 mg of DMCPE and 160 mg of glutaraldehyde-modified enzyme, and the reaction was performed at 30oC and 200 rpm for 52 h. The reusing efficiency of modified Novozyme 435 was further evaluated. Under the optimal conditions, the modified enzyme remained 76.0% of its original yield after 10 times reuse, but the optical purity of the product kept intact; whereas the yield of unmodified enzyme reduced to 20.8% of its initial value and the ee value of product decreased a lot to 90.7% after 7 times recycle. These results showed that the modified Novozyme 435 was more cost-effective for the preparation of S-(+)-DMCPA in industrial application.     

Toward a High‐Performance All‐Plastic Full Battery with a'Single Organic Polymer as Both Cathode and Anode

The design and fabrication of high‐performance all‐plastic batteries is essentially important to achieve future flexible electronics. A major challenge in this field is the lack of stable and reliable soft organic electrodes with satisfactory performance. Here, a novel all‐plastic‐electrode based Li‐ion battery with a single flexible bi‐functional ladderized heterocyclic poly(quinone), (C₆O₂S₂)_n, as both cathode and anode is demonstrated. Benefiting from its unique ladder‐like quinone and dithioether structure, the as‐prepared polymer cathode shows a high energy density of 624 Wh kg^?1 (vs lithium anode) and a stable battery life of 1000 cycles. Moreover, the as‐fabricated symmetric full‐battery delivers a large capacity of 249 mAh g^?1 (at 20 mA g^?1), a good capacity retention of 119 mAh g^?1 after 250 cycles (at 1.0 A g^?1) and a noteworthy energy density up to 276 Wh kg^?1. The superior performance of poly(2,3‐dithiino‐1,4‐benzoquinone)‐based electrode rivals most of the state‐of‐the‐art demonstrations on organic‐based metal‐ion shuttling batteries. The study provides an effective strategy to develop stable bi‐functional electrode materials toward the next‐generation of high performance all‐plastic batteries.     

Selective synthesis of panthenyl esters by a kinetically controlled enzymatic process

Abstract

Panthenyl esters (panthenyl monoacetate and panthenyl diacetate) were synthesized in high yields (≈100%) by a kinetic reaction control using a commercial immobilized Candida Antarctica lipase B (Novozyme 435) in acetonitrile. The enzyme showed excellent synthetic activity, regioselectivity, and operational stability under the conditions used.     

Enzymatic synthesis and antitumor evaluation of mono- and diesters of 3-O-β-D-galactopyranosyl-sn-glycerol

Lipase-catalyzed synthesis of mono- and diesters of 3-O-β-D-galactopyranosyl-sn- glycerol (β-GG) with caproic acid was performed in acetone. The simultaneous production of 1(6’)-monoesters and 1,6’-diesters of β-GG was achieved in this reaction. In order to improve the yield of β-GG esters, four process parameters, enzyme concentration (15?～?25?mg/mL), and substrate molar ratio (caproic acid: β-GG=?1.60?～?2.00?mmol: 0.10?mmol), reaction temperature (40?～?60?°C), and reaction time (8?～?12?h), were optimized via response surface methodology (RSM) employing a three-level-four-variable central composite design. Results showed that enzyme concentration had the most significant (p?β-GG esters. The optimal reaction conditions in acetone were given as follows: Novozyme435 concentration 18.65?mg/mL, molar rate of caproic acid to β-GG 19.46:1, reaction temperature 48?°C, and reaction time 9.83?h. The yield of β-GG esters reached 88.08% under above optimized conditions, which was very close to the predicted value 87.95%. The molar ratio of monoester to diesters was 0.39:0.61. β-GG esters with other fatty acyl chains were synthesized based on the optimized conditions. In vitro antitumor activity indicated that the antitumor activity of β-GG esters was dependent on the nature of fatty acids, such as the length of acyl chain, the degree of saturation, as well as the number of acyl chain.     

Simultaneous conversion of free fatty acids and triglycerides to biodiesel by immobilized Aspergillus oryzae expressing Fusarium heterosporum lipase

The presence of high levels of free fatty acids (FFA) in oil is a barrier to one‐step biodiesel production. Undesirable soaps are formed during conventional chemical methods, and enzyme deactivation occurs when enzymatic methods are used. This work investigates an efficient technique to simultaneously convert a mixture of free fatty acids and triglycerides (TAG). A partial soybean hydrolysate containing 73.04% free fatty acids and 24.81% triglycerides was used as a substrate for the enzymatic production of fatty acid methyl ester (FAME). Whole‐cell Candida antarctica lipase B‐expressing Aspergillus oryzae, and Novozym 435 produced only 75.2 and 73.5% FAME, respectively. Fusarium heterosporum lipase‐expressing A. oryzae produced more than 93% FAME in 72 h using three molar equivalents of methanol. FFA and TAG were converted simultaneously in the presence of increasing water content that resulted from esterification. Therefore, F. heterosporum lipase with a noted high level of tolerance of water could be useful in the industrial production of biodiesel from feedstock that has high proportion of free fatty acids.     

A selective and sensitive fluorescent sensor for cysteine detection based on bi‐8‐carboxamidoquinoline derivative and Cu2+ complex

In this paper, a novel fluorescent sensor 1 for selective and sensitive detection of cysteine was developed based on a complex between bi‐8‐carboxamidoquinoline derivative ligand ( L ) and Cu²⁺. The interaction of Cu²⁺ with the ligand causes a dramatic fluorescence quenching most likely due to its high affinity towards Cu²⁺ and a ligand–metal charge transfer (LMCT) process. The in situ generated L–Cu ² complex was utilized as a chemosensing ensemble for cysteine. In the presence of cysteine, the fluorophore, L , was released from L–Cu ² complex because of the strong affinity of cysteine to Cu²⁺ via the Cu–S bond, leading to the fluorescence recovery of the ligand. The proposed displacement mechanism was confirmed by the results of mass spectrometry (MS) study. Under optimized conditions, the recovered fluorescence intensity is linear with cysteine concentrations in the range 1 × 10^?6 mol/l to 8 × 10^?6 mol/l. The detection limit for cysteine is 1.92 × 10^?7 mol/l. Furthermore, the established method showed a highly sensitive and selective response to cysteine among the 20 fundamental α‐amino acids used as the building blocks of proteins, after Ni²⁺ was used as a masking agent to eliminate the interference of His. The proposed sensor is applicable in monitoring cysteine in practical samples with good recovery rate.     

Hydrolytic properties of a thermostable α‐l‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus

Aims: To characterize of a thermostable recombinant α‐l ‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus for the hydrolysis of arabino‐oligosaccharides to l ‐arabinose. Methods and Results: A recombinant α‐l ‐arabinofuranosidase from C. saccharolyticus was purified by heat treatment and Hi‐Trap anion exchange chromatography with a specific activity of 28·2 U mg^?1. The native enzyme was a 58‐kDa octamer with a molecular mass of 460 kDa, as measured by gel filtration. The catalytic residues and consensus sequences of the glycoside hydrolase 51 family of α‐l ‐arabinofuranosidases were completely conserved in α‐l ‐arabinofuranosidase from C. saccharolyticus. The maximum enzyme activity was observed at pH 5·5 and 80°C with a half‐life of 49 h at 75°C. Among aryl‐glycoside substrates, the enzyme displayed activity only for p‐nitrophenyl‐α‐l ‐arabinofuranoside [maximum k_cat/K_m of 220 m(mol l^?1)^?1 s^?1] and p‐nitrophenyl‐α‐l ‐arabinopyranoside. This substrate specificity differs from those of other α‐l ‐arabinofuranosidases. In a 1 mmol l^?1 solution of each sugar, arabino‐oligosaccharides with 2–5 monomer units were completely hydrolysed to l ‐arabinose within 13 h in the presence of 30 U ml^?1 of enzyme at 75°C. Conclusions: The novel substrate specificity and hydrolytic properties for arabino‐oligosaccharides of α‐l ‐arabinofuranosidase from C. saccharolyticus demonstrate the potential in the commercial production of l ‐arabinose in concert with endoarabinanase and/or xylanase. Significance and Impact of the Study: The findings of this work contribute to the knowledge of hydrolytic properties for arabino‐oligosaccharides performed by thermostable α‐l ‐arabinofuranosidase.     

Lipase‐Catalyzed Kinetic Resolution of Novel Antifungal N‐Substituted Benzimidazole Derivatives

A series of new N‐substituted benzimidazole derivatives was synthesized and their antifungal activity against Candida albicans was evaluated. The chemical step included synthesis of appropriate ketones containing benzimidazole ring, reduction of ketones to the racemic alcohols, and acetylation of alcohols to the esters. All benzimidazole derivatives were obtained with satisfactory yields and in relatively short times. All synthesized compounds exhibit significant antifungal activity against Candida albicans 900028 ATCC (% cell inhibition at 0.25 μg concentration > 98%). Additionally, racemic mixtures of alcohols were separated by lipase‐catalyzed kinetic resolution. In the enzymatic step a transesterification reaction was applied and the influence of a lipase type and solvent on the enantioselectivity of the reaction was studied. The most selective enzymes were Novozyme SP 435 and lipase Amano AK from Pseudomonas fluorescens (E > 100). Chirality 28:347–354, 2016. © 2016 Wiley Periodicals, Inc.     

Enzymatic production of linalool esters in organic and solvent-free system

This work investigated the influence of temperature, enzyme concentration, substrates molar ratio, in the absence and presence of organic solvent, at two molar ratios of the substrates on the enzymatic production of linalil esters using the immobilized lipase Novozym 435 as catalyst, different acids and linalool and Ho-Sho essential oil as substrates. The best reaction conversion was obtained at the highest temperature (70 °C), for both solvent free (3.81%) and with solvent addition (2.25%), for a solvent to substrates molar ratio of 2:1, enzyme concentration of 5 wt% and acid to alcohol molar ratio of 1:1. The reaction kinetics revealed that Ho-Sho essential oil afforded the greatest conversions when compared with pure linalool. Higher linalil esters production were achieved after 10 h reaction (5.58%) in 2:1 solvent to substrates molar ratio, with enzyme concentration of 5 wt%, at 70 °C and anhydride to alcohol molar ratio of 1:1 using Ho-Sho essential oil as substrate.     

Lipase catalytic synthesis of diacylglycerol from tuna oil and its anti-obesity effect in C57BL/6J mice

Diacylglycerol (DAG) production by glycerolysis of glycerol with tuna oil was performed using lipases from Rhizomucor miehei (Lipozyme RMIM) and Candida antarctica (Novozyme 435). Lipozyme RMIM caused a clear estrific positional specificity in HPLC analysis and then the Lipozyme RMIM was chosen for the production of DAG. Moreover, the reaction parameter for DAG synthesis was determined by measuring mole ratio of glycerol/tuna oil, amount of enzyme, temperature and water contents. The optimal mole ratio for glycerol/tuna oil was established to be 3:1. Optimal conditions of lipase, water and temperature were 10%, 10% and 35 °C, respectively. Therefore, we have synthesized DAG from tuna oil under these optimal conditions and investigated the effect of the synthesized DAG on body weight and plasma biochemical markers of obesity in C57BL/6J mice. The consumption of DAG diet has effectively lessened body weight gain and final plasma total cholesterol, triacylglycerol and glucose levels compared to the high triacylglycerol (TAG) group.     

Structure of 6‐diazo‐5‐oxo‐norleucine‐bound human gamma‐glutamyl transpeptidase 1, a novel mechanism of inactivation

Intense efforts are underway to identify inhibitors of the enzyme gamma‐glutamyl transpeptidase 1 (GGT1) which cleaves extracellular gamma‐glutamyl compounds and contributes to the pathology of asthma, reperfusion injury and cancer. The glutamate analog, 6‐diazo‐5‐oxo‐norleucine (DON), inhibits GGT1. DON also inhibits many essential glutamine metabolizing enzymes rendering it too toxic for use in the clinic as a GGT1 inhibitor. We investigated the molecular mechanism of human GGT1 (hGGT1) inhibition by DON to determine possible strategies for increasing its specificity for hGGT1. DON is an irreversible inhibitor of hGGT1. The second order rate constant of inactivation was 0.052 mM ^?1 min^?1 and the K _i was 2.7 ± 0.7 mM . The crystal structure of DON‐inactivated hGGT1 contained a molecule of DON without the diazo‐nitrogen atoms in the active site. The overall structure of the hGGT1‐DON complex resembled the structure of the apo‐enzyme; however, shifts were detected in the loop forming the oxyanion hole and elements of the main chain that form the entrance to the active site. The structure of hGGT1‐DON complex revealed two covalent bonds between the enzyme and inhibitor which were part of a six membered ring. The ring included the OG atom of Thr381, the reactive nucleophile of hGGT1 and the α‐amine of Thr381. The structure of DON‐bound hGGT1 has led to the discovery of a new mechanism of inactivation by DON that differs from its inactivation of other glutamine metabolizing enzymes, and insight into the activation of the catalytic nucleophile that initiates the hGGT1 reaction.     

Heterofunctional Magnetic Metal‐Chelate‐Epoxy Supports for the Purification and Covalent Immobilization of Benzoylformate Decarboxylase From Pseudomonas Putida and Its Carboligation Reactivity

In this study, the combined use of the selectivity of metal chelate affinity chromatography with the capacity of epoxy supports to immobilize poly‐His‐tagged recombinant benzoylformate decarboxylase from Pseudomonas putida (BFD, E.C. 4.1.1.7) via covalent attachment is shown. This was achieved by designing tailor‐made magnetic chelate–epoxy supports. In order to selectively adsorb and then covalently immobilize the poly‐His‐tagged BFD, the epoxy groups (300 µmol epoxy groups/g support) and a very small density of Co²⁺‐chelate groups (38 µmol Co²⁺/g support) was introduced onto magnetic supports. That is, it was possible to accomplish, in a simple manner, the purification and covalent immobilization of a histidine‐tagged recombinant BFD. The magnetically responsive biocatalyst was tested to catalyze the carboligation reactions. The benzoin condensation reactions were performed with this simple and convenient heterogeneous biocatalyst and were comparable to that of a free‐enzyme‐catalyzed reaction. The enantiomeric excess (ee) of (R)‐benzoin was obtained at 99 ± 2% for the free enzyme and 96 ± 3% for the immobilized enzyme. To test the stability of the covalently immobilized enzyme, the immobilized enzyme was reused in five reaction cycles for the formation of chiral 2‐hydroxypropiophenone (2‐HPP) from benzaldehyde and acetaldehyde, and it retained 96% of its original activity after five reaction cycles. Chirality 27:635–642, 2015. © 2015 Wiley Periodicals, Inc.     

Kinetics and mechanism of lipase catalyzed monoacylglycerols synthesis

Monoacylglycerols are increasingly used in several industrial applications as effective and cheap emulsifiers. In the present work monostearin synthesis has been studied, using lipase as a biocatalyst of the esterification reaction of stearic acid with (R,S)-1,2-O-iso-propylidene glycerol (solketal). The lipase from Candida antarctica (CaL B) was immobilized in AOT/isooctane water in oil microemulsions. Optimization of the reaction conditions have shown that the highest production (80% in 30 min) could be achieved at 40 °C, in microemulsions with relatively low water content (w_o = 8). Kinetic studies have shown that the esterification reaction of stearic acid with solketal catalyzed by CaL B occurs via the ordered bi–bi mechanism, in which inhibition by the acid was identified. Moreover, at high fixed solketal concentrations a negative cooperativity is pronounced, which means that binding of the alcohol lowers the affinity of the enzyme for binding of the acid. Values of all kinetic parameters have been determined.