**2.2.2 Water content**

Water content in reaction media is a crucial parameter in lipase-catalyzed synthesis as it alters the thermodynamic equilibrium of the reaction towards hydrolysis or synthesis. Moreover, it is involved in noncovalent interactions which keep the right conformation of an enzyme catalytic site (Foresti et al., 2007). The amount of water required for the catalytic process depends on the enzyme, its form (native or immobilized), the enzyme support, and on the solvent nature (Arroyo et al., 1999; Zaks & Klibanov, 1988). The influence of water content in the reaction system on enzyme activity is variable with various enzymes (lipase from *Rhizomucor miehei*, *Rhizomucor niveus*, *Humicola lanuginosa*, *Candida rugosa*, *Pseudomonas cepacia*).

In general, the water amount which is considered to be optimal for esterifications in organic solvents is 0.2 – 3% (Rocha et al., 1999; Yadav & Piyush, 2003; Iso et al., 2001). The enzymatic esterification of flavonoids in non-aqueous media is greatly influenced by the water content of the reaction system (Ardhaoui et al., 2004b; Gayot et al., 2003; Kontogianni et al., 2003). Ardhaoui et al. (2004b) observed the best enzyme activity when water content was maintained at 200 ppm. Gayot et al. (2003) found that the optimal value of water in an organic reaction medium equaled 0.05% (v/v). Kontogianni et al. (2003) reported that highest flavonoid conversion was reached when initial water activity was 0.11 or less.
