Enzymes as Biocatalysts for Lipid-based Bioproducts Processing - Food and Industrial Bioproducts and Bioprocessing - page 333

temperature and type of reactor used. Lipase, depending on the amount of water present,
catalyzes either the hydrolysis or synthesis of ester bonds in acylglycerols (Angkawidjaja
and Kanaya, 2006). Non-regiospecific lipases have been shown to catalyze often the lipolysis
and liposynthesis to completion, forming either triacylglycerol (TAG) or free fatty acids
(FFA). Meanwhile, 1,3 regiospecific lipases, such as lipases from Rhizomucor meihei
(RML), TLL and Rhizopus sp ., are often used to catalyze the synthesis of partial acylglycerol
(Plou et al ., 1996). Optimum lipase dosage is also equally important in producing partial
acylglycerols at reasonable yield. Although enzyme dosage has a positive correlation with
yield, it is not usually kept at too high a concentration due to mass transfer limitations
between enzymes and substrates. Furthermore, high enzyme concentration is not economi-
cally feasible for industrial application.
The ratio of the reactants has also significant effect on the yield of the desirable product.
For example, in enzymatic glycerolysis to produce DAG, the ideal molar stoichiometric
ratio of oil to glycerol is 2:1. On the other hand, a molar stoichiometric ratio of oil to
glycerol of 1:2 will favor MAG production:

2 TAG 1 glycerol + → 3 DAG (14.13)

1 TAG 2 glycerol + → 3 MAG (14.14)

Various approaches have been used to modify the ratio of reactants during the course of
reactions to shift the reaction equilibrium towards higher partial acylglycerols yield. For
example, Rosu and co-workers (1997) performed a stepwise temperature reduction during
enzymatic glycerolysis, which resulted in a high yield of more than 90% MAG. During
the course of the temperature reduction, the MAG formed, which had a higher melting
temperature than that of the bulk reaction mixture, gradually precipitated and eventually
solidified. This shifted the reaction equilibrium towards production of MAG. Nevertheless,
this approach was found to be ineffective in increasing production of DAG (Kristensen
et al ., 2005). Kristensen and co-workers (2005) deduced that the carrier of the immobilized
lipase might be susceptible to blockage by the paste-like DAG, which hindered contact
among the reactants and lipase. The presence of a significant amount of DAG crystals in the
reaction mixture might have also prompted DAG to act as a seed for co-crystallization with
TAG, which led to the reaction rate stagnating (Cheong and Lai, 2009). Another approach
currently adopted in industrial continuous production of DAG is addition of purified MAG
into the second and subsequent production cycles to drive the esterification forward resulting
in high DAG yield (Minoru et al ., 2006 ).
The reaction media is an important part of enzymatic processing. Conventionally,
solvents such as n -hexane, n -heptane, acetone, tert -butanol and diethyl ether are sometimes
used to improve the miscibility of reaction mixtures and subsequently increasing overall
reaction rate. Tertiary alcohols, namely tert -butanol, tert -pentanol or mixtures of them with
hexane were found to be efficient reaction media for reasonably high yield of MAG
(Damstrup et al ., 2005). Despite that, solvent engineering has been deemed undesirable, as
it poses problems in waste management and, thus, will be environmentally unfriendly. Ionic
liquids, which are considered as eco-friendly solvents due to their negligible vapor pressure,
have emerged as new reaction media. Benefits of ionic liquids include adjustable solubility,
enhanced stability of lipases, positive effects on specificity of enzymes and facilitation of
recoverability and recyclability of lipases. Kahveci and co-workers (2009) found that binary
ionic liquid systems serve as good reaction media with at least 70% DAG yield.