Kinetic resolution of α-lipoic acid via enzymatic differentiation of a remote stereocenter

Kinetic resolution of α-lipoic acid, a case of remote stereocenter discrimination, was accomplished using lipase from Aspergillus oryzae WZ007. Performance of this lipase was investigated for enantioselective esterification of (S)-α-lipoic acid, leaving the target product (R)-α-lipoic acid in unreacted form. The effects of chain length of alcohol, type of solvent, molar ratio of alcohol:acid, and reaction temperature were studied. The optimum reaction conditions were found to be esterification with n-octanol at 50°C in heptane with an alcohol:acid molar ratio of 5:1. The conversion rate of α-lipoic acid was 75.2%, with an enantiomeric excess of 92.5% towards unreacted substrate in a reaction time of 48 h.     

Use of lipases in the resolution of racemic ibuprofen

Summary Resolution of (R,S)-ibuprofen enantiomers by esterification in different organic solvents was studied using Candida cylindracea lipase. This enzyme preparation had high enantiospecificity for S(+)-ibuprofen in the esterification reaction of a racemic ibuprofen with primary alcohols. The esterification yields of secondary alcohols were much lower than those of primary alcohols. Esterification with tertiary alcohols was not observed. The synthesis of esters was profoundly affected by the amount of water in the reaction mixture. C. cylindracea lipase was active only in very hydrophobic solvents. The esterification activity of the lipase was reduced significantly by addition of water. The R- and S-enantiomers of ibuprofen were determined without derivatization by HPLC using a chiral column.     

Effect of Solvents and Kinetic Parameters on Synthesis of Ethyl Propionate Catalysed by Poly (AAc-co-HPMA-cl-MBAm)-Matrix-Immobilized Lipase of Pseudomonas aeruginosa BTS-2.

Summary A purified alkaline thermo-tolerant bacterial lipase from Pseudomonas aeruginosa BTS-2 was immobilized on a poly (AAc-co-HPMA-cl-MBAm) hydrogel network. The hydrogel showed approximately 95% binding efficiency for lipase (specific activity 1.96 U mg⁻¹). The immobilized enzyme achieved 65.1% conversion of ethanol and propionic acid (100 mM each) into ethyl propionate in n-nonane at 65 °C in 9 h. When alkane of C-chain length lower than n-nonane was used as the organic solvent, the conversion of ethanol and propionic acid into ethyl propionate decreased with a decrease in the log P value of alkanes. The immobilized lipase retained approximately 30% of its original catalytic activity after five cycles of reuse for esterification of ethanol and propionic acid into ethyl propionate at temperature 65 °C in 3 h. Addition of a molecular sieve (3 ?) to the reaction mixture enhanced the formation of ethyl propionate to 89.3%. Moreover, ethanol and propionic acid when taken a molar ratio of 3:1 further promoted the conversion rate to 94%. However, an increase in the molar ratio of propionic acid with respect to ethanol resulted in a decline of ethyl propionate synthesis.     

Enantioselective esterification of ibuprofen by a novel thermophilic Biocatalyst: APE1547

The enantioselective esterification of ibuprofen catalyzed by a novel thermophilic esterase (APE1547) from the archaeon Aeropyrum pernix K1 was successfully conducted in organic solvents. The effects of acyl acceptor, substrate ratio, organic solvent, temperature, and water activity were investigated. Under optimum conditions, the highest enantioselectivity (E = 38.1) was obtained with a higher enzyme activity (216.5 μmol/g/h). Celites were added into the reaction mixture to remove the water produced in the esterification. The reaction achieved its equilibrium in approximately 96 h with a conversion of 57 and 99% (ee) of the un-reacted (S)-ibuprofen obtained.     

Stereoselective enzymatic esterification of 3-benzoylthio-2-methylpropanoic acid

Summary A key intermediate, S-(–)-3-benzoylthio-2-methylpropanoic acid (1) was made in high optical purity by the lipase-catalyzed stereoselective esterification of racemic 1 with methanol in an organic solvent system. Among various lipases evaluated, Amano P-30 lipase from Pseudomonas sp. efficiently catalyzed the esterification of 1 to yield R-(+) methyl ester and unreacted S-(–) 1. A reaction yield of 40 mol% and an optical purity of 97.2% were obtained for compound 1 at a substrate concentration of 0.1 m (22 mg/ml). Lipase P-30 was immobilized on Accurel polypropylene (PP) and the immobilized enzyme was reused (23 cycles) in the esterification reaction without loss of enzyme acitivity, productivity or optical purity. Among various solvents evaluated, toluene was found to be the most suitable organic solvent and methanol was the best alcohol for the esterification of racemic 1 by immobilized lipase. Substrate concentrations as high as 1.0 m were used in the esterification reaction. When the temperature was increased from 28° C to 60° C, the reaction time required for the esterification of 0.1 m substrate decreased from 16 h to 2 h. On increasing the methanol to substrate molar ratio from 1:1 to 4:1, the rate of esterification decreased. A lipase fermentation using Pseudomonas sp. ATCC 21 808 was developed. In the batch-fermentation process, 56 units/ml of extracellular lipase activity was obtained. A fed-batch process using soybean oil gave a significant increase in the lipase activity (126 units/ml). Crude lipase recovered from the filtrate by ethanol precipitation and immobilized on Accurel PP was also effective: S-(–) compound 1 was obtained in 35 mol% yield and 95% optical purity. Offsprint requests to: R. N. Patel     

Commercially viable resolution of ibuprofen

Ibuprofen belongs to the non-steroidal anti-inflammatory drug (NSAID) family known as profens. Studies demonstrate that (S-ibuprofen is 160 times more potent than (R-ibuprofen in vitro, while the accumulation of (R-ibuprofen can cause serious side effects such as gastrointestinal pain. Candida rugosa lipase was used to enantioselectively esterify racemic ibuprofen with decan-1-ol and butan-1-ol in cyclohexane with an enantiomeric ratio (E) of 130 and 46, respectively, in up to 46% conversion. Separation by bulb-to-bulb distillation of (R)-ibuprofen and unreacted alcohol from the corresponding (S)-alkyl ibuprofen ester was possible for the decyl but not the butyl case. The enantioselective hydrolysis of (S)-alkyl ibuprofen esters with the same biocatalyst in aqueous phosphate buffer was twice as slow for the decyl alcohol versus the butyl example. The combined environmentally friendly enantioselective esterification and hydrolysis of ibuprofen insured the isolation of (S)-ibuprofen with a greater than 99% enantiomeric excess.     

Optimisation of n-octyl oleate enzymatic synthesis over Rhizomucor miehei lipase

Octyl oleate is a useful organic compound with several applications in cosmetic, lubricant and pharmaceutical industry. At first, the enzymatic synthesis of n-octyl oleate by direct lipase-catalysed esterification of oleic acid and 1-octanol was investigated in a stirred batch reactor in solvent-free system. A systematic screening and optimisation of the reaction parameters were performed to gain insight into the kinetics mechanism. Particularly, enzyme concentration, reaction temperature, stirrer speed, water content, substrates concentration and molar ratio were optimised with respect to the final product concentration and reaction rate. The kinetics mechanism of the reaction was investigated. Finally, a comparison of the experimental results obtained in a solvent free-system with those using two different solvents, supercritical carbon dioxide (SC-CO₂) and n-hexane, was proposed. It resulted that in SC-CO₂ higher concentration of the desired product was attained, requiring lower enzyme concentrations to achieve comparable conversion of free fatty acid into fatty acid ester.     

Enantioselective resolution of 3-phenylthio-2-propanol with Humicola lanuginosa lipase

The acetylation of 3-phenylthio-2-propanol (168 mg) was performed with vinyl acetate (1 ml) using different lipases from 15°C to 51°C. As a result, the (R)-enantiomer was selectively acetylated and the (S)-enantiomer was non-reactive in all the cases. An appropriate choice of conditions can be made to isolate both (R)-alcohol (ee 99%, 36 h, conversion 46%, sub/enz: 1/2) and (S)-alcohol (ee 93%, 38 h, conversion 46%, THF, sub/enz: 1 l^–1) using Humicola lanuginosalipase (Lipolase). Increasing the amount of enzyme increased the ee.     

Synthesis of monoacylglycerol containing pinolenic acid via stepwise esterification using a cold active lipase

High purity monoacylglycerol (MAG) containing pinolenic acid was synthesized via stepwise esterification of glycerol and fatty acids from pine nut oil using a cold active lipase from Penicillium camembertii as a biocatalyst. Effects of temperature, molar ratio, water content, enzyme loading, and vacuum on the synthesis of MAG by lipase‐catalyzed esterification of glycerol and fatty acid from pine nut oil were investigated. Diacylglycerol (DAG) as well as MAG increased significantly when temperature was increased from 20 to 40°C. At a molar ratio of 1:1, MAG content decreased because of the significant increase in DAG content. Water has a profound influence on both MAG and DAG content through the entire course of reaction. The reaction rate increased significantly as enzyme loading increased up to 600 units. Vacuum was an effective method to reduce DAG content. The optimum temperature, molar ratio, water content, enzyme loading, vacuum, and reaction time were 20°C, 1:5 (fatty acid to glycerol), 2%, 600 units, 5 torr, and 24 h, respectively. MAG content further increased via lipase‐catalyzed second step esterification at subzero temperature. P. camembertii lipase exhibited esterification activity up to ?30°C. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Scale-up of the enantioselective reaction for the enzymatic resolution of (R,S)-ibuprofen

The reaction for the resolution of (R,S)-ibuprofen was scaled-up to yield gram quantities of (S)-ibuprofen. This was accomplished through two enantioselective reactions each catalysed by Novozym 435. In the first reaction, starting from 300 g of racemic ibuprofen, 88.9 g of enantioenriched (S)-ibuprofen with an enantiomeric excess of the order of 85% were produced. In the subsequent reaction, 75 g of the 85 % e.e. material were utilized to produce 38.4 g of (S)-ibuprofen with an enantiomeric excess of 97.5 %.     

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.     

Microbial production of (S)-1-phenyl-1,3-propanediol by stereospecific reduction of 3-hydroxy-1-phenylpropane-1-one

A novel microbial method of synthesizing (S)-1-phenyl-1,3-propanediol [(S)-PPD] was developed in this study. Our laboratory stock cultures were screened for microorganisms that stereospecifically produced (S)-PPD from 3-hydroxy-1-phenylpropane-1-one (HPPO) using an intact cell system. Of the 828 strains examined (321 bacteria, 233 yeasts and 274 molds), certain strains of Williopsis saturnus var. mrakii and Cryptococcus albidus were found to produce (S)-PPD with over 99% enantiomeric excess (e.e.). Screening identified W. saturnus var. mrakii AJ-5620 as the most productive strain, and this strain was used for further experiments. The (S)-PPD-producing reaction using intact W. saturnus var. mrakii AJ-5620 cells was carried out by successive feeding of HPPO. A total (S)-PPD yield of 9.9 g/l was produced in 20 h. The molar yield was 81% and the optical purity of the (S)-PPD produced was over 99% e.e.     

Stereoselective synthesis of (R)-1-chloro-3(3,4-difluorophenoxy)-2-propanol using lipases from Pseudomonas aeruginosa in ionic liquid-containing system

Direct transesterification of (R,S)-1-chloro-3-(3,4-difluorophenoxy)-2-propanol (rac-CDPP) (a key intermediate in the synthesis of the chiral drug (S)-lubeluzole) with vinyl butyrate by lipases from Pseudomonas aeruginosa (P. aeruginosa) MTCC 5113 was performed in hexane with ionic liquids (ILs) 1-butyl-3-methyl imidazolium hexafluorophosphate [BMIm][PF₆] and 1-butyl-3-methyl imidazolium tetrafluoroborate [BMIm][BF₄] as co-solvents. The maximum conversion (>49%) and enantiomeric excess (ee > 99.9%) was achieved in 6 h of incubation at 30 °C with [BMIm][PF₆] as co-solvent in a two-phase system. The enzyme was able to perform with the same specificity even at 60 °C in the presence of ILs. It was possible to use lipases repeatedly for more than 10 times while still maintaining absolute enantioselectivity and reactivity. Stability studies on lipases from P. aeruginosa in ILs revealed the fact that the enzyme constancy and the reactivity in catalyzing transesterification of rac-CDPP into (S)-1-chloro-3-(3,4-difluorophenoxy)-2-butanoate was of the order of [BMIm][PF₆] > [BMIm][BF₄] in two-phase system.     

Enzymatic synthesis of betulinic acid ester as an anticancer agent: Optimization study

Abstract

Immobilized Candida antarctica lipase, Novozym 435, was used to catalyze the esterification reaction between betulinic acid and phthalic anhydride to synthesize 3-O-phthalyl betulinic acid in n-hexane/chloroform. Response surface methodology based on a five-level, four-variable central composite rotatable design was employed to evaluate the effects of synthesis parameters such as reaction time, reaction temperature, enzyme amount and substrate molar ratio on the yield of ester. Based on the response surface model, the optimal enzymatic synthesis conditions were predicted to be: reaction time 20.3 h, reaction temperature 53.9°C, enzyme amount 145.6 mg, betulinic acid to phthalic anhydride molar ratio 1:1.11. The predicted yield was 65.8% and the actual yield was 64.7%.     

Production of fructooligosaccharides by crude enzyme preparations of β-fructofuranosidase from <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

Fructooligosaccharides (FOS) were produced from sucrose by using crude enzyme preparations of β-fructofuranosidases (FFases) obtained from sucrose-cultured cells of Aureobasidium pullulans DSM 2404. When the preparation mainly consisted of FFase I, that has high transfructosylating activity, the FOS yield was 62%. When the reaction was carried out with additional commercial glucose isomerase (GI) at an activity ratio of FFase and GI of 1:2, the maximum FOS yield reached 69%. This value was higher than those obtained previously using other Aureobasidium spp. (53–59%).     

Influence of precursors and additives on microbial lipases stabilized by sol-gel entrapment

Sol-gel entrapment of microbial lipases from Candida cylindracea (Cc lipase),Pseudomonas fluorescens (Lipase AK), and Pseudomonas cepacia (Lipase PS), using as precursors tetraethoxysilane (TEOS) and silanes of type R-Si(OEt)₃ with alkyl or aryl R groups, has been investigated. Three different methods using these precursors were tried exhibiting protein immobilization yields in the range of 20–50%. Hydrolysis of emulsified olive oil, esterification of lauric acid with 1-octanol and enantioselective acylation of 2-pentanol have been used as model reactions for testing the properties of the encapsulated lipases. The recovery yields of the enzyme activity in the esterification reaction were between 20–68%, the best performance being achieved with phenyltriethoxysilane and tetraethoxysilane precursors at 3:1 molar ratio. When testing the entrapped Lipase AK in the enantioselective acylation reaction of 2-pentanol, activity recovery yields up to 32% related to the free enzyme were obtained and the immobilization increased the enantioselectivity of the enzyme.     

Optimization of immobilized Candida rugosa lipase LIP2-catalyzed resolution to produce l-menthyl acetate

Esterification of l-menthol by lipase is a highly selective method for the resolution of dl-menthol. The present work focuses on the reaction parameters that affect lipase-catalyzed synthesis of l-menthyl acetate in n-hexane using triacetin as acyl donor. Genetically engineered LIP2, an isoform of Candida rugosa lipase, was used as a biocatalyst in the present study. The main objectives of the work were to develop an approach that would enable a better understanding of relationships between the variables (reaction time, temperature, enzyme amount, substrate molar ratio) and the response (molar conversion) for l-menthyl acetate synthesis, and to obtain the optimum conditions for synthesis. By using central composite rotatable design (CCRD) and response surface methodology (RSM) analysis, we found that substrate molar ratio and enzyme amount were the most important variables for the reaction. Based on ridge max analysis, the optimum synthesis conditions were found to be: reaction time 2.2 days, temperature 34.3°C, enzyme amount 0.09 g and substrate molar ratio (dl-menthol:triacetin) 1:1.9, and molar conversion of dl-menthol to l-menthyl acetate was calculated to be 50%. An experiment under optimum conditions was carried out and molar conversion of 48.3% was obtained.     

Lipase of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 1207: A Potential Biocatalyst for Synthesis of Isoamyl Acetate

Commercial lipase preparations and mycelium bound lipase from Aspergillus niger NCIM 1207 were used for esterification of acetic acid with isoamyl alcohol to obtain isoamyl acetate. The esterification reaction was carried out at 30°C in n-hexane with shaking at 120 rpm. Initial reaction rates, conversion efficiency and isoamyl acetate concentration obtained using Novozyme 435 were the highest. Mycelium bound lipase of A. niger NCIM 1207 produced maximal isoamyl acetate formation at an alcohol/acid ratio of 1.6. Acetic acid at higher concentrations than required for the critical alcohol/acid ratio lower than 1.3 and higher than 1.6 resulted in decreased yields of isoamyl acetate probably owing to lowering of micro-aqueous environmental pH around the enzyme leading to inhibition of enzyme activity. Mycelium bound A. niger lipase produced 80 g/l of isoamyl acetate within 96 h even though extremely less amount of enzyme activity was used for esterification. The presence of sodium sulphate during esterification reaction at higher substrate concentration resulted in increased conversion efficiency when we used mycelium bound enzyme preparations of A. niger NCIM 1207. This could be due to removal of excess water released during esterification reaction by sodium sulphate. High ester concentration (286.5 g/l) and conversion (73.5%) were obtained within 24 h using Novozyme 435 under these conditions.     

Application of central composite rotatable design to lipase catalysed synthesis of m-cresyl acetate

Esterification of m-cresol with acetic acid using porcine pancreas lipase (PPL) was investigated by response surface methodology (RSM). A central composite rotatable design (CCRD) involving 32 experiments of five variables at five levels was employed to analyse the esterification behaviour. The effect of five variables studied namely, m-cresol concentrations (0.005–0.025 mol), enzyme/substrate ratios (0.18–1.22 activity units/millimol, AU/mmol), incubation periods (6–54 h), pH (4–8) and buffer volumes (0–0.2 ml) was useful in arriving at an optimum ester yield. The methodology projected conditions for higher yields up to 6.0 mmol. Validation experiments carried out under these predicated conditions showed good correspondence between experimental and predicted yields. CCRD treatment clearly showed the inhibitory nature of m-cresol in the esterification process. The reaction required the presence of buffer for better conversions and a minimum amount of 0.1 ml buffer was found necessary for this reaction. Buffer pH values around 6.0 and below appeared to favour better esterification than those at pH values in the range 6.0–8.0. However an optimum condition for maximum yield was: incubation period: 54 h; buffer volume: 0.2 ml; pH: 8; E/S ratio: 0.83 AU/mmol; m-cresol: 0.02 mol; predicted yield: 6.0 mmol; experimental yield: 6.4 mmol.     

Berberine bridge enzyme catalyzes the six electron oxidation of (S)-reticuline to dehydroscoulerine

Berberine bridge enzyme catalyzes the stereospecific oxidation and carbon–carbon bond formation of (S)-reticuline to (S)-scoulerine. In addition to this type of reactivity the enzyme can further oxidize (S)-scoulerine to the deeply red protoberberine alkaloid dehydroscoulerine albeit with a much lower rate of conversion. In the course of the four electron oxidation, no dihydroprotoberberine species intermediate was detectable suggesting that the second oxidation step leading to aromatization proceeds at a much faster rate. Performing the reaction in the presence of oxygen and under anoxic conditions did not affect the kinetics of the overall reaction suggesting no strict requirement for oxygen in the oxidation of the unstable dihydroprotoberberine intermediate. In addition to the kinetic characterization of this reaction we also present a structure of the enzyme in complex with the fully oxidized product. Combined with information available for the binding modes of (S)-reticuline and (S)-scoulerine a possible mechanism for the additional oxidation is presented. This is compared to previous reports of enzymes ((S)-tetrahydroprotoberberine oxidase and canadine oxidase) showing a similar type of reactivity in different plant species.