Free Candida Antarctica Lipase Research Articles

Conversion of waste frying palm oil into biodiesel using free lipase A from Candida antarctica as a novel catalyst

Biodiesel, also known as fatty acid methyl ester (FAME), is usually prepared using edible vegetable oils by transesterification. In this work, a clean method for biodiesel production was developed using frying palm oil (FPO) and free Candida antarctica lipase A (CALA) as feedstock and catalyst, respectively. The effect of transesterification conditions (water content, lipase load, substrate ratio, time and temperature) on FAME yield was evaluated and optimized by response surface methodology. Results showed that free lipase CALA had the excellent methanol resistance. And lipase CALA can simultaneously catalyze the transesterification of FPO and the esterification of free fatty acid (FFA) in the presence of excess water. Low mass transfer limitation of the system using lipase CALA as catalyst was found. The effect of reaction conditions on FAME yield decreased in order of water content > reaction time > enzyme load. The optimal reaction conditions were 16.6 wt% water content and 5.5 wt% enzyme load with 7:1 molar ratio of methanol to FPO at 30 °C for 22 h. The maximum FAME yield (94.6 ± 1.4%) was achieved under the optimal conditions. Arrhenius equation and Ea of the transesterification were lnV0 = 9.45–3964.4/T and 32.96 kJ/mol, respectively. K’m and Vmax were 6.75 × 10−1 mol/L and 2.38 × 10−2 mol/(L·min), respectively. This work supplied a clean method to produce biodiesel using waste frying oil with high water and FFA contents as feedstock and a green method using cheap free liquid lipase as catalyst.

Synthesis of fatty acid ethyl esters with conventional and microwave heating systems using the free lipase B from Candida antarctica

Free Candida antarctica lipase B (Lipozyme, CALB L®) was used to produce fatty acid ethyl esters (FAEE) from refined soybean oil in solvent-free media using the conventional (CHS) and microwave (MHS) heating systems. Statistical analyses (95% confidence level) for both reaction products, FAEE and free fatty acids (FFA), were performed. An increase in ethanol:oil molar ratio decreased the catalytic performance of CALB L (p < .05). The best conditions using the microwave radiation were a molar ratio of ethanol:oil of 3:1, a water content of 20.3 wt.% and an enzyme loading of 3 wt.% and this resulted in a total ester content of 64.7% in 15 min, while the same condition using the conventional heating gave only 21.4%. Moreover, the reaction equilibrium was reached 16 times faster with microwave than with conventional heating. High ethanol:oil molar ratios had a negative effect on FAEE synthesis with both CHS and MHS, probably due to the partial inactivation of the enzymes. MHS improved the reaction performance of CALB L, but other process parameters will have to be optimized to enhance the resulting FAEE yields. The recovery and reuse of CALB L using a MHS was demonstrated. Hence, the use of microwave radiation under the conditions applied in this study was not detrimental to the catalytic performance of CALB L for at least one reuse.

Stability of hydrolase enzymes in ionic liquids

AbstractIn this work we attempted to evaluate the stability of penicillin G acylase (PGA) from Escherichia coli in their native form and free Candida antarctica lipase B (CaLB) in ionic liquids (ILs) at low water content. The hydrolysis of penicillin G to 6‐aminopenicillanic acid (6‐APA), and phenyl acetic acid (PAA) catalysed by PGA and the synthesis of butyl butyrate from vinyl butyrate and 1‐butanol catalysed by CaLB were chosen as activity tests. The influence of these new solvents on enzyme stability was studied by incubating the enzyme (PGA or CaLB) in ILs based on dialkylimidazolium cations associated with perfluorinated and dicyanamide anions at a given temperature. Stability studies indicate that CaLB and PGA exhibited greater stability in water‐immiscible ILs than in water‐miscible ILs. Specifically, native PGA shows greater stability in IL media than in organic solvents. For example, a half‐life time of 23 h was obtained in 1‐ethyl‐3‐methylimidazolium bis{(trifluoromethyl)sulfonyl}imide, $[{\rm emim}^ + ][{\rm NTf}_2^ - ]$, which was about 2000‐fold higher than that in 2‐propanol. The higher half‐life time of CaLB was observed in $[{\rm omim}^ + ][{\rm PF}_6^ - ]$ (t1/2 = 84 h).

Synthesis of flavour esters using free Candida antarctica lipase B in ionic liquids

AbstractThe enzymatic synthesis of flavour esters by trans‐esterification from vinyl esters and alcohols catalysed by free Candida antarctica lipase B (CaLB) in ionic liquids has been studied. The influence of different parameters, such us pH, temperature and ionic liquid nature, on activity and selectivity has been carefully analysed, using as reaction model the synthesis of butyl butanoate. Finally, a broad range of flavour esters were sucessfully synthesized using the optimal operation conditions previously established. Copyright © 2008 John Wiley &amp; Sons, Ltd.

Synthesis of esters in ionic liquids: The effect of vinyl esters and alcohols

Free Candida antarctica lipase B (CALB) was successfully applied to catalyzing the synthesis of alkyl esters by transesterification from vinyl esters and alcohols in ionic liquids based on 1-alkyl-3-methylimidazolium cations ([bmim +] and [omim +]) and the hexafluorophosphate anion ([PF 6 −]). First, the variables affecting the performance of CALB in transesterification reactions in ionic liquids, temperature and pH, were studied, choosing the synthesis of butyl butyrate in [bmim +][PF 6 −] as reaction model. An increase in the rate of butyl butyrate synthesis in [bmim +][PF 6 −] was observed as the temperature was increased from 30 to 70 °C, while the optimal pH was 7. Secondly, the role of vinyl esters and alcohols in lipase-catalyzed ester synthesis in ionic liquids was investigated. When the number of carbon atoms of the vinyl ester was increased, the optimum activity was found for six carbon atoms. The influence of the alkyl chain length of the alcohol was tested, the optimal length being four carbon atoms. The rate of catalyzed ester synthesis of esters was found to be closely dependent on the solvent medium, being higher in [omim +][PF 6 −].

Positional Assembly of Enzymes in Polymersome Nanoreactors for Cascade Reactions

In a good position: Nanoreactors can be constructed by the controlled positioning of glucose oxidase (GOX) and horseradish peroxidase (HRP) within the central water pool and block-copolymer membrane of polymersomes. A one-pot multistep reaction sequence is performed with the nanoreactor in combination with free Candida antarctica lipase B (CALB) in the bulk solution (see picture; ABTS: 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)).

On the importance of the supporting material for activity of immobilized Candida antarctica lipase B in ionic liquid/hexane and ionic liquid/supercritical carbon dioxide biphasic media

A commercial solution of free Candida antarctica lipase B (Novozyme 525L) has been immobilized by adsorption onto 12 different silica supports modified with specific side chains (e.g. alkyl, amino, carboxylic, nitrile, etc.). The immobilized derivatives were assayed for the kinetic resolution of rac-1-phenylethanol in both ionic liquid/hexane and ionic liquid/supercritical carbon dioxide biphasic media. The best results were obtained for the supports modified with non-functionalized alkyl chains and when the in water activity increased from 0.33 to 0.90 (e.g. the CALB/butyl-silica activity was enhanced up to five times). Coating immobilized enzyme particles with ionic liquids (butyltrimethylammonium bistriflimide or trioctylmethylammoniun bistriflimide) resulted in a decrease in activity (10 times), although half-life times were enhanced (up to six times) in hexane media at 95 °C. However, immobilized derivatives coated with ionic liquids clearly improved their synthetic activity in supercritical CO 2 by up to six times with respect to the hexane medium, which agrees with the “philicity” between alkyl chain lengths of both the silica support and the cation of ionic liquid.

Performance of free Candida antarctica lipase B in the enantioselective esterification of ( R)-ketoprofen

The performance of free Candida antarctica lipase B (CALB) in a mixed solvent system for enantioselective esterification of ( R)-ketoprofen, leaving the target product ( S)-ketoprofen in unreacted form was investigated. The effects of enzyme loading, substrate concentration, alcohol to acid molar ratio, type of solvents and reaction temperature were studied. The optimum reaction conditions obtained were 200 mg/ml of CALB and racemic ketoprofen concentration of 30 mM, with 20% (v/v) 1,2-dichloropropane–80% (v/v) n-hexane as solvent, alcohol to acid molar ratio of 1:1 at 40 °C. The conversion of ( R)-ketoprofen, X R obtained was 81% with enantioselectivity ( E) value of 10, enantiomeric excess of product (ee p) of 66.4% and enantiomeric excess of substrate (ee s) of 59.4% for the reaction time of 24 h.

Influence of microwave radiation on free Candida antarctica lipase B activity and stability

The influence of microwave heating on free Candida antarctica lipase B activity and stability was studied over the temperature range from 40 to 110 degrees C. Concerning the lipase activity, identical initial rate and conversion yield were obtained under microwave radiation and classical thermal heating for the alcoholysis between ethyl butyrate and butanol in a solvent-free system. On the other hand, the kinetics of the free lipase inactivation in butanol appears to be influenced by the heating mode. The Arrhenius plot obtained under classical heating was linear over all the temperature range studied whereas a biphasic Arrhenius plot was obtained under microwaves. The non-classical effect of the microwave heating on the initial rate of the enzymatic inactivation was thus dependent on the temperature of incubation.

Kinetic resolution of rac-2-pentanol catalyzed by Candida antarctica lipase B in the ionic liquid, 1-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]amide.

The ionic liquid, l-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]amide ([Bmim] [NTf2]), was used as a reaction medium for the kinetic resolution of rac-2-pentanol catalyzed by free Candida antarctica lipase B, using vinyl propionate at 2% (v/v) water content. The synthetic activity of lipase in [Bmim] [NTf2] was up 2.5-times greater than in hexane, and showed high enantioselectivity (ee > 99.99%). The optimal temperature and pH were 60 degrees C and 7, respectively. A decrease in water activity (aw) produced a decay in synthetic activity, and an exponential increase in selectivity.

Enzymatic ester synthesis in ionic liquids

Six different ionic liquids based on dialkylimidazolium or quaternary ammonium cations associated with perfluorinated or bis(trifluoromethyl)sulfonyl amide anions were used as reaction media for ester synthesis catalyzed by both free Candida antarctica lipase B and α-chymotrypsin at 2% (v/v) water content and 50 °C. All the assayed ionic liquids proved adequate media for enzyme-catalyzed transesterification, and in the case of lipase, the synthetic activity was clearly enhanced with respect to that obtained with organic solvents of similar polarity. In general, all the ionic liquids increased the thermal stability of both the enzymes. As for example, 1-ethyl-3-methylimidazolium tetrafluoroborate enhanced 5- and 4-times the synthetic activity and half-life time, respectively, of lipase in comparison to 1-butanol.

Over-stabilization of Candida antarctica lipase B by ionic liquids in ester synthesis

Four different ionic liquids, based on dialkylimidazolium cations associated with perfluorinated and bis(trifluoromethyl)sulfonyl amide anions were used as reaction media for butyl butyrate synthesis catalyzed by free Candida antarctica lipase B at 2% (v/v) water content and 50 °C. Lipase had enhanced synthetic activity in all ionic liquids in comparison with two organic solvents (hexane, and 1-butanol), the enhanced activity being related to the increase in polarity of ionic liquids. The continuous operation of lipase with all the assayed ionic liquids showed over-stabilization of the enzyme. The reuse of free lipase in 1-butyl-3-methylimidazolium hexafluorophosphate in continuous operation cycles showed a half-life time 2300 times greater than that observed when the enzyme was incubated in the absence of substrate (3.2 h), and a selectivity higher than 90%.