Investigation of lipases from various Carica papaya varieties for hydrolysis of olive oil and kinetic resolution of (R,S)-profen 2,2,2-trifluoroethyl thioesters (original) (raw)
Related papers
Biotechnology and Bioengineering, 2005
For the first time, the Carica papaya lipase (CPL) stored in crude papain is explored as a potential enantioselective biocatalyst for obtaining chiral acids from their racemic thioesters. Hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl thioester in water-saturated organic solvents is employed as a model system for studying the effects of temperature and solvents on lipase activity and enantioselectivity. An optimal temperature of 60jC, based on the initial rate of (S)-thioester and a high enantiomeric ratio (i.e., E-value defined as the ratio of initial rates for both substrates) of >100 at 45jC in isooctane, is obtained. Kinetic analysis, considering product inhibition and enzyme deactivation, is also performed, showing agreement between the experimental and best-fit conversions for (S)thioester. A comparison of the kinetic and thermodynamic behaviors of CPL and Candida rugosa lipase (CRL) in isooctane and cyclohexane indicates that both lipases are very similar in terms of thermodynamic parameters ÁÁH and ÁÁS, initial rate of (S)-substrate, and E-value when (R,S)-naproxen 2,2,2-trifluoroethyl thioester or ester is employed as substrate.
Characterization of the lipase from Carica papaya residues
Braz. J. Food. Technol. Preprint Serie, 2008
Lipases from vegetable sources have been the focus of intense and growing research. The use of enzymes from plants has the advantage of employing industrial waste products. Brazil is the world's largest papaya producer and the market for products derived from papaya is growing year after year. In this study, the authors carried out the characterization and partial purification of the lipase obtained from the seeds and skin of Carica papaya. The most active fractions were those obtained using the technique of precipitation with 50% of acetone (EBFL-50 acetone), and the technique of fractioned precipitation with ammonium sulphate 40-80% (EBFL-40-80 sulphate). The EBFL-50 acetone fraction presented an optimum pH at 9.5, with a second peak of activity at pH 4.5, and optimal temperature at 55 ºC. The fraction was stable at pH values between 8.5 and 10.0 and temperatures from 30 to 60 °C and from 90 to 100 °C. The EBFL-40-80 sulphate fraction had an optimum pH at 9.0, with a second peak at pH 6.0, and an optimum temperature at 55 °C, being stable at pH values between 6.0 and 9.0 and temperatures from 60 to 90 °C. The thermal stability of the EBFL-40-80 sulphate fraction changed to the range between 30 and 70 °C in the presence of the protease inhibitor phenylmethanosulfonyl fluorate (PMSF). The thermal stability of the fraction EBFL-50 acetone did not change in the presence of PMSF. The best esterification activity was observed in the presence of stearic acid after 48 h for the EBFL-50 acetone fraction.
Biotechnology and Bioengineering, 2005
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 2phenyl 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 (DDH) and that of activation entropy (DDS), yet the latter was dominated for most of the reacting systems. The postulated active site model was supported from the variation of DDH and DDS 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.
Enzyme and Microbial Technology, 2005
Carica papaya lipase (CPL) stores in the crude papain is explored as a high enantioselective biocatalyst for the hydrolysis resolution of (R,S)-naproxen 2,2,2-trifluoroethyl ester in water-saturated solvents. An optimal temperature of 60 • C for obtaining the maximum initial rate for (S)-naproxen ester in isooctane is found, where the lipase possesses high enantioselectivity with E = 122. Less hydropholic solvents of cyclohexane and MTBE than isooctane are vital on decreasing the lipase activity and enantioselectivity. Comparisons of enzyme performances for CPL and lipase MY indicate that CPL is more enantioselective and active for (S)-naproxen ester, and also more stable in water-saturated isooctane at 60 • C.
Tetrahedron: Asymmetry, 2004
Carica papaya lipase (CPL) stored in the crude papain is found to be enantioselective for the kinetic resolution of (RS)-2-(4-chlorophenoxy)propionic acid via esterification in anhydrous organic solvents. Of the alcohols screened, trimethylsilylmethanol acted as the best acyl acceptor and gave the highest enzyme activity and enantioselectivity in anhydrous cyclohexane. The kinetic analysis at temperatures between 20 and 60°C indicated that CPL is thermally stable, giving a high E value of 113 at 20°C. A change of cyclohexane to other hydrophobic solvents resulted in better lipase performances. In comparison with the performances of other crude Candida rugosa lipases, Carica papaya lipase is more active, enantioselective and thermally stable.
Oilseeds and fats, crops and lipids, 2003
Temperature and water activity (a w) of the reaction medium are two factors that govern enzyme reactions. We studied the influence of these two parameters on the esterification and transesterification activity of Carica papaya lipase in water and solvent free reactions. It was found that over the course of reaction the catalytic activity of C. papaya lipase was dependent on these factors. The best lipase activity for both reactions was at a temperature of 55°C and water activity of 0.22, which corresponds to 2 g of water per 100 g of C. papaya latex.
Carica papaya lipase (CPL): An emerging and versatile biocatalyst
Biotechnology Advances, 2006
In recent years, the Carica papaya lipase (CPL) is attracting more and more interest. This hydrolase, being tightly bonded to the water-insoluble fraction of crude papain, is thus considered as a "naturally immobilized" biocatalyst. To date, several CPL applications have already been described: (i) fats and oils modification, derived from the sn-3 selectivity of CPL as well as from its preference for short-chain fatty acids; (ii) esterification and inter-esterification reactions in organic media, accepting a wide range of acids and alcohols as substrates; (iii) more recently, the asymmetric resolution of different non-steroidal antiinflammatory drugs (NSAIDs), 2-(chlorophenoxy)propionic acids, and non-natural amino acids. Taking into account the novelty and the current interest of the topic, this review aims to highlight the origin, features, and applications of the C. papaya lipase, with the objective to prompt research groups to further investigate the spectra of applications that this emerging and versatile CPL could have in the future.
Biotechnology and Bioengineering, 2002
Adsorption and desorption isotherms of two commercial enzyme preparations of papain and bromelain were determined with a Dynamic Vapor System. The Guggenheim-Anderson-deBoer (GAB) modeling of the obtained sorption isotherms allowed the de®nition of different levels of hydration of those samples. Afterward, these enzyme preparations were used as biocatalysts in water and solvent-free esteri®cation and alcoholysis reactions. The evolution of the obtained fatty acid ester level as a function of the initial hydration level of the biocatalyst, i.e., thermodynamic water activity (a w ) and water content, was studied. The results show an important correlation between the initial hydration level of the biocatalyst and its catalytic activity during the lipasecatalyzed synthesis reactions. Thus, the Carica papaya lipase (crude papain preparation) catalytic activity is highly dependent on the biocatalyst hydration state. The optimized synthesis reaction yield is obtained when the a w value of the enzyme preparation is stabilized at 0.22, which corresponds to 2% water content. This optimal level of hydration occurs on the linear part of the biocatalyst's sorption isotherm, where the water molecules can form a mono-or multiple layer with the protein network. The synthesis reaction yield decreases when the a w of the preparation is higher than 0.22, because the excess water molecules modify the system equilibrium leading to the reverse and competitive reaction, i.e., hydrolysis. These results show also that an optimal storage condition for the highly hydrophilic crude papain preparation is a relative humidity strictly lower than 70% to avoid an irreversible structural transition leading to a useless biocatalyst. Concerning the bromelain preparation, no effect of the hydration level on the catalytic activity during esteri®cation reactions was observed. This biocatalyst has too weak a catalytic activity which makes it dif®cult to observe any differences. Furthermore, the bromelain preparation is far more hydrophobic as it adsorbs only 18 g of water per 100 g of dry material at a w around 0.90. No deliquescence of this enzymatic preparation is oserved at this a w value.