Pentyl valerate was synthesized biocatalytically using
With the emergence of “green chemistry” as an alternative to the conventional chemical methods, biocatalysis has gained a significant position in industrial biotechnology sector. A large number of enzymes have been produced and purified and they are replacing a number of chemical processes involved in production and processing of textiles, food and beverages, cosmetics, pharmaceuticals, and bulk and fine chemical synthesis [
In particular, microbial lipases have proved to be one of the highest studied and applied enzymes in biotechnology [
Although lipases (and other enzymes) function with exceptional efficiency in non-aqueous media, the common drawbacks associated with their use are separation and reusability due to their soluble/powder nature, and solvent induced denaturation caused by change in structure and flexibility of enzyme molecules owing to replacement of bound water molecules in the enzyme molecule by solvent, hence distorting its structure [
One of the areas of lipase directed application is synthesis of short chain esters which are important constituents of flavoring agents. Such biocatalytically synthesized esters have an edge over their chemical counterparts as they excel in their flavor and are considered “natural” making them highly accepted [
Sodium bis-2-(ethylhexyl) sulfosuccinate (AOT), Tween 80, 1-hexanol (>98% purity, GC), 1-pentanol (>99% purity, GC), pentyl valerate (>98% purity, GC), and valeric acid (>98% purity, GC) were obtained from Fluka (Steinheim, Germany). Cetyl trimethyl ammonium bromide (CTAB) and Triton X-100 were obtained from Genei (Bangalore, India) and Hi-Media (Mumbai, India), respectively.
Reverse micelles and MBGs were prepared as described in our previous study [
The reaction mixture consisted of 20 mL of organic solvent containing 100 mM of each substrate (1-pentanol and valeric acid) in 250 mL glass stoppered flasks. To initiate the reaction, the enzyme was added to the reaction mixture and kept on orbital shaker at 37°C and 150 rpm.
surfactant for reverse micelle preparation: AOT, CTAB, Triton X-100, and Tween-80, pH: 5–8.8 of 100 mM strength (acetate buffer, pH 5, sodium phosphate buffer, pH 6, 7, and 8, and Tris-HCl buffer, pH 8.8), Wo: 10–100 (enzyme concentration of 5 mg/mL of reverse micelles), enzyme concentration: 10–100 mg/mL (Wo = 50, pH 7), organic solvent as reaction medium: isooctane, n-hexane, n-heptane, and cyclohexane, cyclic versus acyclic alkanes as reaction medium for ester synthesis, reaction temperature: 20–50°C.
The effects of concentrations of valeric acid and pentanol on the initial rate of pentyl valerate synthesis were studied using free and immobilized lipase keeping the initial concentration of one of the substrates, that is, pentanol/valeric acid constant (100 mM), and varying the initial concentration of the other (50 mM, 75 mM, 100 mM, 125 mM, and 150 mM). One unit of enzyme activity was defined as amount of lipase required for synthesis of 1
Where
Study of the significant effects of various parameters and their interactions on esterification using response surface methodology (RSM) and Box-Behnken design (BBD) is considered. Studying combined effects of various parameters by conventional methods is time consuming and high number of experiments results in wastage of resources as well. Hence, employment of statistical tools such as response surface methodology (RSM) assists in a limited number of runs leading to predicted results followed by confirmatory experiments. In present work, RSM using BBD (Box-Behnken design) which entails full factorial search was employed for studying the simultaneous effects of five important process parameters chosen as independent variables, that is, Wo (
Experimental range and coded values of the variables.
Process variables | Coded values | ||
---|---|---|---|
−1 | 0 | +1 | |
Enzyme concentration (mg/mL) ( |
30 | 55 | 80 |
Time (days) ( |
3 | 6 | 9 |
Substrate molar ratio ( |
1 : 2 | 1 : 1 | 3 : 2 |
Wo ( |
40 | 60 | 80 |
Solvent for MBG preparation ( |
n-Hexane | n-Heptane | Isooctane |
Full factorial Box-Behnken design for synthesis of pentyl valerate using MBGs in cyclohexane.
Block | Number | Enzyme concentration | Time | Substrate ratio | Wo | Solvent | Actual (%) | Predicted (%) |
---|---|---|---|---|---|---|---|---|
1 | 1 | −1 | −1 | 0 | 0 | 0 | 22.49 | 22.99 |
2 | 1 | −1 | 0 | 0 | 0 | 53.41 | 55.58 | |
3 | −1 | 1 | 0 | 0 | 0 | 53.29 | 52.90 | |
4 | 1 | 1 | 0 | 0 | 0 |
|
86.25 | |
5 | −1 | 0 | −1 | 0 | 0 | 41.83 | 32.72 | |
6 | 1 | 0 | −1 | 0 | 0 | 42.81 | 43.90 | |
7 | −1 | 0 | 1 | 0 | 0 | 36.88 | 31.12 | |
8 | 1 | 0 | 1 | 0 | 0 |
|
87.78 | |
9 | 0 | 0 | 0 | 0 | 0 | 66.18 | 65.66 | |
|
||||||||
2 | 10 | −1 | 0 | 0 | −1 | 0 | 31.97 | 29.45 |
11 | 1 | 0 | 0 | −1 | 0 | 80.18 | 72.64 | |
12 | −1 | 0 | 0 | 1 | 0 | 54.05 | 58.89 | |
13 | 1 | 0 | 0 | 1 | 0 | 78.15 | 82.89 | |
14 | −1 | 0 | 0 | 0 | −1 | 38.09 | 42.96 | |
15 | 1 | 0 | 0 | 0 | −1 | 82.51 | 79.75 | |
16 | −1 | 0 | 0 | 0 | 1 | 32.97 | 38.30 | |
17 | 1 | 0 | 0 | 0 | 1 | 71.86 | 72.25 | |
18 | 0 | 0 | 0 | 0 | 0 | 63.8 | 66.73 | |
|
||||||||
3 | 19 | 0 | −1 | −1 | 0 | 0 | 30.3 | 20.85 |
20 | 0 | 1 | −1 | 0 | 0 | 36.08 | 34.65 | |
21 | 0 | −1 | 1 | 0 | 0 | 27.31 | 25.19 | |
22 | 0 | 1 | 1 | 0 | 0 | 70.85 | 71.96 | |
23 | 0 | −1 | 0 | −1 | 0 | 24.16 | 21.67 | |
24 | 0 | 1 | 0 | −1 | 0 | 61.08 | 56.58 | |
25 | 0 | −1 | 0 | 1 | 0 | 41.48 | 46.14 | |
26 | 0 | 1 | 0 | 1 | 0 | 71.83 | 71.79 | |
27 | 0 | 0 | 0 | 0 | 0 | 62.5 | 59.52 | |
|
||||||||
4 | 28 | 0 | −1 | 0 | 0 | −1 | 40.27 | 41.17 |
29 | 0 | 1 | 0 | 0 | −1 | 67.36 | 67.74 | |
30 | 0 | −1 | 0 | 0 | 1 | 32.92 | 31.38 | |
31 | 0 | 1 | 0 | 0 | 1 | 68.21 | 64.01 | |
32 | 0 | 0 | −1 | −1 | 0 | 33.38 | 33.85 | |
33 | 0 | 0 | 1 | −1 | 0 | 41.13 | 43.02 | |
34 | 0 | 0 | −1 | 1 | 0 | 38.11 | 42.04 | |
35 | 0 | 0 | 1 | 1 | 0 | 73.67 | 74.53 | |
36 | 0 | 0 | 0 | 0 | 0 | 61.87 | 61.68 | |
|
||||||||
5 | 37 | 0 | 0 | −1 | 0 | −1 | 41.11 | 41.37 |
38 | 0 | 0 | 1 | 0 | 1 | 59.57 | 56.45 | |
39 | 0 | 0 | −1 | 0 | 1 | 37.76 | 42.85 | |
40 | 0 | 0 | 1 | 0 | 1 | 55.77 | 56.45 | |
41 | 0 | 0 | 0 | −1 | −1 | 57.6 | 60.57 | |
42 | 0 | 0 | 0 | 1 | −1 |
|
75.99 | |
43 | 0 | 0 | 0 | −1 | 1 | 41.33 | 49.72 | |
44 | 0 | 0 | 0 | 1 | 1 |
|
74.00 | |
45 | 0 | 0 | 0 | 0 | 0 | 64.48 | 69.86 |
For statistical calculations the independent variables were coded as
This response surface methodology allows creating a model of a second order equation that illustrates the response (in present work pentyl valerate yield) as a function of the five variables studied. Subsequently, pentyl valerate synthesis data was analyzed and response surface model given by the following equation was fitted. Consider
After completion of one reaction cycle, the MBGs were separated from reaction mixture by filtration using Whatman filter paper and washed with cyclohexane 2-3 times for complete removal of residual substrates and product which might have diffused into/been adsorbed onto the organogel. They were then air-dried and reused for ester synthesis. After 3-4 cycles, when the ester production was observed to decrease considerably, the MBGs were treated with dehydrant solution (1 M AOT/isooctane) for 24 h for extraction of excess water (by-product of esterification reaction), given 2-3 solvent washes of neat isooctane for complete removal of AOT and used for next cycle of esterification.
After initiation of the reaction, 100
Water content of the three types or organogels (i) fresh MBGs (unused), (ii) MBGs reused for 8 cycles, and (iii) reused MBGs subjected to dehydrating agent (1 M AOT/isooctane) was determined by the Karl-Fischer method using Hydranal-E reagent by volumetric titration.
Dynamic thermogravimetric experiments were carried out using a Mettler Toledo (Switzerland) TGA/SDTA/821e thermal analyzer, allowing measurement of mass change. The system employed for this work was equipped with a PtRh furnace capable of operating from 25°C to 1500°C, the temperature being measured using type R thermocouple. The system is vacuum tight, allowing measurements to be conducted under controlled atmosphere. TGA-DSC analysis was performed on small samples (0.1–2.5 mg) and taken in alumina crucible without lid, in argon atmosphere (flow rate of argon gas, 200 mL/min). The temperature range was varied from room temperature to 300°C and with heating rate of 20°C/min. For isothermal analysis, the temperature was maintained at 100°C for 30 min.
Present study details the process of immobilization of the enzyme
Surfactants are amphiphilic molecules that are used for preparation of reverse micelles containing an aqueous core in a nonaqueous environment. One of the vastly studied surfactants used for microemulsion preparation is AOT [
(a) Effect of surfactant on pentyl valerate synthesis. Free enzyme and immobilized enzyme are represented by dotted and solid lines, respectively. The MBGs consisted of surfactant/isooctane/CRL (70 mg/mL),
The fact that enzymes exhibit an optimum pH value and the unusual properties exhibited by the “water pool” of reverse micelles necessitated the study of effect of pH on ester synthesis. In present work, the results showed that the highest ester yield was obtained in MBGs containing enzyme dissolved in buffer of neutral pH (pH 6-7), whereas it started declining as the pH was increased to 8 and 8.8. From Figure
The water activity Wo (also known as R sometimes) is one of the most important parameters contributing to the stability of reverse micelles and consequently to organogels prepared using them. Also, the concept of “superactivity” of lipases within reverse micelles makes it an important parameter to be observed. Present study exhibited optimum
(a) Effect of Wo on pentyl valerate synthesis. Reaction system consisted of AOT/isooctane organogels prepared using varying Wo values and 5 mg/mL of reverse micelles lipase solution. (b) Effect of enzyme concentration on ester synthesis. MBGs containing lipase (10–100 mg/mL) of the combination AOT/isooctane at
In enzyme catalyzed reactions, there is a strong correlation between the concentration of enzyme and final product yield. In present study, the concentration of enzyme within the reverse micelles was varied while keeping the
Nonaqueous biocatalysis has been popularized in the past few decades due to numerous advantages [
(a) Effect of organic solvents as medium on pentyl valerate synthesis. The reaction system comprised AOT/isooctane organogels (pH 7,
A similar pattern was observed in our previous study [
As temperature is another important physical parameter for biological and chemical reactions, this study was performed in the range of 20–50°C and highest yield (99.52%) was observed at 37°C (Figure
One of the most important aspects of enzyme catalyzed reactions is the study of reaction kinetics. Various workers have studied the reaction kinetics of esterification reactions [
This was in close agreement with the effects of substrate concentration on reaction rates (Figures
Dependence of initial reaction rate of ester synthesis on concentration of (a) valeric acid concentration (while keeping pentanol concentration constant at 100 mM) and (b) pentanol (while keeping valeric acid concentration constant at 100 mM). The reaction was carried out at 37°C and 150 rpm in cyclohexane as reaction medium.
Response surface methodology (RSM) has been applied for various processes in the field of biocatalysis such as for optimization of enzyme immobilization onto/into supports [
The results of the 45-run BBD of the five variables, namely, enzyme concentration, time, substrate ratio,
RSM conjugated 5-factor-3-level BBD was found to be significantly efficient in explaining the interactions of the five process parameters studied and their cumulative effects on ester yield. Blocking was employed to facilitate experimentation and study the variation created (if any) in product yield when the experiments were performed at different points of time. The 45 experiments were run in 5 blocks of 9 experiments each (Table
Using the statistical tool, the predicted values were obtained and found to be satisfactorily correlated to observed values (Table
Estimated regression coefficients for esterification (%) for enzyme concentration (
Term | Coefficient | Standard error coefficient (SE) |
|
|
---|---|---|---|---|
|
16.7997 | 1.338 | 12.557 |
|
|
15.1419 | 1.338 | 11.318 |
|
|
10.4122 | 1.409 | 7.390 |
|
|
9.9234 | 1.338 | 7.418 |
|
|
−3.0408 | 1.390 | −2.187 |
|
|
−3.7261 | 4.338 | −0.859 | 0.394 |
|
−7.8221 | 2.906 | −2.692 |
|
|
−13.5324 | 2.257 | −5.996 |
|
|
−1.1522 | 2.209 | −0.521 | 0.604 |
|
−3.8029 | 2.898 | −1.312 | 0.194 |
|
0.1887 | 2.676 | 0.071 | 0.944 |
|
11.5275 | 2.676 | 4.308 |
|
|
−5.6825 | 2.676 | −2.124 |
|
|
−0.7100 | 2.676 | −0.265 | 0.792 |
|
8.2425 | 2.676 | 3.081 |
|
|
−2.3162 | 2.676 | −0.866 | 0.390 |
|
1.8575 | 2.676 | 0.694 | 0.490 |
|
5.8313 | 2.676 | 2.179 |
|
|
−3.7794 | 3.072 | −1.230 | 0.223 |
|
2.2137 | 2.676 | 0.827 | 0.411 |
The coefficients of interactions showed that only four interactions, that is, enzyme concentration-substrate ratio (
The analysis of variance (ANOVA) for the model is shown in Table S2. This analysis shows that all the regression terms, that is, linear, square, and interaction, were statistically highly significant (
Contour plots were prepared for better understanding of the effect of interactions between the variables on the response (esterification). As stated in previous section, the interactions of all parameters (except solvent) with substrate ratio were significant and, hence, these interactions, namely, substrate ratio:
Figure
Contour plot for the esterification (%) as a function of (a) substrate ratio and Wo using n-hexane as solvent used for MBG preparation with hold values of time at 7.5 days (coded value 0.5) and enzyme concentration at 77.5 mg/mL (coded value 0.9). (b) Enzyme concentration and substrate ratio using n-hexane as solvent used for MBG preparation with hold values of time at 7.5 days (coded value 0.5) and enzyme concentration at 80 mg/mL (coded value 1). (c) Time and substrate ratio using n-hexane as solvent used for MBG preparation with hold values of Wo = 80 (coded value 1) and enzyme concentration at 70 mg/mL (coded value 0.6).
Figure
Figure
In a nutshell, it is evident from the three contour plots (Figures
Two confirmatory experiments were carried out using the optimum values of variables for validation of the model. Here, the values of the five variables were taken as follows.
Enzyme concentration is of 77.5 mg/mL, time 7 days, 4 h, and 48 min, substrate ratio = 2.8 : 2,
Enzyme concentration is of 70 mg/mL, time 8 days, 7 h, and 6 min, substrate ratio= 2.9 : 2,
Hence, both of these experiments fully justified the model and showed that it could be applied for pentyl valerate synthesis using MBGs in organic solvents.
The primary aim of immobilization is to use the enzyme repeatedly, apart from easy downstream processing of the product. It was observed that the activity of the enzyme was quite high for first two reaction cycles but decreased gradually thereafter (Figure
Operational stability of the MBGs. (a) Graph showing number of reusability runs. The organogels were given solvent washes after every run and reused for pentyl valerate synthesis at 37°C and 150 rpm. Down arrows indicate AOT/isooctane treatment given to the organogels for extraction of accumulated water formed as by-product of esterification reaction. (b) TGA weight loss curves for fresh (blue curve), reused (red curve), and reused and treated with 1 M AOT/isooctane (black curve).
Further, to confirm the action of AOT/isooctane solution for extracting excess water, the three types of MBGs were analyzed by TGA weight loss (Figure
Hence, regarding the reusability studies, two important findings were observed. Firstly, there is an accumulation of water within the MBGs which acts as the substrate and hence drives the equilibrium towards the hydrolysis resulting in reduced ester formation. This excess water can be extracted by a simple treatment by incubating with a dehydrant solution such as AOT/solvent. Secondly, the reduced ester formation after 3-4 runs of esterification is mainly contributed to the shifting of equilibrium towards hydrolysis due to water accumulation (and by a small factor due to enzyme denaturation) which can be restored by a large factor using dehydrant solution.
These two findings support the fact that indeed immobilization of enzymes within MBGs not only facilitates easy separation and renders them reusable, but also offers protection to a great extent and simple methods of recovery of enzyme activity. Our previous studies have shown that the enzyme within MBGs can resist high temperatures up to 60°C for 10 h and 70°C for 4 h indicating the thermostability offered by immobilization matrix [
Present study outlines the synthesis of the flavor ester pentyl valerate using
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors acknowledge University Grants Commission, New Delhi, for the financial support for the major research project entitled “Biotechnological process for synthesis of food esters in organic solvents using microemulsion based organogel entrapped lipases,” Project no. F. 33-240/2007 (SR).