Developing a high-temperature solvent-free system for efficient biosynthesis of octyl ferulate

Ferulic acid esters have been suggested as a group of natural chemicals with sunscreen function. The study aimed to utilize an environment-friendly enzymatic method to produce octyl ferulate by esterification of ferulic acid with octanol. The Box-Behnken design with response surface methodology (RSM) was adopted to evaluate the effects of synthesis variables, including reaction temperature (70–90 °C), enzyme amount (1,000–2,000 PLU) and stir speed (50–150 rpm), on the molar conversion of octyl ferulate. According to the joint test, both the reaction temperature and enzyme amount had great impacts on the molar conversion. RSM-developed second-order polynomial equation further showed great ability on data-fitting. Based on ridge max analysis, the optimum parameters for the biocatalyzed reaction were: 72 h reaction time, 92.2 °C reaction temperature, 1,831 PLU enzyme amount and 92.4 rpm stir speed, respectively. Finally, the molar conversion of octyl ferulate under optimum condition was verified to be 93.2 ± 1.5%. In conclusion, high yield of octyl ferulate synthesized by commercial immobilized lipase under elevated temperature conditions has been suggested, which our findings could broaden the utilization of the lipase and provide a biocatalytic approach, instead of the chemical method, for ferulic acid ester synthesis.


Introduction
Ferulic acid (FA) is one of the phenolic acids and abundantly exists in fruits and vegetables.FA has been demonstrated having many bioactivities such as antioxidation [1], excellent ultraviolet-absorbing activity [2,3], and potential health benefits against cardiovascular diseases, inflammatory diseases and cancers [4], etc.However, FA is a small and polar compound but only with limited solubility in oils.Due to the poor solubility of FA in a lipophilic medium, the applications of FA in food, cosmetics, and other nutraceutical industries were commonly limited owing to their low dissolution rate [5,6].Thus, it is essential to improve the solubility of FA to increase its practicality as food antioxidants.A related study has been reported that the ester form of FA has better antioxidant activity than the acid form itself as well as the antioxidant activity of ferulate ester as compared to BHT [7].To overcome the problem of poor solubility, pharmaceutical particle technology of chemical modifications has attracted much attention [8][9][10][11][12][13].However, it is much difficult to synthesize such derivatives chemically, as FA is susceptible to oxidation under certain pH conditions [7,14,15].Thus, enzymatic biosynthesis of FA lipids has been considered as an attractive alternative to conventional chemical processes [16].However, environment-friendly preparation of hydrophobic derivatives of FA is still a challenge for researchers.
The hydrophilic feruloylated derivatives have been synthesized by the esterification of FA with monosaccharides [17].The water-soluble derivative of glyceryl ferulate biosynthesized by esterification of FA with glycerol using pectinase [18], and the solvent-free enzymatic transesterification of ethyl ferulate (EF) with triolein having a higher EF conversion of 77% [19], which were also reported.In additional, hydrophobic feruloylated derivatives can be produced by the esterification of FA with alcohols.The strategy of esterification of hydrophilic FA with lipophilic molecules, such as aliphatic alcohols, can be used to alter its solubility in a lipophilic medium.However, the reaction rate of FA condensation with alcoholic substrates is slow and might be that FA could conjugate with a carboxyl group and a bulky noncarboxylic region [20].
Lipases are the most commonly used among the biocatalysts in synthetic organic chemistry [21,22].It has been extensively used to obtain chiral alcohols and carboxylic acids, due to their excellent chiral recognition [23].The isoform B of the lipase from Candida Antarctica (CAL-B) is one of the most employed among them and possesses the high enantioselectivity for a broad range of substrates [24,25].Novozym® 435 is a commercial enzyme prepared by immobilization of CAL-B onto a macroporous acrylic polymer resin.Novozym® 435 is a versatile biocatalyst, mainly used to perform the hydrolysis of oils and fats, under specific conditions, also capable of catalyzing esterification, transesterification, and interesterification reactions [26].3D-structures of the Novozym® 435 have shown the presence of a short oligopeptide helix that may act as a lid, and it has been found to adopt different conformations as a function of detergent concentration, suggesting high mobility of the active environment [27].The expression of catalytic activity requires that the enzyme active site should be flexible.Novozym® 435 is very active in organic solvents, which is needed to react under the condition of lower water activity in esterification process [28], suggesting that the enzyme could be adsorbed on this biocatalyst mainly by hydrophobic interactions [29,30].Evidence that an increase in enzyme hydration is accompanied by an increase in the molecular mobility of enzymes [31,32].Although Novozym® 435 had been used in the lipasecatalyzed synthesis of ferulate esters, the yield remained low (17%) after the reaction by several days [33].Moreover, it has been reported that during the synthesis of ferulic esters by direct esterification with ferulic acid, the catalytic activity of the enzyme might be severely affected by the hydrophilicity of ferulic acid [34,35].In fact, solvent-free reaction systems, an eco-friendly method minimizing the environmental pollution, could be applied in the lipase-catalyzed synthesis of ferulate esters, which might decrease the reaction time but increase the yield of synthesized esters [36,37].
Additionally, the solvent-free reaction system is a condition commonly with a simple mixture of substrates and offers some reaction advantages including maximization of substrate concentration, higher volumetric productiveness, less environmental hazard, and cost savings in reactor design in large-scale process and chemical product separation/purification [38].However, in the case of FA esterification, developing a solvent-free system operated at high temperature was required to increase the yield of FA esters.
As mentioned above, ferulic acid (FA) has been suggested as one of the natural nutraceuticals with biological function, but FA exhibits poor solubility and low stability in various solvent systems might limit its application.The aim of the present work was tending to develop a simple and environmentally-friendly biocatalytic method, instead of a chemical catalytic method, for the preparation of FA esters.In this study, the lipasecatalyzed esterification of FA with octanol to form octyl ferulate under solvent-free condition was conducted, followed by optimizing processes with an experimental design using RSM to evaluate the best experimental conditions.Finally, the thermodynamic and optimum effects on solvent-free reaction system of lipasecatalyzed biosynthesis of FA was evaluated.

Enzymatic synthesis of octyl ferulate
All materials were dehydrated by a molecular sieve 4 Å for 24 h before use.Ferulic acid (20 mM) and Novozym ® 435 were well mixed with octanol (1 mL) in the sealed 10 mL dark vials, and placed in a water bath for 72 h, under different experimental conditions of temperatures, enzyme amount and stir speed as Table 1 design.The liquid samples were further analyzed by high-performance liquid chromatography (HPLC).
Samples were centrifuged and diluted with hexanol/2-methyl-2-butanol (1:100).The analysis was done according to the procedure modified by Huang et al. [38].The flow rate was 1.0 mL/min, and sampling was detected at the wavelength of 325 nm.The molar conversion was calculated based on the peak areas of the sample.

Experimental design
A 3-level-3-factor Box-Behnken requiring 15 experiments was employed in this study.To avoid bias, the 15 runs were performed in a random order.The variables and their levels selected for the study of octyl ferulate biosynthesis were: reaction

Statistical analysis
Each data was analyzed by the response surface regression (RSREG) procedure with SAS software to fit the equation of second-order polynomial shown in Equation 1: where Y is the molar conversion of octyl ferulate; β0 is a constant, βi, βii and βij are constant coefficients; and xi and xj are uncoded independent variables.The suffixes i and j are seen in the above equation with the three independent variables denoting: x1 for reaction temperature, x2 for enzyme amount and x3 for stir speed.The option of a ridge max option in the SAS software was employed to compute the estimated ridge of maximum response when increasing the radius from the center of the original design.

Primary experiment
Lipase-catalyzed synthesis of octyl ferulate from ferulic acid (20 mM), Novozym ® Also, the higher the reaction temperature, the faster the reaction could be observed without deterioration of enantioselectivities [39].It is a known fact that the reaction temperature is a crucial parameter in biocatalysis.However, the elevated temperature may result in inactive enzymes.From the report, enzymatic transesterification of ethyl ferulate (EF) and monostearin for feruloylated lipids production was investigated, which the optimum reaction temperature was 74°C [9].A novel enzymatic route of feruloylated structured lipids synthesis by the transesterification of EF with castor oil in a solvent-free system was investigated.The study indicated that high EF conversion was obtained under the reaction temperature of 90°C [22].The reaction temperature also has a considerable effect on the thermodynamic equilibrium of the reversible reaction.Therefore, the experiments at three different temperatures (70, 80 and 90°C) were carried out to investigate the influence of temperature on biosynthesis reaction.
As shown in Figure 2, the molar conversion increased with elevating reaction temperature.At a reaction temperature of 90°C, molar conversion reached the highest value of a molar conversion of 88.4%, and the fastest reaction rate (reflected by the slope of the linear part of the curves) was also observed.When the reaction time surpassed 72 h, the time course was leveled-off.The reason may be that the viscosity of the reaction mixture was gradually increased over reaction time, leading to a decrease in mutual solubility and inhibit the diffusion of substrates [40].Thus, reducing mass transfer and unfavorable interactions between enzyme particles and substrates might occur in the process.However, longer reaction time could lead to a higher molar conversion.Thus, the following experimental design was conducted on the reaction time of 72 h.

Model fitting
In order to systematically understand the relationships between reaction temperature, enzyme amount and stir speed for the synthesis of octyl ferulate, a 3-level-3-factor Box-Behnken design (BBD) was applied to carry out a total of 15 treatments (experiments), followed by determining the experimental data for process optimization of octyl ferulate bioproduction, using statistical RSM modelling.The aim of this study was the development and evaluation of a statistical approach to understanding the relationship between experimental variables and lipase-catalyzed responses well.Thus, the process could be optimized before the scaling-up procedure to save the production costs and time to obtain a high-quality product.Compared with the single-factor-at-atime design, which has been adopted most often in literature, the RSM combined with a BBD employed in this study was more efficient in reducing experimental runs and time for investigating the optimized biosynthesis of octyl ferulate.
The experimental conditions and results of a 3-level, 3-factor BBD are shown in Table 1.The RSREG procedure for SAS was used to fit the second-order polynomial Equation ( 1) to the experimental data-percent mole conversions.Among these various treatments, the highest molar conversion (88. Further, the analysis of variance (ANOVA) indicated that the second-order polynomial equation showed a highly significant correlation and was adequate to represent the relationship between the experimental response (molar conversion) and significant variables, having a very small p-value (0.0001) as well as a satisfactory coefficient of determination (R 2 = 0.9887).Furthermore, the overall effects of the three synthesis variables on the molar conversion were further analyzed by a joint test.The results revealed that reaction temperature (X1) and enzyme amount (X2) were the most important factors, exerting a statistically significant overall effect (p < 0.001) on the responded molar conversion.The stirring speed (X3) was insignificant statistically on the responded molar conversion.

Optimal synthesis conditions
The optimum biosynthesis of octyl ferulate was further determined by the ridge max analysis (Figure 3), indicating that the maximum molar conversion was 91.74 ± 2.3% at 72 h, temperature 92.2°C, enzyme amount 1,831 PLU and stir speed 92.4 rpm.

Figure 4 represents the correlation between the experimental values and the predicted
values of the RSM model for average cutting speed.A verification experiment performed at the suggested optimum conditions could obtain a molar conversion of 93.2 ± 1.5%, which was close to the RSM-predicted molar conversion; thus, the predicted model was successfully established.In this study, the lipase-catalyzed biosynthesis of octyl ferulate could be carried out with an easy method.
Additionally, to confirm the enzyme reusability, the immobilized lipase for octyl ferulate synthesis was also determined under optimum conditions.The lipase was recovered from the medium after the reaction, followed by being reused directly in the next batch.When the batch of immobilized lipase was reused five times, the molar conversion of octyl ferulate remained higher than 90% with very little loss of enzymatic activity (Figure 5).The data indicated that the immobilized lipase could keep enzymatic stability under the conditions of long-term octanol exposure and high reaction temperature (90°C).Thus, the result confirmed that the lipase could be effectively applied for the synthesis of octyl ferulate and that the enzymatic stability was high enough to permit reuse.

Conclusions
In this study, the esterification of ferulate with octanol catalyzed by lipase in a solvent-free medium was investigated.The immobilized Novozym ® 435 could be well used to synthesize octyl ferulate.Both 3-level-3-factor BBD and RSM were employed successfully for the experimental design and data analysis.An environmentally friendly experimental model for the octyl ferulate synthesis was well built, and the optimal conditions for biosynthesis were respectively in a reaction time of 72 h, a reaction temperature of 92.2°C, enzyme amount of 1,831 PLU and stir speed of 92.4 rpm.
Synthesis of octyl ferulate under this optimal condition was performed to obtain a molar conversion of 93.2 ± 1.5%.Notably, our results indicated that the high reaction temperature significantly affected the efficiency of lipase-catalyzed ester synthesis under the solvent-free system.Overall, as compared to the chemical methods, our present study suggests that the lipase-catalyzed environmental-friendly synthesis of esters under solvent-free reaction system at higher reaction temperature without marked loss of enzymatic activity could offer a significant reference for industrial preparation process.

435 ( 1 ,
500 PLU) and octanol were carried out in a water bath at a reaction time of 72 h.The octyl ferulate catalyzed by Novozym ® 435, and the liquid samples analyzed by high-performance liquid chromatography (HPLC) are shown in Figure 1.Moreover, the effects of reaction temperature and reaction time on molar conversion of octyl ferulate are shown in Figure 2, illustrating that a higher molar conversion was obtained after the reaction time of 24 hours as well as by an increase of reaction temperature.

Figure 1 .
Figure 1.Scheme of octyl ferulate biosynthesis and HPLC chromatogram of ferulic

Figure 2 .
Figure 2. Effects of reaction time and temperature on Novozym ® 435-catalyzed

Figure 3 .
Figure 3. Response surface plots are showing the relationships between the molar

Figure 4 .
Figure 4. Comparison of the experimental data with those predicted by the RSM model.

Figure 5 .
Figure 5. Reusable cycles of Novozym ® 435 in the synthetic process of octyl ferulate

Table 1
Box-Behnken design experiments and observed data of molar conversion.Molar conversion for octyl ferulate showing means ± SD of duplicated experiments.
a The treatments were run in a random order.b Preprints (www.