- Research article
- Open Access
Enhancement of lipase activity in non-aqueous media upon immobilization on multi-walled carbon nanotubes
© Shah et al; 2007
Received: 04 August 2007
Accepted: 29 November 2007
Published: 29 November 2007
Immobilization of biologically active proteins on nanosized surfaces is a key process in bionanofabrication. Carbon nanotubes with their high surface areas, as well as useful electronic, thermal and mechanical properties, constitute important building blocks in the fabrication of novel functional materials.
Lipases from Candida rugosa (CRL) were found to be adsorbed on the multiwalled carbon nanotubes with very high retention of their biological activity (97%). The immobilized biocatalyst showed 2.2- and 14-fold increases in the initial rates of transesterification activity in nearly anhydrous hexane and water immiscible ionic liquid [Bmim] [PF6] respectively, as compared to the lyophilized powdered enzyme. It is presumed that the interaction with the hydrophobic surface of the nanotubes resulted in conformational changes leading to the 'open lid' structure of CRL. The immobilized enzyme was found to give 64% conversion over 24 h (as opposed to 14% with free enzyme) in the formation of butylbutyrate in nearly anhydrous hexane. Similarly, with ionic liquid [Bmim] [PF6], the immobilized enzyme allowed 71% conversion as compared to 16% with the free enzyme. The immobilized lipase also showed high enantioselectivity as determined by kinetic resolution of (±) 1-phenylethanol in [Bmim] [PF6]. While free CRL gave only 5% conversion after 36 h, the immobilized enzyme resulted in 37% conversion with > 99% enantiomeric excess. TEM studies on the immobilized biocatalyst showed that the enzyme is attached to the multiwalled nanotubes.
Successful immobilization of enzymes on nanosized carriers could pave the way for reduced reactor volumes required for biotransformations, as well as having a use in the construction of miniaturized biosensensor devices.
There has been considerable interest in the use of nanomaterials in biochemical applications [1–4], in particular the immobilization of proteins on nanomaterials [3–8], with nanotubes having lately attracted considerable attention [3, 5–8]. Both single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs) have been used as matrices.
Immobilization is carried out using a variety of approaches [9, 10], including adsorption [5, 7] and covalent immobilization [6, 8], techniques on which various studies have been carried out. A key parameter in any immobilization is the retention of the protein's biological activity. In this respect adsorption is assumed to be superior to other approaches, which generally utilize harsher conditions [9, 10]. In most cases reported hitherto [5–8], the retention of enzymic activity upon immobilization on nanotubes, has not been high, with reports describing enzymes retaining 1–20% of their biological activity [7, 8]. This is similar to the early work on macroscopic matrices and it is expected that better results would be obtained once better experience/strategies with respect to nanotubes (as matrices) were in place. It is noteworthy, however, that two recent studies report retention of biological activities over the range 55–100% [11, 12].
There do not, however, appear to be any reports on the evaluation of enzymes immobilized on carbon nanotubes in non-aqueous media, which, both in the contexts of biocatalysis and bioanalysis, offer great potential [13–17].
This study reports the preparation and characterization of a high activity Candida rugosa lipase (CRL) immobilized on multi-walled carbon nanotubes by adsorption. Numerous applications have been found for lipases in non-aqueous media [13–17], with the Candida rugosa lipase one of the most frequently used in biotechnology . Yet there is another reason for which a lipase was chosen for this study. Most lipases have a 'lid', which opens up upon interaction with hydrophobic surfaces, enabling them to assume an 'active conformation'. Given that the surface of nanotubes is hydrophobic , the choice of a lipase was considered worthwhile in developing a nanobiocatalyst with high activity.
Results and Discussion
Adsorption of lipases on MWNT
Immobilization of Candida rugosa lipase on MWNT. Different units of CRL were immobilized on 1 mg of MWNTs. The hydrolytic activity was determined on MWNTs, supernatant and washings. All experiments were carried out in duplicate and the results within each pair differed by < 5%.
Supernatant + washings
Expressed activity (B)
Theoretical activity (A)
Immobilization of Candida rugosa lipase on MWNTs in presence of BSA. CRL (3 units) were immobilized on 1 mg of MWNTs in presence of varying amount of BSA. The hydrolytic activity was determined on MWNTs, supernatant and washings. All experiments were carried out in duplicate and the results within each pair differed by < 5%.
Supernatant + washings
Expressed activity (B)
Theoretical activity (A)
Catalytic performance in non-aqueous media
Initial transesterification rates exhibited by Candida rugosa lipase immobilized MWNTs in different reaction media. All experiments were carried out in duplicate and the results within each pair differed by < 5%.
Initial rates (μmoles min-1 h-1)
Immobilized on MWNTs (In presence of BSA)
Immobilized on MWNTs
Immobilized on MWNTs (In presence of BSA)
Immobilized on MWNTs
Interactions with the hydrophobic surfaces of nanotubes are expected to result in the 'open lid', as well as more active lipase conformations. Another observation was that in such media, the lipase immobilized in the absence of BSA gave somewhat higher initial rates. This may be explained by the fact that enzymes in nearly anhydrous media are extremely rigid molecules. In fact, it has been reported that some enhancement of flexibility either through urea denaturation  or three-phase partitioning  results in higher enzymic biological activity. It is not unlikely that the conformational changes leading to limited denaturation as a result of interactions with the carbon nanotube had acquired the necessary conformational flexibility. The interactions of the enzyme with the carbon nanotube in the presence of BSA, on the other hand, had a very close to native, and hence normal, rigid structure.
Lately, ionic liquids have also emerged as an alternative reaction medium for enzyme catalysis [24, 25]. Described as 'green solvents' and 'designer solvents', these room temperature ionic liquids release practically no volatile organic compounds into the atmosphere [24–26]. The water immiscible ionic liquids have generally been reported as being better reaction media than water miscible ionic liquids for enzyme catalysis [24, 25], with [Bmim] [PF6] being one of the most frequently used water immiscible ionic liquids for enzyme catalysis [24, 25, 27, 28].
The same reaction, that is, formation of butylbutyrate, was also carried out in [Bmim] [PF6]. The lipase immobilized on MWNT (in the absence of BSA) showed a 14-fold increase in the initial rates of transesterification over those shown by pH tuned lyophilized powders (Table 3).
Biotransformations with the lipase immobilized on MWNT
Enantioselectivity of lipase immobilized on MWNT
Transesterification of (±) 1-phenyl ethanol in ionic liquid with Candida rugosa lipase immobilized on MWNTs. All experiments were carried out in duplicate and the results within each pair differed by < 5%.
CRL adsorbed on MWNTs
CRL adsorbed on MWNTs
CRL adsorbed on MWNTs
TEM of CRL immobilized on MWNT
There is increasing interest in the miniaturization of technological devices, with studies on the immobilization of enzymes on nanotubes, part of this trend. The present work illustrates that lipase-nanotube systems have some unique features, for instance immobilization on nanotubes actually led to considerable enhancement of enzymic activity in non-aqueous media. In this study, we have reported for the time the performance of an enzyme on carbon nanotubes in non-aqueous media.
CRL was a gift from Amano Enzyme Inc., Nagoya, Japan. Multi-walled carbonnanotubes (OD = 10–30 nm, ID = 3–10 nm, length = 1–10 μm, purity 90 + %, Cat. No. 636517) and p-Nitrophenylpalmitate (p-NPP) were obtained from Sigma Chemical Co., St. Louis, USA. The ionic liquid was from Acros Organics, USA. The purity of [Bmim] [PF6] as specified by the vendor was 99.6% (by HPLC) with water content of 0.05% v/v by Karl-Fisher. All solvents were of low water grade (< 0.005% water (vv-1)) and obtained from J. T. Baker, USA. These solvents were further dried by gentle shaking with 3 Å molecular sieves (E. Merck, Mumbai, India).
Adsorbtion of Lipase on MWNTs
The MWNTs (1 mg) were dispersed by sonication (Elma transsonic digital ultrasonic unit, model T 490 DH, Germany) at a fixed frequency of 40 kHz and at 110 W power rating) in 0.5 ml of 50 mM phosphate buffer pH 7.0 for 30 min, followed by addition of 0.5 ml of the lipase solution (containing varying amount of the lipase). The mixture was kept at 20°C with constant shaking at 200 rpm for 3 h. After, incubation, mixture was centrifuged at 8,000 g for 10 min at 20°C. The supernatant was removed and MWNTs were washed with 1 ml of 50 mM phosphate buffer at pH 7.0 until no hydrolytic activity was detected in the washings.
Ethyl butyrate (60 mM) and n-butanol (120 mM) were added to a vial containing 1 ml of hexane/ionic liquid followed by addition of free lipase/lipase adsorbed (10 mg) on MWNTs. The reaction mixture was incubated at 35°C at 200 rpm. The aliquots were withdrawn at different time periods and analyzed by GC. A control was run in which MWNTs were added without lipase.
The protein concentration was determined according to Bradford's method, using bovine serum albumin as the standard protein .
The hydrolytic activity of lipase was monitored by following the rate of hydrolysis of p-nitrophenylpalmitate spectrophotometrically at 410 nm .
The alkylesters were analyzed on Agilent Technologies 6890 N network GC systems, USA, with a flame ionization detector. The capillary specifications were: a column length of 30 m, internal diameter of 0.25 mm. A nitrogen carrier gas at a constant pressure of 4 Kg/cm2 was used. The column oven temperature was programmed over the range of 150 to 250°C at 10°C min-1 with injector and detector temperatures at 240 and 250°C, respectively.
Kinetic resolution of (±)-1-phenylethanol
The alcoholysis between (±)-1-phenylethanol (1 mmol) and vinyl acetate (1 mmol) was carried out in 1 ml ionic liquid followed by addition of CRL/CRL immobilized on MWNTs at 30°C with constant shaking at 250 rpm. The samples were taken out at different time intervals and analyzed by HPLC. A control was run in which MWNTs were added without lipase.
For HPLC analysis, 50 μl of the reaction mixture was extracted with 500 μl n-hexane-propan-2-ol (97.5:2.5) mixture. The extract was analyzed by HPLC using Chiracel OD12 RH column (Diacel, Japan). The eluent consisted of 96.5% (vv-1) n-hexane, 3% (vv-1) propan-2-ol and 0.5% (vv-1) ethanol with a flow rate of 1 ml min-1 and detection was carried out with a UV detector at 254 nm.
Transmission electron microscopy (TEM)
Transmission electron micrographs were recorded on a FEI Philips Tecnai F20 (200 kV, FEG) instrument. A drop of MWNTs/lipase adsorbed on MWNTs dispersed in distilled water was placed on a copper grid and dried.
This work was supported by funds obtained from Department of Science and Technology, Govt. of India (DST) in the form of a 'Core group grant for applied Biocatalysis'.
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