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A facile enzymatic synthesis of geranyl propionate by physically adsorbed Candida rugosa lipase onto multi-walled carbon nanotubes
Enzyme and Microbial Technology 72 (2015) 49–55
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A facile enzymatic synthesis of geranyl propionate by physically adsorbed Candida rugosa lipase onto multi-walled carbon nanotubes Nur Royhaila Mohamad a , Nor Aziah Buang a,∗∗ , Naji Arafat Mahat a , Yen Yen Lok a , Fahrul Huyop b , Hassan Y. Aboul-Enein c , Roswanira Abdul Wahab a,∗ a
Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81310 UTM Skudai, Johor, Malaysia Department of Biotechnology and Medical Engineering, Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81310 UTM Skudai, Johor, Malaysia c Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Cairo 12311, Egypt b
a r t i c l e
i n f o
Article history: Received 5 November 2014 Received in revised form 17 February 2015 Accepted 25 February 2015 Available online 6 March 2015 Keywords: Candida rugosa lipase Immobilization Carbon nanotubes Esterification Geranyl propionate Geraniol
a b s t r a c t In view of several disadvantages as well as adverse effects associated with the use of chemical processes for producing esters, alternative techniques such as the utilization of enzymes on multi-walled carbon nanotubes (MWCNTs), have been suggested. In this study, the oxidative MWCNTs prepared using a mixture of HNO3 and H2 SO4 (1:3 v/v) were used as a supportive material for the immobilization of Candida rugosa lipase (CRL) through physical adsorption process. The resulting CRL-MWCNTs biocatalysts were utilized for synthesizing geranyl propionate, an important ester for flavoring agent as well as in fragrances. Enzymatic esterification of geraniol with propionic acid was carried out using heptane as a solvent and the efficiency of CRL-MWCNTs as a biocatalyst was compared with the free CRL, considering the incubation time, temperature, molar ratio of acid:alcohol, presence of desiccant as well as its reusability. It was found that the CRL-MWCNTs resulted in a 2-fold improvement in the percentage of conversion of geranyl propionate when compared with the free CRL, demonstrating the highest yield of geranyl propionate at 6 h at 55 ◦ C, molar ratio acid: alcohol of 1:5 and with the presence of 1.0 g desiccant. It was evident that the CRL-MWCNTs biocatalyst could be reused for up to 6 times before a 50% reduction in catalytic efficiency was observed. Hence, it appears that the facile physical adsorption of CRL onto F-MWCNTs has improved the activity and stability of CRL as well as served as an alternative method for the synthesis of geranyl propionate. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Terpene esters of short-chain fatty acids are essential oils that present a great deal of interest in food, cosmetics and pharmaceutical industries as flavors and fragrances; acetate, propionates and butyrates of acyclic terpene alcohols such as geraniol and citronellol, being the main components of essential oil [1]. Traditionally, these esters are obtained by various methods viz. chemical synthesis, extraction from natural products as well as fermentation [2]. Despite the common use of chemical synthesis for producing esters, the process requires harsh reaction conditions such as high temperature and pressure, involvement of strong acid
∗ Corresponding author. Tel.: +60 7 5534148; fax: +60 7 5566162. ∗∗ Corresponding author. Tel.: +60 7 5534163; fax: +60 7 5566162. E-mail addresses: [email protected] (N.A. Buang), [email protected] (R. Abdul Wahab). http://dx.doi.org/10.1016/j.enzmictec.2015.02.007 0141-0229/© 2015 Elsevier Inc. All rights reserved.
catalyst and hazardous chemicals, considerably long reaction time while providing low conversion rate [1]. Moreover, the chemical synthesis has been associated with tedious separation processes, extreme exposure of toxicants as well as unwanted harmful reaction byproducts [3]. Hence, the enzymatic production of flavors and fragrances using natural raw materials may prove useful for scientific and industrial settings, considering the ever arising demands for such products [4]. Due to the fact that lipases (triacylglycerol ester hydrolysis EC 3.1.1.3) may catalyze esterification reactions without providing high temperature and pressure conditions and since the procedure remains relatively uncomplicated when compared with the chemical synthesis, such enzymes have acquired popularity as biocatalysts for the reactions [5]. In this context, Candida rugosa lipase (CRL), a stable mesophilic lipase, has been commonly used due to its high activity and broad specificity in reaction medium [6]. Considering that the CRL is often unstable in its free form, demonstrating low activity in organic solvents, high tendency of deactivation in
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prolonged exposure toward high temperature and extreme pH [6], immobilization of CRL onto carbon nanotubes may be one of the probable solutions. In addition, advances in enzyme immobilization techniques have enabled the use of a wide range of biocatalysts for various reactions under extreme pH, temperature and pressure conditions [7,8]. Owing to its excellent binding capacity due to large surface area to volume ratio, physicochemical properties as well as biological compatibility, carbon nanotubes have been frequently utilized as the support materials for immobilization of enzymes [9,10]. Enzyme immobilization confers a multitude of advantages such as structural stability, improved activity, specificity and selectivity, reduction of inhibition [11], increased flexibility with enzyme/substrate contact using various reactor configurations [12] and longer half-life of the enzyme [13]. Since these enzymes are expensive and are required in large volumes for industrial needs [14], immobilization of such enzymes onto solid support materials would be helpful in maintaining the catalytically active tertiary structure of enzymes [15], which contributes to its reusability. In this context, the physical adsorption method may have higher commercial values than other methods [16] because it is one of the simplest and cheapest immobilization methods available and importantly, in most cases, the enzyme productivity remains unaffected [17]. This present study was aimed at investigating the application of physically adsorbed CRL on the surface of acid functionalized MWCNTs (CRL-MWCNTs) as potential economical biocatalysts. The CRL-MWCNTs were compared with the free CRL for synthesizing geranyl propionate. In addition, its effects on the incubation time, temperature, molar ratio of acid to alcohol, presence of desiccant and reusability in rendering the highest conversion of geranyl propionate were evaluated. 2. Materials and methods 2.1. Chemicals Multi-walled carbon nanotubes (MWCNTs) prepared by the chemical vapor deposition method were provided by one of the co-authors (Assoc. Prof. Dr. Nor Aziah Buang). Lipase Type VII of C. rugosa (EC 3.1.1.3) with measured activity of 1410 units mg1 , substrates, geraniol (98%), propionic acid (99%), phosphate buffer, Bradford reagent, phenolftalein and molecular sieves were all purchased from the Sigma Chemical Co. (St. Louis, USA). Other chemicals that include sodium hydroxide and n-heptane were of analytical grade and used without further purification. Distilled water was prepared in our laboratory and used in all experiments. 2.2. Purification and functionalization of MWCNTs The raw MWCNTs (0.5 g) were transferred into a 100 mL flask containing 4 M HCl (20 mL) and refluxed with stirring at 80 ◦ C for 5 h. After cooling to room temperature, the liquid was decanted, the MWCNTs were washed with distilled water until no residual acid was detected and dried in an oven at 60 ◦ C for 24 h. The purified MWCNTs were refluxed at 120 ◦ C by stirring in a mixture of concentrated HNO3 and H2 SO4 with ratio 1:3 (v/v) for 24 h. After cooling to room temperature, the mixture was decanted, washed with distilled water until no residual acid was present and dried at 60 ◦ C for 24 h. 2.3. Adsorption immobilization of lipase and its characterization The free CRL was immobilized onto the acid functionalized MWCNTs (FMWCNTs). The MWCNTs (10 mg/mL) were first sonicated in aqueous buffer (pH 7) for 30 min to ensure homogeneous dispersion. Then, the MWCNTs were suspended in a 50 mL flask of phosphate buffer (50 mM, solution pH 7) containing the CRL (10 mg/mL) and incubated at 20 ◦ C with constant stirring at 150 rpm. After 3 h of incubation, the flask containing CRL-MWCNTs was stored at 4 ◦ C for 24 h. Upon completion, the unbound protein was removed by washing with phosphate buffer (pH 7) until no hydrolytic activity was detected in the washing. The supernatant was then subjected to protein analysis. Similar CRL immobilization treatments were also performed to unfunctionalized MWCNTs. 2.3.1. Fourier transform infrared (FTIR) A ratio 1:100 mass of sample was ground thoroughly with potassium bromide and the resulting powder was pressed into a transparent pellet by a hydraulic press.
The FTIR spectra obtained using a BOMEM spectrophotometer in transmission mode between 400 and 4000 cm−1 at a resolution of 4 cm were analyzed. 2.3.2. Field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM) A field emission scanning electron microscope (FESEM) (JEOL JEM-6700F) was used to study the surface morphology of MWCNTs, F-MWCNTs and CRL-MWCNTs. For FESEM analysis, the sample was prepared by taking one drop of acetone containing the dispersed MWCNTs on a silicon wafer and allowing it to dry in a vacuum oven for 30 min. For TEM analysis, electron source was used as W-emitter and LaB6, operating at an accelerating voltage of 200 kV. The analysis utilized an objective lens (S-Twin) with a point resolution of 2.0 nm or better with a 25× to 7500, 200× or higher magnification and a single tilt holder with LCD camera. The MWCNTs, F-MWCNTs and CRL-MWCNTs were dispersed in deionized water and a drop was placed on a copper grid and observed after drying in vacuum. 2.4. Determination of protein loading Protein content of the enzyme solution, before and after immobilization, in the washing buffer was determined by Bradford method using BioRad protein dye reagent concentrate and bovine serum albumin (BSA) as the protein standard [18] as well as in the reaction mixture. 2.5. Enzymatic synthesis of geranyl propionate catalyzed by free CRL and CRL-MWCNTs The reaction medium that consisted of 0.25 M propionic acid and 1.25 M geraniol was dissolved in n-heptane in a flask followed by the addition of free CRL (10 mg/mL) and CRL-MWCNTs (5 mg/mL), respectively. The reaction mixture was refluxed with continuous stirring at 200 rpm in a paraffin oil bath. The geranyl propionate obtained was expressed in terms of percent conversion i.e. percent of propionic acid converted versus the total acid in the reaction mixture by titrating aliquots of liquid sample (1 mL) withdrawn periodically with 0.05 M NaOH using phenolphthalein as an indicator. Each measurement was performed in triplicates and the standard error was calculated. The percent conversion was calculated according to the equation prescribed by previous researchers [1] detailed below:
% Conversion =
(Vo − Vt ) Vo
× 100
whereby, Vo is the volume of NaOH at initial time (t = 0) and Vt is the volume of NaOH at each hour (t = t1 , t2 , t3 , . . .). 2.5.1. Effect of incubation time Time course study is a good indicator of enzyme performance as well as product yield and a good performance enzyme requires relatively shorter duration to obtain good yields when compared with the poor ones [19]. The effect of incubation was monitored up to 24 h with sampling intervals of 0, 4, 8, 12, 16, 20 and 24 h, respectively. The reactions were carried out in 50 mL round bottom flasks, stirred constantly at 200 rpm. Upon completion of preliminary screening tests, the reaction temperature was maintained at 40 ◦ C. 2.5.2. Effect of temperature It has been reported that temperature has a significant effect on the equilibrium of the reaction as well as the activity and stability of lipase [20]. The effect of temperature on the enzymatic synthesis of geranyl propionate of both free CRL and CRL-MWCNT was determined at varying temperatures ranging from 40 to 60 ◦ C, at increasing intervals of 5 ◦ C each. 2.5.3. Effect of the presence of desiccant Water is a byproduct of esterification that needs to be removed for obtaining higher yield of ester [21]. Following the successful employment as desiccant in many enzymatic esterification reactions [22,23], in this present study too, molecular sieves ˚ were chosen. They act as absorbents that are replaceable with new ones when (4 A) saturated with water [24]. By removing water in media, the reaction equilibrium would shift toward the synthesis of the desired ester hence, higher conversion of ester products [24]. The effects of desiccant on the free CRL and CRL-MWCNTs were evaluated in the presence (1.0 g) or absence (0 g) of molecular sieves. 2.5.4. Reusability of CRL-MWCNTs The reusability of the CRL-MWCNTs was examined by reusing the recovered lipase for repeated reaction cycles. After each cycle, the enzyme was filtered, washed with similar solvent and allowed to dry before reuse. For investigating the reusability, the reaction mixture that consisted of propionic acid and geraniol dissolved in n-heptane with the presence of immobilized lipase was used. Each reaction was monitored for a period of 8 h at 30–50 ◦ C with constant stirring (Table 1).
N.R. Mohamad et al. / Enzyme and Microbial Technology 72 (2015) 49–55 Table 1 FTIR frequency table (a) raw MWCNTs, (b) F-MWCNTs and (c) CRL-MWCNTs. Frequency (cm−1 )
Functional group
(a)
3428.01 1630.82
O H stretching Conjugated C C bonds
(b)
3445.22 1112.73 1634.70 1202.46
O C C C
(c)
1032.96 3420.59 2929.41 1116.03
C N stretching N H stretching CH2 bending C O stretching
H stretching O stretching O stretching O stretching
3. Results and discussion 3.1. Functionalization of MWCNTs for the adsorption of CRL protein Refluxing the MWCNTs in the mixture of H2 SO4 and HNO3 , introduces the polar groups (COO− ) to the non-polar surface and tubular ends of the MWCNTs supports, hence, the surface of the MWCNTs is primed for the attachment with other polar moieties (NH2 , O H) present on the CRL protein. The role of the introduced COO− group is to anchor the CRL protein to the MWCNTs through attractions between the oppositely charged carboxyl moiety (electron rich) and hydrogen (electron poor) of the back-bone and side-chain of polar amino acids present on the outer surface of the CRL protein. Conversely, attachment of the CRL proteins to the non-polar unfunctionalized MWCNTs, was found to be insignificant. The physical adsorption of CRL to the F-MWCNTs was reasonably strong as the amount of detached protein in the reaction mixture was either negligible or not detected. The activity of the adsorbed CRL is 592 Umg−1 is relatively high, though a little lower than that of the free CRL. However, when used as a biocatalyst under extended reaction period, the former is anticipated to be more stable and its initial enzyme activity is retained. Furthermore, the acid functionalized CRL-MWCNTs were observed to be significantly better dispersed and were easily suspended when stirred in the reaction vessel as compared to the unfunctionalized ones. The ability of CRL-MWCNTs to remain longer in suspension is somewhat desirable for an enzymecatalyzed reaction, therefore, favoring increased possibility of effective enzyme-substrate collision as well as reduced tendency of the CRL-MWCNTs to precipitate out of the reaction. 3.2. Characterization of immobilized lipase (CRL-MWCNTs) 3.2.1. Spectroscopy (FTIR) The FTIR spectra obtained for the raw MWCNTs, F-MWCNTs and CRL-MWCNTs are presented in Fig. 1. The appearance of a broad band at 3428.01 cm−1 in Fig. 1a was assigned to the O H stretching of the surface group of the raw MWCNTs as received. This could be attributed to ambient atmospheric moisture that was tightly bound to the MWCNTs [25]. The spectrum of raw MWCNTs showed a weak band at 1630.82 cm−1 is assigned to the conjugated C C bonds [26]. After oxidation, the stretch from O H group shifted from 3428.01 cm−1 to 3445.22 cm−1 (Fig. 1b), which indicated the hydroxyl group had covalently bonded to the sidewalls of the MWCNTs [27]. The presence of a new peak at 1112.73 cm−1 is assigned to a C O stretching that clearly represented successful oxidation of sp2 hybridized carbon in raw MWCNTs to sp3 [28]. Whereas, a peak at 1634.70 cm−1 was observed which arise from the C O stretching vibration of the carboxylic group. Conjugation
51
of O C with the C C resulted in the C O to adapt a more single bond character through resonance. This phenomenon ultimately leads to a lower vibration frequency of carbonyl group. A new peak appeared at 1202.46 cm−1 corresponded to the C O of the COOH moiety [29]. Fig. 1c exhibited the absence of band at 1634.70 cm1 and the corresponding appearance of band at lower wavelength, 1620.71 cm−1 . This band represented the C O stretching of amide which is the most outstanding adsorption bands that confirm the presence of proteins [26]. In addition, the presence of a new band at 1032.96 cm−1 was attributed to the C N bond that corroborated the existence of the amide functional group. Meanwhile, the N H and CH2 bands appeared at 3420.59 cm−1 and 2929.41 cm−1 , respectively. Distinction in the spectra of the raw MWCNTs and CRL-MWCNTs was observed in the range of 900–1200 cm−1 , the spectral region was for carbohydrate functional group bands [30]. Following immobilization, an intense peak at 1125.04 cm−1 assigned to vibrations the C O of the carbohydrate moiety for spectrum of free lipase was reduced, expanded and moved to a lower frequency at 1116.03 cm−1 [26]. It can be concluded that the CRL was successfully immobilized onto the outer face of the F-MWCNTs. Fig. 1c shows the appearance of an absorption band at 2929.41 cm−1 which implied the successful attachment of CRL to the surface of the MWCNTs, a band not observed in the spectrum of F-MWCNTs. 3.2.2. Field emission scanning electron microscope (FESEM) and transmission electron microscopy (TEM) The morphologies of raw MWCNTs, F-MWCNTs and CRLMWCNTs observable through FESEM and TEM are presented in Fig. 2. It was observed that the raw MWCNTs demonstrated openended tubular structures with smooth surfaces that were closely attached to each other with some contaminants on the carbon fiber surface prior to purification (Fig. 2a). Due to the possibility of impeding the potential characters of carbon nanotubes, these contaminants need to be removed [31]. Following treatment with H2 SO4 and HNO3 , the diameter of MWCNTs was increased with observable rough surfaces probably due to surface erosion [29]. The increased diameter of MWCNTs strongly indicated the successful attachment of acid functional groups on the surface of F-MWCNTs (Fig. 2b), similarly to the findings reported by previous work [32]. Furthermore, the length of the F-MWCNTs was evidently shorter following treatment with those acids, in consistency with the previously reported studies [31,33] indicating that acid treatment would change the highly tangled long MWCNTs into shorter, open ended tubular MWCNTs with numerous carboxylic groups at the open ends. This is due to the fact that elevated temperatures and increased oxidation time tend to destroy the structure of MWCNTs [34], leading to surface imperfections on the MWCNTs as well as affecting the properties of the resulting material. Fig. 2c clearly shows that the CRL was physically present on the surface MWCNTs, rendering increased diameter of CRL-MWCNTs when compared with the F-MWCNTs. Hence, this implies that the surface of the MWCNTs was covered by enzyme molecules [35]. 3.3. Optimization of esterification activity of CRL-MWCNT 3.3.1. Effect of incubation time Fig. 3 represents the reaction time profile for the free CRL and CRL-MWCNTs in catalyzing esterification of propionic acid and geraniol over a 12 h period, indicating the increased conversion of geranyl propionate with increasing reaction time. Interestingly, CRL-MWCNTs were found to increase the production of ester, attaining the highest percent conversion (47.3%) at 6 h of incubation time when compared with that observed for the free CRL (28.1%); beyond which the conversion of geranyl propionate started to decline. The higher conversion of geranyl propionate in reactions
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Fig. 1. FT-IR spectra of (a) raw MWCNTs, (b) F-MWCNTs and (c) CRL-MWCNTs.
catalyzed by the CRL-MWCNTs can be attributed to the formation of stable multipoint interactions (e.g. hydrophobic interaction, hydrogen bond, electrostatic and van der Waals forces) between CRL molecules and MWCNTs, resulting in the immobilized CRL being more stable than the free CRL [36]. Under such conditions, the enzyme is less susceptible to denaturation by solvents, temperature or substrates in the reaction vessel. The decline percent conversion of geranyl propionate may be attributable to increased production of water molecules as byproducts of the esterification reaction, a counterproductive reverse reaction hydrolyzing the geranyl propionate formed during the reaction [37], apart from diminished substrates that resulted in reduced degree of enzyme substrate saturation [38].
3.3.2. Effect of temperature Considering that temperature would significantly affect the equilibrium as well as the activity and stability of the enzymes in the reactions [39], such factor was investigated in this present study and the relevant data are tabulated in Fig. 4. It was revealed that the optimum temperature for the free CRL and CRL-MWCNTs were 40 ◦ C and 55 ◦ C, respectively, a 15 ◦ C increase from the observed temperature in the free CRL catalyzed reactions. Since similar shifts in optimal temperature for Talaromyces thermophilus immobilized onto chitosan as well as CRL immobilized on styrene divinylbenzene copolymer have been reported [16,40]. Therefore, such deviation observed in the optimum temperature recorded for the free CRL and CRL-MWCNTs in this study too prove common, implying higher stability of the CRL-MWCNTs at higher reaction temperatures. The increase in optimum temperature of the CRL-MWCNTs over the free CRL may be due to the network of hydrophobic and van der Waals interactions, as well as hydrogen bonds between CRL molecules and MWCNTs which altered the physical and chemical properties of the free CRL following adsorption onto the MWCNTs [36,41]. These additional interactions increase the structural
rigidity of the adsorbed CRL but somewhat, tend to denature its catalytically active structure. The CRL-MWCNTs is made more flexible and its catalytically active form is restored upon increasing the reaction temperature, hence, the observed higher optimum temperature of the CRL-MWCNTs over the free CRL. It has been reported that at higher temperature, the immobilized CRL has greater structural stability conferred by the attachment of lipase onto the functionalized surface of the MWCNTs [41], better resistance of the CRL-MWCNTs toward thermal denaturation at 40–60 ◦ C than that of free CRL. Correspondingly, it was observed that at 55 ◦ C the production of geranyl propionate by CRL-MWCNTs was higher (51.3%) when compared with that of the free CRL (16%), potentially afforded by hydrogen bonds and electrostatic interactions between the enzyme and the support [42].
3.3.3. Effect of the presence of desiccant Although adequate amount of water content would keep the enzyme in its active configuration, excessive amount of water would impede the equilibrium shift toward product [43]. It was observed that the molecular sieves as desiccant quickly dispersed in the reaction medium within a few minutes of stirring and caused the medium to turn slightly viscous. Fig. 5 illustrates the effect of desiccant on the enzymatic production of geranyl propionate. It was found that reactions catalyzed by both free CRL and CRL-MWCNTs supplemented with molecular sieves resulted in higher conversion of ester, yielding up to 24.3% and 47.3%, respectively; considerably higher percent conversion than that of 12.1% and 32.5% recorded for the free CRL and CRL-MWCNTs in the absence of such desiccant. In this context, it is pertinent to indicate that water is an unwanted byproduct of esterification reaction and the addition of desiccant may shift the equilibrium toward the formation of product [44]. The role of the molecular sieves was to absorb the excess water liberated in the free CRL and CRL-MWCNTs catalyzed esterification reaction and avoid the counterproductive hydrolysis
N.R. Mohamad et al. / Enzyme and Microbial Technology 72 (2015) 49–55
53
62.5 Free CRL-CNT
% Conversion
50
37.5
25
12.5
0
1
2
3
4
5
6
7
8
10
12
Time (h) Fig. 3. The effect of incubation time for the synthesis of geranyl propionate. Reaction conditions: propionic acid/geraniol (1:5) molar ratio, enzyme amount 5 mg/mL for CRL-MWCNTs and 10 mg/mL free CRL, molecular sieves 100 mg, agitation speed 200 rpm, temperature 40 ◦ C and heptane as solvent. 60
Free CRL-CNT
% Conversion
45
30
15
0
40
45
50 Temperature (°C)
55
60
Fig. 4. The effect of temperature (a) free CRL (b) CRL-MWCNTs in the synthesis of geranyl propionate. Reaction conditions: propionic acid/geraniol (1:5) molar ratio, enzyme amount 5 mg/mL for CRL-MWCNTs and 10 mg/mL free CRL, molecular sieves 100 mg, agitation speed 200 rpm, temperature (40–60 ◦ C) and heptane as solvent.
3.3.4. Reusability of immobilized lipase The reusability of CRL-MWCNT biocatalyst is an important aspect in determining the efficiency of the immobilized lipase as well as for use in industrial settings. In this study, reusability of the immobilized lipase was investigated by centrifuging after 62.5
CRL-CNT Free CRL
Conversion (%)
50
Fig. 2. Field emission scanning electron microscope (FESEM) and corresponding transmission electron microscope (TEM) images of (a) raw MWCNTs, (b) F-MWCNTs and (c) CRL-MWCNTs.
37.5
25
12.5
0
A bsenc e
Presence Molecular sieves
of geranyl propionate back into its alcohol and fatty components. In this study, direct addition of molecular sieves into the reaction clearly favored this process and was by far a simple method to remove the generated water [45].
Fig. 5. The effect of dessicant on (a) free CRL (b) CRL-MWCNTs on the synthesis of geranyl propionate. Reaction conditions: propionic acid/geraniol (1:5) molar ratio, enzyme amount 5 mg/mL for CRL-MWCNTs and 10 mg/mL free CRL, temperature 40 ◦ C, agitation speed 200 rpm and n-heptane as solvent.
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CRL-MWCNTs is particularly useful when the biocatalysts are used for an extended period of time during prolonged esterification reactions under extreme temperatures and acidic conditions. Since (a) the enzymatic activity was improved by as much as 2-fold for the CRL-MWCNTs over the free CRL, (b) geranyl propionate was successfully synthesized under mild conditions with reasonably high yield within a short period of reaction and (c) the CRL-MWCNTs could be reused for up to 6 cycles; this approach proved to be a facile, mild and environmental friendly method for synthesizing geranyl propionate. Acknowledgments
Fig. 6. Reusability of CRL-MWCNTs as biocatalysts for the enzymatic synthesis of geranyl propionate evaluated at 30 ◦ C, 40 ◦ C and 50 ◦ C.
the completion of a catalysis run. The biocatalysts were repeatedly reintroduced into a fresh reaction mixture for another round of esterification at 55 ◦ C and the data are simplified in Fig. 6. It was found that the CRL-MWCNTs were able to retain at least 50% of its initial activity even after they were reused for up to 2–6 times corresponding to a descending reaction temperature of 50–30 ◦ C, indicating fairly good durability of the biocatalysts when used at lower reaction temperatures. Beyond which, substantial decrease in the lipase activity was evident at 50 ◦ C, probably due to several factors that included enzyme leakage from the carbon nanotube supports as well as denaturation of the CRL protein during centrifuging, drying and regular handling [46]. Although the reusability of the CRL-MWCNTs may not be impressive, however, the simplicity of the immobilization technique, the requirement of considerably lower enzyme amount, as well as the ability to synthesize a relatively high yield of geranyl propionate in a short period of time, utilization the CRL-MWCNTs as biocatalysts is potentially more economical. Furthermore, the CRLMWCNTs can easily be regenerated using a facile two-step method (acid treatment and CRL re-immobilization by stirring) before the biocatalysts can be used again. The cost of commercially available enzymes to which industries have to pay are currently >USD900 and >USD400 for a 10 g of Novozyme and Lipozyme, respectively. The expenditure on purchasing the biocatalysts will be increased for larger reaction batches, hence, the reluctance of many industrial manufacturers of commercial esters to switch over to the enzymatically based methods. 4. Conclusions Besides addressing disadvantages associated with the chemical catalyzed route in the production of geranyl propionate, physical immobilization of free CRL onto F-MWCNTs served as a feasible platform to improve the activity and stability of CRLMWCNTs when compared with CRL in its free form. The surfaces of the F-MWCNTs consist of the COO− moiety that can interact with the charged side-chains, back-bone amino groups of the CRL protein through ionic interactions. While the predominantly hydrophobic/non-polar MWCNTs that are made up of carbon atoms are attracted to the hydrophobic amino acids found on the outer surface of the CRL through hydrophobic and van der Waals interactions. The additional network of interactions between the MWCNTs and CRL protein subsequently improve the structural integrity and the mechanical strength of the CRL protein. Thus, the CRL protein became more rigid in resisting premature unraveling and maintaining its catalytically active form. This enhance feature of
This work was supported by the Exploratory Research Grant Scheme from the Ministry of Higher Education Malaysia (ERGS R.J130000.7826.4L132) and the Research University Grant Scheme (GUP 08J13) from the Universiti Teknologi Malaysia, Skudai, Johor. We would also like to acknowledge valuable help and suggestions provided by our colleagues. References [1] Radzi SM, Mustafa WAF, Othman SS, Noor HM. Green synthesis of butyl acetate: a pineapple flavour via lipase catalyzed reaction. World Acad Sci Eng Technol 2011;59:677–80. [2] Malcata FX, Reyes HR, Garcia HS, Hill CG, Amundson CH. Immobilized lipase reactor for modification of fats and oils: a review. J Am Oil Chem Soc 1990;67:890–910. [3] Charpe TW, Rathod VK. Biodiesel production using waste frying oil. Waste Manag 2011;31:85–90. [4] Paroul N, Grzegozeski LP, Ciaradia V, Treichel H, Cansian RL, Vladimir Oliveira J, et al. Production of geranyl propionate by enzymatic esterification of geraniol and propionic in solvent free system. Chem Technol Biotechnol 2010;12(85):1636–41. [5] Salah RB, Ghamghui H, Miled N, Mejdoub H, Gargouri Y. Production of butyl acetate ester by lipase from novel strain of Rhizopus oryzae. J Biosci Bioeng 2007;103:368–72. [6] Zhou G, Wu C, Jiang X, Ma J, Zhang H, Song H. Active biocatalysts based on Candida rugosa lipase immobilized in vesicular silica. Process Biochem 2012;47:953–9. [7] Ansorge-Schumacher MB, Thum O. Immobilised lipases in the cosmetics industry. Chem Soc Rev 2013;42:6475–90. [8] Jaeger KE, Eggert T. Lipases for biotechnology. Curr Opin Biotechnol 2001;13:390–7. [9] Shim M, Kam NWS, Chen RJ, Li YM, Dai HJ. Functionalization of carbon nanotubes for compatibility and biomolecular recognition. Nano Lett 2002; 22:28–58. [10] Zhang P, Henthorn DB. Synthesis of PEGylated single wall carbon nanotubes by a photoinitiated graft from polymerization. AIChE J 2010;56:1610–5. [11] Cesar M, Jose MP, Gloria FL, Jose MG, Roberto FL. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb Technol 2007;40:1451–63. [12] Zorica K, Nenad M, Dejan B, Zivana J, Radivoje P. Immobilization of lipase from Candida rugosa on Eupergit® C supports by covalent attachment. Biochem Eng J 2006;30:269–78. [13] Afag S, Iqbal J. Immobilization and stabilization of papain on chelating sepharose: a metal chelate regenerable carrier. Electron J Biotech 2001;4(3), http://dx.doi.org/10.2225/vd4-issue3-fulltext-1. [14] Tischer W, Kascher V. Immobilized enzymes: crystals or carriers? Trends Biotechnol 1999;17:326–35. [15] Goel MK. Immobilized enzymes; 1994 http://www.rpi.edu/dept/chemeng/ BiotechEnviron/goel.html (accessed April 201). [16] de Oliveira PC, Alves GM, de Castro HF. Immobilisation studies and catalytic properties of microbial lipase onto styrene divinylbenzene copolymer. Biochem Eng J 2000;5:63–71. [17] Kumar N [thesis] Studies of glucose oxidase immobilized carbon nanotube polyaniline composites. India: Thapar University; 2009. [18] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 1976;72:248–54. [19] Abdul Rahman MB, Chaibakhsh N, Basri M, Abdul Rahman RNZ, Salleh AB, Md Radzi S. Modelling and optimization of lipase-catalysed synthesis of dilauryl adipate ester by response surface methodology. J Chem Technol Biotechnol 2008;83:1534–40. [20] Gogoi S, Dutta NN. Kinetics and mechanism of esterification of isoamyl alcohol with acetic acid by immobilized lipase. Indian J Chem Technol 2009;16:209–15. [21] Meier WM, Uytterhoeven JB. Molecular sieves. Part 1. Advances in chemistry series, vol. 121. Washington: Am. Chem. Soc.; 1973.
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Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
A facile enzymatic synthesis of geranyl propionate by physically adsorbed Candida rugosa lipase onto multi-walled carbon nanotubes Nur Royhaila Mohamad a , Nor Aziah Buang a,∗∗ , Naji Arafat Mahat a , Yen Yen Lok a , Fahrul Huyop b , Hassan Y. Aboul-Enein c , Roswanira Abdul Wahab a,∗ a
Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81310 UTM Skudai, Johor, Malaysia Department of Biotechnology and Medical Engineering, Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81310 UTM Skudai, Johor, Malaysia c Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Cairo 12311, Egypt b
a r t i c l e
i n f o
Article history: Received 5 November 2014 Received in revised form 17 February 2015 Accepted 25 February 2015 Available online 6 March 2015 Keywords: Candida rugosa lipase Immobilization Carbon nanotubes Esterification Geranyl propionate Geraniol
a b s t r a c t In view of several disadvantages as well as adverse effects associated with the use of chemical processes for producing esters, alternative techniques such as the utilization of enzymes on multi-walled carbon nanotubes (MWCNTs), have been suggested. In this study, the oxidative MWCNTs prepared using a mixture of HNO3 and H2 SO4 (1:3 v/v) were used as a supportive material for the immobilization of Candida rugosa lipase (CRL) through physical adsorption process. The resulting CRL-MWCNTs biocatalysts were utilized for synthesizing geranyl propionate, an important ester for flavoring agent as well as in fragrances. Enzymatic esterification of geraniol with propionic acid was carried out using heptane as a solvent and the efficiency of CRL-MWCNTs as a biocatalyst was compared with the free CRL, considering the incubation time, temperature, molar ratio of acid:alcohol, presence of desiccant as well as its reusability. It was found that the CRL-MWCNTs resulted in a 2-fold improvement in the percentage of conversion of geranyl propionate when compared with the free CRL, demonstrating the highest yield of geranyl propionate at 6 h at 55 ◦ C, molar ratio acid: alcohol of 1:5 and with the presence of 1.0 g desiccant. It was evident that the CRL-MWCNTs biocatalyst could be reused for up to 6 times before a 50% reduction in catalytic efficiency was observed. Hence, it appears that the facile physical adsorption of CRL onto F-MWCNTs has improved the activity and stability of CRL as well as served as an alternative method for the synthesis of geranyl propionate. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Terpene esters of short-chain fatty acids are essential oils that present a great deal of interest in food, cosmetics and pharmaceutical industries as flavors and fragrances; acetate, propionates and butyrates of acyclic terpene alcohols such as geraniol and citronellol, being the main components of essential oil [1]. Traditionally, these esters are obtained by various methods viz. chemical synthesis, extraction from natural products as well as fermentation [2]. Despite the common use of chemical synthesis for producing esters, the process requires harsh reaction conditions such as high temperature and pressure, involvement of strong acid
∗ Corresponding author. Tel.: +60 7 5534148; fax: +60 7 5566162. ∗∗ Corresponding author. Tel.: +60 7 5534163; fax: +60 7 5566162. E-mail addresses: [email protected] (N.A. Buang), [email protected] (R. Abdul Wahab). http://dx.doi.org/10.1016/j.enzmictec.2015.02.007 0141-0229/© 2015 Elsevier Inc. All rights reserved.
catalyst and hazardous chemicals, considerably long reaction time while providing low conversion rate [1]. Moreover, the chemical synthesis has been associated with tedious separation processes, extreme exposure of toxicants as well as unwanted harmful reaction byproducts [3]. Hence, the enzymatic production of flavors and fragrances using natural raw materials may prove useful for scientific and industrial settings, considering the ever arising demands for such products [4]. Due to the fact that lipases (triacylglycerol ester hydrolysis EC 3.1.1.3) may catalyze esterification reactions without providing high temperature and pressure conditions and since the procedure remains relatively uncomplicated when compared with the chemical synthesis, such enzymes have acquired popularity as biocatalysts for the reactions [5]. In this context, Candida rugosa lipase (CRL), a stable mesophilic lipase, has been commonly used due to its high activity and broad specificity in reaction medium [6]. Considering that the CRL is often unstable in its free form, demonstrating low activity in organic solvents, high tendency of deactivation in
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prolonged exposure toward high temperature and extreme pH [6], immobilization of CRL onto carbon nanotubes may be one of the probable solutions. In addition, advances in enzyme immobilization techniques have enabled the use of a wide range of biocatalysts for various reactions under extreme pH, temperature and pressure conditions [7,8]. Owing to its excellent binding capacity due to large surface area to volume ratio, physicochemical properties as well as biological compatibility, carbon nanotubes have been frequently utilized as the support materials for immobilization of enzymes [9,10]. Enzyme immobilization confers a multitude of advantages such as structural stability, improved activity, specificity and selectivity, reduction of inhibition [11], increased flexibility with enzyme/substrate contact using various reactor configurations [12] and longer half-life of the enzyme [13]. Since these enzymes are expensive and are required in large volumes for industrial needs [14], immobilization of such enzymes onto solid support materials would be helpful in maintaining the catalytically active tertiary structure of enzymes [15], which contributes to its reusability. In this context, the physical adsorption method may have higher commercial values than other methods [16] because it is one of the simplest and cheapest immobilization methods available and importantly, in most cases, the enzyme productivity remains unaffected [17]. This present study was aimed at investigating the application of physically adsorbed CRL on the surface of acid functionalized MWCNTs (CRL-MWCNTs) as potential economical biocatalysts. The CRL-MWCNTs were compared with the free CRL for synthesizing geranyl propionate. In addition, its effects on the incubation time, temperature, molar ratio of acid to alcohol, presence of desiccant and reusability in rendering the highest conversion of geranyl propionate were evaluated. 2. Materials and methods 2.1. Chemicals Multi-walled carbon nanotubes (MWCNTs) prepared by the chemical vapor deposition method were provided by one of the co-authors (Assoc. Prof. Dr. Nor Aziah Buang). Lipase Type VII of C. rugosa (EC 3.1.1.3) with measured activity of 1410 units mg1 , substrates, geraniol (98%), propionic acid (99%), phosphate buffer, Bradford reagent, phenolftalein and molecular sieves were all purchased from the Sigma Chemical Co. (St. Louis, USA). Other chemicals that include sodium hydroxide and n-heptane were of analytical grade and used without further purification. Distilled water was prepared in our laboratory and used in all experiments. 2.2. Purification and functionalization of MWCNTs The raw MWCNTs (0.5 g) were transferred into a 100 mL flask containing 4 M HCl (20 mL) and refluxed with stirring at 80 ◦ C for 5 h. After cooling to room temperature, the liquid was decanted, the MWCNTs were washed with distilled water until no residual acid was detected and dried in an oven at 60 ◦ C for 24 h. The purified MWCNTs were refluxed at 120 ◦ C by stirring in a mixture of concentrated HNO3 and H2 SO4 with ratio 1:3 (v/v) for 24 h. After cooling to room temperature, the mixture was decanted, washed with distilled water until no residual acid was present and dried at 60 ◦ C for 24 h. 2.3. Adsorption immobilization of lipase and its characterization The free CRL was immobilized onto the acid functionalized MWCNTs (FMWCNTs). The MWCNTs (10 mg/mL) were first sonicated in aqueous buffer (pH 7) for 30 min to ensure homogeneous dispersion. Then, the MWCNTs were suspended in a 50 mL flask of phosphate buffer (50 mM, solution pH 7) containing the CRL (10 mg/mL) and incubated at 20 ◦ C with constant stirring at 150 rpm. After 3 h of incubation, the flask containing CRL-MWCNTs was stored at 4 ◦ C for 24 h. Upon completion, the unbound protein was removed by washing with phosphate buffer (pH 7) until no hydrolytic activity was detected in the washing. The supernatant was then subjected to protein analysis. Similar CRL immobilization treatments were also performed to unfunctionalized MWCNTs. 2.3.1. Fourier transform infrared (FTIR) A ratio 1:100 mass of sample was ground thoroughly with potassium bromide and the resulting powder was pressed into a transparent pellet by a hydraulic press.
The FTIR spectra obtained using a BOMEM spectrophotometer in transmission mode between 400 and 4000 cm−1 at a resolution of 4 cm were analyzed. 2.3.2. Field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM) A field emission scanning electron microscope (FESEM) (JEOL JEM-6700F) was used to study the surface morphology of MWCNTs, F-MWCNTs and CRL-MWCNTs. For FESEM analysis, the sample was prepared by taking one drop of acetone containing the dispersed MWCNTs on a silicon wafer and allowing it to dry in a vacuum oven for 30 min. For TEM analysis, electron source was used as W-emitter and LaB6, operating at an accelerating voltage of 200 kV. The analysis utilized an objective lens (S-Twin) with a point resolution of 2.0 nm or better with a 25× to 7500, 200× or higher magnification and a single tilt holder with LCD camera. The MWCNTs, F-MWCNTs and CRL-MWCNTs were dispersed in deionized water and a drop was placed on a copper grid and observed after drying in vacuum. 2.4. Determination of protein loading Protein content of the enzyme solution, before and after immobilization, in the washing buffer was determined by Bradford method using BioRad protein dye reagent concentrate and bovine serum albumin (BSA) as the protein standard [18] as well as in the reaction mixture. 2.5. Enzymatic synthesis of geranyl propionate catalyzed by free CRL and CRL-MWCNTs The reaction medium that consisted of 0.25 M propionic acid and 1.25 M geraniol was dissolved in n-heptane in a flask followed by the addition of free CRL (10 mg/mL) and CRL-MWCNTs (5 mg/mL), respectively. The reaction mixture was refluxed with continuous stirring at 200 rpm in a paraffin oil bath. The geranyl propionate obtained was expressed in terms of percent conversion i.e. percent of propionic acid converted versus the total acid in the reaction mixture by titrating aliquots of liquid sample (1 mL) withdrawn periodically with 0.05 M NaOH using phenolphthalein as an indicator. Each measurement was performed in triplicates and the standard error was calculated. The percent conversion was calculated according to the equation prescribed by previous researchers [1] detailed below:
% Conversion =
(Vo − Vt ) Vo
× 100
whereby, Vo is the volume of NaOH at initial time (t = 0) and Vt is the volume of NaOH at each hour (t = t1 , t2 , t3 , . . .). 2.5.1. Effect of incubation time Time course study is a good indicator of enzyme performance as well as product yield and a good performance enzyme requires relatively shorter duration to obtain good yields when compared with the poor ones [19]. The effect of incubation was monitored up to 24 h with sampling intervals of 0, 4, 8, 12, 16, 20 and 24 h, respectively. The reactions were carried out in 50 mL round bottom flasks, stirred constantly at 200 rpm. Upon completion of preliminary screening tests, the reaction temperature was maintained at 40 ◦ C. 2.5.2. Effect of temperature It has been reported that temperature has a significant effect on the equilibrium of the reaction as well as the activity and stability of lipase [20]. The effect of temperature on the enzymatic synthesis of geranyl propionate of both free CRL and CRL-MWCNT was determined at varying temperatures ranging from 40 to 60 ◦ C, at increasing intervals of 5 ◦ C each. 2.5.3. Effect of the presence of desiccant Water is a byproduct of esterification that needs to be removed for obtaining higher yield of ester [21]. Following the successful employment as desiccant in many enzymatic esterification reactions [22,23], in this present study too, molecular sieves ˚ were chosen. They act as absorbents that are replaceable with new ones when (4 A) saturated with water [24]. By removing water in media, the reaction equilibrium would shift toward the synthesis of the desired ester hence, higher conversion of ester products [24]. The effects of desiccant on the free CRL and CRL-MWCNTs were evaluated in the presence (1.0 g) or absence (0 g) of molecular sieves. 2.5.4. Reusability of CRL-MWCNTs The reusability of the CRL-MWCNTs was examined by reusing the recovered lipase for repeated reaction cycles. After each cycle, the enzyme was filtered, washed with similar solvent and allowed to dry before reuse. For investigating the reusability, the reaction mixture that consisted of propionic acid and geraniol dissolved in n-heptane with the presence of immobilized lipase was used. Each reaction was monitored for a period of 8 h at 30–50 ◦ C with constant stirring (Table 1).
N.R. Mohamad et al. / Enzyme and Microbial Technology 72 (2015) 49–55 Table 1 FTIR frequency table (a) raw MWCNTs, (b) F-MWCNTs and (c) CRL-MWCNTs. Frequency (cm−1 )
Functional group
(a)
3428.01 1630.82
O H stretching Conjugated C C bonds
(b)
3445.22 1112.73 1634.70 1202.46
O C C C
(c)
1032.96 3420.59 2929.41 1116.03
C N stretching N H stretching CH2 bending C O stretching
H stretching O stretching O stretching O stretching
3. Results and discussion 3.1. Functionalization of MWCNTs for the adsorption of CRL protein Refluxing the MWCNTs in the mixture of H2 SO4 and HNO3 , introduces the polar groups (COO− ) to the non-polar surface and tubular ends of the MWCNTs supports, hence, the surface of the MWCNTs is primed for the attachment with other polar moieties (NH2 , O H) present on the CRL protein. The role of the introduced COO− group is to anchor the CRL protein to the MWCNTs through attractions between the oppositely charged carboxyl moiety (electron rich) and hydrogen (electron poor) of the back-bone and side-chain of polar amino acids present on the outer surface of the CRL protein. Conversely, attachment of the CRL proteins to the non-polar unfunctionalized MWCNTs, was found to be insignificant. The physical adsorption of CRL to the F-MWCNTs was reasonably strong as the amount of detached protein in the reaction mixture was either negligible or not detected. The activity of the adsorbed CRL is 592 Umg−1 is relatively high, though a little lower than that of the free CRL. However, when used as a biocatalyst under extended reaction period, the former is anticipated to be more stable and its initial enzyme activity is retained. Furthermore, the acid functionalized CRL-MWCNTs were observed to be significantly better dispersed and were easily suspended when stirred in the reaction vessel as compared to the unfunctionalized ones. The ability of CRL-MWCNTs to remain longer in suspension is somewhat desirable for an enzymecatalyzed reaction, therefore, favoring increased possibility of effective enzyme-substrate collision as well as reduced tendency of the CRL-MWCNTs to precipitate out of the reaction. 3.2. Characterization of immobilized lipase (CRL-MWCNTs) 3.2.1. Spectroscopy (FTIR) The FTIR spectra obtained for the raw MWCNTs, F-MWCNTs and CRL-MWCNTs are presented in Fig. 1. The appearance of a broad band at 3428.01 cm−1 in Fig. 1a was assigned to the O H stretching of the surface group of the raw MWCNTs as received. This could be attributed to ambient atmospheric moisture that was tightly bound to the MWCNTs [25]. The spectrum of raw MWCNTs showed a weak band at 1630.82 cm−1 is assigned to the conjugated C C bonds [26]. After oxidation, the stretch from O H group shifted from 3428.01 cm−1 to 3445.22 cm−1 (Fig. 1b), which indicated the hydroxyl group had covalently bonded to the sidewalls of the MWCNTs [27]. The presence of a new peak at 1112.73 cm−1 is assigned to a C O stretching that clearly represented successful oxidation of sp2 hybridized carbon in raw MWCNTs to sp3 [28]. Whereas, a peak at 1634.70 cm−1 was observed which arise from the C O stretching vibration of the carboxylic group. Conjugation
51
of O C with the C C resulted in the C O to adapt a more single bond character through resonance. This phenomenon ultimately leads to a lower vibration frequency of carbonyl group. A new peak appeared at 1202.46 cm−1 corresponded to the C O of the COOH moiety [29]. Fig. 1c exhibited the absence of band at 1634.70 cm1 and the corresponding appearance of band at lower wavelength, 1620.71 cm−1 . This band represented the C O stretching of amide which is the most outstanding adsorption bands that confirm the presence of proteins [26]. In addition, the presence of a new band at 1032.96 cm−1 was attributed to the C N bond that corroborated the existence of the amide functional group. Meanwhile, the N H and CH2 bands appeared at 3420.59 cm−1 and 2929.41 cm−1 , respectively. Distinction in the spectra of the raw MWCNTs and CRL-MWCNTs was observed in the range of 900–1200 cm−1 , the spectral region was for carbohydrate functional group bands [30]. Following immobilization, an intense peak at 1125.04 cm−1 assigned to vibrations the C O of the carbohydrate moiety for spectrum of free lipase was reduced, expanded and moved to a lower frequency at 1116.03 cm−1 [26]. It can be concluded that the CRL was successfully immobilized onto the outer face of the F-MWCNTs. Fig. 1c shows the appearance of an absorption band at 2929.41 cm−1 which implied the successful attachment of CRL to the surface of the MWCNTs, a band not observed in the spectrum of F-MWCNTs. 3.2.2. Field emission scanning electron microscope (FESEM) and transmission electron microscopy (TEM) The morphologies of raw MWCNTs, F-MWCNTs and CRLMWCNTs observable through FESEM and TEM are presented in Fig. 2. It was observed that the raw MWCNTs demonstrated openended tubular structures with smooth surfaces that were closely attached to each other with some contaminants on the carbon fiber surface prior to purification (Fig. 2a). Due to the possibility of impeding the potential characters of carbon nanotubes, these contaminants need to be removed [31]. Following treatment with H2 SO4 and HNO3 , the diameter of MWCNTs was increased with observable rough surfaces probably due to surface erosion [29]. The increased diameter of MWCNTs strongly indicated the successful attachment of acid functional groups on the surface of F-MWCNTs (Fig. 2b), similarly to the findings reported by previous work [32]. Furthermore, the length of the F-MWCNTs was evidently shorter following treatment with those acids, in consistency with the previously reported studies [31,33] indicating that acid treatment would change the highly tangled long MWCNTs into shorter, open ended tubular MWCNTs with numerous carboxylic groups at the open ends. This is due to the fact that elevated temperatures and increased oxidation time tend to destroy the structure of MWCNTs [34], leading to surface imperfections on the MWCNTs as well as affecting the properties of the resulting material. Fig. 2c clearly shows that the CRL was physically present on the surface MWCNTs, rendering increased diameter of CRL-MWCNTs when compared with the F-MWCNTs. Hence, this implies that the surface of the MWCNTs was covered by enzyme molecules [35]. 3.3. Optimization of esterification activity of CRL-MWCNT 3.3.1. Effect of incubation time Fig. 3 represents the reaction time profile for the free CRL and CRL-MWCNTs in catalyzing esterification of propionic acid and geraniol over a 12 h period, indicating the increased conversion of geranyl propionate with increasing reaction time. Interestingly, CRL-MWCNTs were found to increase the production of ester, attaining the highest percent conversion (47.3%) at 6 h of incubation time when compared with that observed for the free CRL (28.1%); beyond which the conversion of geranyl propionate started to decline. The higher conversion of geranyl propionate in reactions
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Fig. 1. FT-IR spectra of (a) raw MWCNTs, (b) F-MWCNTs and (c) CRL-MWCNTs.
catalyzed by the CRL-MWCNTs can be attributed to the formation of stable multipoint interactions (e.g. hydrophobic interaction, hydrogen bond, electrostatic and van der Waals forces) between CRL molecules and MWCNTs, resulting in the immobilized CRL being more stable than the free CRL [36]. Under such conditions, the enzyme is less susceptible to denaturation by solvents, temperature or substrates in the reaction vessel. The decline percent conversion of geranyl propionate may be attributable to increased production of water molecules as byproducts of the esterification reaction, a counterproductive reverse reaction hydrolyzing the geranyl propionate formed during the reaction [37], apart from diminished substrates that resulted in reduced degree of enzyme substrate saturation [38].
3.3.2. Effect of temperature Considering that temperature would significantly affect the equilibrium as well as the activity and stability of the enzymes in the reactions [39], such factor was investigated in this present study and the relevant data are tabulated in Fig. 4. It was revealed that the optimum temperature for the free CRL and CRL-MWCNTs were 40 ◦ C and 55 ◦ C, respectively, a 15 ◦ C increase from the observed temperature in the free CRL catalyzed reactions. Since similar shifts in optimal temperature for Talaromyces thermophilus immobilized onto chitosan as well as CRL immobilized on styrene divinylbenzene copolymer have been reported [16,40]. Therefore, such deviation observed in the optimum temperature recorded for the free CRL and CRL-MWCNTs in this study too prove common, implying higher stability of the CRL-MWCNTs at higher reaction temperatures. The increase in optimum temperature of the CRL-MWCNTs over the free CRL may be due to the network of hydrophobic and van der Waals interactions, as well as hydrogen bonds between CRL molecules and MWCNTs which altered the physical and chemical properties of the free CRL following adsorption onto the MWCNTs [36,41]. These additional interactions increase the structural
rigidity of the adsorbed CRL but somewhat, tend to denature its catalytically active structure. The CRL-MWCNTs is made more flexible and its catalytically active form is restored upon increasing the reaction temperature, hence, the observed higher optimum temperature of the CRL-MWCNTs over the free CRL. It has been reported that at higher temperature, the immobilized CRL has greater structural stability conferred by the attachment of lipase onto the functionalized surface of the MWCNTs [41], better resistance of the CRL-MWCNTs toward thermal denaturation at 40–60 ◦ C than that of free CRL. Correspondingly, it was observed that at 55 ◦ C the production of geranyl propionate by CRL-MWCNTs was higher (51.3%) when compared with that of the free CRL (16%), potentially afforded by hydrogen bonds and electrostatic interactions between the enzyme and the support [42].
3.3.3. Effect of the presence of desiccant Although adequate amount of water content would keep the enzyme in its active configuration, excessive amount of water would impede the equilibrium shift toward product [43]. It was observed that the molecular sieves as desiccant quickly dispersed in the reaction medium within a few minutes of stirring and caused the medium to turn slightly viscous. Fig. 5 illustrates the effect of desiccant on the enzymatic production of geranyl propionate. It was found that reactions catalyzed by both free CRL and CRL-MWCNTs supplemented with molecular sieves resulted in higher conversion of ester, yielding up to 24.3% and 47.3%, respectively; considerably higher percent conversion than that of 12.1% and 32.5% recorded for the free CRL and CRL-MWCNTs in the absence of such desiccant. In this context, it is pertinent to indicate that water is an unwanted byproduct of esterification reaction and the addition of desiccant may shift the equilibrium toward the formation of product [44]. The role of the molecular sieves was to absorb the excess water liberated in the free CRL and CRL-MWCNTs catalyzed esterification reaction and avoid the counterproductive hydrolysis
N.R. Mohamad et al. / Enzyme and Microbial Technology 72 (2015) 49–55
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62.5 Free CRL-CNT
% Conversion
50
37.5
25
12.5
0
1
2
3
4
5
6
7
8
10
12
Time (h) Fig. 3. The effect of incubation time for the synthesis of geranyl propionate. Reaction conditions: propionic acid/geraniol (1:5) molar ratio, enzyme amount 5 mg/mL for CRL-MWCNTs and 10 mg/mL free CRL, molecular sieves 100 mg, agitation speed 200 rpm, temperature 40 ◦ C and heptane as solvent. 60
Free CRL-CNT
% Conversion
45
30
15
0
40
45
50 Temperature (°C)
55
60
Fig. 4. The effect of temperature (a) free CRL (b) CRL-MWCNTs in the synthesis of geranyl propionate. Reaction conditions: propionic acid/geraniol (1:5) molar ratio, enzyme amount 5 mg/mL for CRL-MWCNTs and 10 mg/mL free CRL, molecular sieves 100 mg, agitation speed 200 rpm, temperature (40–60 ◦ C) and heptane as solvent.
3.3.4. Reusability of immobilized lipase The reusability of CRL-MWCNT biocatalyst is an important aspect in determining the efficiency of the immobilized lipase as well as for use in industrial settings. In this study, reusability of the immobilized lipase was investigated by centrifuging after 62.5
CRL-CNT Free CRL
Conversion (%)
50
Fig. 2. Field emission scanning electron microscope (FESEM) and corresponding transmission electron microscope (TEM) images of (a) raw MWCNTs, (b) F-MWCNTs and (c) CRL-MWCNTs.
37.5
25
12.5
0
A bsenc e
Presence Molecular sieves
of geranyl propionate back into its alcohol and fatty components. In this study, direct addition of molecular sieves into the reaction clearly favored this process and was by far a simple method to remove the generated water [45].
Fig. 5. The effect of dessicant on (a) free CRL (b) CRL-MWCNTs on the synthesis of geranyl propionate. Reaction conditions: propionic acid/geraniol (1:5) molar ratio, enzyme amount 5 mg/mL for CRL-MWCNTs and 10 mg/mL free CRL, temperature 40 ◦ C, agitation speed 200 rpm and n-heptane as solvent.
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CRL-MWCNTs is particularly useful when the biocatalysts are used for an extended period of time during prolonged esterification reactions under extreme temperatures and acidic conditions. Since (a) the enzymatic activity was improved by as much as 2-fold for the CRL-MWCNTs over the free CRL, (b) geranyl propionate was successfully synthesized under mild conditions with reasonably high yield within a short period of reaction and (c) the CRL-MWCNTs could be reused for up to 6 cycles; this approach proved to be a facile, mild and environmental friendly method for synthesizing geranyl propionate. Acknowledgments
Fig. 6. Reusability of CRL-MWCNTs as biocatalysts for the enzymatic synthesis of geranyl propionate evaluated at 30 ◦ C, 40 ◦ C and 50 ◦ C.
the completion of a catalysis run. The biocatalysts were repeatedly reintroduced into a fresh reaction mixture for another round of esterification at 55 ◦ C and the data are simplified in Fig. 6. It was found that the CRL-MWCNTs were able to retain at least 50% of its initial activity even after they were reused for up to 2–6 times corresponding to a descending reaction temperature of 50–30 ◦ C, indicating fairly good durability of the biocatalysts when used at lower reaction temperatures. Beyond which, substantial decrease in the lipase activity was evident at 50 ◦ C, probably due to several factors that included enzyme leakage from the carbon nanotube supports as well as denaturation of the CRL protein during centrifuging, drying and regular handling [46]. Although the reusability of the CRL-MWCNTs may not be impressive, however, the simplicity of the immobilization technique, the requirement of considerably lower enzyme amount, as well as the ability to synthesize a relatively high yield of geranyl propionate in a short period of time, utilization the CRL-MWCNTs as biocatalysts is potentially more economical. Furthermore, the CRLMWCNTs can easily be regenerated using a facile two-step method (acid treatment and CRL re-immobilization by stirring) before the biocatalysts can be used again. The cost of commercially available enzymes to which industries have to pay are currently >USD900 and >USD400 for a 10 g of Novozyme and Lipozyme, respectively. The expenditure on purchasing the biocatalysts will be increased for larger reaction batches, hence, the reluctance of many industrial manufacturers of commercial esters to switch over to the enzymatically based methods. 4. Conclusions Besides addressing disadvantages associated with the chemical catalyzed route in the production of geranyl propionate, physical immobilization of free CRL onto F-MWCNTs served as a feasible platform to improve the activity and stability of CRLMWCNTs when compared with CRL in its free form. The surfaces of the F-MWCNTs consist of the COO− moiety that can interact with the charged side-chains, back-bone amino groups of the CRL protein through ionic interactions. While the predominantly hydrophobic/non-polar MWCNTs that are made up of carbon atoms are attracted to the hydrophobic amino acids found on the outer surface of the CRL through hydrophobic and van der Waals interactions. The additional network of interactions between the MWCNTs and CRL protein subsequently improve the structural integrity and the mechanical strength of the CRL protein. Thus, the CRL protein became more rigid in resisting premature unraveling and maintaining its catalytically active form. This enhance feature of
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