Comparative Characterization of Peanut β-Amylase Immobilization

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Comparative Characterization of Peanut β‑Amylase Immobilization onto Graphene Oxide and Graphene Oxide Carbon Nanotubes by Solid-State NMR Ranjana Das,†,§ Renuka Ranjan,‡,§ Neeraj Sinha,*,‡ and Arvind M. Kayastha*,† †

School of Biotechnology, Institute of Science, Banaras Hindu University, Varanasi 221005, India Centre of Biomedical Research, Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS) Campus, Raebarelly Road, Lucknow 226014, India

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‡

S Supporting Information *

ABSTRACT: In this Article, we describe solid-state NMR experiments on a model biocatalyst system consisting of the enzyme β-amylase covalently immobilized on graphene oxide nanosheets (GO) and graphene oxide−carbon nanotube composites (GO−CNT). Onedimensional magic angle spinning (MAS) NMR technique was employed on carbon nuclei (13C) in natural abundance. The support systems (GO and GO−CNTs) were characterized first, and it was possible to assign carbon species. The difference in the 13C spectrum between GO and GO−CNT indicated that CNT rods were successfully incorporated within GO sheets, producing sharp peaks. The shifts in the spectrum of enzyme-immobilized support systems indicated immobilization. Many more changes were observed in the 13C MAS NMR spectra during the immobilization process, which arose from cross-linking of the surface carbon species via glutaraldehyde with the amino group of enzyme. This study showed the potential of natural abundance 13C MAS NMR for comparative characterization of the two nanobiocatalyst systems and supported the results of our previous finding that GO−CNT composites are better platforms for enzyme immobilization owing to their large surface area. In addition, this study is the first report on 13C NMR spectra of GO−CNT nanocomposites.

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INTRODUCTION β-Amylase or 4-α-D-glucanmaltohydrolase is an enzyme of industrial importance in food and pharmaceutics. It is found in higher plants and microorganisms, attacking alternate glycosidic linkages in starch and related polysaccharides producing maltose. Maltose production from cereal grains by the action of β-amylase makes its role important in mashing and brewing process. The ability of β-amylase to produce maltose exclusively is utilized in structural analysis of starch and glycogen. The enzyme is also exploited as an exclusive source of carbon in the production of Diptheria Pertussis Tetanus vaccine.1,2 Most of the chemical and industrial applications including fine and green chemistry, diagnosis, decontamination, drug delivery, biosensing, textile, food and pharmaceutics, etc. depend upon the use of immobilized enzymes.3−5 The immobilization of proteins/enzymes holds importance as they carry out various catalytical reactions under moderate physiological conditions, thereby lowering the trend of chemical procedures.6,7 Enzyme/ protein immobilization onto diverse insoluble matrices leads to increased stability and reaction catalysis under extremes of pH and temperatures, which is often the case with industrial enzymes. Furthermore, the immobilized enzyme becomes heterogeneous (insoluble) and can be easily separated from the reaction mixture, thereby reducing the problem of using homogeneous enzyme (soluble) catalysis in solution in an industrial scheme.8,9 This enables the costly enzyme catalyst to © XXXX American Chemical Society

be restored and reused and abandons product stream contamination by the protein.10 There exist several modes of protein immobilization,11 mainly categorized as adsorption,12 covalent linkage,13 encapsulation, and entrapment within a matrix.14 Among the covalent linkage methods, one very interesting approach is the immobilization of protein onto nanomatrices by using glutaraldehyde as a linker. The method ensures greater immobilization efficiency than mere adsorption.3 In addition, the enzymes immobilized through this method have been reported to have greater stability over a period of a few months. In the past few years, NMR of heterogeneous protein systems has been extensively harnessed for detailed structural analysis. Solution as well as solid-state NMR utilizes different pulse sequences to track down alterations in different types of coupling energy constants of NMR-active nuclei. Interactions between NMR-active nuclei in proteins such as 1H, 13C, and 15N are detected through NMR, which proves its utility to measure distances, bonding, and alignment of the atoms.15 Owing to advanced NMR technologies and increased efficacy of the NMR hardware over time, it is now easier to detect NMR-active nuclei in natural abundance of a material.16−19 Many NMR methods Received: June 29, 2018 Revised: August 7, 2018 Published: August 7, 2018 A

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toward tracking changes in functionalized graphene oxide materials when an enzyme is immobilized onto these materials using simple solid-state NMR techniques.

facilitate determination of the insoluble protein dynamics for different time scales at atomic resolution. Several advanced NMR experiments could track changes in the microenvironment of the biological systems, and these can also provide information about motion of individual molecular structural sites on a picosecond timescale.20−22 Cross-polarization and direct-polarization NMR of 13C nuclei have been used as basic protocols to study any new material. There have been several reports of the use of these NMR methods applied on graphene oxide, carbon nanotubes, and several variants of nanostructures formed by graphene oxides.23−33 This study utilizes simple solid-state NMR methods such as 13C cross-polarization and 13C direct polarization (onepulse experiment) to track changes in the molecular environment of graphene oxide materials when immobilization of enzymes using covalent cross-linking is carried out on these materials. The immobilized proteins are noncrystalline in nature, therefore lacking long-range order.34 Considering this, solid-state NMR could be applied to a heterogeneous enzyme system because the technique does not require long-range order of the sample. There have been several studies conducted on immobilized enzymes on other materials where labeled enzyme has been used in the NMR experiments.10,35,36 In this study, we focus on the structural changes induced by the phenomenon of immobilization on the graphene oxide materials. Graphene and its derivative carbon nanotubes are extensively studied for use in electrochemical sensing as well as in biotechnology and biomedical applications, etc. Graphene oxide (GO) is a two-dimensional sheet of one-atom thick hexagonally arrayed sp2 bonded carbon atoms. It has attracted attention owing to its unique structural, thermal, mechanical, optical, and electrical properties. However, the tendency of GO to revert to its agglomerated form because of its π−π cloud limits its application. One-dimensional carbon nanotubes (CNTs) are nanoscopic structures with natural and tunable properties, which are predicted to impact many areas of our lives. The incorporation of CNT rods into GO nanosheets separates the sheets, accompanying larger surface area, and prevents agglomeration. This three-dimensional GO−CNT composite pursues the properties of both GO and CNTs, making them better than either of the two. The composite of GO−CNT is least worked and hence offers a novel support for various biotechnological applications.37,38 In our previous work,39 we have discussed in detail immobilization of β-amylase from peanut (Arachis hypogaea) onto GO nanosheets and GO−CNT composite through glutaraldehyde as a covalent linker. β-Amylase from peanut (30 kDa) catalyzes the release of maltose from starch and related polysaccharides by attacking alternate α-1,4-glycosidic linkages. Immobilized β-amylase has an industrial relevance in mashing and brewing process owing to its hydrolysis of cereal grain starch and production of maltose. The nanostructures were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared (FTIR), atomic force microscopy (AFM), and fluorescence microscopy before and after enzyme immobilization. The results showed that GO−CNTs were better substrates for enzyme immobilization owing to the separation of GO nanosheets by CNT rods, which increases the surface area for enzyme immobilization, thereby leading to higher immobilization efficiency than GO. Herein, an attempt has been made to characterize and support the same through natural abundance solid-state NMR. This study is directed

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MATERIALS AND METHODS Materials. Peanuts were purchased from an agricultural store. All the chemicals for buffer preparation were of analytical or electrophoretic grade from Merck Eurolab GmbH Damstadt, Germany. The rest of the chemicals (for protein purification and nanomaterial synthesis) were purchased from Sigma Chem. Co, St. Louis, U.S.A. Milli Q (MQ) water (Millipore, Bedford, MA, U.S.A.) with resistance > 18 MΩ.cm was used throughout the experiment. Enzyme Preparation. β-Amylase enzyme was purified from soaked peanuts (Arachis hypogaea) by a combination of solvent extractions and chromatographic techniques.40 The steady-state kinetics of the purified enzyme has been discussed in our previous work.40 Synthesis of Functionalized Nanostructures and Covalent Immobilization of β-Amylase and Enzymatic Assay. The synthesis and functionalization of graphene oxide nanosheets (GO) and graphene oxide−carbon nanotube composites (GO−CNT) have been discussed earlier.39 βAmylase enzyme purified from peanuts (Arachis hypogaea) was immobilized onto the nanostructures through covalent linkage with the oxygen- and nitrogen-containing functional groups, using glutaraldehyde as linker.20 The soluble and immobilized enzyme systems were assayed by following Bernfeld’s method using 3,5-dinitrosalicylic acid.41 Immobilization Efficiency. The proficiency of immobilization on the two supports was determined by specific activity of the immobilized enzyme with respect to soluble enzyme. Immobilization efficiency is given by the following formula: immobilization efficiency specific activity of immobilized enzyme = × 100% specific activity of soluble enzyme

Solid-State NMR Experiments. Experiments were performed on 600 MHz solid-state NMR spectrometer (Avance III, Bruker Biospin, Switzerland) as reported earlier.15,42 To carry out solid-state NMR experiments, GO nanosheet solid flakes were packed manually in 3.2 mm zirconia rotor. GO−CNT solid powder was also packed in the rotor using the same method. For 13C one-pulse experiments on graphene oxide nanosheets, different MAS had been utilized for differentiating between sideband and center-band resonances with 2560 transients. GO nanosheets and GO−CNTs (native as well as enzymeimmobilized) were subjected to 13C one-pulse acquisition at MAS of 10 kHz. 13C π/2 pulse length was 5 μs, and 1H pulse length during small phase incremental alteration (SPINAL-64) decoupling sequence was 6.5 μs. A recycle delay for 8 s was used with an acquisition time of 15 ms for 5120 transients. 13 C cross-polarization was carried out with a linear ramp of 100% on 1H channel for a contact time of 1 ms and SPINAL-64 decoupling at a MAS spin rate of 10 kHz. The pulse length for a π/2 pulse at 1H is 2.5 μs, and the pulse length during SPINAL-64 decoupling sequence was 6.5 μs with 2560 transients.

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RESULTS AND DISCUSSION Immobilization of β-Amylase onto GO and GO−CNT Nanostructures. β-Amylase was successfully immobilized onto the two nanostructures, GO and GO−CNT, with high

B

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Figure 1. 13C one-pulse spectra at MAS 10 kHz showing peaks of functionalized GO nanosheets (A) in native state and (B) β-amylase immobilized on GO nanosheets using glutaraldehyde as a linker.

Figure 2. 13C one-pulse spectra at MAS 10 kHz showing peaks of functionalized GO−CNTs (A) in native state and (B) β-amylase immobilized onto them using glutaraldehyde as a linker.

ultimately increases the surface area, and a large amount of enzyme was immobilized onto GO nanosheets along with CNT rods. The reusability of enzyme was also improved, with 70%

immobilization efficiencies, 88% and 90%, respectively. GO− CNT showed better immobilization efficiency as compared to GO, due to the separation of GO sheets by CNT rods. This C

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Figure 3. 13C one-pulse spectrum of aqueous glutaraldehyde (without MAS).

and 50% residual activities after 10 reuses for β-amylase immobilized onto GO−CNT and GO, respectively. The residual activities of the nanobiocatalysts over a period of 90 days for GO and GO−CNT were 67% and 70%, respectively, whereas free enzyme had only 25% residual activity for the same time period. Other kinetic parameters have been discussed earlier.39 Solid-State NMR. Functionalized GO nanosheets were subjected to 13C one-pulse and 13C cross-polarization experiments. A comparison between simple 13C one-pulse spectra for functionalized GO nanosheets and β-amylase immobilized onto them at a spinning rate of 10 kHz is shown in Figure 1. The natural abundance 13C spectra for functionalized GO nanosheets showed two broad resonances around 60 ppm, which corresponds to alcoholic carbon and epoxide groups, and 130 ppm, which corresponds to sp2 carbon of graphite, along with their respective side bands, which is in agreement with the previous studies.27,31,32 The 13C resonances broaden as the enzyme was immobilized onto functionalized GO nanosheets, showing no sharp peaks. This phenomenon can be explained by the fact that there are several moieties, bound as well as unbound after immobilization of the enzyme onto the graphene oxide nanosheets where unbound moieties might be causing the 13C resonance peaks of graphene oxide to broaden. On the contrary, NMR spectra for functionalized GO−CNT showed a difference in natural abundance 13C chemical shifts from that of nanosheets, in native state as well as with β-amylase immobilized onto them, which is evident from Figure 2. The difference between GO nanosheets and GO−CNT 13C spectra is due to the packing arrangement of graphene oxide in these two materials, which has arisen due to different methods of preparation.39 Sharp peaks in the 13C spectrum for functionalized GO−CNT in the native state were obtained around 30, 70, and 175 ppm, which are not very different from that of the chemical shifts reported for graphite oxide in previous studies.33,43−46 These changes in chemical shifts are attributed to the structure of GO−CNT and functionalization of GO with L-cystine during preparation of material for immobilization, which is more clear in the spectra for GO−CNT than that of GO

nanosheets. In addition, the peaks are different from 1D CNT as well.29,44 The natural abundance 13C spectrum for immobilized β-amylase cross-linked by glutaraldehyde on functionalized GO−CNT showed resonance peaks at 24.5 and 61 ppm, two resonances around 70 ppm, and a single peak at 181 ppm. The chemical shifts in the NMR spectra for immobilized β-amylase on functionalized GO−CNT showed differences from that of the native functionalized GO−CNT. The single peaks at 175 ppm (which is assigned to −COOH group in GO−CNT) and around 30 ppm (assigned to Cγ of cystine)47 in functionalized GO−CNT experience a shift of 6−7 ppm when β-amylase is immobilized on functionalized GO−CNT (Supporting Information, Figure S4). This change in chemical shifts occurs when enzymes are covalently cross-linked by glutaraldehyde to functionalized GO−CNT. The changes in chemical shifts in these two peaks also indicate that these sites are interacting with the glutaraldehyde cross-linked β-amylase. Aqueous glutaraldehyde was also subjected to 13C one-pulse experiment without MAS. The spectrum in Figure 3 showed sharp peaks at 17 and 31 ppm, a broad resonance at 91.5 ppm, and peaks at 179 ppm as well as 208 ppm, which showed a lower intensity as compared to the rest.48 The peaks at 17 and 31 ppm correspond to C3 carbon and C2 and C4 carbon of glutaraldehyde, respectively. The peak at 91.5 ppm indicates the aldehyde group of glutarladehyde. The chemical shifts in NMR spectra of the glutaraldehyde also vary when used for immobilization of the enzyme onto both carbon-based nanomatrices, as evident from their spectra.These peaks are in accordance with previously mentioned reports of 13C NMR of glutaraldehyde.48 Sharp peaks in GO−CNT suggest that there are a lower number of freely moving protons in the vicinity after immobilization of enzyme, and therefore 13C peaks were not affected by 1H resonances, suggesting that the functional groups are bound well by the enzyme. There is also a clear difference between native functionalized GO−CNT and the native GO nanosheets. 13C one-pulse NMR spectra for native functionalized GO−CNT showed sharp peaks as compared to GO nanosheets, which showed broad resonances, as mentioned D

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Figure 4. 13C cross-polarization spectrum at MAS 10 kHz for β-amylase immobilized on functionalized graphene oxide nanosheets.

of 13C atoms for cross-polarization to occur. When functionalized GO−CNTs were treated with glutaraldehyde, there may have been free protons available for polarization transfer. When enzyme binds to these protons, it leaves no free 1H for crosspolarization to 13C, implying an increase in immobilization efficiency. This shows that GO−CNTs are more efficient nanomaterials than GO nanosheets for immobilization of βamylase, which supports the finding in our previous section.

earlier. This indicated that functionalized GO−CNTs have a larger surface area than that of the GO nanosheets. Figure 4 shows 13C cross-polarization spectra of immobilized enzyme onto GO nanosheets. Sharp resonances at 35, 53, 72, and 175 ppm were obtained. Peaks around 175 ppm indicate the −COOH group of graphene oxide. The 13C resonances around 60−80 ppm are assigned to the −C−OH and −C−O−C groups of graphene oxide, and 13C resonances obtained around 35 and 53 ppm appear to be C2 and C4 carbons of glutaraldehyde, respectively. Other smaller and broad resonances from 20 to 60 ppm might be due to other alkyl groups in the enzyme as well as due to cysteine functionalization.33 Cross-polarization experiments were also performed on other samples of GO nanosheets and GO−CNTs (native as well as immobilized). In native functionalized GO nanosheets, cross-polarization did not yield a clear spectra (Supporting Information, Figure S3), which indicated lack of free protons in the vicinity of 13C nuclei for polarization transfer from 1H to 13C, or the protons in the vicinity to the carbon are highly mobile so that cross-polarization is ineffective. When functionalized GO nanosheets are treated with glutaraldehyde, it may contribute to an increase in the number of free protons available for polarization transfer in the vicinity. The 13C cross-polarization MAS (CPMAS) works due to polarization transfer to 13C nuclei from its surrounding 1H. Several studies report 13C CPMAS spectra for only graphene oxide, while the materials used in this study are graphene oxide nanosheets and graphene oxide−carbon nanotubes. These materials are prepared in such a way that the packing arrangement of 13C nuclei and 1H nuclei are not facilitating any polarization transfer and thus are not producing any peaks in 13 C CPMAS spectra. When enzyme binds to a glutaraldehyde-treated surface, it bonds with most free protons while leaving some free 1H unbound with enzyme in the vicinity of the 13C atoms of GO. This phenomenon may have caused the 13C CPMAS to work on these samples. Functionalized GO−CNTs (in native as well as immobilized state) subjected to the 13C CPMAS experiments did not yield a clear spectra. In the native state, these may have an abundance of highly mobile 1H or no protons in the vicinity

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CONCLUSION Solid-state NMR was employed to characterize the support (native and enzyme-immobilized GO and GO−CNT) used in immobilization of model biocatalyst β-amylase from peanuts. NMR experiments were performed on 13C nuclei in natural abundance, and these proved to be useful as an indicator of the immobilization phenomenon. These methods could be used directly to the dried samples without any need of 13C or 15N labeling, thereby reducing cost and time. The results suggest that these NMR spectra could be used to track the disturbances arising due to the interaction of the enzyme with the glutaraldehyde-activated nanomatrices.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.8b06219. Sidebands appearing in 13C one-pulse spectrum of GO nanosheets, 13C one-pulse deconvolution spectra of GO nanosheets, comparison of 13C CPMAS spectra of graphene oxide materials in native and immobilized form, immobilization of peanut β-amylase on GO−CNT (DOCX)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone:+91-522-2495034. *E-mail: [email protected]. Phone: +91-542-2368331. ORCID

Arvind M. Kayastha: 0000-0002-5090-7159 E

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R.D. and R.R. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS R.D. acknowledges the scholarship from Indian Council of Medical Research (New Delhi, India) in the form of JRF and SRF (Ref. no. 3/1/3/JRF-2012/HRD). R.R. acknowledges Senior Research fellowship from Council of Scientific & Industrial Research, India (File no. 09/916(0085)/2015EMR-I). N.S. acknowledges financial support from SERB India (Grant no. EMR/2015/001758).

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DOI: 10.1021/acs.jpcc.8b06219 J. Phys. Chem. C XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jpcc.8b06219 J. Phys. Chem. C XXXX, XXX, XXX−XXX