Sustainable production of the emulsifier methyl oleate by Candida rugosa lipase nanoconjugates

food and bioproducts processing 9 6 ( 2 0 1 5 ) 211–220

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Food and Bioproducts Processing journal homepage: www.elsevier.com/locate/fbp

Sustainable production of the emulsifier methyl oleate by Candida rugosa lipase nanoconjugates Nur Haziqah Che Marzuki a , Naji A. Mahat 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 Johor Bahru, Johor, Malaysia b Department of Biotechnology and Medical Engineering, Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia c Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Giza 12622, Egypt

a r t i c l e

i n f o

a b s t r a c t

Article history:

Acid functionalization of multi-walled carbon nanotubes (F-MWCNTs) using a mixture of

Received 22 April 2015

HNO3 and H2 SO4 (1:3, v:v) was used as support materials for the adsorption of Candida rugosa

Received in revised form

lipase (CRL) as nanoconjugates (CRL-MWCNTs) for producing methyl oleate. To evaluate the

15 August 2015

competency of the CRL-MWCNTs nanoconjugates, parameters viz. reaction time, surfactant

Accepted 18 August 2015

as well as thermostability and reusability were investigated. The characterization of CRL-

Available online 28 August 2015

MWCNTs nanoconjugates using Fourier transform infrared spectroscopy, Field Scanning

Keywords:

of CRL onto the F-MWCNTs. Utilization of CRL-MWCNTs nanoconjugates resulted in a higher

Candida rugosa lipase

acid conversion in the synthesis of methyl oleate (79.85% at 11 h of reaction time) when

Electron Microscopy and Transmission Electron Microscopy revealed successful attachment

Nanoconjugates

compared with the free CRL i.e. an approximately 1.5-fold improvement over the free CRL.

Esterification

The highest percentage of esterification (83.62%) was observed following the use of non-

Immobilization

ionic surfactant when compared with the anionic and cationic ones. The CRL-MWCNTs

Multi-walled carbon nanotubes

nanoconjugates could be used up to 5 cycles, retaining 50% of its residual activity. Since the

Methyl oleate

preparation of the CRL-MWCNTs nanoconjugates was facile and cheap while producing reasonable yield, the CRL-MWCNTs nanoconjugates developed here were found as promising biocatalysts for the production of methyl oleate. © 2015 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

1.

Introduction

Being one of the main components for manufacturing emulsifiers, detergents, intermediate stabilizers and wetting agents, esters such as methyl oleate are functionally important compounds in many industrial sectors (Long et al., 2013). However, the means of extracting such esters from plants and other natural sources are often too scarce or expensive for commercial use (Long et al., 2013). Pertinently, many disadvantages of utilizing the current chemical processes such as the use

∗

of corrosive acids and hazardous chemicals, requirement for high energy as well as counterproductive degradation of the produced ester under prolonged reaction time (Aranda et al., 2008) have been reported. Therefore, alternative methods that would overcome such disadvantages but at the same time enhance productivity need to be suggested. In this context, the enzymatic esterification using lipase for synthesizing methyl oleate may prove promising as an alternative route to the prevailing chemical production of esters (Pecnik and Knez, 1992; Mahmood et al., 2013). This is because enzymatic synthesis

Corresponding author. Tel.: +60 75534148; fax: +60 75566162. E-mail address: [email protected] (R.A. Wahab) .

http://dx.doi.org/10.1016/j.fbp.2015.08.005 0960-3085/© 2015 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

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can be performed at ambient condition and the fact that improved production technologies and engineered enzymatic properties are two key elements desirable by industrial manufacturers (Iyer and Ananthanarayan, 2008). Lipases (triacylglycerol ester hydrolysis EC 3.1.1.3) are one of the most adaptable classes of industrial enzymes with catalyzing capability in aqueous and organic solvents. Moreover, the enzymes can act on a broad spectrum of substrates, enantioselective as well as performing specific biotransformation (Treichel et al., 2010). Among others, Candida rugosa lipase (CRL) has been prevalently used in oil hydrolysis, transesterification, esterification and interesterification as well as catalyzing long chain of fatty acid esters (Abdul Rahman et al., 2012). However, the free form of CRL is (a) often unstable, (b) exhibiting low activity in organic solvents and (c) prone to deactivation when exposed to prolonged exposure of high temperature and extreme pH (Zhou et al., 2012). Therefore, immobilization of CRL onto multi-walled carbon nanotubes (MWCNTs) as supportive materials may prove useful for enhancing its activity and stability (Radzi et al., 2011). Currently, the focus in enzyme immobilization technology is shifting toward the use of nanosized materials as supports due to their high specific surface areas (Radzi et al., 2011). An enzyme immobilization process involves attachment of free or soluble enzymes onto different types of supports (Khan and Alzohairy, 2010; Mohamad et al., 2015a) to enhance structural stability, activity, specificity and selectivity (Cesar et al., 2007). Immobilization of CRL would extend reaction life and lead to the excellent binding capacity, favorable physicochemical properties as well as biological compatibility of MWCNTs (Zhang and Henthorn, 2010). The role of the multiwalled carbon nanotubes (MWCNTs) as support materials in enhancing activity and stability of enzymes has been clearly indicated in literature (Tavares et al., 2015). The improved stability of immobilized enzymes is due to the formation of stable multipoint interactions between lipase molecules and support materials (Metin, 2013). Such process may result in such enzymes becoming too stable or even loss of activity (Metin, 2013). In this context, a relatively simple cost-effective physical adsorption resulting from weaker unspecific forces is preferred for facilitating easy separation of the biocatalyst from the reaction mixture, without compromising its productivity (Mohamad et al., 2015a). Due to its outstanding mechanical, electrical and thermal properties as well as biocompatibility to CRL (Tan et al., 2012), the surface of acid functionalized MWCNTs (F-MWCNTs) was used for immobilizing the free CRL. Since other studies reported about the use of chemically mediated processes for enzyme immobilization that may be harmful to human and associated with high production costs, while the CRL-MWCNTs evaluated here remained cost-effective and environmentally friendly; its application deserves consideration. Considering a variety of experimental conditions for optimizing the production of methyl oleate, productivity and stability of the physically adsorbed CRL (CRL-MWCNTs) with that of free CRL alone were duly examined.

2.

Experimental

2.1.

Materials

MWCNTs were prepared using a chemical vapor deposition method. Lipase from C. rugosa lipase Type VII (1140 units/mg),

sodium hydroxide, sulphuric acid (99%), nitric acid (99%), potassium phosphate buffer pH 7.0 and phenolphthalein were purchased from Sigma–Aldrich (St. Louis, USA). Surfactants, Triton X-100, Tween-80, hexadecyltrimethylammonium bromide (HTAB), cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS) and dioctylsulfosuccinate sodium salt (AOT) were also acquired from Sigma–Aldrich (St. Louis, USA). Analytical grade methanol, oleic acid and isooctane were procured from QReC Chemicals (New Zealand). Distilled and deionized water was produced in our lab and used without further purification.

2.2.

Methods

2.2.1.

Purification and functionalization of MWCNTs

For purifying the MWCNTs, the as-synthesized MWCNTs (1 g) were transferred into a 100 mL round bottom flask containing 4.0 M HCl (60 mL) and refluxed with stirring for 24 h at 100 ◦ C. Upon cooling to room temperature, the liquid was decanted and the MWCNTs were washed repeatedly with distilled water. The suspension of MWCNTs was pelleted down by centrifuging (6000 rpm) for 5 min and the liquid was decanted. The process was repeated until no residual acid was detected after which the purified MWCNTs were dried overnight in an oven at 60 ◦ C (Yudianti et al., 2011). For functionalizing the MWCNTs, the purified MWCNTs were refluxed in a mixture of concentrated H2 SO4 :HNO3 (3:1, v/v) for 6 h at 100 ◦ C. The mixture was left overnight to ensure deposition of the F-MWCNTs. Then, the suspension was diluted and rinsed with distilled water until no residual acid was detected and subsequently dried at 80 ◦ C (Yudianti et al., 2011).

2.2.2. Immobilization of CRL onto F-MWCNTs by physical adsorption The produced F-MWCNTs (100 mg) were suspended in a 50 mL flask of potassium phosphate buffer pH 7.0 (20 mL) that contained CRL (3 mg/mL) and incubated at 4 ◦ C for 3 h with constant stirring at 150 rpm. The suspension was centrifuged for 10 min at 6000 rpm, the liquid decanted and the unbound proteins were removed by repeated washing with potassium phosphate buffer pH 7.0 until no evidence of hydrolytic activity was detected in the washings. The CRL-MWCNTs nanoconjugates were lyophilized overnight and stored at 4 ◦ C until further use (Peng et al., 2013).

2.2.3.

Characterization of CRL-MWCNTs

The CRL-MWCNTs were characterized using Fouriertransform infrared (FT-IR) and Field Emission Scanning Electron Microscope (FESEM). For infrared analysis, a mass of sample was ground thoroughly with potassium bromide (1:100 ratios) and the resulting powder was pressed onto a transparent pellet using a hydraulic press. The FT-IR spectra were obtained using a spectrophotometer (Perkin Elmer) in transmission mode between 400 and 4000 cm−1 with a resolution of 4 cm−1 . Morphology of the synthesized MWCNTs, F-MWCNTs and CRL-MWCNTs were examined using FESEM (JEOL JEM-6700F), which operated at an accelerating voltage of 5 kV and electric current of 10 ␮A. Prior to examination, a sample was mounted on the surface of a silicon wafer and sputter-coated with a thin film of gold to avoid charging under the electron beam.

food and bioproducts processing 9 6 ( 2 0 1 5 ) 211–220

2.2.4. Determination of protein concentration and lipase activity

2.2.7.

The concentration of protein content in the enzyme solution, before and after immobilization, in the washing buffer was determined by the Bradford method using BioRad protein dye reagent concentrate and bovine serum albumin (BSA) as the protein standard (Bradford, 1976). Different concentrations of BSA were prepared by using a stock solution (0.1 mg BSA in 10 mL of deionized water) and the absorbance was measured at 595 nm in a spectrophotometer (HITACHI U-3210) using preparations without BSA as blank. All determinations were performed in triplicate. The activity of the CRL-MWCNTs was determined using the method suggested by Langone and Sant’anna (1999). The lipase activity was quantitated at OD715 by measuring the consumption of lauric acid in the esterification reaction with glycerol (lauric acid/glycerol, 1:3) at 50 ◦ C following the use of 5 mg/mL of enzyme. One esterification unit of the lipase (U) was defined as 1 ␮ mole of lauric acid consumed/min. The determinations were performed in triplicate for all samples. Catalytic activity of the CRL-MWCNTs was found to be 374 U/mg, while the free CRL as per indicated by manufacturer was 847 U/mg.

2.2.5. Effect of reaction conditions on enzymatic production of methyl oleate A standard esterification reaction was executed in a 100 mL round-bottom flask that consisted of a mixture of methanol (26.16 mmole, 1 M), oleic acid (3.154 mmole, 1 M) (1:1 molar ratio) and iso-octane as solvent in a made up volume of 30 mL. For ascertaining the suitable solvent with the highest acid conversion, several different solvents (diethyl ether, toluene, n-hexane, n-heptane and decane) were investigated. Our results revealed that iso-octane being the best one and therefore, such solvent used in the subsequent reactions. The reaction was initiated by addition of free CRL or CRL-MWCNTs nanoconjugates and the mixture was stirred at 200 rpm in a paraffin oil bath. Ester accumulation was monitored by removing aliquots (1 mL) of the reaction mixture at designated intervals and titrated with NaOH (0.02 M) using phenolphthalein as the indicator (Abdul Rahman et al., 2012). The methyl oleate obtained was expressed in percentage conversion i.e. percent of oleic acid converted versus the total acid in the reaction mixture. Each measurement was performed in triplicate and the standard error was calculated. The percentage conversion was calculated according to the prescribed equation (Eq. (1)) (Radzi et al., 2011). Vo − Vt % Conversion = × 100% Vo

(1)

whereby, Vo is volume of NaOH at initial time (t = 0) and Vt is volume of NaOH at a particular interval (t = t1 , t2 , t3 , . . .).

2.2.6.

Effects of reaction time

The effect of reaction time was monitored at every 1 h interval for up to 12 h. The reactions were carried out in 50 mL round bottom flasks consisting of methanol and oleic acid (1:1 molar ratio). The reaction was initiated by adding free CRL (3 mg/mL) or CRL-MWCNTs nanoconjugates (3 mg/mL) into each flask and stirred at 200 rpm at 50 ◦ C. The F-MWCNTs were used as negative controls for monitoring catalysis (if any).

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Effect of surfactants

The effects of surfactants on CRL-MWCNTs nanoconjugates were investigated for their application in enhancing the yield of esters in enzyme assisted esterification reactions (Thakar and Madamwar, 2005). In this study, the effects of various surfactants on esterification were evaluated for: anionic (AOT and SDS), cationic (CTAB and HTAB) and nonionic (Triton X-100 and Tween-80) surfactants. The reaction consisted of surfactant (1 mM) and oleic acid and methanol (molar ratio of 1:1) and iso-octane as solvent. The free CRL (3 mg/mL) or CRL-MWCNTs nanoconjugates (3 mg/mL) was added to the respective flasks and the mixture stirred at 200 rpm at 50 ◦ C for continuous 12 h. The F-MWCNTs were retained as negative controls for monitoring catalysis (if any).

2.2.8.

Thermostability

The thermal stability of CRL-MWCNTs was examined by incubating the biocatalysts in sealed vials for 1 h at various temperatures ranging from 50 to 70 ◦ C, at increasing intervals of 10 ◦ C each. After the stipulated period, the CRL-MWCNTs (3 mg/mL) were left to cool to room temperature. Such evaluation was attempted by introducing the thermally treated CRL-MWCNTs into the reaction mixture that consisted of oleic acid and methanol dissolved in n-heptane and incubated at the stipulated temperatures with constant stirring. The relative activities were determined by comparing the activities of both the free CRL and CRL-MWCNTs without prior incubation and the ones incubated under various temperatures. The relative activities of both free CRL and CRL-MWCNTs were expressed as percentage of the enzyme activity at different temperature (Raghavendra et al., 2010).

2.2.9.

Reusability

Reusability is one of the pivotal factors that influence the increasing application of immobilized lipases in industrial sectors when compared with that of free lipase (Raghavendra et al., 2013). The reusability of CRL-MWCNTs nanoconjugates (3 mg/mL) was examined by reusing the recovered CRL-MWCNTs over 10 repeated cycles in a mixture of oleic acid and methanol (1:1) with iso-octane as the solvent. The CRLMWCNTs nanoconjugates were washed with iso-octane after each reaction, centrifuged (6000 rpm), and lyophilized before reuse. Each reaction cycle was monitored at 50 ◦ C for 11 h.

3.

Results and discussion

3.1.

Rationale for the functionalization of MWCNTs

Refluxing the MWCNTs in a mixture of H2 SO4 and HNO3 introduces the polar COO− group to the non-polar surface and tubular ends of the MWCNTs supports (Mohamad et al., 2015b). The attachment of COO− moiety onto the MWCNTs increases the interaction compatibility between amino acids having polar functional groups the surface of enzyme molecules and MWCNTs, hence, enabling formation of hydrogen bonds with hydrogen atoms of amino and hydroxyl groups of CRL (Mohamad et al., 2015b). Conversely, the physical adsorption of CRL onto the F-MWCNTs was reasonably strong as the amount of detached proteins was observed as insignificant following 6 h of stirring. Although the CRLMWCNT nanoconjugates recorded lower activity than the free CRL, they were expected to be more stable and less likely to be affected by temperature or chemical related premature enzyme deactivation, thereby, increasing their operational

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stability (Mohamad et al., 2015a). It was also observed in our experiment that the CRL-MWCNTs nanoconjugates were well dispersed when stirred during reaction as opposed to the MWCNTs. The facile dispersibility of the CRL-MWCNTs nanoconjugates is advantageous in ensuring effective mixing between the biocatalysts and the substrates. This enhances effective collisions between enzyme and substrate molecules and can potentially improve the reaction yield. It is pertinent to indicate that during the pre-trial experiment the optimum concentration of protein for the free CRL was found at 3 mg/mL, beyond which higher viscosity and lower formation of methyl oleate were observed. Therefore, this concentration of protein was used in the subsequent experiments dealing with the free CRL alone. However, whenever the same concentration of protein (3 mg/mL) was attempted for the CRL-MWCNTs, the amount of ballast was found to substantially increase, leading to higher viscosity of the reaction mixture. It has been indicated that higher viscosity would result in lower production of the ester. This is attributable to problems relating to the mass transfer, poor integration of the substrates (Miranda et al., 2014) and the non-active involvement of the excess biocatalysts in the reaction (Badgujar and Bhanage, 2015), therefore, such aspects must be kept at minimum. The fact that to sustain such amount of enzyme protein in this study, adequate amount of MWCNTs must also be added and this may not be favorable especially when it comes into costing. Considering such problem, lower concentration of protein (0.6 mg/mL) was used for the reactions catalyzed by the CRL-MWCNTs although the overall mass was maintained at 3 mg/mL for both the free CRL and CRL-MWCNTs in the reactions. Despite the lower amount of CRL protein used in the reactions catalyzed by the CRLMWCNTs, it is paramount to indicate that the overall yield of methyl oleate was evidently higher, up to 1.5-fold than that of the free CRL (3 mg/mL of protein). Since lower amount of protein used for the CRL-MWCNTs had resulted in higher production of methyl oleate, this aspect proves its applicability over the use of larger amount of the free CRL protein; an interesting factor for consideration especially in industrial settings.

3.2. Characterization of immobilized lipase (CRL-MWCNTs) The FT-IR spectra obtained for the MWCNTs, CRL-MWCNTs and F-MWCNTs are presented in Fig. 1. Qualitative evaluation through FT-IR analysis (Chopra et al., 2005) revealed the presence of specific functional groups in CRL-MWCNTs; those functional groups were not observed in both the MWCNTs and F-MWCNTs. Since the as-synthesized MWCNTs were less sensitive to FTIR, the number of observable peaks remained limited (Fig. 1a) and the findings are detailed below. The appearance of a band at 3432 cm−1 represented the O H stretching that can be assigned to ambient moisture that was tightly bound to the MWCNTs (Ramanathan et al., 2005). Another band at 1632 cm−1 was assigned to conjugated C O bonds, possibly attributed to induced internal defect of the MWCNTs (Fig. 1(a)). Following the acidic treatment (H2 SO4 :HNO3 ) of MWCNTs, the oxygenated acidic groups were introduced onto the carbon surface of the MWCNTs. This resulted in the emergence of a new band seen in the region of 1610−1730 cm−1 that corresponded with the existence of carboxylic groups (Tan et al., 2012). The band that would indicate the presence of the C O of the carboxylic group in MWCNTs (1632 cm−1 ) was observed

shifted to a lower wave number (1629 cm−1 ) in F-MWCNTs (Fig. 1(b)), consistent with the conjugation effect due to attachment of OH in the benzene rings both to the walls of the MWCNTs and/or to the benzene rings at the ends of the tube (Abdul Majid et al., 2010). The band representing the C O stretching of amide observed at 1634 cm−1 in CRL-MWCNTs (Fig. 1(c)), strongly indicated successful adsorption of the polypeptide chains of CRL onto MWCNTs. It was found that the contrasting spectra for MWCNTs and CRL-MWCNTs were observed within the range of 900−1200 cm−1 , indicating the spectral region for carboxylate functional groups (Fig. 1(c)). Hence, the presence of a medium intensity band at 1121 cm−1 for the CRL-MWCNTs (Fig. 1(c)) may be attributable to vibrations of C O carbohydrate moiety. Such observations were consistent with the findings reported by previous researchers (Prlainovic et al., 2013). The FT-IR results further indicated that after immobilization of CRL, the vibrations of C O of the carboxylate moiety were observed to be reduced, expanded, and moved to a lower frequency range (1121–1110 cm−1 ). Since such changes to the C O adsorption band were observed in the CRL-MWCNTs alone (Fig. 1(c)), it can be inferred that the immobilization process of CRL onto the surface of the F-MWCNTs has been successful (Prlainovic et al., 2013). Morphology of the as-synthesized MWCNTs, F-MWCNTs and CRL-MWCNTs observable using FESEM are presented in Fig. 2 (a1, b1, c1). Apparently, acidic treatment (H2 SO4 and HNO3 ) and immobilization of CRL protein onto MWCNTs had increased the diameter of the MWCNTs. It was observed that the diameters for the as-synthesized MWCNTs (Fig. 2a1), F-MWCNTs (Fig. 2b1) and CRL-MWCNTs (Fig. 2c1) ranged between 14 to 23 nm, 23.5 to 32.3 nm and 33.9 to 57.4 nm, respectively. It was found that the acid functionalization process altered the morphology of the as-synthesized MWCNTs (Fig. 2a1) whereby the F-MWCNTs (Fig. 2b1) were observably much shorter than the spiral structures of the assynthesized MWCNTs (Boncel et al., 2013). Refluxing in the H2 SO4 /HNO3 acid mixture had cut the tangled long fiber of the as-synthesized MWCNTs into shorter, open-ended pipes as well as producing numerous carboxylic groups at the open ends of the nanotubes (Fig. 2b1) (Tan et al., 2012; Liu et al., 1998). Following immobilization of the CRL protein, the diameter of the carbon nanotubes was further increased from 23.5−32.3 nm to 33.9−57.4 nm. Increment in the thickness of the sidewall of the F-MWCNTs after physical adsorption of enzymes confirmed the existence and successful attachment of CRL to the surface of F-MWCNTs. Likewise, the observed rough surfaces of the CRL-MWCNTs (Fig. 2c1) illustrated that the CRL protein had covered the surface of the nanotubes, reflecting the evenness of the enzyme ‘coating’ (Shah et al., 2007; Johan et al., 2014). Similar observations of uniform coating of other lipases to the MWCNTs via physical adsorption were also reported (Shah et al., 2007; Verma et al., 2013). The TEM analyses revealed differences in the morphology of the as-synthesized MWCNTs, F-MWCNTs and CRL-MWCNTs, respectively (Fig. 2). The F-MWCNTs were observably shorter and the fibers remained relatively defected (Fig. 2b2) as a result from a stronger acid treatment (H2 SO4 :HNO3 ) during functionalization process (Tan et al., 2012; Osorio et al., 2008). The open-ended pipes on the FMWCNTs hypothetically increase the surface area for the attachment of carboxylic groups at the open ends (Liu et al., 1998), thereby, renders the surface accessible for biological and chemical reactions (Tan et al., 2012). The TEM image of the CRL-MWCNTs (Fig. 2c2) depicted a layer of lipase protein that

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Fig. 1 – FTIR spectra of (a) as synthesized MWCNTs, (b) f-MWCNTs and (c) immobilized CRL-MWCNTs. coated surface of the MWCNTs, confirming successful immobilization of CRL. Pertinently, studies reported by Che Marzuki et al. indicated that for any weight of the CRL-MWCNTs used, the free CRL protein would constitute approximately 20% (w/w) of the developed biocatalysts (Che Marzuki et al., 2015).

3.3. Effect of reaction conditions on enzymatic production of methyl oleate 3.3.1.

Effects of reaction time

One of the parameters for measuring performance of an enzymatic reaction is the reaction time; a factor that determines the attainment of a reasonable percentage yield with minimum production cost (Syamsul et al., 2010). Profile of the reaction time for synthesizing methyl oleate catalyzed by the free CRL and CRL-MWCNTs nanoconjugates was monitored over a 12 h period and the data are presented in Fig. 3. It was found that the CRL-MWCNTs nanoconjugates afforded higher percentage of acid conversion (79.85%), a noteworthy 1.1-fold improvement over the free CRL (66.96%). Interestingly, the free CRL reached the maximum percent conversion of methyl oleate at 8 h of incubation when compared with the CRL-MWCNTs, beyond which the percent conversion started to decline (Fig. 3). In contrast, the esterification yields continued to increase for additional 3 h for the CRL-MWCNTs and the declining pattern was only observed after 11 h of reaction, hence producing generally higher quantities of such ester (Fig. 3). Methyl oleate was not produce when using the FMWCNTs alone (negative control). Pavilidis et al. (2012) reported that the activity of hydrolases was significantly enhanced upon immobilization on CNTs as opposed to other nanomaterials such as graphene oxide, indicating the suitability and biocompatibility of CNTs as supports for enzyme immobilization. In this study, immobilization onto F-MWCNTs has been observed to improve the catalytic properties of CRL due to the thermostability of MWCNTs at high

temperatures while providing a robust environment for CRL linkage (Raghavendra et al., 2010), in concurrence with a previous work reported by Min et al. (2012). Apart from conserving the structure of the CRL as well as preventing enzyme degradation during long reaction periods at high temperature (Ramos et al., 2014), the use of MWCNTs is providing a hydrophobic microenvironment suitable for CRL activity (Miranda et al., 2011). However, the decrease of conversion rate of the CRL-MWCNTs after 11 h could be attributed to the adverse alteration on the conformation of lipase active site and reduced lipase activity (Ramos et al., 2014). Under extensive incubation period, the free CRL are exposed longer to heat that increases propensity of un-raveling and unfolding of their active structure and subsequently gradual loss of enzymatic activity (Abdul Rahman et al., 2005). In addition, studies reported about better suitability of the non-polar solvents in lipase catalyzed synthetic reactions (Badgujar and Bhanage, 2015). Therefore, such a considerable yield of methyl oleate observed this present study can be attributed to use of the non-polar iso-octane as solvent. Further studies for exploring the suitability of other solvents for enhancing the production of this ester deserve consideration.

3.3.2.

Effect of surfactants

In general, the use of surfactants can either enhance the complexity of the reactions as well as equilibria involved in the enzymatic reaction or inhibit the enzyme itself (Delorme et al., 2011). They also indicated that depending on the differences in their chemical properties (i.e. nonionic, cationic, anionic and zwitterionic), surfactants are useful for modulating certain interactions. Therefore, the effect of surfactant on lipase interactions would depend strongly on the choice of both lipase and surfactant included in the reactions (Kamiya et al., 1995). It was observed that after 11 h of reaction, the Triton X-100 (non-ionic) yielded the highest percentage of acid conversion for CRL-MWCNTs (to 83.62%) when compared with that of free

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It appears that, in reactions supplemented with non-ionic surfactants (Triton X-100 and Tween 80), the activity of CRLMWCNTs was more pronounced than the free CRL. This may be due to the formation of soft interactions (hydrogen-bonding and hydrophobic interactions) between the surfactant and lipase that led to better catalytic activity than that of supplemented with cationic and anionic surfactants (Mahmood et al., 2013). A study focusing on the formation of mesoporous bioactive glasses indicated that supplementing the immobilization of CRL with nonionic surfactant would retain the catalytic activity of lipase (Min et al., 2012). It has been indicated that the non-ionic surfactants promote accumulation of the CRL-MWCNTs at interface, in so changing the surface charge density at the interfacial region via hydrophobic modification of enzyme (Mahmood et al., 2013). In addition, the hydrophobic surfaces of MWCNTs may further activate the catalytic activity of CRL due to its interaction with the amphiphilic alpha-helix peptide covering the active site (‘open lid’ effect) (Dave and Madamwar, 2008). Since the use of such surfactant would promote formation of higher number of lipase conformations (de María et al., 2006; Al-Duri et al., 1995), improvement in the yield of esterification may be attributable to the shift in the equilibrium of CRL-MWCNTs toward the open conformation by amphiphilic chains of the non-ionic surfactants. One possible explanation for the lesser activity in the reaction supplemented with anionic and cationic surfactants when compared with the nonionic surfactants is the formation of electrostatic interaction between the surfactants with the charge of the protein (Delorme et al., 2011). It has been reported that the strong interaction between the cationic head group within the molecule of a cationic surfactant with the negatively charged lipase would induce alteration in the three dimensional structure of lipase protein (Thakar and Madamwar, 2005; Shah et al., 2007).

3.3.3.

Fig. 2 – Field emission spectroscopy (1) and transmission electron microscopy (2) images of (a) as synthesized MWCNTs, (b) MWCNTs functionalized with HNO3 :H2 SO4 (1:3 v/v) and (c) CRL-MWCNTs, respectively. CRL (55.28%), demonstrating almost a 1.5-fold improvement in methyl oleate synthesis (Fig. 4). In addition, a remarkable increased in the yield of methyl oleate was observed in CRL-MWCNTs (78.38%) versus the free CRL (34.03%) catalyzed reactions, both supplemented with Tween 80 (non-ionic) surfactant (Fig. 4). In contrast, significant improvement in the yields of methyl oleate catalyzed by the free CRL and CRLMWCNTs supplemented with both the anionic (AOT and SDS) and cationic surfactants (HTAB and CTAB) was not observed (Fig. 4). Hence, Triton X-100 was found as the best surfactant in the enzyme assisted esterification for producing methyl oleate in this present study.

Thermostability

Enzymes are sensitive toward harsh environments especially at high temperatures (Raghavendra et al., 2010). Therefore, alteration of spatial structure of many enzymes that renders denaturation and loss of activity (Sheldon and Van Pelt, 2013) may be possible, should the reactions and/or productions occurring at unfavorable conditions. As lipases are involved in fat-splitting and synthetic reactions related to hydrophobic substrates, application of high temperatures is often required to keep such solid/semi solid fats in their liquid forms. Therefore, specific studies focusing on the effect of temperature on the activity of an enzyme would provide empirical evidence on its optimum working temperature and tolerance to high temperatures (Raghavendra et al., 2013). Such aspects may prove pertinent for consideration in industrial as well as laboratory settings. This study evaluated the thermal stability of free CRL and CRL-MWCNTs nanoconjugates and data consolidated revealed an appreciable efficiency for CRL-MWCNTs in catalyzing the production of methyl oleate over a wide range of temperature settings. The CRL-MWCNTs nanoconjugates were noticeably stable at 40–60 ◦ C, affording significantly higher percentages of esterification than the free CRL (Fig. 5). At 40 ◦ C, the free CRL and the CRL-MWCNTs nanoconjugates afforded 74.25% and 45.16% of methyl oleate yields, respectively. The high percentage of esterification attained by the CRL-MWCNTs nanoconjugates can be attributed to the enhanced rigidity of its structure, owing to the multipoint interactions (hydrogen

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Fig. 3 – Effect of reaction time during esterification reaction for the synthesis of methyl oleate with free lipase from Candida rugosa and immobilized C. rugosa lipase onto MWCNTs. Reaction condition: molar ratio acid:alcohol 1:1, solvent: iso-octane, enzyme loading: 3 mg/ml (w/v), 200 rpm.

Fig. 4 – Effect of surfactant during esterification reaction for the synthesis of methyl oleate with free lipase from Candida rugosa and immobilized C. rugosa lipase onto MWCNTs. Reaction condition: molar ratio acid:alcohol 1:1, solvent: iso-octane, enzyme loading: 3 mg/ml (w/v), 200 rpm, time: 1 h. bonding, ionic bonding and van der Waals) of CRL to the support matrix (Min et al., 2012; Asuri et al., 2006). The immobilization process may preserve the enzyme activity even at higher temperature, while providing a number of processing improvements. The improvements in the process range from reduced risk of contamination, lower viscosity to improved transfer rates and substrate solubility (Mahmood et al., 2013; Delorme et al., 2011; Asuri et al., 2006; Nirprit and Jagdeep, 2002) due to a robust environment for enzyme attachment by MWCNTs (Raghavendra et al., 2013). However, a considerable decrease in the activity of both CRL and CRL-MWCNTs was observed when the reaction temperature was kept at 70 ◦ C, attaining only 24.19% and 42.11% of methyl oleate production, respectively. The steep declined in esterification beyond 60 ◦ C can be attributed to probable loss of enzyme active structure due to breakage of the multipoint interactions between CRL and the MWCNTs (Raghavendra et al.,

2013), making them less rigid and more susceptible to protein unfolding.

3.3.4.

Reusability

Considering that the ability to repeatedly use a catalyst in a reaction may have profound economic importance, such aspect was investigated on the newly developed CRL-MWCNTs in producing methyl oleate. The CRL-MWCNTs nanoconjugates were washed with hexane and reused in fresh reaction medium. Despite its benefit of easy operation that does not require harsh reaction condition as well as additive during the immobilization process, physical adsorption was chosen due to easy regenerations of matrix for enhanced reusability (Zhao et al., 2015). In general, the CRL-MWCNTs nanoconjugates showed better stability over the free CRL, attributable to their physically adsorbed proteins onto the nanoscale support matrix, thereby

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Fig. 5 – Thermostability studies for the esterification reaction for the synthesis of methyl oleate with free lipase from Candida rugosa and CRL-MWCNTs. Reaction condition: molar ratio acid:alcohol 1:1, solvent: iso-octane, enzyme loading: 3 mg/ml (w/v), 200 rpm, time: 11 h. aiding in preserving or enhancing enzyme bioactivity (AlDuri et al., 1995). However, the esterification yield plunged below 50% beyond the 5th cycles of reaction (Fig. 6), which may be caused by accumulation of excess water as byproduct of the esterification (Raghavendra et al., 2010). Nevertheless, the findings reported in this study is considered comparable to previous work detailing about the CRL coated in CTAB immobilized in sodium bis-2-(ethylhexyl)sulfosuccinate based organogels that were significantly unaffected up to 5 cycles of reaction (Dandavate and Madamwar, 2008). Moreover, Miranda et al. had reported reusability up to 5 cycles following physical adsorption of the Thermomyces lanuginosus lipase on mesoporous poly-hydroxybutyrate particles with the lipase retaining approximately 70% of its activity (Miranda et al., 2014). Badgujar and Bhanage advocated good retention of lipase activity by such immobilization approach, reported that the Burkholderia cepacia lipase was efficiently recycled up to six times following immobilization on polylactic acid,

chitosan and polyvinyl alcohol matrix hybrid (Badgujar and Bhanage, 2015). Although the reusability of the developed CRL-MWCNTs may not be particularly remarkable, nonetheless, the utilization the CRL-MWCNTs as biocatalysts is possibly more economical due to the effortlessness immobilization technique and considerably low amount of enzyme amount that was used. In addition to the ability of the to synthesize a relatively high yield of methyl oleate, the activity of the CRL-MWCNTs biocatalysts can simply be restored using a facile two-step method involving acid treatment and CRL re-immobilization by stirring, prior to re-use (Mohamad et al., 2015b). In actual fact, in comparison to the cost of commercially available enzymes to which industries are paying currently, namely, >USD900 and >USD400 for a 10 g of Novozyme and Lipozyme (Mohamad et al., 2015b), respectively, CRL-MWCNTs biocatalysts is relatively cheap and the larger amount of the biocatalysts can be employed to catalyzed larger reaction batches. However, the reluctance of many industrial manufacturers of commercial esters to switch over to the enzymatically based methods is expected. An important point to highlight here that the mass of the free CRL in the CRL-MWCNTs used to catalyze the esterification reaction was significantly lower than that of the free CRL; but yet the CRL-MWCNTs yielded considerably higher conversion of methyl oleate than reactions catalyzed by the free CRL. Such low concentration of CRL immobilized on the surface of MWCNTs to afford considerably high yield of methyl oleate is considerably desirable in terms of cost saving and feasibility to produce cheaper biocatalysts.

4.

Conclusion

This study showed that the physically immobilized CRLMWCNTs nanoconjugates were more efficient in the esterification production of methyl oleate when compared with the free CRL. The physically adsorbed method of enzyme immobilization afforded much benefits i.e. improved stability of the free CRL and facilitated easy recovery of the CRL in nanoscales supports matrix. Hence, the CRL-MWCNTs nanoconjugates (1)

Fig. 6 – Reusability studies for esterification reaction for the synthesis of methyl oleate with immobilized Candida rugosa lipase onto MWCNTs. Reaction condition: molar ratio acid:alcohol 1:1, solvent: iso-octane, enzyme loading: 3 mg/ml (w/v), 200 rpm.

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demonstrated the optimum reaction time of 11 h, (2) catalyzed the esterification reaction at its best with the presence of Triton X-100 as surfactant, (3) were noticeably stable at 40–60 ◦ C and (4) could be reused for up to 5 cycles before a significant decline in activity was observed. Therefore, it can be construed that the CRL-MWCNTs nanoconjugates developed here were promisingly cheap and useful biocatalysts for producing methyl oleate for industrial applications.

Acknowledgments This work was supported by the Exploratory Research Grant Scheme from the Ministry of Higher Education Malaysia (ERGS R.J130000.7826.4L132). We would also like to acknowledge valuable help and suggestions provided by our colleagues.

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