*2.5.3. Selectivity and specificity of lipase*

It is beneficial to have knowledge about lipase selectivity/specificity to guide research to the best choice of lipase for particular fatty acid or for synthesis of PL containing ester of a specific fatty acid. Specificity generally refers to the ability of enzyme to differentiate between several substrates. Lipases can be divided according to their specificity into three groups: (i) nonspecific lipases, (ii) acyl-group specific lipases, and (iii) positional specific lipases. Nonspecific lipase can catalyze the release of FA from any position on the glycerol molecule. Acyl-group specific lipases catalyze the release of a particular type of FA from the TAG molecules, while positional lipases attack sn-1,3 positions on the TAG molecule. The use of positional specific lipases has led to the production of useful TAG mixtures whose composition could not be produced by simple chemical transesterification. In recent years, positional specific lipases have been intensively used in research purposes and food industry sectors [77–79].

### **2.6. Enzyme reactions in organic solvent media (OSM)**

Enzymes in organic solvents have manifested good selectivity and stability; however, catalytic activities in this environment are generally lower than in aqueous solutions. This could be partly explained by the fact that in low water environments, enzymes are less flexible. On

*2.5.2.2. Esterification*

98 Phenolic Compounds - Natural Sources, Importance and Applications

removal of water [72].

*2.5.2.3. Transesterification*

are described clearly in **Figure 6**.

*2.5.3. Selectivity and specificity of lipase*

Esterification is the reverse reaction of hydrolysis and is used to synthesize selected products under appropriate reaction conditions [70]. The products of an esterification reaction are usually an ester and water. The water content of esterification reaction system strongly effects on lipase activity. Low water content shifts the equilibrium of the reaction to favor the synthesis of lipids. So that additional techniques were used to drive synthesis reaction including removal of water that formed during the process by evaporation under reduced pressure [71] or by adding molecular sieves to adsorb the water. Direct enzymatic esterification of some primary alcohols and selected carboxylic acids was catalyzed by the *Candida antarctica* and *Rhizomucor miehei* lipases. The reactions were performed in solvent-free medium with the

Transesterification is a process of acyl exchange between two molecules. This process normally takes place between an ester and alcohol (alcoholysis), an ester and an acid (acidolysis), or an ester with another ester (interesterification), and no water is involved in the reaction. Acidolysis is one of the most frequently used reactions to incorporate novel fatty acids into TAG in several researches [13, 73, 74]. Interesterification involving hydrolysis and esterification, firstly hydrolysis of the TAG molecule, then followed by re-synthesis of the liberated fatty acids onto the glycerol molecule. Interesterification is another main strategy to incorporate PUFAs into TAGs. The literature reported extensive research work on the interesterification reaction [75, 76]. Lipase-catalyzed alcoholysis, acidolysis, and interesterification reactions

It is beneficial to have knowledge about lipase selectivity/specificity to guide research to the best choice of lipase for particular fatty acid or for synthesis of PL containing ester of a specific fatty acid. Specificity generally refers to the ability of enzyme to differentiate between several substrates. Lipases can be divided according to their specificity into three groups: (i) nonspecific lipases, (ii) acyl-group specific lipases, and (iii) positional specific lipases. Nonspecific lipase can catalyze the release of FA from any position on the glycerol molecule. Acyl-group specific lipases catalyze the release of a particular type of FA from the TAG molecules, while positional lipases attack sn-1,3 positions on the TAG molecule. The use of positional specific lipases has led to the production of useful TAG mixtures whose composition could not be produced by simple chemical transesterification. In recent years, positional specific lipases have

Enzymes in organic solvents have manifested good selectivity and stability; however, catalytic activities in this environment are generally lower than in aqueous solutions. This could be partly explained by the fact that in low water environments, enzymes are less flexible. On

been intensively used in research purposes and food industry sectors [77–79].

**2.6. Enzyme reactions in organic solvent media (OSM)**

**Figure 6.** Lipase-catalyzed transesterification reactions. R1, R2, and R3 are different acyl groups.

the other hand, the activities of enzymes also depend on the type of organic solvent, since some are known to inactivate or denature biocatalysts. Meanwhile, the advantages of using organic solvent media are increased solubility of hydrophobic compounds that permits for greater interactions between substrates and enzymes as well as advantageous, partitioning of substrates and products; specifically, this is because partitioning of products away from the enzyme can decrease the possibility of inhibition due to excess product around the biocatalyst [51, 72].

When enzymes are placed in OSM, they exhibit novel characteristics such as altered chemoand stereoselectivity, enhanced stability, increased rigidity, insolubility, and high thermal stability [80]. It has also been reported that the thermal stability of lipases can be improved in organic solvent systems since the lack of water prevents the unfolding of the lipase at high temperatures [81]. The activity of lipase in OSM depends on the nature and concentration of the substrate and source of the enzyme. Moreover, the organic solvent used can dramatically affect the activity of the lipase. Lipases are more active in n-hexane, n-heptane, and isooctane as compared to other solvents, such as toluene, ethyl acetate, and acetonitrile [82, 83]. It has been reported that the hydrophobicity of the solvent can affect the degree of acyl migration during transesterification using a 1,3-specific lipase [84]. Since the choice of organic solvents based on minimization of acyl migration may conflict with maximization of transesterification, acyl migration is usually minimized by reducing reaction times [85]. With increasing concern for the environment, synthesis of PL in solvent-free systems [86–88] and ionic liquids systems [89] has been extensively studied.

The enzymatic synthesis of vanillyl-PUFA esters from fish oil and vanillyl alcohol in acetone solvent medium was studied by [10]. Lipase-catalyzed esterification of vanillyl alcohol with different fatty acids was carried out by [51] to the synthesis of capsiate analogs. Equimolar concentration of vanillyl alcohol and fatty acid was solubilized in *tert*-butanol and esterified using *C. antartica* lipase (Novozym 435) at 55°C for 4 h.
