**2.1.2 Esterases**

Esterases (carboxyl esterases, EC 3.1.1.1) represent a diverse group of hydrolases catalyzing the cleavage and formation of ester bonds with wide distribution in animals, plants and microorganisms. A classification scheme for esterases is based on the specificity of the enzymes for the acid moiety of the substrate, such as the carboxyl esterases, aryl esterases, acetyl esterases, cholin esterases, cholesterol esterases, etc. (Jeager et al., 1999). Esterases show high regio- and stereospecificity, which makes them attractive biocatalysts for the production of optically pure compounds in fine-chemicals synthesis (reviewed in Bornscheuer, 2002).

They have the same reaction mechanism as lipases, but differ from them by their substrate specificity, since they prefer short-chain fatty acids, whereas lipases usually prefer longchain fatty acids. Another difference lies in the interfacial activation (Hidalgo & Bornscheuer, 2006). In contrast to lipases, only a few esterases have commercial applications in organic synthesis because lipases are generally more entantioselective and resistant to organic solvents. The most widely used esterase is the preparation isolated from pig liver (Hidalgo & Bornscheuer, 2006). The practical applications of esterases in enzymatic transformation of flavonoids are not very attractive as it enables the implementation only of the molecule of a short aliphatic chain length, such as acetate, propionate and butyrate (Sakai et al., 1994).

### **2.1.3 Lipases**

Today lipases stand amongst the most important biocatalysts in industry. Among them, microbial lipases find the biggest application use. They can be classified according to sequence alignment into three major groups: mammalian lipases (e.g. porcine pancreatic lipase), fungal lipases (*Candida rugosa* and *Rhizomucor* family) and bacterial lipases (*Staphylococcus* and *Pseudomonas* family) (Hidalgo & Bornscheuer, 2006). More than 50% of the reported lipases are produced by yeast in the forms of various isozymes (Vakhlu & Kour, 2006).

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) belong to the class of serine hydrolases. They catalyze a wide range of reactions, including hydrolysis, interesterification, alcoholysis, acidolysis, esterification and aminolysis (Vakhlu & Kour, 2006). Under natural conditions, they catalyze the hydrolysis of ester bonds at the hydrophilichydrophobic interface. At this interface, lipases exhibit a phenomenon termed interfacial activation, which causes a remarkable increase in activity upon contact with a hydrophobic surface. The catalytic process involves a series of differentiated stages: contact with the interface, conformational change, penetration in the interface, and finally the catalysis itself (Hidalgo & Bornscheuer, 2006). Under certain experimental conditions, such as in the absence of water, they are capable of reversing the reaction. The reverse reaction leads to esterification and formation of glycerides from fatty acids and glycerol (Saxena et al., 1999). This synthetic activity of lipases is being successfully utilized also in flavonoid ester production.

serine proteases (subtilisin) have been used in ester synthesis (Danieli et al., 1989, 1990;

Esterases (carboxyl esterases, EC 3.1.1.1) represent a diverse group of hydrolases catalyzing the cleavage and formation of ester bonds with wide distribution in animals, plants and microorganisms. A classification scheme for esterases is based on the specificity of the enzymes for the acid moiety of the substrate, such as the carboxyl esterases, aryl esterases, acetyl esterases, cholin esterases, cholesterol esterases, etc. (Jeager et al., 1999). Esterases show high regio- and stereospecificity, which makes them attractive biocatalysts for the production of optically pure compounds in fine-chemicals synthesis (reviewed in

They have the same reaction mechanism as lipases, but differ from them by their substrate specificity, since they prefer short-chain fatty acids, whereas lipases usually prefer longchain fatty acids. Another difference lies in the interfacial activation (Hidalgo & Bornscheuer, 2006). In contrast to lipases, only a few esterases have commercial applications in organic synthesis because lipases are generally more entantioselective and resistant to organic solvents. The most widely used esterase is the preparation isolated from pig liver (Hidalgo & Bornscheuer, 2006). The practical applications of esterases in enzymatic transformation of flavonoids are not very attractive as it enables the implementation only of the molecule of a short aliphatic chain length, such as acetate, propionate and butyrate

Today lipases stand amongst the most important biocatalysts in industry. Among them, microbial lipases find the biggest application use. They can be classified according to sequence alignment into three major groups: mammalian lipases (e.g. porcine pancreatic lipase), fungal lipases (*Candida rugosa* and *Rhizomucor* family) and bacterial lipases (*Staphylococcus* and *Pseudomonas* family) (Hidalgo & Bornscheuer, 2006). More than 50% of the reported lipases are produced by yeast in the forms of various isozymes (Vakhlu &

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) belong to the class of serine hydrolases. They catalyze a wide range of reactions, including hydrolysis, interesterification, alcoholysis, acidolysis, esterification and aminolysis (Vakhlu & Kour, 2006). Under natural conditions, they catalyze the hydrolysis of ester bonds at the hydrophilichydrophobic interface. At this interface, lipases exhibit a phenomenon termed interfacial activation, which causes a remarkable increase in activity upon contact with a hydrophobic surface. The catalytic process involves a series of differentiated stages: contact with the interface, conformational change, penetration in the interface, and finally the catalysis itself (Hidalgo & Bornscheuer, 2006). Under certain experimental conditions, such as in the absence of water, they are capable of reversing the reaction. The reverse reaction leads to esterification and formation of glycerides from fatty acids and glycerol (Saxena et al., 1999). This synthetic activity of lipases is being successfully utilized also in

Kodelia et al., 1994).

**2.1.2 Esterases** 

Bornscheuer, 2002).

(Sakai et al., 1994).

**2.1.3 Lipases** 

Kour, 2006).

flavonoid ester production.

*Candida antarctica* lipase B (CALB) is one of the most widely used biocatalysts in organic synthesis on both the laboratory and the commercial scale (Anderson et al., 1998; Uppenberg et al., 1995) due to its ability to accept a wide range of substrates, its non-aqueous medium tolerance and thermal deactivation resistance (Degn et al., 1999; Anderson et al., 1998; Cordova et al., 1998; Drouin et al., 1997). CALB belongs to the α/β hydrolase-fold superfamily with a conserved catalytic triad consisting of Ser105-His224-Asp187 (Uppenberg et al., 1995). It comprises 317 amino acid residues. The active site contains an oxyanion hole which stabilizes the transition state and the oxyanion in the reaction intermediate (Haeffner et al., 1998). Reaction mechanism of CALB follows the bi-bi pingpong mechanism, illustrated in Fig.1 (Kwon et al., 2007). The substrate molecule reacts with serine of the active site forming a tetrahedral intermediate which is stabilized by catalytic residues of His and Asp. In the next step alcohol is released and the acyl-enzyme complex is created. A nucleophilic attack (water in hydrolysis, alcohol in transesterification) causes another tetrahydral intermediate formation. In the last step, the intermediate is split into product and enzyme and is recovered for the next catalytic cycle (Patel, 2006).

Fig. 1. Reaction mechanism catalyzed by *Candida antarctica* lipase (Kwon et al., 2007).

The active site of CALB consists of a substrate-nonspecific acyl-binding site and a substrate specific alcohol-binding site (Cygler & Schrag, 1997; Uppenberg et al., 1995). It is selective for secondary alcohols (Uppenberg et al., 1995), as reflected by the geometry of the alcoholbinding site (Lutz, 2004). In contrast to most lipases, CALB has no lid covering the entrance to the active site and shows no interfacial activation (Martinelle et al., 1995). CALB is being frequently used in acylation of various natural compounds such as saccharides, steroids and natural glycosides, including flavonoids (Riva, 2002; Davis & Boyer, 2001). The proper enzyme selection plays multiple roles in flavonoid acylation. The biocatalyst significantly influences the regioselectivity of the reaction. Information is available mainly on the use of lipases for flavonoid ester synthesis; especially the use of lipase B from *Candida antarctica*, which is preferred due to its acceptance of a wide range of substrates, good catalytic activity and a high degree of regioselectivity (Viskupicova et al., 2010; Katsoura et al., 2006, 2007; Ghoul et al., 2006; Mellou et al., 2005, 2006; Stevenson et al., 2006; Ardhaoui et al., 2004a, 2004b, 2004c; Passicos et al., 2004; Moussou et al., 2004; Gayot et al., 2003; Ishihara & Nakajima, 2003; Ishihara et al., 2002; Kontogianni et al., 2001, 2003; Nakajima et al., 1999, 2003; Gao et al., 2001; Otto et al., 2001; Danieli et al., 1997).

As for flavonoid aglycons, only two enzymes have been reported to be capable of acylating this skeleton – lipase from *Pseudomonas cepacia* and carboxyl esterase. Lambusta et al. (1993) investigated the use of *P. cepacia* lipase for catechin modification. They discovered that the acylation took place on the C5 and C7 hydroxyls. Sakai et al. (1994) observed that carboxyl esterase showed regioselectivity towards C3-OH of catechin. Sakai et al. (1994) explored the use of carboxyl esterase from *Streptomyces rochei* and *Aspergillus niger* for the 3-*O*-acylated catechin production.
