Papaya Lipase Research Articles

Efficient enzymatic synthesis of d-α-tocopherol acetate by Carica papaya lipase-catalyzed acetylation of d-α-tocopherol in a solvent-free system

Enzymatic synthesis of d-α-tocopherol acetate is a promising approach for cost-effective production of liposoluble food and pharmaceutical antioxidant. In this study, we developed an efficient and environmentally friendly process that employed plant-derived Carica papaya lipase (CPL) as biocatalyst for the synthesis of d-α-tocopherol acetate in a solvent-free system. The optimum conditions were: 400 mg/mL d-α-tocopherol, 125 mg/mL CPL (44 U, aw = 0.328), with acetic anhydride as acyl donor, at 60°C and 200 rpm for 30 h. Interestingly, yields of 90.7%, 77.7%, 60.3% and 48.1% of d-α-tocopherol acetate were achieved after the “naturally immobilized” CPL recycled for 4 times, indicating a novel enzyme immobilization mechanism. Moreover, the synthetic product was purified from scale-up reaction, and confirmed by FTIR and GC-MS analysis, providing a total of 97.5% purity (91.1% d-α-tocopherol acetate plus 6.4% d-α-tocopherol). Overall, this work provided an efficient alternative method for green production of d-α-tocopherol acetate probably being applied in food and pharmaceutical industries, and this could also enhance the comprehensive utilization of Carica papaya.

Green Synthesis Optimization of Glucose Palm Oleate and Its Potential Use as Natural Surfactant in Cosmetic Emulsion

This study aimed to optimize the green synthesis of glucose palm oleate catalyzed by Carica papaya Lipase (CPL) through transesterification in a solvent-free system. Palm olein was used as a fatty acid donor for transesterification reactant and was also employed as a reaction medium. Reaction optimization was performed by using response surface methodology (RSM). Seventeen synthesis conditions were generated by a Box–Behnken design and the products were further determined by ultra-performance liquid chromatography (UPLC). Fatty acid compositions of palm olein identified by gas chromatography-mass spectrometry (GC-MS) found that oleic acid (51.77 ± 0.67%) and palmitic acid (37.22 ± 0.48%) were major components. The synthesis variable factors of 50 °C, 45 h reaction time, and 1400 U of CPL were predicted by the RSM to be optimum conditions and thus provided the highest glucose palm oleate of 0.3542 mmol/g. Conjugation between palm olein fatty acids and glucose via transesterification resulted in glucose palm oleate being obviously verified by UPLC, Fourier-transform infrared spectroscopy (FTIR), and thin-layer chromatography (TLC) analyses. The synthesized sugar fatty acid ester revealed an HLB value of 6.20 represented by the lowest % creaming index (%CI) of 35.40 ± 3.21%. It also exhibited a critical micelle concentration (CMC) of 3.16 × 10−5 M. This study is the first report to reveal the transesterification of glucose and palm olein catalyzed by CPL in a system without using any solvent. Glucose palm oleate has been shown to be derived from an environmentally friendly synthesis process and would be promising as a potential alternative natural surfactant for cosmetic application.

Biological evaluations of decellularized extracellular matrix collagen microparticles prepared based on plant enzymes and aqueous two-phase method.

For patients with extensive full-thickness burns who do not have sufficient autologous split-thickness skin for skin grafts, the application of biological skin substitutes may be considered. The aim of this study was to find an optimal new type method for the production of a biovital skin substitute based on acellular dermal matrix (ADM) and preclinical evaluations. In this work, 25 methods of ADM production were assessed. The proposed methods are based on the use of the following enzymes: papain, Carica papaya lipase (CPL), and purification using a polymer/salt aqueous two-phase system. The obtained ADM samples were characterized via scanning electron microscopy (SEM), porosity measurement and water vapor transmission test. Results showed that the collagen bundles of ADM microparticles were intact and orderly. Through differential scanning calorimetry (DSC), thermo gravimetric analysis (TGA) and biocompatibility tests, the results indicated that the proportion of papain and CPL was the same and 5 h processing time are the optimum conditions for ADM preparation and the material showed good biocompatibility. Our results suggested that the potential of developing this kind of decellularization process to manufacture ADM scaffolds for clinical application.

Production of MLM Type Structured Lipids From Grapeseed Oil Catalyzed by Non‐Commercial Lipases

Low calorie triacylglycerols (TAG) presenting medium‐chain fatty acids (M) at positions sn‐1,3 and long‐chain fatty acids (L) at position sn‐2 are known as MLM. This study aims at the production of MLM by acidolysis of grapeseed oil with medium‐chain caprylic (C8:0) or capric (C10:0) acids. Grapeseed oil is used as source of long‐chain polyunsaturated fatty acids (FAs), especially linoleic acid, at sn‐2 position in TAG. Reactions are performed in batch, in solvent‐free systems, during 48 h. Novel non‐commercial sn‐1,3 regioselective lipases are used as alternative to high‐cost commercial biocatalysts, namely, the heterologous lipase from Rhizopus oryzae (rROL) immobilized in Amberlite™ IRA 96 and Carica papaya lipase (CPL) self‐immobilized in papaya latex. The highest productions of new TAG are achieved at 40 °C, molar ratio TAG:M of 1:2, after 48 h, with both biocatalysts, with yields varying between 38 and 69%. rROL immobilized in Amberlite IRA 96 shows a preference toward caprylic acid while CPL shows no preference toward caprylic or capric acid. First‐order deactivation kinetics is observed for both biocatalysts. Half‐lives at 40 °C are 166 and 118 h for rROL and 96 and 81 h for CPL, in the acidolysis of grapeseed oil with C8:0 or C10:0, respectively.Practical Applications: Grapeseeds of Vitis vinifera L. are a by‐product of the wine industry. Using grapeseed oil to produce added‐value functional oils rich in linoleic acid (essential fatty acid) may be a way of improving the revenues of the enological and oil sectors. Both lipases, and mainly Carica papaya lipase self‐immobilized in papaya latex, due to its low‐cost production and easy preparation, are promising non‐commercial biocatalysts for the synthesis of MLM in solvent‐free media. The use of a solvent‐free system, in addition of being a green option, is also preferred for economic reasons, avoiding the costs with solvent and solvent recovery. Also, the use of the stoichiometric molar ratio TAG:M of 1:2 will 1) decrease costs related with the recovery and reutilization of medium chain fatty acids in excess, product recovery and purification, and 2) avoid biocatalyst deactivation by high amounts of free fatty acids in reaction media.Both Carica papaya lipase (CPL) self‐immobilized in papaya latex and rROL in Amberlite IRA 96 catalyze the synthesis of MLM from grapeseed oil in solvent‐free medium. The highest productions of new TAG are achieved at 40 °C, molar ratio TAG:M of 1:2, after 48 h, with both biocatalysts, with yields varying between 38 and 69%. rROL immobilized in Amberlite IRA96 shows a preference toward caprylic acid while CPL shows no preference toward caprylic or capric acid.

Functional expression, extracellular production, purification, structure modeling and biochemical characterization of Carica papaya lipase 1

Abstract Carica papaya latex has been reported to contain lipolytic activity since 1925, nevertheless the efforts to isolate lipolytic enzymes directly from the latex matrix have been unsuccessful. Nowadays papaya genome is known and heterologous expression is an alternative to overcome this problem. Therefore, in this study, Carica papaya lipase 1 sequence (CpLip1) has been identified in papaya genome and for the first time, functionally expressed using Pichia pastoris as host system. Purification of the recombinant enzyme was carried out by affinity chromatography and reached a 7-fold purification factor with 25 U/mg in the purified fraction. Interestingly, homology modeling with lipases of known structure revealed homology with microbial lipases. The biochemical characterization of the purified enzyme shows that CpLip1 hydrolyzed preferentially long-chain triglycerides, it has an optimal pH of 8.5 and an optimal temperature of 35 °C. Finally, the study of its stability in organic solvents showed that, as many lipases, CpLip1 activity is affected in polar solvents. This contribution opens the possibility of studying the catalytic performance of pure CpLip1 in several reactions, and a better understanding of the role of lipases in Carica papaya .

Biodiesel production from crude Jatropha oil catalyzed by non-commercial immobilized heterologous Rhizopus oryzae and Carica papaya lipases

The aim of this study was to evaluate the feasibility of biodiesel production by transesterification of Jatropha oil with methanol, catalyzed by non-commercial sn-1,3-regioselective lipases. Using these lipases, fatty acid methyl esters (FAME) and monoacylglycerols are produced, avoiding the formation of glycerol as byproduct. Heterologous Rhizopus oryzae lipase (rROL) immobilized on different synthetic resins and Carica papaya lipase (rCPL) immobilized on Lewatit VP OC 1600 were tested. Reactions were performed at 30°C, with seven stepwise methanol additions.For all biocatalysts, 51–65% FAME (theoretical maximum=67%, w/w) was obtained after 4h transesterification. Stability tests were performed in 8 or 10 successive 4h-batches, either with or without rehydration of the biocatalyst between each two consecutive batches. Activity loss was much faster when biocatalysts were rehydrated. For rROL, half-life times varied from 16 to 579h. rROL on Lewatit VPOC 1600 was more stable than for rCPL on the same support.

Carica papaya by-products as new biocatalysts for the synthesis of oleic acid esters

Carica papaya lipase is a versatile biocatalyst that is employed for many biotechnological purposes. Its lipase activity was first observed to be tightly linked to the insoluble fraction of latex. Nevertheless, recent studies have shown that this activity is also present in the fruit peel and seeds, suggesting that the lipase activity occurs in other parts of the plant. In the present work, the hydrolytic activity on trioctanoin was determined in various plant by-products, including latex, leafs, petioles, meristems, fruits, and the stem. The most hydrolytic activity was found in the latex (11 U/mL), followed by the petioles (1.7 U/mL). The hydrolytic selectivity was determined using triacetin, tripropionin, tributyrin, and trioctanoin. The enzymes present in the latex showed a higher rate of hydrolysis of tributyrin, while those present in the petioles had a preference for tripropionin, possibly indicating the occurrence of at least two different triacylglycerol hydrolases. Five self-immobilized biocatalysts were obtained: lyophilized latex (LL), lyophilized petioles (LP), bagasse from petioles (BP), and, after a simple cold water washing treatment, treated lyophilized latex (TLL), and treated lyophilized petioles (TLP). This procedure yielded a 5- and 10-fold increase in the latex and petiole activity, respectively, on tributyrin. The selected biocatalysts, TLL and BP, were tested for the synthesis of oleic acid esters (OAE), reaching conversions over 80%. Unexpectedly, only BP preferentially synthesized dodecyl oleate (DO) and showed the highest thermostability. Therefore, BP was further assayed for DO synthesis in a packed bed reactor (PBR), achieving 96% conversion over 40 h. This study shows the great potential of C. papaya by-products, particularly BP, as biocatalysts for the synthesis of OAE.

Carica papaya Lipase Catalysed Resolution of β‐Amino Esters for the Highly Enantioselective Synthesis of (S)‐Dapoxetine

AbstractAn efficient synthesis of the (S)‐3‐amino‐3‐phenylpropanoic acid enantiomer has been achieved by Carica papaya lipase (CPL) catalysed enantioselective alcoholysis of the corresponding racemic N‐protected 2,2,2‐trifluoroethyl esters in an organic solvent. A high enantioselectivity (E > 200) was achieved by two strategies that involved engineering of the substrates and optimization of the reaction conditions. Based on the resolution of a series of amino acids, it was found that the structure of the substrate has a profound effect on the CPL‐catalysed resolution. The enantioselectivity and reaction rate were significantly enhanced by switching the conventional methyl ester to an activated trifluoroethyl ester. When considering steric effects, the substituted phenyl and amino groups should not both be large for the CPL‐catalysed resolution. The mechanism of the CPL‐catalysed enantioselective alcoholoysis of the amino acids is discussed to delineate the substrate requirements for CPL‐catalysed resolution. Finally, the reaction was scaled up, and the products were separated and obtained in good yields (≥ 80 %). The (S)‐3‐amino‐3‐phenylpropanoic acid obtained was used as a key chiral intermediate in the synthesis of (S)‐dapoxetine with very high enantiomeric excess (> 99 %).

Low papaya consumption increases risk for low high‐density lipoprotein cholesterol in Mexican college applicants

Raising high‐density lipoprotein cholesterol (HDL‐C) via nutrition intervention is a challenge. Papaya fruit contains Carica papaya lipase, which may influence lipid metabolism. Our objective was to determine whether individuals consuming less papaya were at greater risk for low HDL‐C or other dyslipidemias. We focused on a subset of 339 Mexican college applicants (50.7% females) aged 18–25 years with complete data on a Mexican‐adapted Willett food frequency questionnaire and blood lipid profiles. Overall prevalence of low HDL‐C was 48.4%. Subjects were split into two consumption groups: <3 or >3 weekly papaya servings (mean intake 0.38 vs. 4.3 servings/week, P<0.0001). There were no differences in blood lipid profiles between the two groups; however, females who consumed less than 3 servings of papaya per week were at 1.52 times greater risk (95%CI: 0.99–2.32, P=0.05) of having low HDL‐C after adjustment for age and family history of cardiovascular disease and diabetes. These findings were not observed in males. We did not observe risk for other at‐risk blood lipid measures. Consumption of at least 3 servings of papaya per week may prevent low HDL‐C in female Mexican college applicants.Grant Funding Source: This study was sponsored by C09‐PIFI‐030606 (UASLP); UIUC Center for Health Aging and Disability grant; and USDA NIFA Hatch Projects.

Carica papaya latex: A low‐cost biocatalyst for human milk fat substitutes production

AbstractThis work aims at evaluating the potential of Carica papaya lipase (CPL) self‐immobilized in papaya latex as a biocatalyst for the synthesis of human milk fat substitutes (HMFS), to be used as a low‐cost alternative to commercial lipases. Two different CPL preparations, one extracted from the papaya fruit (CPL I) and the other from petiole leaves (CPL II) of papaya tree, were tested as catalysts for the acidolysis between tripalmitin and (i) oleic acid or (ii) omega‐3 PUFA, batchwise, at 60°C, in solvent‐free media. After 24 h, molar incorporation was higher for oleic acid (22.1 mol%) when CPL I was used. This biocatalyst was selected for further studies. RSM was used to model reaction conditions: medium formulation (molar ratio oleic acid/tripalmitin, MR, 1.2:1–6.8:1) and temperature (58–72°C). Acyl migration decreased with MR increase. In batch operational stability assays at 60°C, using MR of 2:1 and 6:1, the highest stability was observed for a MR of 2:1.Practical applications: The use of this biocatalyst is a feasible way to valorize papaya agro‐residues which represent an important environmental problem in the producing countries. The obtained results were rather promising since, with this almost zero‐cost biocatalyst, it was possible to produce a high added‐value product (HMFS). Under optimized conditions, the obtained results were comparable with those obtained with expensive immobilized commercial lipases.

Enantioselective esterification of (R,S)-2-methylalkanoic acid with Carica papaya lipase in organic solvents

Isooctane was the best reaction medium for the enantioselective esterification of (R,S)-2-methylalkanoic acid with n-butanol using Carica papaya lipase as catalyst. Increasing linear alkyl-chain length of racemic 2-methylalkanoic acids from ethyl to hexyl increased the enantioselectivity (E) from 2.1 to 98.2 for the esterification of racemic 2-methylalkanoic acids with n-butanol at 35°C. Decreasing reaction temperature from 40 to 20°C increased the enantioselectivity (E) from 14 to 33 for the esterification of racemic 2-methylhexanoic acids with n-butanol. We obtained a maximum enantioselectivity, of E = 24.3, for the enantioselective esterification of racemic 2-methylhexanoic acids with n-butanol in isooctane at water activity 0.33, and at 35°C.

Carica papaya lipase-catalyzed synthesis of terpene esters

An efficient approach to synthesize terpene esters using plant lipase-Carica papaya lipase (CPL) as the biocatalyst was developed in this work. The effects of chain length of acyl donor and solvent type on the CPL-catalyzed transesterification reaction were investigated firstly. It was found that CPL showed the highest activity in n-hexane with vinyl octanoate as the best acyl donor. To obtain high yield of terpene esters, the main reaction parameters were studied and further optimized by response surface methodology. Ping–Pong Bi–Bi mechanism with dead end complex of citronellol was found to fit the initial rate data and the kinetic parameters were obtained by regression analysis. The optimal conditions were: 55 °C, 9% (w/w) of CPL based on substrate, equimolar ratio of substrates. Under these conditions, yield of more than 99% was achieved after 8 h reaction. Ionic liquids (ILs) were used to improve the operational stability because the CPL was found to lose its activity markedly during the repeated runs, it showed that the stability of CPL increased about 5 times when it was coated with ionic liquids. The CPL is low cost yet effective, thus the process developed here shows obvious potential for the production of terpene esters industrially.

Carica papaya Lipase: A Naturally Immobilized Enzyme with Interesting Biochemical Properties

Triacylglycerol (TAG) lipases have been thoroughly characterized in mammals and microorganisms, whereas very little is known about plant TAG lipases. The lipolytic activity occurring in all the laticies is known to be associated with sedimentable particles, and all attempts to solubilize the lipolytic activity of Carica papaya latex have been unsuccessful so far. However, some of the biochemical properties of the lipase from Carica papaya latex (CPL) were determined from the insoluble fraction of the latex. The activity was optimum at a temperature of 37°C and a pH of 9.0, and the specific activities of CPL were found to be 2,000 ± 185 and 256 ± 8 U/g when tributyrin and olive oil were used as substrates, respectively. CPL was found to be active in the absence of any detergent, whereas many lipases require detergent to prevent the occurrence of interfacial denaturation. CPL was inactive in the presence of micellar concentrations of Triton X-100, sodium dodecyl sulfate (SDS) and tetradecyl trimethylammonium bromide (TTAB), and still showed high levels of activity in the presence of sodium taurodeoxycholate (NaTDC) and the zwitterionic Chaps detergent. The effects of various proteases on the lipolytic activity of CPL were studied, and CPL was found to be resistant to treatment with various enzymes, except in the presence of trypsin. All these properties suggest that CPL may be a good candidate for various biotechnological applications.

Carica papaya lipase-catalyzed transesterification resolution of secondary alcohols in organic solvents

Aside from the capability in lipids bioconversion and kinetic resolution of chiral acids, Carica papaya lipase (CPL) as a naturally immobilized enzyme is explored as a potential biocatalyst for the transesterification resolution of chiral secondary alcohols with vinyl esters as the acyl donor in anhydrous solvents. A substrate-type model was proposed for interpreting the enzyme stereochemical preference, showing low enantioselectivity for all tested secondary alcohols except for ( R, S)-methyl 3-phenyllactate ( 2). The kinetic analysis further revealed that the high enantioselectivity was mainly due to the prompt proton transfer from fast-reacting ( S)- 2 to the imidazole moiety of catalytic histidine in the deacylation step, as well as the low enzyme specific activity for ( R)- 1 or ( S)- 1 was owing to the difficult substrate affinity to the active site. Effects of changing the solvent, vinyl ester, and enzyme pretreatments on the lipase performance were also reported.

In vitro comparisons between Carica papaya and pancreatic lipases during test meal lipolysis: Potential use of CPL in enzyme replacement therapy

High levels of lipase activity are known to occur in Carica papaya latex, and this activity is being used in some biotechnological applications. The lipolytic activity of C. papaya lipase (CPL) on dietary triacylglycerols (TAG) has not yet been studied. Hence, the aim of this study was to characterise the specific activity of CPL on dietary TAG present in a crude preparation. Also, we have determined its stability during the lipolysis of a test meal at various pH values mimicking those occurring in the gastro-intestinal tract, with or without bile, and have compared these properties with those of porcine pancreatic extract (PPE) and human pancreatic lipase (HPL). CPL showed maximum stability at pH 6.0, both with and without bile. Some residual activity was still observed at pH 2 (20%), whereas the pancreatic lipases tested were immediately completely inactivated at this pH. In the absence of bile, the highest specific activities were measured at pH 6 in the case of CPL, PPE and HPL. Adding bile slightly decreased the CPL activity in the 4-6 pH range, thus shifting the optimum CPL activity to pH 7, where the presence of bile had no effect. Lipolysis levels decreased with the pH, but CPL was still more active than PPE at pH 5 on a relative basis. These results suggest that CPL might be a promising candidate for use as a therapeutic tool on patients with pancreatic exocrine insufficiency.

Lipase-catalyzed enantioselective hydrolysis of methyl 2-fluoro-2-arylpropionates in water-saturated isooctane

Lipases from Candida rugosa, Candida antartica B and Carica papaya are employed as the biocatalyst for the hydrolytic resolution of methyl 2-fluoro-2-arylpropionates in water-saturated isooctane, in which excellent to good enantioselectivity without the formation of byproducts is obtained for the papaya lipase when using ( R, S)-2-fluoronaproxen methyl ester ( 1) and methyl ( R, S)-2-fluoro-2-(4-methoxyphenyl)propionate ( 2), but not methyl ( R, S)-2-fluoro-2-(naphth-1-yl)propionate ( 3) as the substrates. The thermodynamic analysis indicates that the enantiomer discrimination for the papaya lipase is driven by the difference in activation enthalpy for compound 1, 2 or ( R, S)-naproxen methyl ester ( 4). The kinetic analysis also demonstrates that in comparison with ( S)- 4, the insertion of the 2-fluorine moiety in ( R)- 1 has increased k 2, but not K m, and consequently the lipase activity.

Altering lipase activity and enantioselectivity in organic media using organo‐soluble bases: Implication for rate‐limiting proton transfer in acylation step

With the hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl esters via a partially purified papaya lipase (PCPL) in water-saturated isooctane as the model system, the enzyme activity, and enantioselectivty is altered by adding a variety of organo-soluble bases that act as either enzyme activators (i.e., TEA, MP, TOA, DPA, PY, and DMA) or enzyme inhibitors (i.e., PDP, DMAP, and PP). Triethylamine (TEA) is selected as the best enzyme activator as 2.24-fold increase of the initial rate for the (S)-ester is obtained when adding 120 mM of the base. By using an expanded Michaelis-Menten mechanism for the acylation step, the kinetic analysis indicates that the proton transfer for the breakdown of tetrahedral intermediates to acyl-enzyme intermediates is the rate-limiting step, or more sensitive than that for the formation of tetrahedral intermediates when the enzyme activators of different pKa are added. However, no correlation for the proton transfers in the acylation step is found when adding the bases acting as enzyme deactivators.

Chemoenzymatic synthesis of structured triacylglycerols with conjugated linoleic acids (CLA) in central position

A chemoenzymatic process for the production of structured triacylglycerols (TAG) containing CLA at sn2 position and lauric acid at external ones is proposed. First, castor bean oil was chemically dehydrated and isomerised to obtain a new modified oil with very high proportion of CLA (>95%). Then, this new oil was used for enzymatic transesterification allowing the grafting of lauric acid at external positions of the TAG backbone by using 1,3 regioselective enzymes. Among these, Aspergillus niger lipase was not satisfactory giving very low lauroyl incorporation (<5%) On the contrary, lipases from Thermomyces lanuginosa (Lipozyme TL IM) and from Carica papaya latex allowed good reaction yields. The effect of the type of acyl donor was studied. With alkyl esters T. lanuginosa lipase provided a final incorporation of 58.9% after 72 h corresponding to 88.4% transesterification yield. Concerning C. papaya lipase, incorporation of lauroyl residues was lower than Lipozyme TL IM. This lipase exhibited higher performance with lauric acid accounting for 44.7% lauroyl incorporation at the end of reaction for a 67.1% transesterification yield. The effect of the substrates mole ratio was also evaluated. It was observed that a 1:3 TAG/acyl donor mole ratio was the most efficient for both lipases. Finally, fatty acids regiodistribution of the newly formed structured TAG was determined. With Lipozyme TL IM, the proportion of lauric acid incorporated at the sn2 position did not exceed 5.4% after 72 h while with C. papaya lipase a more pronounced incorporation of lauroyl residues at the central position (8.8%) was observed.

Partially purifiedCarica papayalipase: a versatile biocatalyst for the hydrolytic resolution of (R,S)-2-arylpropionic thioesters in water-saturated organic solvents

With the hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl thioesters in water-saturated isooctane as a model system, improvements of the specific lipase activity and thermal stability were found when a crude Carica papaya lipase (CPL) was partially purified and employed as the biocatalyst. The partially purified Carica papaya lipase (PCPL) was furthermore explored as an effective enantioselective biocatalyst for the hydrolytic resolution of (R,S)-profen thioesters in water-saturated organic solvents. The kinetic analysis in water-saturated isooctane indicated that both acyl donor and acyl acceptor have profound influences on the lipase activity, E-value, and enantioselectivity. Inversion of the enantioselectivity from (S)- to (R)-thioester was found for (R,S)-fenoprofen and (R,S)-ketoprofen thioesters that contained a bulky substituent at the meta-position of 2-phenyl moiety of the acyl part. Kinetic constants for the acylation step were furthermore estimated for elucidating the kinetic data and postulating an active site model. The thermodynamic analysis indicated that the enantiomer discrimination was driven by the difference of activation enthalpy (DeltaDeltaH) and that of activation entropy (DeltaDeltaS), yet the latter was dominated for most of the reacting systems. The postulated active site model was supported from the variation of DeltaDeltaH and DeltaDeltaS with the acyl moiety, in which a good linear enthalpy-entropy compensation relationship was also illustrated. A comparison of the performances between Candida rugosa lipase (CRL) and PCPL indicated that PCPL was superior to CRL in terms of the better thermal stability, similar or better lipase activity for the fast-reacting substrate, time-course-stability, and lower enzyme cost.

Enantioselective hydrolysis of ( R, S)-naproxen 2,2,2-trifluoroethyl ester in water-saturated solvents via lipases from Carica pentagona Heilborn and Carica papaya

A crude lipase prepared from Carica pentagona Heilborn latex was explored as an effective enantioselective biocatalyst for the hydrolytic resolution of ( R, S)-naproxen 2,2,2-trifluoroethyl ester in water-saturated organic solvents. Comparisons of the enzyme performance with that from Carica papaya lipase indicated that both lipases showed low tolerance to the hydrophilic solvent and were inhibited by ( S)-naproxen and 2,2,2-trifluoroethanol. Improvements on the enzyme activity and enantioselectivty were demonstrated when both lipases in partially purified forms were employed. By using the thermodynamic analysis, the enantiomeric discrimination was mainly driven by the difference of activation enthalpy for all reaction systems except for employing Carica papaya lipase as the biocatalyst for ( R, S)-fenoprofen 2,2,2-trifluoroethyl thioester.