2. LITERATURE REVIEW
2.8 Industrially important applications
2.8.3 Cutinase in ester synthesis
Cutinase has also been used for ester synthesis (Sebastião et al., 1993; Sebastião et al., 1992).
The esters produced from short-chain fatty acids have applications as flavoring agents in food industry. Methyl and ethyl esters of long-chain acids have been used to enrich diesel fuels.
Although, lipases can be used for the hydrolysis and synthesis of esters, are active in organic solvents and have wide substrate specificity, still they have a number of shortcomings. The most important shortcoming of lipases is their relatively large size and instability under industrial process conditions. Despite the known ability of cutinases to catalyse the hydrolysis of esters, use of cutinases have been suggested only for very limited purposes until now, viz.,
successful utilization as catalyst for synthesis of esters in laboratory scale. Cutinases can be used for the hydrolysis of a variety of ester substrates including monoesters, such as p- nitrophenylbutyrate, and triglycerides, such as triolein and tributyrin.
Table 2.3 Reported biocatalytic applications of cutinase.
Type of reaction
Substrates Enzymatic Preparation
Reference
Hydrolysis Triglyceride Triolein Reverse micelle AOT/isooctane
Melo et al., 1995b Aqueous/triolein
biphasic medium
Flipsen et al., 1996 Free enzyme Chen et
al., 2008 Tricaprylin Immobilization onto
zeolites
Gonçalves et al., 1996a Entrapment in
calcium alginate
Gonçalves et al., 1995 Covalent binding on
porous silica
Gonçalves et al.,1995
Esters
p-nitrophenyl valerate Micelles of SDS, Triton
X-100
Pocalyko and Tallman, 1998 p-nitrophenyl
palmitate
Immobilization on dextran and derivatized silica supports
Gonçalves et al., 1998
Free enzyme Chen et al., 2008, Sebastian and
Kolattukudy 1988 Methyl-,ethyl-,
propylpropionate
Gas/solid system Lamare et al., 1997
Type of reaction
Substrates Enzymatic Preparation
Reference
Synthesis Esterification Oleic acid+hexanol Reversed micelles of AOT/isooctane
Sebastião et al., 1993,Sebas tião et al., 1992 Caprylic
acid+butanol
Substrates in organic media
Sarazin et al., 1992, Sarazin et al., 1995 Butyric acid+2-
butanol
Phosphatidylcholine/
isooctane reversed micelles
Pinto- Sousa et al., 1994 Oleic acid+glycerol Organic solvents Melo et al.,
1995a Hexanoic
acid+hexanol
CTAB reversed micelles
Cunnah et al., 1996 Immobilization onto
Accurel
EP 100 in SC CO2
Sereti et al., 1997 Lauric acid+pentanol Reversed micelles of
AOT/isooctane
Papadimitr iou et al., 1996 Short chain fatty acid
(C4-C18) + ethanol
Lyophilized enzyme in organic media
De Barros et al 2009a Caproic acid+ ethanol Lyophilized enzyme
in organic media
De Barros et al., 2010a De Barros et al., 2010b Methyl
propionate+propanol
Gas/solid system Lamare and Legoy, 1995 ,Lamare et al., 1997 Transestrification Butyl
acetate+hexanol
Reversed micelles of AOT/isooctane
Carvalho et al., 1997a;
Carvalho et al., 1998b Reversed micelles of
CTAB/isooctane
Cunnah et al., 1996
Type of reaction
Substrates Enzymatic Preparation
Reference
Immobilization onto zeolites
Serralha et al., 1998 Methanol + Triolien Reversed micelles of
AOT/isooctane
Badenes et al., 2010a Reversed micelles of
AOT/isooctane
Badenes et al., 2010b Reversed micelles of
AOT/isooctane
Badenes et al., 2011a
Unlike a specific esterase, cutinases can also catalyse the rapid hydrolysis of long-chain triglycerides, such as triolein, in emulsion form. The substrate dependence profile of the catalytic activity of cutinases on tributyrine showed (Longhi et al., 1999) that its enzymatic activity was not influenced by the formation of an interface. At the substrate concentration where aggregates of tributyrine are formed, no appreciable increase in enzyme activity could be noticed, quite in contrast to what is known for lipases (Fleet et al., 1983; Verger et al., 1976). Cutinases can also be used for the direct synthesis of esters starting from alcohols and organic acids (e.g., fatty acids). This derives from the reversibility of the cutinase reaction.
The synthesis of fatty acid esters by cutinase has been analyzed in reversed micelles of both anionic, AOT, (Sebastião et al., 1993; Sebastião et al., 1992) and cationic, CTAB, (Cunnah et al., 1996) surfactants. The oleic acid esterification with aliphatic alcohols by microencapsulated cutinase in AOT reversed micelles showed that cutinase has a preference for C5 to C6 alcohols, reflecting both the intrinsic selectivity of the enzyme and the different accessibility of the alcohol substrates to the cutinase active site (Sebastião et al., 1993;
Sebastião et al., 1992). The effect of the fatty acid chain length on the esterification of
hexanol was also evaluated and the maximum activity was obtained with butyric acid, confirming the cutinase selectivity for short chain substrates. The same reactions were also performed with cutinase encapsulated in CTAB reversed micelles (Cunnah et al., 1996) and similar conclusions can be drawn; the cutinase activity was maximal for the esterification of hexanol with butyric acid. However, the cutinase activity in AOT reversed micelles was higher than in CTAB reversed micelles. The esterification of hexanol with butyric acid was also investigated by other authors (Carvalho et al., 1998a) using cutinase adsorbed on a macroporous polypropylene support in both water-immiscible (hexane and diisopropyl ether) and water-miscible (acetonitrile) solvents. This reaction was used as a model system to evaluate the reactants solvation effects on cutinase, namely solvation of reactants. The results showed that the optimum water activity decreased with increasing polarity of the solvent.
Substrate solvation was found to be most strongly affected by water in acetonitrile. To relate to solvation, kinetic parameters were determined at two water activities in each solvent. A Ping-Pong model with competitive inhibition by hexanol and butyric acid was developed. The Michaelis constants for hexanol in hexane at two aw values of 0.44 and 0.69 were 299 and 231 mM, respectively (Carvalho et al., 1998a). These values are higher than the Km=86 mM, obtained for hexanol in the reaction catalysed in AOT reversed micelles (Sebastião et al., 1993). This may result from higher diffusion effects present in the supported enzyme when compared with the microencapsulated cutinase. The esterification of hexanoic acid with hexanol has also been performed in some studies (Cunnah et al., 1996; Sereti et al., 1997). In supercritical CO2 (Sereti et al., 1997), this reaction was very slow when compared with a CTAB reversed micellar system (Cunnah et al., 1996). The initial reaction rate in supercritical CO2 is considerably lower (around 1.1 nmol min-1 mg-1 protein) than that in CTAB reversed
micelles, (14 mmol min-1 mg-1 protein). Also the equilibrium in the supercritical reaction medium was reached after 5 days. The esterification of caprylic acid with butanol was also performed with lyophilized cutinase (Sarazin et al., 1992; Sarazin et al., 1995) using a NMR tube as a probe of the spectrometer. Around 80% of esterification could be achieved after 7 hours. The ester synthesis of butyric acid and 2-butanol was carried out by cutinase microencapsulated in nonionic surfactant, phosphotidylcholine (Pinto-Sousa et al., 1994). The enzymatic activity for the synthesis of butyl butyrate increased with increasing substrates concentrations according to a Michaelis-Menten kinetics. However, the inhibition of cutinase was observed at higher than 500 mM and 200 mM for 2-butanol and butyric acid, respectively. The apparent Michaelis constants obtained for 2-butanol and butyric acid were 47.2 and 38.8 mM, respectively. The esterification of lauric acid and pentanol with cutinase microencapsulated in AOT reversed micelles was performed as a model system to study the structural and catalytic properties of the enzyme by using EPR spectroscopy of the labeled active site (Papadimitriou et al., 1996). The effect of water content on cutinase activity was assessed, the maximum being at wo=9. Up to wo 9, there was an increase of both activity and active site mobility. As the water content of the system became higher, the mobility of the bound spin label stabilized, whereas the enzymatic activity dropped considerably. Kinetic studies allowed the determination of the apparent kinetic parameter, Km208 mM and 60 mM for pentanol and lauric acid, respectively. The synthesis of oleoyl glycerides, monoolein, diolein and triolein, catalysed by lyophilized cutinase was demonstrated (Melo et al., 1995a) using the monomolecular film technique previously used to study the kinetics of lipase hydrolysis. The water sub phase was replaced by glycerol and a film of oleic acid was initially spread on the glycerol surface. More than 50% of the oleic acid film was acylated after 7
minutes of reaction. De Barros et al., (2009a; 2009b; 2010a; 2010b) studied the ability of cutinase to catalyse the esterification of short chain alkyl esters in isooctane, miniemulsion system. De Barros et al., (2009a) observed higher ester yields and initial reaction rates in the esterification of butyric (C4) and valeric acid (C5) as compared with the shorter or longer chain length acid. De Barros et al., (2009b) observed that cutinase has substrate specificity towards short- chain fatty acids (C4-C5) for synthesis of ethyl esters by esterification in iso- octane, where as the specificity shifted to C10-C18 when cutinase was used in miniemulsion system. De Barros et al., (2010b) obtained higher stability of cutinase, when esterification reactions were carried out in fed batch mode using consecutive feeding pulse of substrate (alcohol).