Enzyme Immobilization on Carboxyl-Functionalized Graphene Oxide

Subscriber access provided by University of British Columbia Library

Article

Enzyme Immobilization on Carboxyl Functionalized Graphene Oxide for Catalysis in Organic Solvent Qingzhong Li, Fei Fan, Yang Wang, Wei Feng, and Peijun Ji Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/ie400558u • Publication Date (Web): 22 Apr 2013 Downloaded from http://pubs.acs.org on April 28, 2013

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Industrial & Engineering Chemistry Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Industrial & Engineering Chemistry Research

Enzyme Immobilization on Carboxyl Functionalized Graphene Oxide for Catalysis in Organic Solvent Qingzhong Li+, Fei Fan+, Yang Wang, Wei Feng*, Peijun Ji* Department of Biochemical Engineering, Beijing University of Chemical Technology, Beijing, 100029, China

ABSTRACT Carboxyl functionalized graphene oxide (GO-COOH) was utilized to immobilize lipase. The spectra of FTIR, UV-vis and XPS were measured to characterize the lipase immobilization. At the optimal temperature 40 °C, the immobilized lipase retains 80% of the hydrolytic activity of the native lipase. For catalyzing the enantioseletive reaction in the organic solvent heptane, at 50 °C (optimal), the catalysis efficiency of the immobilized lipase is 1.6 times that of native lipase, and the immobilized lipase retains the selectivity of the native lipase. This work demonstrates that graphene oxide is a suitable support for immobilization of lipase for catalysis in organic solvent.

1. Introduction Lipase is produced in high yields and show high chemo- and stereo-selectivity,1 they have been widely used as biocatalysts in organic synthesis. Generally, the catalytic activity displayed by lipases in organic solvents is lower than in water. The reason for which it is thought to generate closed lids.2 The conformational rearrangements of the lid have been reported to be closely related to the interfacial activation of lipases.3 Upon interaction with a hydrophobic interface, the lid undergoes movement in such a way that the active site is exposed, providing free access for the substrate.4

* Corresponding author. +86 10 64446249; Email: [email protected]; [email protected] + Both authors contributed equally to this work.

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 24

To improve lipase activity and stability, lipases have generally been studied with the enzymes immobilized on a solid support.5,6 Immobilization of enzymes can enhance catalytic activity, stability and selectivity as well as facilitate recycling the enzymes.7 Various materials have been utilized for enzyme immobilization, including hydrophobic magnetic nanoparticles,8 carbon nanotubes,9 silica-based material,10 cellulose-based membrane,11 polymers12 and resins.13,14 Graphene is a one-atom-thick 2D carbon nanomaterial with extraordinary electronic, thermal, and mechanical properties.15 Graphene oxide (GO) is a water dispersible version of graphene presenting oxygen-containing functional groups such as hydroxyl, carboxyl, and epoxy groups. Graphene oxide and functionalized graphene nanomaterials16-18 have been studied for the immobilization of enzyme, such as glucose oxidase. The GO-immobilized enzymes so far studied are for electrochemical

applications,

they

are

in

aqueous

reaction

media.18 The

physicochemical properties and structure15,19 suggest graphene has great potential for applying in non-aqueous reaction media. In this work, we explore the application of graphene oxide in biocatalysis in organic solvent. Carboxyl functionalized grapheme oxide is utilized as the support to immobilize Yarrowia Lipolytica lipase. The resulting immobilized lipase has been demonstrated to exhibit a high efficiency for the resolution of racemic compound in organic solvent. The GO-immobilized lipase also showed good reproducibility and operation stability, suggesting great potential for practical applications, especially for kinetic resolution of racemic mixtures in organic media.

2. METHODS 2.1. Materials. The lipase was obtained from Beijing Key Laboratory of Bioprocess. The lipase was purified by ion exchange chromatography, as described in


Page 3 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the paper.19 The analysis on SDS-PAGE showed that the lipase is pure, and the lipase has

a

molecular

weight

of

about

N-ethyl-N-(3-(dimethylamino)propyl)

38

kDa.

(R,

carbodiimide

S)-1-Phenyl

hydrochloride

ethanol, (EDC),

2-(N-morpholino) ethanesulfonic acid (MES), N-hydroxysuccinimide (NHS), was purchased from Sigma-Aldrich Chemical Co., Shanghai. Heptane and vinyl acetate, sulfuric acid, hydrochloric acid, and nitric acid were purchased from Sinopharm Chemical Reagent Co., Beijing. Natural flake graphite (42 m particle size) was provided by Heilongjiang Aoyu Graphite Co., China. All materials were used as received. 2.2. Synthesis of carboxyl functionalized graphene oxide. Graphene oxide (GO) was prepared according to the method as described in literature20 from natural flake graphite. Typically, sulfuric acid (80 mL) and nitric acid (40 mL) were stirred together in an ice bath for 10 min. Graphite particles (5 g) were added to the acids mixture under vigorous stirring, and the suspension was cooled for 30 min. Potassium chlorate (55 g) was slowly added with a flow rate of 1.0 g/min, while keeping the reaction vessel inside an ice bath, making sure the temperature not exceeding 20 °C. Oxidation was carried out for 100 h. The suspension was washed with an aqueous hydrochloric acid solution (10 vol %), and then washed repeatedly with deionized water until neutral pH. The reaction mixture was precipitated by centrifugation at 11813 × g and washed with distilled water. The precipitation was then dispersed in water by sonication in a sonicating water bath, which is 250W rated with a 40 kHz transducer. Then centrifugation at 5250 × g for 30 min was done for three times to remove small GO pieces. The GO was further oxidized in the 3:1 concentrated H2SO4:HNO3 mixture for 3 h in an ice bath under sonication. After these treatments, the resulting product carboxyl



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

functionalized graphene oxide (GO-COOH) was neutralized with dilute hydrochloric acid and purified by repeated rinsing and centrifugation until the product was well-dispersed in deionized water. Then, the GO-COOH suspension was dialyzed against distilled water for over 48 h to remove any ions. 2.3. Lipase covalently immobilized onto GO-COOH. The covalent attachment of lipase onto GO-COOH is based on the method described in the article.21 Scheme S1 schematically presents the covalent approach of lipase immobilization. Brief description is as following. 200 mg of GO-COOH were dispersed in 200 ml of MES buffer (50 mM, pH 6.2), and then the mixture was added to the solution of NHS in MES buffer. The mixture was sonicated for 15 min followed by addition of EDC (20 mmol/L). The resulting mixture was shaken at 200 rpm for 60 min. The activated GO-COOH solution was then filtered through a polycarbonate membrane (0.45 µm) and rinsed thoroughly with MES buffer to remove excess EDC and NHS. The filtered GO-COOH was transferred to a solution of lipase (5.0 mg/ml) and sonicated to redisperse the GO-COOH. The mixture was then shaken at 150 rpm and 25 °C for 10 hours. After conjugation, the mixture was freeze centrifuged (4 °C) for 10 min at 5250 × g and the supernatant was removed. Typically, six washes were performed, with fresh buffer added each time to remove unbound lipase. The amount of immobilized enzyme was obtained by standard BCA protein assays (Pierce Biotechnology) of the original lipase solution, the supernatants, and washing solutions after immobilization, respectively. The amount of lipase immobilized was 0.75 ± 0.04 mg lipase/ mg GO-COOH. 2.4. Spectrum measurement. Shimadzu UV2550-PC spectrophotometer was used to measure the ultraviolet-visible spectra a using 1-cm-path length quartz cuvette. Spectra were collected within a range of 190-800 nm.


Page 4 of 24

Page 5 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


XPS spectra were acquired using a Thermo VG ESCALAB250 X-ray photoelectron spectrometer, which was operated at the pressure of 2 × 10-9 Pa using Mg Ka X-ray as the excitation source. Analysis of the data was carried out with Thermo Avantage XPS software. All XPS spectra were referenced to the main C 1s hydrocarbon peak at 284.9 eV binding energy. Raman spectroscopy measurements were recorded using a Renishaw InVia equipment (514.5 nm, Elaser = 2.41 eV). The X-ray diffraction (XRD) patterns were obtained with a powder diffractometer of Rigaku D/Max 2500 VBZ+/PC using a Gu target at 35kV, 30mA. The powder diffractograms were operated at a scan rate of 2θ = 1°/min from 2θ=5° to 2θ = 90°. 2.5. Hydrolytic activity determination using olive oil. The concentration of the native lipase was 0.15 mg/ml, and that of the GO-lipase was 0.35 mg/ml. The enzymes were dissolved in 50 mM phosphate buffer (pH 8) to make the enzyme solutions. Olive oil (4 ml) was added to 8 ml of the enzyme solutions, and the hydrolysis reactions were carried out in an incubator at 200 rpm and, in a temperature range of 35 °C to 60 °C. Each reaction was carried out for 15 min. And then, toluene was used to stop the reaction and extract the fatty acid. After centrifugation at 1313 × g for 10 min, the free fatty acid was determined in the upper organic phase. The activities of the native lipase and GO-lipase for the olive oil hydrolysis were measured using a Shimadzu spectrophotometer (model UV 2550) at an absorbance of 715 nm.22 2.6. Enantioselective resolution of (R,S)-1-phenyl ethanol. Scheme S2 presents the reaction catalyzed by lipase.23 Racemic (R, S)-1-phenyl ethanol (25 mol/ml) and acyl donor (100 mol/ml) were mixed with 10 ml heptane in a reaction vessel of 50 ml. 21.43 mg of the native lipase and 50.0 mg of GO-lipase were separately used for



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 24

the catalysis. The vessel was tightly sealed. The free and immobilized lipases were dispersed in the organic solvent by shaking at 100 rpm at a certain temperature. The reaction temperatures were from 35 °C to 60 °C, and the temperature increment was 5 °C. At each temperature, the reaction was carried out three times. The enantiomeric excess and conversion were analyzed by high-performance liquid chromatography (HPLC). The HPLC analysis was performed in triplicate using Shimadzu 10AVP instrument equipped with a UV detector on a Chiracel OJ-H column (0.46 mm diameter, 250 mm long, 5µm, Chiracel). The mobile phase consists of hexane–isopropyl alcohol at 99:01 (v/v) with a flow rate of 1.0 ml/min. At 220 nm, the substrate and product were detected. The retention time of (R)-1-phenylethyl acetate is 9.5 min and that of (S)- and (R)-alcohol enantiomers are 27.4 and 29.1 min, respectively.

The

enantiomeric

excess

(ees)

is

defined

as

the

ratio

ees  [S ]  [ R] [S ]  [ R] 100% , where [R] and [S ] are the concentrations of R- and S-alcohol, respectively. The conversion is calculated by ees/ ees  eep  ,

eep  [ P]  [Q] [ P]  [Q] , where [P] and [Q] are the concentrations of R- and Sphenylethyl acetate, respectively. 3. RESULTS 3.1. Preparation of GO-COOH. The TEM image provides more detailed morphological information on the resulting GO (Figure 1). It exhibits the typical wrinkle morphology of GO and is exfoliated into single or very thin layers. Figure 2 shows the XRD patterns of graphite and GO. Graphite shows a sharp characteristic peak at 2θ = 26.2°. After oxidation, the sharp diffraction peak in graphite disappeared, GO exhibited a new flat diffraction peak at 2θ = 22.9°, indicating the complete oxidation of graphite. This is a prerequisite to successfully exfoliating GO and obtaining single-layer or few-layer grapheme sheets.12, 24


Page 7 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Graphite and GO are further characterized with Raman and XPS measurements. In Raman spectra of carbon materials, such as carbon nanotubes and graphite, the G-band, in the 1500-1600 cm-1 region, results from the tangential C-C stretching vibrations.25,26 In the spectra of graphite (Figure 3), the G-band peaks at 1580 cm-1. After oxidation, this band shifts by 5 cm-1 from 1580 to 1585 cm-1. The disorder peak, also known as the D-band, can be found in the 1300-1400 cm-1 region.21 This peak is attributed to scattering from sp2 carbons containing defects. The strength of this peak is related to the amount of disordered graphite and the degree of conjugation disruption in the graphene sheet. The D-band of graphite peaks at 1350 cm-1. It is noted that, after oxidation, the D-band shifts by 4 cm-1 from 1350 to 1354 cm-1. The intensity ratio of D to G bands of GO is much greater than those of graphite, indicating oxidation of graphite. The C1s XPS spectra of the GO (Figure 4) suggest that oxygen atoms are bound to surface carbon mainly through C-OH (285.4 eV), C=O (286.4 eV) and O-C=O (288.8 eV) bonds. 3.2. Characterization of the immobilized lipase. The solution of the GO-lipase was monitored by UV-vis spectroscopy, using a Shimadzu spectrophotometer (model UV 2550) operated at a resolution of 1 nm. Figure 5 presents the UV-vis spectra. The GO-COOH exhibits a flat line (black). The native lipase shows a shoulder peak around 253 nm. The GO-lipase presents a peak absorbance at 253 nm, which is due to the immobilized lipase. Figure 6 shows the XPS spectra. Compared to that of GO, in the spectrum of GO-lipase, the nitrogen from the lipase is presented, confirming the immobilization of lipase. Figure 7 shows XRD patterns for the native lipase, GO-lipase, and GO-COOH. The XRD pattern of the GO-lipase (in olive) presents peaks around 2θ = 23.08° and 2θ = 31.92°, while the native lipase exhibits much more peaks (in blue). The results



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

indicate that the immobilization of lipase on GO-COOH has changed the crystalline form of lipase. Circular dichroism (CD) spectra were analyzed for the change of secondary structure of the lipase immobilized on GO-COOH. The CD spectra (not shown) demonstrated that 73.6% of the a-helix content of the native lipase are retained. CDPro software (http://lamar.colostate.edu) was used for analyzing the circular dichroism spectra of lipase. 3.3. Hydrolytic activity determination using olive oil. Figure 8 shows the effect of temperature on the hydrolytic activity retained by GO-lipase. At optimal temperature 40 °C, the immobilized lipase retained 80 % of the hydrolytic activity of the free lipase. The loss of hydrolytic activity is due to the covalent attachment of lipase on GO, which caused the secondary structure change of the lipase. The activity loss can be compensated by using the immobilized lipase repeatedly (4 cycles have been tested with small decrease in the activity). This demonstrates that the GO-bound lipase has a greater hydrolytic potential than the free lipase. 3.4. Catalyzing the resolution of (R,S)-1-Phenyl ethanol in heptane. For the resolution of (R,S)-1-Phenyl ethanol by lipase, (R)-1-Phenyl ethanol is converted to (R)-1-Phenylethyl acetate, while (S)-1-Phenyl ethanol is unconverted, as illustrated in scheme S1. Therefore, for this reaction, the maximum conversion is 50%. The experimental results of the resolution of (R, S)-1-Phenyl ethanol with the native lipase and the immobilized lipase at different temperatures are presented in Figure S1. The reaction time is 38 h. In each experiment, the amount of the native lipase used was equivalent to that of lipase immobilized on GO-COOH. For the catalysis by GO-lipase, the conversions reached 49.8 % at 50 °C. For the native lipase catalysis, the conversion was increased from 35 °C to 55 °C. The change of conversion and


Page 8 of 24

Page 9 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


enantiomeric excess with reaction time is shown in Figure 9. As can be seen, the conversions achieved by the GO-lipase are much higher than that by the native lipase. The results demonstrate that the immobilization of lipase on GO can greatly improve the catalysis efficiency for the resolution of (R, S)-1-Phenyl ethanol in the organic solvent. The eep values of the immobilized lipase are over 99 %, which are comparable to that of the naive lipase (>99 %). This means that the immobilized lipase has an excellent resolution selectivity for (R)-1-phenyl ethanol. Two control experiments were carried out. In one control experiment, GO was used to resolve (R, S)-1-Phenyl ethanol in heptane. After 40 h, it was found that GO can not resolve (R, S)-1-Phenyl ethanol. In another control experiment, the conjugate of GO-lipase was added into the solution of (R, S)-1-Phenyl ethanol in heptane, but with no vinyl acetate. This control experiment is to measure the adsorption of (R, S)-1-Phenyl ethanol on the GO in the presence of immobilized lipase. After 40 h, it was found that 1.56 % of (R, S)-1-Phenyl ethanol was adsorbed by the conjugate. This adsorption value has been taken into account when calculating the conversions. The results in Figure S2 are for the catalysis in the organic solvent. The Vmax and Km values were determined by the Lineweaver−Burk plot derived from a series of experimental determinations of the enzyme activity. The kinetic parameters of the immobilized lipase were compared to that of the free lipase. The immobilized lipase demonstrated Km value slightly lower than that of the free lipase. It means that the lipase after immobilization improves its affinity towards the substrate, possibly because the immobilization promotes the lid opening as suggested by the molecular dynamics simulation.27 The Kcat value for the immobilized lipase was higher than that of the free lipase. The Kcat/Km ratio is a measure of the catalytic efficiency of enzymes and was used to compare the apparent kinetic parameters of the immobilized



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

lipase (Table S1). Compared to the native lipase, a higher Kcat/Km value was observed for the immobilized lipase. This result supports the observation of Figure 9, confirming that in the organic solvent system the immobilized lipase shows increased catalytic specificity. It can be noticed that the immobilized lipase has a larger specificity constant expressed by the ratio Vmax/Km. It is demonstrated that the immobilized lipase shows a higher catalytic specificity than the free lipase in organic solvent. CD spectra show that the lipase loses its secondary structure after immobilization, however the immobilized lipase exhibits a higher catalysis efficiency. The reason can be explained by the above kinetic parameters obtained.

4. CONCLUSIONS The lipase was immobilized on GO-COOH through the reaction of the amino groups of the lipase with the carboxylic acid groups on GO-COOH. However, due to the strong affinity and spontaneity of adsorption, the adsorption of the lipase on GO-COOH is also possible.27 The basal surfaces GO-COOH contain hydroxyl (-OH), epoxide (-O-), and carboxyl (-COOH) functional groups. The basal planes also include unmodified graphenic domains. Thus the GO-COOH sheets are capable of hydrophobic interactions with the residues of lipases, which are covalently immobilized and adsorbed. The immobilization caused the loss of the secondary structure of the lipase. As a result, the hydrolytic activity of the immobilized lipase is reduced compared to that of free lipase. While in the organic solvent the enzymatic activity by the immobilized lipase is significantly larger than that of free lipase. Previous study27 on the interaction mechanism of carbon nanotube with lipase can help to understand the increase of catalysis efficiency by GO-lipase. Possibly the hydrophobic interaction between the residues and the GO sheet can be propagated to the remote lid region of the lipase and induce the structural change of the lid, making


Page 10 of 24

Page 11 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the lid become more open. This work demonstrates that the lipase immobilized on carboxyl functionalized grapheme oxide is an efficient catalyst in organic medium.

Acknowledgements This work was supported by the National Science Foundation of China (21076018). Supporting Information Available: One table, two schemes, and two additional figures are provided as supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

REFERENCE (1) Mahmood, I.; Guo, C.; Xia, H.; Ma, J.; Jiang, Y.; Liu, H. Lipase lmmobilization on oleic acid-pluronic (L-64) block copolymer coated magnetic nanoparticles, for hydrolysis at the oil/water interface. Ind. Eng. Chem. Res. 2008, 47, 6379. (2) Verger, R. Interfacial activation of lipases: facts and artifacts. Trends Biotechnol. 1997, 15, 32. (3) Ferrato, F.; Carriere, F.; Verger, R. Acriticalreevaluation of the phenomenon of interfacial activation. Method Enzymol. 1997, 286, 327. (4) Grochulski, P.; Li, Y.; Schrag, J. D.; Bouthillier, F.; Smithllier, P.; Harrison, D.; Rubin, B.; Cygler, M. Insights into interfacial activation from an open structure of Candida rugosa lipase. J. Biol. Chem. 1993, 268, 12843. (5) Ren, M. Y.; Bai, S.; Zhang, D. H.; Sun, Y. pH memory of immobilized lipase for (±)-menthol resolution in ionic liquid. J. Agric. Food Chem. 2008, 56, 2388. (6) Laurent, N.; Haddoub, R.; Flitsch, S. L. Enzyme catalysis on solid surfaces. Trends Biotechnol. 2008, 26, 328. (7) Pundir, C. S.; Chauhan, N. Coimmobilization of detergent enzymes onto a plastic bucket and brush for their application in cloth washing. Ind. Eng. Chem. Res. 2012, 51, 3556.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 24

(8) Lee, D. G.; Ponvel, K. M.; Kim, M.; Hwang, S.; Ahn, I. S.; Lee, C. H. Immobilization of lipase on hydrophobic nano-sized magnetite particles. J. Mol. Catal. B: Enzym. 2009, 57, 62. (9) Feng, W.; Ji, P. Enzymes immobilized on carbon nanotubes. Biotechnol. Adv. 2011, 29, 889. (10) Shakeri, M.; Engstrom, K.; Sandstrom, A. G.; Backvall, J.E. Highly Enantioselective resolution of b-amino esters by candida antarctica lipase a immobilized in mesocellular foam: application to dynamic kinetic resolution. ChemCatChem 2010, 2, 534. (11)

Kumar,

A.;

Kanwar,

S.S.

Catalytic

potential

of

a

nitrocellulose

membrane-immobilized lipase in aqueous and organic media. J. Appl. Polym. Sci. 2012, 124, E37–E44. (12) Miletić, N.; Vuković, Z.; Nastasović, A.; Loos, K. effect of Candida Antarctica lipase B immobilization on the porous structure of the carrier. Macromol. Biosci. 2011, 11, 1537. (13) Vorhaben, T.; Botcher, D.; Jasinski, D.; Menyes, U.; Bruser, V.; Schroder, K.; Bornscheuer, U.T. Plasma-modified polypropylene as carrier for the immobilization of candida antarctica lipase B and pyrobaculum calidifontis esterase. ChemCatChem 2010, 2, 992. (14) Quintana, P. G.; Guillén, M.; Marciello, M.; Valero, F.; Palomo, J. M.; Baldessari, A. Immobilized heterologous Rhizopus Oryzae lipase as an efficient catalyst in the acetylation of cortexolone. Eur. J. Org. Chem. 2012, 4306. (15) Du, D.; Wang, L.; Shao, Y.; Wang, J.; Engelhard, M. H. Lin, Y. Functionalized graphene oxide as a nanocarrier in a multienzyme labeling amplification strategy for ultrasensitive electrochemical immunoassay of phosphorylated p53 (S392). Anal.


Page 13 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Chem. 2011, 83, 746. (16) Yang, S.; Guo, D.; Su, L.; Yu, P.; Li, D.; Ye, J.; Mao, L. Electrochemical sensing and biosensing platform based on chemically reduced graphene oxide. Anal. Chem. 2009, 81, 5603. (17) Shan, C.; Yang, H.; Song, J.; Han, D.; Ivaska, A.; Niu, L. Direct electrochemistry of glucose oxidase and biosensing for glucose based on grapheme. Anal. Chem. 2009, 81, 2378. (18) Liu, Y.; Yu, D.; Zeng, C.; Miao, Z.; Dai, L. Biocompatible graphene oxide-based glucose biosensors. Langmuir 2010, 26, 6158. (19) Fu, D.; Yu, M.; Tan, T.; Zhou, X. Separation, characterization and catalytic properties of Lip2 isoforms from Candida sp. 99-125. J. Mol. Catal. B: Enzym. 2009, 56, 115. (20) Herrera-Alonso, M.; Abdala, A. A.; McAllister, M. J.; Aksay, I. A.; Prud’homme, R. K. Intercalation and stitching of graphite oxide with diaminoalkanes. Langmuir 2007, 23, 10644. (21) Gao, Y, Kyratzis I. Covalent immobilization of proteins on carbon nanotubes using the cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide — a critical assessment. Bioconjugat Chem. 2008, 19, 1945. (22) Lee, S. Y.; Rhee, J. S. Production and partial purification of a lipase from Pseudomonas putida 3SK. Enzym. Microb. Technol. 1993, 15, 617. (23) Chen, C. S., Fujimoto, Y., Girdaukas, G., Sih, C. J. Quantitative analyses of biochemical kinetic resolution of enantiomers. J. Am. Chem. Soc. 1982, 104, 7294. (24) McAllister, M. J.; Li, J. L.; Adamson, D. H.; Schniepp, H. C.; Abdala, A. A.; Liu, J.; Herrera-Alonso, M.; Milius, D. L.; Car, R.; Prud'homme, R. K.; Aksay, I. A. Single sheet functionalized graphene by oxidation and thermal expansion of graphite.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chem. Mater. 2007, 19, 4396. (25) Sinani, V. A., Gheith, M. K., Yaroslavov, A. A., Rakhnyanskaya, A. A., Sun, K., Mamedov, A. A., Wicksted, J. P., Kotov, N. A. Aqueous Dispersions of Single-wall and Multiwall Carbon Nanotubes with Designed Amphiphilic Polycations. J. Am. Chem. Soc. 2005, 127, 3463. (26) Holzinger, M., Abraham, J., Whelan, P., Graupner, R., Ley, L., Hennrichl, F., Kappes, M., Hirsch, A. Functionalization of single-walled carbon nanotubes with (r-)oxycarbonyl nitrenes. J. Am. Chem. Soc. 2003, 125, 8566. (27) Wei Feng, Xiuchan Sun, Peijun Ji. Activation mechanism of Yarrowia Lipolytica lipase immobilized on carbon nanotubes. Soft Matter 2012, 8, 7143.


Page 14 of 24

Page 15 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure captions:

Figure 1. TEM image of grapheme oxide Figure 2. XRD patterns of graphite and GO-COOH Figure 3. Raman spectra of graphite and GO-COOH Figure 4. XPS spectra of graphite and GO-COOH (a) Graphite, (b) GO, (c) GO-COOH Figure 5. UV-vis spectra of native lipase, GO-lipase and GO-COOH Figure 6. XPS spectra of GO-lipase, GO-COOH, and graphite Figure 7. XRD pattern of native lipase, GO-lipase and GO-COOH Figure 8. Hydrolytic activity retained by the immobilized lipase at different temperature Figure 9. Enantiomeric excess and conversion by the immobilized lipase (a) and native lipase (b). The reaction temperature is 50 °C



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1.


Page 16 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Intensity

Page 17 of 24

Graphite

GO-COOH

20

40

60

2 (deg)

Figure 2.


80


12000

GO-COOH

Graphite

10000

8000

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 24

6000

4000

2000

0

1000

1200

1400

1600

1800

-1

Raman shift (cm )

Figure 3.


2000

Page 19 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a Graphite

291

288

285

282

Binding energy (eV)

b GO

291

288

285

282

Binding energy (eV)

c GO-COOH

291

288

285

282

Binding energy (eV)

Figure 4.



2.0

1.5

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1.0

Native lipase GO-lipase GO-COOH

0.5

0.0 240 300 360 420 480 540 600 660 720 780

Wavelength (nm)

Figure 5.


Page 20 of 24

Page 21 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


GO-lipase

GO-COOH

Graphite

C1s

O1s N1s

600

550

500

450

400

350

300

Biding Energy/eV

Figure 6.


250

200


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

GO-COOH GO-lipase Native lipase

20

40

60

2 (deg))

Figure 7.


80

Page 22 of 24

Page 23 of 24

100

Retained hydrolitic activity by the immobilized lipase (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


90 80 70 60 50 40 30 20 10 0 30

35

40

45

0

50

Reaction temperature ( C)

Figure 8.


55

Page 24 of 24

a

100 GO-lipase 90 80 70 Enantiomeric excess (ees) Conversion

60 50 40 30 5

10

15

20

25

30

35

40

45

Reaction time /h

Conversion and Enantiomeric excess (ees) / %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Conversion and Enantiomeric excess (ees) / %


100

b

Native lipase

90 80

Enantiomeric excess (ees) Conversion

70 60 50 40 30 20 10 5

10

15

20

25

30

35

Reaction time /h

Figure 9.


40

45

Enzyme Immobilization on Carboxyl-Functionalized Graphene Oxide

Recommend Documents