*2.8.5. Substrate composition and concentration (molar ratio)*

hydrolysis or ester synthesis [101]. While ester synthesis depends on low water content, too low water activity prevents all reaction from occurring. The monolayer of water on the surface of enzyme is required to maintain the three-dimensional structure of the enzyme, which is essential to enzymatic activity [102]. This layer acts as a buffer between the enzyme surface and the bulk reaction medium. However, too much water can cause hydrolysis of the TAG [14]. The activity of lipases at different water activities is dependent on the source of the enzyme and the type of solvent and immobilization support used [103]. Lipases from molds have shown to be more tolerant to low water activity than bacterial lipases. The optimal water content for most interesterification reactions by different lipases has been reported to be in the

However, the amount of water in the system should be minimized in order to decrease the byproducts. Lipases tend to retain the greatest degree of original activity, when immobilized on hydrophobic supports. Therefore, when the immobilized lipase contacts with oil in water emulsion, the oil phase tends to associate with and permeate the support, which can be assumed that an ordered hydrophobic network of lipid molecules will surround the support. Any water that reaches the enzyme for participation in the reaction must diffuse from the bulk emulsion. Thus, to avoid diffusional limitations, the oil phase must be well saturated with water [105].

Zhao et al. [106] investigated the effect of different reaction parameters on the enzymatic acidolysis of lard with capric acid catalyzed by Lipozyme TL-IM. They achieved the highest incorporation of capric acid (35.56 mol%) without added water. The amount of incorporation was almost constant up to 10% added water but decreased significantly above this amount. The current research work shows that Lipozyme TL-IM-catalyzed interesterification can easily be moved to the industrial sector for commercial exploitation. Both stirred tank reactors [107] and PBR [108, 109] can be used for the production of plastic fats, and the control of water activity in the system presents no particular difficulty, as is often the case in other lipase-application systems, in which the lipase activity was not affected by the reduction of water content in the

In order to promote the synthesis of phenolic lipids by shifting the reaction toward synthesis rather than hydrolysis, a reduction of water content in the reaction mixture can be accomplished through the addition of molecular sieve pellets as dehydrating agents. Li et al. [111] reported that the addition of molecular sieves increased the rate and conversion yield; this is due to the effect of the molecular sieves to sequester the water layer from the enzyme molecule which is essential for the water-enzyme interaction. Mellou et al. [112] found that the conversion yield of rutin during esterification reaction with oleic acid catalyzed by immobilized *C. antarctica* lipase B in different solvents was varied from 37 to 71% under the use of molecular sieves (100 mg/ ml). However, Karboune et al. [113] observed 28 and 35% decrease in the maximum conversion yield upon the addition of 10 mg/ml of molecular sieves to the lipase-catalyzed biosynthesis of cinnamoylated lipids. This could be explained by the fact that molecular sieves promote the lipase-catalyzed synthesis reactions by dehydrating; however, excess of molecular sieves will

capture the necessary water of enzyme, which may inhibit the enzyme activity.

range of 0.04 to 11% (w/v) [104].

102 Phenolic Compounds - Natural Sources, Importance and Applications

system [107, 110].

*2.8.4. Molecular sieve*

Chemical structures of the phenolic compounds have an effect on the conversion yield of the end products. Different studies presented the effect of chemical structure of phenolic compounds which are hydroxylated or methoxylated derivatives of cinnamic, phenyl acetic and benzoic acids on the conversion yield [34, 35, 42, 44]. The presence of a hydroxyl group in the sn-2 position has a negative inductive effect. Thus, TAG is hydrolyzed at a faster rate as compared to DAGs, which are hydrolyzed faster than MAGs. Substrate conformation can also affect the reaction rate, since the hydrophobic tunnel in the lipase accepts aliphatic chains and aromatic rings easier than branched structures. Moreover, oxidation of substrates, such as PUFAs, could cause inhibition and decrease in lipase activity due to the production of hydroperoxides and their consequent breakdown to free radicals.

Substrate concentration has an effect on the rate of enzyme hydrolysis and transesterification. So, the selection of a suitable substrate molar ratio in terms of reaction efficiency (incorporation level of acyl donors per unit time) and productivity (product quantity per unit time) in a reaction system is very important. The choice of the proper substrate molar ratio is also related to the downstream processing expenses and associated difficulties of separating FFAs or acyl donors by evaporation and/or distillation. Previous studies have shown that high substrate molar ratio would require a shorter reaction time, move the reaction equilibrium to the product formation, and improve the acyl incorporation [114, 115]. Yang et al. [114] reported the positive effect of substrate molar ratio on the interesterification reaction between EPA and DHA ethyl esters and tripalmitin. They indicated that the optimization results suggested a molar ratio of 6 along with an enzyme load of 20% (Lipozyme TL-IM) and a 17.9 h reaction time would provide the highest incorporation. However, due to the downstream purification expenses, they decided to select the optimal conditions to be a molar ratio of 5 along with a 20% enzyme load and 20 h reaction time. Lee et al. [115] investigated the synthesis of 1,3-dioleoyl-2-palmitoyl glycerol-rich HMFS from tripalmitin-rich fraction and ethyl oleate by lipase-catalyzed interesterification. Similarly, these authors reported an increase of OPO content (25.7%) with an increase of substrate molar ratio up to a ratio of 1:6 of tripalmitin-rich fraction to ethyl oleate.

The study of Sabally et al. [32] investigated the enzymatic transesterification of selected PAs with TAGs, including trilinolein and trilinolenin in organic solvent media (OSM), and reported that the affinity of Novozym 435 was found to be greater for DHCA than that for ferulic acid; these authors suggested that the presence of both the methoxyl substituent and the double bond on the side of the aromatic ring of the ferulic acid could explain its lower affinity for the transesterification reactions with TAG.

Karboune [42] study the effect of PA structure on the bioconversion yield (BY) of phenolic lipids (PLs) obtained by acidolysis of FSO with selected PAs, including hydroxylated and/ or methoxylated derivatives of cinnamic, phenyl acetic and benzoic acids in OSM, using Novozym 435 as biocatalyst. The overall findings showed that the BY of PL was dependent on the structural characteristics of PAs. The highest BY was obtained with cinnamic acid (74%). In addition, Karboune et al. [42] concluded that the presence of p-OH groups on the benzene cycle of cinnamic acid derivatives may have an inhibitory effect on the lipase activity, since the BY decreased to 45 and 11%, respectively, when *p*-coumaric and caffeic acids were used as substrates. The inhibitory effect of p-OH substituent was most likely due to their electronic donating effect rather than to their steric hindrance in the enzyme-active site as the inhibition was much less significant (56%) in the presence of a double bond on the side chain conjugated with the aromatic ring of DHPA.
