**3. Results and discussion**

#### **3.1. Influence of water removal techniques**

The presence of a minimum amount of water is necessary for maintaining the catalytic activity of lipase. But, high concentration of water can favor the hydrolysis reaction.

Different water removal techniques were investigated. In the presence of molecular sieves, two alternatives have been tested. The molecular sieves were introduced either in the bulk medium or in the outer loop of the reactor. The performance of these reactions carried out with or without water removal were evaluated and compared.

The results are reported in **Figure 3a**–**c**. The reaction carried out with high water content leads to the lowest conversion yield, initial rate, and productivity while, reactions conducted with water removal are characterized by high conversion yield, initial rate, and productivity. The highest conversion rates of rutin were almost similar for the two configurations of outer loop, 79 and 68%, respectively. In the presence of high water content, only monorutin formation was observed, while at low water content both mono‐ and di‐rutin esters were synthesized. HPLC and NMR analyses showed that the synthesized mono and dirutin esters are respec‐ tively rutin 4‴‐hexadecanedioate and dirutin 4‴, 4‴‐hexadecanedioate (**Figure 2**).

The improved performance of the acylation reaction with a water removal system has already been described by several authors [24–26], but its influence on the selectivity of the reaction has never been mentioned before. The water removal by the two outer loop configurations gave similar results and presents the advantage of avoiding the abrasion of the enzyme.

Due to its implementation facility, the water removal by vapor and liquid phase (**Figure 3b**) was selected as a standard method to investigate the effect of the other factors on this reaction.

#### **3.2. Effect of the temperature on the rutin solubility and esters synthesis**

#### *3.2.1. Effect on the solubility*

The solubility of substrates is one of the main factors that affect the performance of the acyla‐ tion reaction. This solubility is drastically influenced by the temperature. For this reason, the effect of the temperature (60, 80, and 90°C) on the solubility of rutin in *tert*‐amyl alcohol was evaluated in the first step. In all cases, solubility increases with temperature. At the equilibrium

**Figure 2.** Synthesis of rutin hexadecanedioate and dirutin hexadecanedioate.

Biocatalysis of Rutin Hexadecanedioate Derivatives: Effect of Operating Conditions on Acylation Performance and... http://dx.doi.org/10.5772/67621 119

The results are reported in **Figure 3a**–**c**. The reaction carried out with high water content leads to the lowest conversion yield, initial rate, and productivity while, reactions conducted with water removal are characterized by high conversion yield, initial rate, and productivity. The highest conversion rates of rutin were almost similar for the two configurations of outer loop, 79 and 68%, respectively. In the presence of high water content, only monorutin formation was observed, while at low water content both mono‐ and di‐rutin esters were synthesized. HPLC and NMR analyses showed that the synthesized mono and dirutin esters are respec‐

The improved performance of the acylation reaction with a water removal system has already been described by several authors [24–26], but its influence on the selectivity of the reaction has never been mentioned before. The water removal by the two outer loop configurations gave similar results and presents the advantage of avoiding the abrasion of the enzyme.

Due to its implementation facility, the water removal by vapor and liquid phase (**Figure 3b**) was selected as a standard method to investigate the effect of the other factors on this reaction.

The solubility of substrates is one of the main factors that affect the performance of the acyla‐ tion reaction. This solubility is drastically influenced by the temperature. For this reason, the effect of the temperature (60, 80, and 90°C) on the solubility of rutin in *tert*‐amyl alcohol was evaluated in the first step. In all cases, solubility increases with temperature. At the equilibrium

tively rutin 4‴‐hexadecanedioate and dirutin 4‴, 4‴‐hexadecanedioate (**Figure 2**).

**3.2. Effect of the temperature on the rutin solubility and esters synthesis**

**Figure 2.** Synthesis of rutin hexadecanedioate and dirutin hexadecanedioate.

*3.2.1. Effect on the solubility*

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**Figure 3.** Effect of drying techniques on the esterification of rutin. Reactions carried out with 131 mM of rutin, 117.9 mM of acid, 30 g/L of Novozym 435 at 90°C. (a) Conversion rate of rutin at 50 h. (b) Selectivity of the reaction at 50 h. (c) Initial rate of rutin monoester formation and productivity at 50 h.

(10 h), the concentrations of rutin in the bulk medium are 42.3 ± 2.1 mM, 81.5 ± 4.0 mM, and 102.2 ± 5.1 mM, respectively for 60, 80, and 90°C. Fatty acids are totally soluble in *tert*‐amyl alcohol at the studied concentrations and temperatures.

### *3.2.2. Effect on rutin esters synthesis*

To investigate the effect of temperature on conversion rate, initial rate, and productivity, three set points of temperature (60, 80, and 90°C) were studied. Meanwhile, the other factors were kept constant at 65 mM of rutin, 58.5 mM of diacid, and 30 g/L of Novozym 435. NMR analy‐ sis showed that the two formed products are rutin 4‴‐hexadecanedioate and dirutin 4‴, 4‴‐ hexadecanedioate (**Figure 2**).

Theodosiou et al. [23] reported similar results during their studies of enzymatic acylation of sily‐ bin by different dicarboxilic acids. They observed that at 50°C and after 96 h of incubation, both mono‐ and di‐esters of silybin were synthetized but they identified only the monoester by NMR.

The behavior of the kinetics of rutin esters formation and the productivity of the reaction are summarized in **Figure 4a** and **b**. The highest performances were obtained at 90°C with 38.4 mM of total esters and a productivity of 0.78 g/L/h at 50 h, while the lowest values were found at 60°C with 20.8 mM and 0.38 g/L/h, respectively. Using esculin and palmitic acid as substrates, Lue et al. [16] reported similar effect of temperature. The observed results could be explained by the increase of rutin solubility, the decrease of the medium viscosity, and thus the increase of the mass transfer rate at high temperature. The increase of the temperature favors also the water removal and then the shift of the equilibrium to the product formation. These results suggested that the temperature has to be maintained as high as possible (≥80°C).

### **3.3. Effect of rutin concentration**

During the acylation reaction of rutin by hexadecanedioate acid, three concentrations of rutin were tested (65, 131, and 196 mM) in the presence of hexadecanedioate acid (58.5, 117.9, and 176.4 mM, respectively), and 30 g/L of Novozym 435, in *tert*‐amyl‐alcohol at 90°C.

**Figure 5** reports the obtained results concerning conversion yields (**Figure 5a**), selectivity, productivity (**Figure 5b**), and initial rate of rutin acylation (**Figure 5c**).

It appears that the initial rates of rutin acylation as well as the productivity increase with substrate concentrations (from 2.88 to 5.61 mM/h and from 0.78 to 2.56 g/(L/h), respectively), while conversion yields of both substrates (70% of rutin and 66% of hexadecanedioic acid) and selectivity of the reaction (76%) remain almost unchanged.

At 90°C, whatever the initial concentration, the rutin is totally soluble. Therefore, the behavior of the conversion rate and selectivity is rather due to the effect of molar ratio and not due to the solubility.

### **3.4. Effect of diacid/rutin molar ratio**

The hydrophobicity of the reaction medium varies depending on the diacid/rutin molar ratio. Consequently, the selectivity of the reaction could be affected. This assumption was checked

Biocatalysis of Rutin Hexadecanedioate Derivatives: Effect of Operating Conditions on Acylation Performance and... http://dx.doi.org/10.5772/67621 121

(10 h), the concentrations of rutin in the bulk medium are 42.3 ± 2.1 mM, 81.5 ± 4.0 mM, and 102.2 ± 5.1 mM, respectively for 60, 80, and 90°C. Fatty acids are totally soluble in *tert*‐amyl

To investigate the effect of temperature on conversion rate, initial rate, and productivity, three set points of temperature (60, 80, and 90°C) were studied. Meanwhile, the other factors were kept constant at 65 mM of rutin, 58.5 mM of diacid, and 30 g/L of Novozym 435. NMR analy‐ sis showed that the two formed products are rutin 4‴‐hexadecanedioate and dirutin 4‴, 4‴‐

Theodosiou et al. [23] reported similar results during their studies of enzymatic acylation of sily‐ bin by different dicarboxilic acids. They observed that at 50°C and after 96 h of incubation, both mono‐ and di‐esters of silybin were synthetized but they identified only the monoester by NMR. The behavior of the kinetics of rutin esters formation and the productivity of the reaction are summarized in **Figure 4a** and **b**. The highest performances were obtained at 90°C with 38.4 mM of total esters and a productivity of 0.78 g/L/h at 50 h, while the lowest values were found at 60°C with 20.8 mM and 0.38 g/L/h, respectively. Using esculin and palmitic acid as substrates, Lue et al. [16] reported similar effect of temperature. The observed results could be explained by the increase of rutin solubility, the decrease of the medium viscosity, and thus the increase of the mass transfer rate at high temperature. The increase of the temperature favors also the water removal and then the shift of the equilibrium to the product formation. These results suggested that the temperature has to be maintained as high as possible (≥80°C).

During the acylation reaction of rutin by hexadecanedioate acid, three concentrations of rutin were tested (65, 131, and 196 mM) in the presence of hexadecanedioate acid (58.5, 117.9, and

**Figure 5** reports the obtained results concerning conversion yields (**Figure 5a**), selectivity,

It appears that the initial rates of rutin acylation as well as the productivity increase with substrate concentrations (from 2.88 to 5.61 mM/h and from 0.78 to 2.56 g/(L/h), respectively), while conversion yields of both substrates (70% of rutin and 66% of hexadecanedioic acid)

At 90°C, whatever the initial concentration, the rutin is totally soluble. Therefore, the behavior of the conversion rate and selectivity is rather due to the effect of molar ratio and not due to

The hydrophobicity of the reaction medium varies depending on the diacid/rutin molar ratio. Consequently, the selectivity of the reaction could be affected. This assumption was checked

176.4 mM, respectively), and 30 g/L of Novozym 435, in *tert*‐amyl‐alcohol at 90°C.

productivity (**Figure 5b**), and initial rate of rutin acylation (**Figure 5c**).

and selectivity of the reaction (76%) remain almost unchanged.

alcohol at the studied concentrations and temperatures.

*3.2.2. Effect on rutin esters synthesis*

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hexadecanedioate (**Figure 2**).

**3.3. Effect of rutin concentration**

**3.4. Effect of diacid/rutin molar ratio**

the solubility.

**Figure 4.** Effect of temperature on the performance of rutin hexadecanedioate synthesis. Reactions carried out with 65 mM of rutin, 58.5 mM of acid, and 30 g/L of Novozym 435 at 60, 80, and 90°C. (a) Effect of temperature on the conversion rate of rutin and hexadecanedioic acid at 50 h. (b) Effect of temperature on the initial rate (■) and productivity (□) at 50 h.

during a preliminary study with a diacid/rutin molar ratio of 0.05, 0.9, and 20. The obtained results showed that only rutin monoester was synthesized in the presence of a diacid/rutin molar ratio of 20 and both mono‐ and di‐esters are produced with the two other lower ratios. In all cases, the rutin diester formation appears only after 5 h of incubation while the acid was com‐ pletely depleted from the medium after 30–40 hours independently to molar ratio values. After the depletion of the fatty acid concentration, the monorutin ester became a substrate for the diru‐ tin ester synthesis. At the end of the reaction (50 h), a plateau was reached (**Figure 6a**–**c**). These results suggested that rutin and diacid are better substrates to the enzyme than monorutin ester.

The effect of the molar ratio was investigated in several works. Similar results were observed by Ma et al. [27] during the acylation of isoorientin and isovitexin by Novozym 435.

**Figure 5.** Effect of rutin concentration on the performance of rutin acylation. Reactions performed at 65, 131, 196 mM of rutin with 58.5, 117.9 and 176.4 mM, respectively of acid with 30 g/L of Novozym 435 at 90°C. (a) Rutin and hexadecanedioic acid conversion rate at 50 h. (b) Selectivity (□) and productivity at (■) 50 h. (c) Initial rate of rutin acylation.

Biocatalysis of Rutin Hexadecanedioate Derivatives: Effect of Operating Conditions on Acylation Performance and... http://dx.doi.org/10.5772/67621 123

**Figure 6.** Kinetics of rutin esterification using different initial rutin concentrations. (a) Kinetics of rutin (131 mM) esterification reaction with hexadecanedioic acid (0.05 eq) by Novozym 435 (30 g/L) at 90°C, acid (▲), rutin hexadecanedioate (■) dirutin hexadecanedioate (♦). (b) Kinetics of rutin (131 mM) esterification reaction with hexadecanedioic acid (0.9 eq) by Novozym 435 (30 g/L) at 90°C, rutin (●) acid, (▲) rutin hexadecanedioate (■), dirutin hexadecanedioate (♦). (c) Kinetics of rutin (65 mM) esterification reaction with hexadecanedioic acid (20 eq) by Novozym 435 (30 g/L) at 90°C, rutin (●), rutin hexadecanedioate (■).

**Figure 5.** Effect of rutin concentration on the performance of rutin acylation. Reactions performed at 65, 131, 196 mM of rutin with 58.5, 117.9 and 176.4 mM, respectively of acid with 30 g/L of Novozym 435 at 90°C. (a) Rutin and hexadecanedioic acid conversion rate at 50 h. (b) Selectivity (□) and productivity at (■) 50 h. (c) Initial rate of rutin

acylation.

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