Preparation of S-( )-ketoprofen by coupling the enantioselective hydrolysis of ketoprofen methyl ester with the photo-oxidation of methanol

A novel method for preparation of S-(+)-ketoprofen is presented involving coupling enantioselective hydrolysis of ketoprofen methyl ester catalyzed by a surfactant-coated-lipase with the photo-oxidation of methanol in a water-saturated organic solvent. The effect of photocatalytic conversion of methanol into water and carbon dioxide on the hydrolysis of ketoprofen methyl ester and the stability of the enzyme was investigated. The photo-oxidation of methanol shifted the equilibrium of the hydrolysis toward the formation of ketoprofen, increasing the equilibrium conversion ratio and improving the enantioselectivity. Because the surfactant-coated lipase and ketoprofen methyl ester dissolved in the organic solvent and ketoprofen was absorbed on the TiO₂ photocatalyst particles, the separation procedures could be simplified and the stability of the enzyme was increased.     

Preparation of S-(+)-ketoprofen by coupling the enantioselective hydrolysis of ketoprofen methyl ester with the photo-oxidation of methanol

A novel method for preparation of S-(+)-ketoprofen is presented involving coupling enantioselective hydrolysis of ketoprofen methyl ester catalyzed by a surfactant-coated-lipase with the photo-oxidation of methanol in a water-saturated organic solvent. The effect of photocatalytic conversion of methanol into water and carbon dioxide on the hydrolysis of ketoprofen methyl ester and the stability of the enzyme was investigated. The photo-oxidation of methanol shifted the equilibrium of the hydrolysis toward the formation of ketoprofen, increasing the equilibrium conversion ratio and improving the enantioselectivity. Because the surfactant-coated lipase and ketoprofen methyl ester dissolved in the organic solvent and ketoprofen was absorbed on the TiO₂ photocatalyst particles, the separation procedures could be simplified and the stability of the enzyme was increased.     

Resolution of 2-cyano-2-methylalkanoic acids via porcine pancreatic lipase-catalyzed enantioselective ester hydrolysis: effect of the alcohol moiety of the substrate ester on enantioselectivity

2-Cyano-2-methylalkanoic acids were resolved via porcine pancreatic lipase-catalyzed enantioselective ester hydrolysis. The importance of the alcohol moiety of the substrate ester on enantioselectivity was confirmed: the E value was increased up to 9-fold by using the n-butyl ester instead of the conventional methyl ester. The maximum E value was 180.     

HPLC studies on leukotriene A4 obtained from the hydrolysis of its methyl ester

Alkaline hydrolysis of leukotriene A4 methyl ester to leukotriene A4 was studied in either methanol or acetone. Hydrolysis in acetone yielded larger amounts of leukotriene A4 than similar hydrolysis in methanol. The maximum amount was obtained 60 minutes after the beginning of the hydrolysis. Leukotriene A4, as well as leukotriene B4 methoxy isomers were obtained from hydrolysis of leukotriene A4 methyl ester in methanol. It was found that initial leukotriene A4 methyl ester concentration affected the amount of LTA4 produced during the hydrolysis. The maximum concentration of leukotriene A4 was obtained by hydrolyzing solutions of 0.25 mg/ml leukotriene methyl ester in acetone. Spontaneous degradation of leukotriene A4 occurred when it was diluted with tris buffer. Addition of bovine serum albumin to the tris buffer significantly prolonged the half life of leukotriene A4.     

Effect of several factors on soluble lipase-mediated biodiesel preparation in the biphasic aqueous-oil systems

Biodiesel is increasingly perceived as an important component of solutions to the important current issues of fossil fuel shortages and environmental pollution. Utilization of soluble lipase offers an alternative approach to lipase-catalyzed biodiesel production using immobilized enzyme or whole-cell catalysis. Soluble lipase NS81020, produced by submerged fermentation of genetically modified Aspergillus oryzae microorganism, was first proposed here as the catalyst of biodiesel preparation with oleic acid in the biphasic aqueous-oil systems. The effect factors such as enzyme concentration, water content, temperature, molar ratio of methanol to oil, stirring rate and pH of buffer solution on the esterification rate were investigated systematically. The reaction time could be shortened with the increasing of enzyme concentration as long as the maximum enzyme absorptive capacity on the interface in the biphasic aqueous-oil systems was not achieved. The optimal water content in the biphasic aqueous-oil systems was 10 wt% by oleic acid weight. The reaction rate was enhanced with the increasing molar ratio of methanol to oil, the increasing stirring rate or the decreasing temperature. Although soluble lipase NS81020 had lower activity at pH 10.55, hydroxyl ion conduced to restrain hydrolysis of methyl ester and facilitated the reaction toward the methyl ester formation.     

Optimization of whole cell-catalyzed methanolysis of soybean oil for biodiesel production using response surface methodology

Utilizing whole cell biocatalyst instead of free or immobilized enzyme is a potential way to reduce the cost of catalyst in lipase-catalyzed biodiesel production. Rhizopus oryzae (R. oryzae) IFO4697 whole cell immobilized within biomass support particles (BSPs) was used for the methanolysis of soybean oil for biodiesel production in this paper. tert-Butanol was demonstrated to be an ideal reaction medium, in which the negative effects caused by substrate methanol could be eliminated effectively. A central composite design was adopted to study the effect of tert-butanol quantity, methanol quantity, water content and dry biomass of the immobilized cell on biodiesel (methyl ester) yield. Each factor was studied in five levels. Using response surface methodology, a quadratic polynomial equation was obtained for methyl ester yield by multiple regression analysis. Biodiesel yield of 72% could be obtained under the optimal conditions and further verification experiments confirmed the validity of the predicted model.     

Kinetics of alpha-chymotrypsin catalyzed hydrolysis in equilibrium. II. Comparison of ester and amide substrates

Kinetic of the alpha-chymotrypsin catalyzed reversible hydrolytic reaction of methyl N-acetyl-L-phenylalaninate and N-acetyl-L-phenylalanylglycinamide at pH 5.5 and equilibrium conditions has been studied. The rates of the labeled reaction products incorporated into the substrate a different methanol concentrations shows that the reaction proceeds by a compulsory mechanism with the formation of N-acetyl-L-phenylalanine-alpha-chymotrypsin complex. For the amide substrate the data obtained are also in agreement with the compulsory mechanism of its hydrolysis. Equilibrium kinetics of ester and amide substrates hydrolysis has been compared.     

Lipase-catalyzed naproxen methyl ester hydrolysis in water-saturated ionic liquid: significantly enhanced enantioselectivity and stability

The lipase selective hydrolysis of Naproxen methyl ester was explored in both water-saturated isooctane and water-saturated ionic liquid 1-butyl-3-methylimidazolium hexafluoro-phoshate ([bmim]PF₆) to see any significant differences in terms of enantioselectivity and stability between two different classes of reaction media. It is shown that polar and hydrophobic of [bmim]PF₆ made it an unearthly reaction medium for hydrolysis of Naproxen methyl ester. It not only decreases the equilibrium constant (K) and enhances the enantiomeric ratio (E), consequently improves the equilibrium conversion (C_Eq) of the hydrolysis reaction and enantiomeric excess of product (ee_p), but also maintains the lipase activity. Because the lipase would not dissolve in the 1-butyl-3-methylimidazolium hexafluoro-phoshate, it can be filtrated up from 1-butyl-3-methylimidazolium hexafluoro-phoshate and recycled for several runs. The stability of lipase was improved due to the higher solubility of methanol in 1-butyl-3-methylimidazolium hexafluoro-phoshate than in isooctane.     

Transesterification of canola oil in mixed methanol/ethanol system and use of esters as lubricity additive

Transesterification of canola oil was carried out with methanol, ethanol, and various mixtures of methanol/ethanol, keeping the molar ratio of oil to alcohol 1:6 and using KOH as a catalyst. Mixtures of alcohol increased the rate of transesterification reaction and produced methyl as well as ethyl esters. The increased rate was result of better solubility of oil in reaction mixture due to better solvent properties of ethanol than methanol and equilibrium due to methanol. With 3:3 molar ratio of methanol to ethanol {MEE (3:3)} the amount of ethyl ester formed was 50% that of methyl ester. Properties (acid value, viscosity, density) of all esters including mixed esters were within the limits of ASTM standards. Lubricities of these esters are in the order: ethyl ester>methyl ethyl ester>methyl ester.     

Lipase-catalyzed biodiesel production with methyl acetate as acyl acceptor

Biodiesel is an alternative diesel fuel made from renewable biological resources. During the process of biodiesel production, lipase-catalyzed transesterification is a crucial step. However, current techniques using methanol as acyl acceptor have lower enzymatic activity; this limits the application of such techniques in large-scale biodiesel production. Furthermore, the lipid feedstock of currently available techniques is limited. In this paper, the technique of lipase-catalyzed transesterification of five different oils for biodiesel production with methyl acetate as acyl acceptor was investigated, and the transesterification reaction conditions were optimized. The operation stability of lipase under the obtained optimal conditions was further examined. The results showed that under optimal transesterification conditions, both plant oils and animal fats led to high yields of methyl ester: cotton-seed oil, 98%; rapeseed oil, 95%; soybean oil, 91%; tea-seed oil, 92%; and lard, 95%. Crude and refined cottonseed oil or lard made no significant difference in yields of methyl ester. No loss of enzymatic activity was detected for lipase after being repeatedly used for 40 cycles (ca. 800 h), which indicates that the operational stability of lipase was fairly good under these conditions. Our results suggest that cotton-seed oil, rape-seed oil and lard might substitute soybean oil as suitable lipid feedstock for biodiesel production. Our results also show that our technique is fit for various lipid feedstocks both from plants and animals, and presents a very promising way for the large-scale biodiesel production.     

Ester synthesis in aqueous media in the presence of various lipases

Summary The ability of seven lipase preparations to catalyse methyl ester synthesis in aqueous media was compared and the synthesis reaction (esterification or alcoholysis) determined. Three behaviours were observed: three enzymes catalysed ester synthesis by esterification of free fatty acids and one enzyme catalysed alcoholysis but the other three lipases did not catalyse a net ester synthesis under the conditions tested. The three groups also differed by the influence of methanol on the hydrolysis reaction. The first group was not significantly inhibited up to the highest methanol concentration tested (5 M). Hydrolysis in the presence of the enzyme of the second group was increasingly inhibited with increasing methanol concentrations. In the presence of the third group, hydrolysis was 40 to 50% inhibited for all the concentrations tested (0.2–5 M).     

Efficient microbial elimination of methanol inhibition for Naproxen resolution by a lipase

To eliminate methanol inhibition of the activity of a lipase, methanotrophic bacteria, which can convert methanol into water and CO₂, were introduced to the reaction of enantioselective hydrolysis of Naproxen methyl ester catalysed by lipase from Candida rugosa. Both the activity and stability of lipase were improved by the removal of methanol by the bacteria.     

Optical resolution of racemic 2-hydroxy octanoic acid by lipase-catalyzed hydrolysis in a biphasic membrane reactor

Racemic 2-hydroxy octanoic acid methyl ester was optically resolved by lipase-catalyzed hydrolysis in a biphasic membrane reactor using hydrophilic/hydrophobic capillary membranes. In a buffer/hexane biphasic membrane reactor using hydrophilic ultrafiltration membranes, (S)-2-hydroxy octanoic acid was recovered from the aqueous phase at 59–67% yield and 0.9–0.92 enantiomeric excess (ee), and the ester of (R)-isomer was recovered from the organic phase at 73–75% yield and 0.92–0.99 ee.     

A magnetically separable biocatalyst for resolution of racemic naproxen methyl ester

Candida rugosa lipase (CRL) was encapsulated via the sol–gel method, using 5, 11, 17, 23-tetra-tert-butyl-25,27-bis(2-aminopyridine)carbonylmethoxy-26, 28-dihydroxy-calix[4]arene-grafted magnetic Fe₃O₄ nanoparticles (Calix-M-E). The catalytic activity of encapsulated lipase (Calix-M-E) was tested both in the hydrolysis of p-nitrophenyl palmitate (p-NPP) and the enantioselective hydrolysis of racemic naproxen methyl ester. The present study demonstrated that the calixarene-based compound has the potential to enhance both reaction rate and enantioselectivity of the lipase-catalyzed hydrolysis of racemic naproxen methyl ester. The encapsulated lipase (Calix-M-E) had great catalytic activity and enantioselectivity (E > 400), as well as remarkable reusability as compared to the encapsulated lipase without supports (E = 137) for S-Naproxen.     

Incorporation of methanol into pectic substance

Others have shown that l-methionine is utilized in the biosynthesis of methyl ester groups in pectic substance. Methanol, like l-methionine, is used for methyl ester biosynthesis by detached parsley leaves (Petroselinum crispum). When a combination of methanol-³H and methanol-¹⁴C is given to parsley leaves, methanol recovered from pectic substance by alkaline hydrolysis has a ³H/¹⁴C ratio about one-fourth that of the mixture administered. Unlike l-methionine, methanol is oxidized prior to its utilization as a carbon source for methyl ester biosynthesis.     

Ipolearoside: A new glycoside from Ipomoea leari with anti-cancer activity

Ipolearoside, a new glycoside with anticancer activity, has been isolated from Ipomoea leari Paxt. On acid hydrolysis in methanol, it gave the aglycone methyl ester, characterised as methyl 3,11-dihydroxyhexadecanoate. Ipolearoside is a complex glycoside of 3,11-dihydroxyhexadecanoic acid and glucose, rhamnose and fucose.     

Lipase-catalyzed hydrolysis of 2-naphtyl esters in biphasic system

The authors measured the rate of hydrolysis of the homologs of 2-naphtyl ester by using a Lewis cell with constant interfacial area to elucidate the kinetic mechanism of the lipase-catalyzed hydrolysis in biphasic system. On the basis of the two-film model, it was found from the analysis of experimental results that the hydrolysis of these substrates proceeds at the interface between the aqueous and organic phases. The interfacial reaction rate could be correlated by Michaelis-Menten mechanism. The values of the rate constant and the Michaelis constant were almost independent of the kinds of 2-naphtyl ester. The values of the interfacial kinetic parameters for 2-naphtyl ester were much greater than those for the hydrolysis in the aqueous phase.     

Chemical synthesis and growth-promoting activity of all-trans-retinyl beta-D-glucuronide.

All-trans-retinol reacts with methyl (2,3,4-tri-O-acetyl-1-bromo-1-deoxy-beta-D-glucopyran)uronate in the presence of Ag2CO3 to give the triacetate methyl ester of retinyl beta-glucuronide. Hydrolysis of this ester with sodium methylate in methanol gives retinyl beta-D-glucuronide in about 15% yield. The water-soluble retinyl beta-D-glucuronide was characterized by u.v.-visible, n.m.r. and mass spectra, by elemental analysis and by its susceptibility to hydrolysis by bacterial beta-glucuronidase. Retinyl beta-glucuronide, when administered intraperitoneally in saline (0.9% NaCl), supports well the growth of vitamin A-deficient rats.     

The catalytic serine of meta-cleavage product hydrolases is activated differently for C-O bond cleavage than for C-C bond cleavage

meta-Cleavage product (MCP) hydrolases catalyze C-C bond fission in the aerobic catabolism of aromatic compounds by bacteria. These enzymes utilize a Ser-His-Asp triad to catalyze hydrolysis via an acyl-enzyme intermediate. BphD, which catalyzes the hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) in biphenyl degradation, catalyzed the hydrolysis of an ester analogue, p-nitrophenyl benzoate (pNPB), with a k(cat) value (6.3 ± 0.5 s(-1)) similar to that of HOPDA (6.5 ± 0.5 s(-1)). Consistent with the breakdown of a shared intermediate, product analyses revealed that BphD catalyzed the methanolysis of both HOPDA and pNPB, partitioning the products to benzoic acid and methyl benzoate in similar ratios. Turnover of HOPDA was accelerated up to 4-fold in the presence of short, primary alcohols (methanol > ethanol > n-propanol), suggesting that deacylation is rate-limiting during catalysis. In the steady-state hydrolysis of HOPDA, k(cat)/K(m) values were independent of methanol concentration, while both k(cat) and K(m) values increased with methanol concentration. This result was consistent with a simple model of nucleophilic catalysis. Although the enzyme could not be saturated with pNPB at methanol concentrations of >250 mM, k(obs) values from the steady-state turnover of pNPB at low methanol concentrations were also consistent with a nucleophilic mechanism of catalysis. Finally, transient-state kinetic analysis of pNPB hydrolysis by BphD variants established that substitution of the catalytic His reduced the rate of acylation by more than 3 orders of magnitude. This suggests that for pNPB hydrolysis, the serine nucleophile is activated by the His-Asp dyad. In contrast, rapid acylation of the H265Q variant during C-C bond cleavage suggests that the serinate forms via a substrate-assisted mechanism. Overall, the data indicate that ester hydrolysis proceeds via the same acyl-enzyme intermediate as that of the physiological substrate but that the serine nucleophile is activated via a different mechanism.     

Graft copolymerization of methyl methacrylate onto mercaptochitin and some properties of the resulting hybrid materials

The graft copolymerization of methyl methacrylate onto mercaptochitin and some properties of the resulting graft copolymers have been studied. Methyl methacrylate was efficiently graft copolymerized onto mercaptochitin in dimethyl sulfoxide, and the grafting percentage reached 1300% under appropriate conditions. Although the side-chain ester groups were resistant to aqueous alkali, hydrolysis could be achieved with a mixture of aqueous sodium hydroxide and dimethyl sulfoxide. Subsequent treatment with acetic anhydride in methanol transformed the sodium carboxylate groups into carboxyl groups. Although the graft copolymers exhibited an improved affinity for organic solvents, those having sodium carboxylate or carboxyl units were characterized by a much more enhanced solubility and were soluble in common solvents. The hygroscopic nature of chitin decreased with an increase in the grafting extent but increased significantly upon hydrolysis of the ester groups. The enzymatic degradability of the graft copolymers, as evaluated with lysozyme, was also dependent on the grafting extent and much higher than that of the original chitin. DSC measurements revealed the presence of a glass transition phenomenon, which could be ascribed to the poly(methyl methacrylate) side chain.