2. LITERATURE REVIEW
2.10 Cutinase function and applications
2.10.3 Cutinase in ester synthesis
Cutinase has been used for synthesis of short chain fatty acid esters that has applications as flavoring agents in food industry (Sebastião et al., 1992; Sebastião et al., 1993). 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,
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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-nitrophenyl butyrate, and triglycerides, such as triolein and tributyrin.
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., 1992; Sebastião et al., 1993) 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., 1992;
Sebastião et al., 1993). 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. The esterification of hexanoic acid with hexanol has also been performed in some studies (Cunnah et al., 1996; Sereti et al., 1997). However,
TH-1214_KHEGDE
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the initial reaction rate in supercritical CO2 was considerably lower (around 1.1 nmol min-1 mg-1 protein) than that in CTAB reversed micelles, (14 mmol min-1 mg-1 protein) (Cunnah et al., 1996; Sereti et al., 1997). In addition, 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 h. 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. Dutta and Dasu (2011) also studied the synthesis of short chain alkyl esters catalysed by P. cepacia cutinase. In their study, ability of P. cepacia cutinase to synthesize butyl esters of various chain lengths and the specificity for acid chain length and alcohol was evaluated. The maximum conversion (%) during synthesis of ester was obtained for butyric acid (C4) and valeric acid (C5) with butanol reflecting the specificity of the enzyme for short-chain length fatty acids. In case of alcohol specificity, butanol was found to be most preferred substrate by the enzyme and conversion (%) decreased with increasing carbon chain length of alcohol used in the esterification reaction. 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
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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, Km 208 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. It was also reported by De Barros et al., (2009b) that cutinase has substrate specificity towards short chain fatty acids (C4-C5) for synthesis of ethyl esters by esterification in iso-octane, whereas the specificity shifted to C10-C18 when cutinase was used in miniemulsion system. In another study, De Barros et al., (2010b) observed that a higher stability of cutinase can be achieved when esterification reactions were carried out in fed batch mode using consecutive feeding pulse of substrate (alcohol).