Enzymatic transesterification of TG by lipases (3.1.1.3) is a good alter­native over a chemical process due to its eco-friendly, selective nature and low temperature requirement. Lipases break down the TAG into FFA and glycerol that exhibits maximum activity at the oil-water inter­face. Under low-water conditions, the hydrolysis reaction is reversible,

i. e., the ester bond is synthesized rather than hydrolyzed. Scientists are interested in the development of lipase applications to the inter­esterification reactions of vegetable oils for production of biodiesel.

Nag has reported [43] celite-immobilized commercial Candida rugosa lipase and its isoenzyme lipase 4 efficiently catalyzed alcoholysis (dry ethanol) of various TG and soybean oil (see Fig. 6.14). This process has many advantages over chemical processes such as (a) low reaction tem­perature, (b) no restriction on organic solvents, (c) substrate specificity on enzymatic reactions, (d) efficient reactivity requiring only the mixing of the reactants, and (e) easy separation of the product.

Kaieda et al. have developed [44] a solvent-free method for methanol — ysis of soybean oil using Rhizopus oryzae lipase in the presence of 4-30 wt%

water in the starting materials. Oda et al. [45] have reported methanol — ysis of the same oil using whole-cell biocatalyst, where R. oryzae cells were immobilized within porous biomass support particles (BSP). Kose et al. [46] have reported the lipase-catalyzed synthesis of alkyl esters of fatty acids from refined cottonseed oil using primary and secondary alcohols in the presence of an immobilized enzyme from C. antarctica, commercially called Novozym-435 in a solvent-free medium. Under the same conditions, with short-chain primary and secondary alcohols, cot­tonseed oil was converted into its corresponding esters.

Alcoholysis of soybean oil with methanol and ethanol using several lipases has been investigated. The immobilized lipase from Pseudomonas cepacia was the most efficient for synthesis of alkyl esters, where 67 and 65 mol% of methyl and ethyl esters, respectively, were obtained by Noureddini et al. [47]. Shimada et al. [48] have reported transesterifi­cation of waste oil with stepwise addition of methanol using immo­bilized C. antarctica lipase, where they have successfully converted more than 90% of the oil to fatty acid ME. They have also implemented the same technique for ethanolysis of tuna oil.

Dossat et al. [49] have found that hexane was not a good solvent as the glycerol formed after the reaction was insoluble in n-hexane and adsorbed onto the enzyme, leading to a drastic decrease in enzymatic activity. Enzymatic transesterification of cottonseed oil has been stud­ied using immobilized C. antarctica lipase as catalyst in t-butanol sol­vent by Royon et al. [50].

Sometimes, gums present in the oils used inhibit alcoholysis reactions due to interference in the interaction of the lipase molecule with sub­strates by the phospholipids present in the oil gum. Crude soybean oil cannot be transesterified by immobilized C. antarctica lipase. So, Watanabe et al. [51] have used degummed oil as a substrate for a trans­esterification reaction, in order to minimize this problem, and have effec­tively achieved conversion of 93.8% oil to biodiesel.

Methanol is insoluble in the oil, so it inhibits the lipases, thereby decreasing its catalytic activity toward the transesterification reaction. Du et al. [52] transesterified soybean oil using methyl acetate in the presence of Novozym-435 (see Fig. 6.15). Further, glycerol was also insol­uble in the oil and adsorbed easily onto the surface of the immobilized lipase, leading to a negative effect on lipase activity. They have suggested that methyl acetate was a novel acceptor for biodiesel production and no glycerol was produced in that process, as shown below:

CH,-OOC-R1 . RrCOOCH3 CH2-OOCCH4

j Lipase | “

CH-OOC-R2 + ЗСН3СООСН3 R2-COOCH3 + CH-OOCCH3

CH — OOC-R R3-COOCH3 CH2OOCCH3

They found 92% yield with a methyl acetate-oil molar ratio of 12:1, and methyl acetate showed no negative effect on enzyme activity.

The comparison of biodiesel production by acid, alkali, and enzyme is given in Table 6.2.