Water content plays a key role in enzymatic transes­terification as it is vital to sustain the three­dimensional conformations of enzyme catalytic site. Presence of an oil—water interface creates a favorable environment for the conformation of active site (Al — Zuhair et al., 2006, 2003). Water interacts with the enzyme hydrophilic groups located on surface, and changes the conformation of hydrogen bond interac­tions inside enzyme, leading to transformation of lipase active (Gao et al., 2006). Generally lipase activity in­creases with increase in water content up to 15% (w/w of oils). Beyond 15%, the conversion rate decreased slightly. But 20% of water content also efficiently cata­lyzed alcoholysis using lipases from Rhizopus delemar and Rhizomucor miehei (Tweddell et al., 1998). About 5% of initial water content was suggested as optimum for biodiesel production from jatropha oil using various lipases (Shah and Gupta, 2007). Thus, the optimum level of initial water (moisture) is based on the type of biocat­alyst and reaction conditions.

ACYL ACCEPTORS

Generally transesterification reactions are conducted using straight — and branched-chain alcohols. Because of abundant availability and low cost, methanol is the widely used short-chain alcohol acyl acceptor for bio­diesel production (Fan, 2012). The negative effect of methanol on enzyme activity alleviates by stepwise addition of alcohols. Ethanol, n-butanol and i-butanol, n-amylalcohol and i-amylalcohol, and n-propanol were also used during transesterification. But increase in C number of the alcohols has not significantly influenced fatty acid ester contents and shown the negative effect (Soumanou and Bornscheuer, 2003a, b). Also, it is gener­ally believed that primary alcohols are more suitable than secondary alcohols and alcohols with less than eight carbon atoms can be used under the conditions that gave the highest conversion of the oils to FAME. Methyl acetate had no negative effect on enzymatic ac­tivity. No changes were detected in lipase activity even after being continuously used for 100 batches (Sulaiman,

2007) . Recently, ethyl acetate, methyl acetate, butyl acetate, vinyl acetate and dimethyl carbonate (DMC) are considered as novel acyl acceptors. The work revealed by Er-Zheng et al. (2007) proved that DMC gives two — to threefold higher conversion than those of conventional acyl acceptors (methanol and methyl ace­tate) and is also ecofriendly, neutral, odorless, cheap, noncorrosive, nontoxic, and exhibits good solvent properties.

SOLVENTS

In the above section (molar ratio) discussed that excess amount of alcohols increases FAME yield. In order to in­crease the solubility of alcohol (not the enzyme), solvents are used and they alleviate negative effect of methanol on the catalyst and precede the transesterification. Enzyme should be insoluble in solvent; otherwise, it will not be active (Kanerva et al., 1990; Antczak et al., 2009). Various hydrophilic and hydrophobic organic solvents such as cyclohexane, n-hexane, tert-butanol, petroleum ether, isooctane and 1,4-dioxane are mainly studied organic sol­vents in enzymatic biodiesel production. If organic sol­vent is used as medium, overall alcohol is added at the beginning of the reaction. In solvent-free reaction me­dium, alcohol is added stepwise to prevent enzyme activ­ity with high alcohol concentration (Sevil et al., 2012).