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C: Physical Processes in Nanomaterials and Nanostructures

Comparative Characterization of Peanut #-Amylase Immobilization Onto Graphene Oxide and Graphene Oxide Carbon Nanotubes by Solid State NMR Ranjana Das, Renuka Ranjan, Neeraj Sinha, and Arvind M. Kayastha J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b06219 • Publication Date (Web): 07 Aug 2018 Downloaded from http://pubs.acs.org on August 8, 2018

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The Journal of Physical Chemistry

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Comparative Characterization of Peanut β-Amylase Immobilization onto Graphene Oxide

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and Graphene Oxide Carbon Nanotubes by Solid State NMR

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Ranjana Das¶1, Renuka Ranjan¶2, Neeraj Sinha2*, Arvind M. Kayastha1*

5

1

6

221005.

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2

8

(SGPGIMS) Campus, Raebarelly Road, Lucknow 226014, India

School of Biotechnology, Institute of Science, Banaras Hindu University, Varanasi, India –

Centre of Biomedical Research, Sanjay Gandhi Post Graduate Institute of Medical Sciences

¶ Authors contributed equally 9

* Corresponding Authors

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E-mail id: [email protected] (Prof. Arvind M. Kayastha)

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Ph.: +91-542-2368331 (Prof. Arvind M. Kayastha)

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[email protected] (Dr.Neeraj Sinha)

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Ph.:+91-522-2495034 (Dr. Neeraj Sinha)

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15

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1 ACS Paragon Plus Environment

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ABSTRACT

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In this paper, we describe solid-state NMR experiments on a model biocatalyst system consisting

3

of the enzyme β-amylase covalently immobilized on graphene oxide nanosheets (GO) and

4

graphene oxide-carbon nanotube composites (GO-CNT). One-dimensional magic angle spinning

5

(MAS) NMR technique was employed on carbon nuclei (13C) in natural abundance. The support

6

systems (GO and GO-CNTs) were characterized first and it was possible to assign carbon

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species. The difference in the 13C spectrum between GO and GO-CNT indicated that CNT rods

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were successfully incorporated within GO sheets, producing sharp peaks. The shifts in the

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spectrum of enzyme immobilized support systems indicated immobilization. Many more changes 13

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were observed in the

C MAS NMR spectra during the immobilization process, which arose

11

from cross-linking of the surface carbon species via glutaraldehyde with the amino group of

12

enzyme. This study showed the potential of natural abundance 13C MAS NMR for comparative

13

characterization of the two nanobiocatalyst systems and supported the results of our previous

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finding that GO-CNT composites are better platform for enzyme immobilization owing to their

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large surface area. In addition, this study is the first report on

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nanocomposites.

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13

C NMR spectra of GO-CNT

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INTRODUCTION

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β-Amylase or 4-α-D-glucanmaltohydrolase is an enzyme of industrial importance in food and

3

pharmaceutics. It is found in higher plants and microorganisms, attacking alternate glycosidic

4

linkages in starch and related polysaccharides producing maltose. Maltose production from

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cereal grains by the action of β-amylase makes its role important in mashing and brewing

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process. The ability of β-amylase to produce maltose, exclusively, is utilized in structural

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analysis of starch and glycogen. The enzyme is also exploited as exclusive source of carbon in

8

the production of Diptheria Pertussis Tetanus vaccine.1-2

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Most of the chemical and industrial applications including fine and green chemistry, diagnosis,

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decontamination, drug delivery, biosensing, textile, food and pharmaceutics, etc. depend upon

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the use of immobilized enzymes.3-5 The immobilization of proteins/enzymes holds importance as

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they carry out various catalytical reactions under moderate physiological conditions, thereby

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lowering the trend of chemical procedures.6-7 Enzyme/Protein immobilization onto diverse

14

insoluble matrices, leads to increased stability and reaction catalysis under extremes of pH and

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temperatures, which is often the case with industrial enzymes. Furthermore, the immobilized

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enzyme becomes heterogeneous (insoluble), which can be easily separated from reaction

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mixture, thereby reducing the problem of using homogeneous enzyme (soluble) catalysis in

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solution, in an industrial scheme.8-9 Thus enabling the costly enzyme catalyst to be restored and

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reused and abandons product stream contamination by the protein.10

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There exist several modes of protein immobilization,11 mainly categorized as adsorption,12

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covalent linkage,13 encapsulation and entrapment within a matrix.14 Among the covalent linkage

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method, one very interesting approach is the immobilization of protein onto nanomatrices by



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using glutaraldehyde as a linker. The method ensures greater immobilization efficiency than

2

mere adsorption.3 In addition, the enzymes immobilized through this method have been reported

3

to have greater stability, over a period of few months.

4

In the past few years, NMR of heterogeneous protein systems has been extensively harnessed for

5

detailed structural analysis. Solution as well as solid state NMR utilizes different pulse sequences

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to track down alterations in different types of coupling energy constants of NMR active nuclei.

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Interactions between NMR active nuclei in proteins such as 1H, 13C and 15N are detected through

8

NMR that prove its utility to measure distances, bonding as well as alignment of the atoms.15

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Owing to advanced NMR technologies and increased efficacy of the NMR hardware over time, it

10

is now easier to detect NMR active nuclei in natural abundance of a material.16-19 Many NMR

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methods facilitate determination of the insoluble protein dynamics for different time scale at

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atomic

13

microenvironment of the biological systems and these can also provide information about motion

14

of individual molecular structural sites on a picosecond time-scale.20-22

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Cross-polarization and direct polarization NMR of 13C nuclei have been used as basic protocols

16

to study any new material. There had been several reports of use of these NMR methods applied

17

on graphene oxide, carbon nanotubes and several variants of nanostructures formed by graphene

18

oxides.23-33 This study utilizes simple solid state NMR methods such as

19

and 13C direct polarization (one pulse experiment) to track changes in the molecular environment

20

of graphene oxide materials when immobilization of enzymes using covalent cross-linking is

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carried out on these materials. The immobilized proteins are non-crystalline in nature, therefore

22

lack long-range order.34 Considering this, solid state NMR could be applied to heterogeneous

23

enzyme system as the technique does not require long-range order of the sample. There had been

resolution.

Several

advanced

NMR

experiments


could

track

changes

in

13

C cross-polarization

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several studies conducted on immobilized enzymes on other materials where labeled enzyme has

2

been used in the NMR experiments.10, 35-36 In this study, we focus on the structural changes

3

induced by the phenomenon of immobilization on the graphene oxide materials.

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Graphene along with its derivative carbon nanotubes are extensively studied for electrochemical

5

sensing as well as in biotechnology and biomedical applications etc. Graphene oxide (GO) is a

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two dimensional sheet, one-atom thick hexagonally arrayed sp2 bonded carbon atoms. It has

7

attracted attention owing to its unique structural, thermal, mechanical, optical and electrical

8

properties. However, the tendency of GO to revert to its agglomerated form because of its π-π

9

cloud, limits its application. One dimensional carbon nanotubes (CNTs) are nanoscopic

10

structures with natural and tunable properties, which are predicted to impact many areas of our

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lives. The incorporation of CNT rods into GO nanosheets separates the sheets, accompanying

12

larger surface area and prevents agglomeration. This three dimensional GO-CNT composite

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pursues the properties of both GO and CNTs, making them better than either of the two. The

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composite of GO-CNT is least worked and hence offers a novel support for various

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biotechnological applications.37-38

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In our previous work,39 we have discussed in detail immobilization of β-amylase from peanut

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(Arachis hypogaea) onto GO nanosheets and GO-CNT composite through glutaraldehyde as a

18

covalent linker. β-Amylase from peanut (30 kDa) catalyzes the release of maltose from starch

19

and related polysaccharides by attacking alternate α-1,4-glycosidic linkages. Immobilized β-

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amylase has an industrial relevance in mashing and brewing process owing to its hydrolysis of

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cereal grain starch and production of maltose.



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The nanostructures were characterized by SEM, TEM, FTIR, AFM and fluorescence microscopy

2

before and after enzyme immobilization. The results showed that GO-CNTs were better substrate

3

for enzyme immobilization owing to the separation of GO nanosheets by CNT rods, which

4

increases the surface area for enzyme immobilization. Thereby, leading to higher immobilization

5

efficiency than GO. Herein, attempt has been made to characterize and support the same through

6

natural abundance solid state NMR. This study is directed towards tracking changes in

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functionalized graphene oxide materials when an enzyme is immobilized onto these materials

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using simple solid state NMR techniques.

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MATERIALS AND METHOD

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Material

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Peanuts were purchased from agricultural store. All the chemicals for buffer preparation were of

12

analytical or electrophoretic grade from Merck Eurolab GmbH Damstadt, Germany. Rest of the

13

chemicals (for protein purification and nanomaterial synthesis) was purchased from Sigma

14

Chem. Co St. Louis, USA. Milli Q (MQ) water (Millipore, Bedford, MA, USA) with resistance

15

˃ 18 Ω was used throughout the experiment.

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Enzyme preparation

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β-Amylase enzyme was purified from soaked peanuts (Arachis hypogaea) by a combination of

18

solvent extractions and chromatographic techniques.40

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The steady state kinetics of the purified enzyme has been discussed in our previous work.40

20

Synthesis of functionalized nanostructures and covalent immobilization of β-amylase and

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enzymatic assay 6 ACS Paragon Plus Environment

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The synthesis and functionalization of graphene oxide nanosheets (GO) and graphene oxide-

2

carbon nanotube composites (GO-CNT) have been discussed earlier.39 β-Amylase enzyme

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purified from peanuts (Arachis hypogaea) was immobilized onto the nanostructures through

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covalent linkage with the oxygen and nitrogen containing functional groups, using

5

glutaraldehyde as linker.20 The soluble and immobilized enzyme systems were assayed by

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following Bernfeld’s method using 3,5 dinitrosalicylic acid.41

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Immobilization efficiency

8

The proficiency of immobilization on the two supports was determined by specific activity of the

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immobilized enzyme with respect to soluble enzyme. Immobilization efficiency is given by the

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formula:

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Immobilization efficiency =

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Solid-state NMR experiments

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Experiments were performed on 600 MHz solid-state NMR spectrometer (Avance III, Bruker

14

Biospin, Switzerland) as reported earlier.15, 42

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To carry out solid state NMR experiments, GO nanosheets solid flakes were packed manually in

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3.2 mm zirconia rotor. GO-CNT solid powder was also packed in the rotor using same method.

17

For

18

for differentiating between side bands and center band resonances with 2560 transients. GO

19

nanosheets and GO-CNTs (native as well as with enzyme immobilized) were subjected to

20

one pulse acquisition at MAS of 10 kHz.

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x 100%

C one pulse experiments on graphene oxide nanosheets, different MAS had been utilized

13

13

C

C π/2 pulse length was 5 µs and 1H pulse length



Page 8 of 22

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during Small Phase Incremental Alteration (SPINAL-64) decoupling sequence was 6.5 µs. A

2

recycle delay for 8s was used with an acquisition time of 15 ms for 5120 transients.

3

13

4

time of 1ms and SPINAL-64 decoupling at a MAS spin rate 10 kHz. Pulse length for a π/2 pulse

5

at 1H is 2.5 µs and pulse length during SPINAL-64 decoupling sequence was 6.5 µs with 2560

6

transients.

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RESULTS AND DISCUSSION

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Immobilization of β-amylase onto GO and GO-CNT nanostructures

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β-Amylase was successfully immobilized onto the two nanostructures, GO and GO-CNT with

10

high immobilization efficiency, 88% and 90%, respectively. GO-CNT showed better

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immobilization efficiency as compared to GO, due to the separation of GO sheets by CNT rods.

12

This ultimately increases the surface area and large amount of enzyme was immobilized onto GO

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nanosheets along with CNT rods. The reusability of enzyme was also improved, being 70% and

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50% residual activity after 10 reuses for β-amylase immobilized onto GO-CNT and GO,

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respectively. The residual activity of the nanobiocatalysts over a period of 90 days for GO and

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GO-CNT was 67% and 70%, respectively, whereas free enzyme had only 25% residual activity

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for the same time-period. Other kinetic parameters have been discussed earlier.39

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Solid-state NMR

19

Functionalized GO nanosheets were subjected to

20

experiments. A comparison between simple

21

nanosheets and β-amylase immobilized onto them at a spinning rate of 10 kHz is shown in Fig.

C Cross-polarization was carried out with a linear ramp of 100% on 1H channel for a contact

13

13

C one pulse and

13

C cross-polarization

C one pulse spectra for functionalized GO


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13

1

1. The natural abundance

C spectra for functionalized GO nanosheets showed two broad

2

resonances around 60 ppm, which corresponds to alcoholic carbon and epoxide groups, and 130

3

ppm, which corresponds to sp2 carbon of graphite, along with their respective side bands, which

4

is in agreement with the previous studies.27, 31-32 The 13C resonances broaden as the enzyme was

5

immobilized onto functionalized GO nanosheets, showing no sharp peaks. This phenomenon can

6

be explained by the fact that there are several moieties bound as well as unbound after

7

immobilization of the enzyme onto the graphene oxide nanosheets where unbound moieties

8

might be causing the 13C resonance peaks of graphene oxide to broaden.

9

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Fig. 1 13C one pulse spectra at MAS 10 kHz showing peaks of functionalized GO nanosheets (A)

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in native state, and (B) β-amylase immobilized on GO nanosheets using glutaraldehyde as a

12

linker



1

On the contrary, NMR spectra for functionalized GO-CNT showed a difference in natural

2

abundance 13C chemical shifts from that of nanosheets, in native state as well as with β-amylase

3

immobilized onto them, which is evident from Fig. 2. Difference between GO nanosheets and

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GO-CNT 13C spectra is due to the packing arrangement of graphene oxide in these two materials

5

which has arisen due to different methods of preparation.39 Sharp peaks in the 13C spectrum for

6

functionalized GO-CNT in native state were obtained around 30 ppm, 70 ppm, and 175 ppm,

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which are not very different from that of the chemical shifts reported for graphite oxide in

8

previous studies.33, 43-46 These changes in chemical shifts are attributed to the structure of GO-

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CNT and functionalization of GO with L-cystine during preparation of material for

10

immobilization, which is more clear in the spectra for GO-CNT than that of GO nanosheets. In

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addition, the peaks are different from 1D CNT as well.29, 44 The natural abundance 13C spectrum

12

for immobilized β-amylase cross-linked by glutaraldehyde on functionalized GO-CNT showed

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resonance peaks at 24.5 ppm, 61 ppm, two resonances around 70 ppm and a single peak at 181

14

ppm. The chemical shifts in the NMR spectra for immobilized β-amylase on functionalized GO-

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CNT showed differences from that of the native functionalized GO-CNT. The single peaks at

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175 ppm (which is assigned to -COOH group in GO-CNT) and around 30 ppm (assigned to Cγ

17

of cystine)47 in functionalized GO-CNT experience a shift of 6-7 ppm when β-amylase is

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immobilized on functionalized GO-CNT (Supplementary Information Fig. S4). This change in

19

chemical shifts occurs when enzymes covalently cross-linked by glutaraldehyde to

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functionalized GO-CNT. The changes in chemical shifts in these two peaks also indicate that

21

these sites are interacting with the glutaraldehyde cross-linked β-amylase.

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23 10 ACS Paragon Plus Environment

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1 13

C one pulse spectra at MAS 10 kHz showing peaks of functionalized GO-CNTs (A) in

2

Fig. 2

3

native state, and (B) β-amylase immobilized onto them using glutaraldehyde as a linker.

4 13

5

Aqueous glutaraldehyde was also subjected to

C one pulse experiment without MAS. The

6

spectrum in Fig. 3 showed sharp peaks at 17 ppm, 31 ppm, a broad resonance at 91.5 ppm and

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peaks at 179 ppm as well as 208 ppm, which showed a lesser intensity as compared to the rest.48

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The peaks at 17 ppm and 31 ppm corresponds to C3 carbon and C2 and C4 carbon of

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glutaraldehyde, respectively. Peak at 91.5 ppm indicates aldehyde group of glutarladehyde. The

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chemical shifts in NMR spectra of the glutaraldehyde also vary when used for immobilization of



1

the enzyme onto both carbon based nanomatrices, as evident from their spectra.These peaks are

2

in accordance with previous mentioned reports of 13C NMR of glutaraldehyde.48

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Sharp peaks in GO-CNT suggest that there are lesser number of freely moving protons in the

4

vicinity after immobilization of enzyme and therefore

5

resonances, suggesting that the functional groups are bound well by the enzyme. There is also a

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clear difference between native functionalized GO-CNT and the native GO nanosheets. 13C one

7

pulse NMR spectra for native functionalized GO-CNT showed sharp peaks as compared to GO

8

nanosheets which showed broad resonances, as mentioned earlier. This indicated that

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functionalized GO-CNT have a larger surface area than that of the GO nanosheets.

13

C peaks were not affected by 1H

10

11

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

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Fig. 4 shows 13C cross-polarization spectra of immobilized enzyme onto GO nanosheets. Sharp

13

resonances at 35 ppm, 53 ppm, 72 ppm and 175 ppm were obtained. Peaks around 175 ppm


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indicate -COOH group of graphene oxide. The 13C resonances around 60-80 ppm are assigned to

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the -C-OH and -C-O-C groups of graphene oxide and

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and 53 ppm appear to be C2 and C4 carbons of glutaraldehyde, respectively. Other smaller and

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broad resonances from 20-60 ppm might be due to other alkyl groups in the enzyme as well as

5

due to cysteine functionalization.33 Cross-polarization experiments were also performed on other

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samples of GO nanosheets, and GO-CNTs (native as well as immobilized). In native

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functionalized GO nanosheets, cross-polarization did not yield a clear spectra (Supplementary

8

Information Fig. S3), which indicated lack of free protons in the vicinity of

9

polarization transfer from 1H to 13C, or the protons in the vicinity to the carbon are highly mobile

10

so that cross-polarization is ineffective. When functionalized GO nanosheets were treated with

11

glutaraldehyde, it may contribute to increase in number of free protons available for polarization

12

transfer in the vicinity.

13

C resonances obtained around 35 ppm

13

C nuclei for

13 14

Fig. 4 13C Cross-polarization spectrum at MAS 10 kHz for β-amylase immobilized on

15

functionalized graphene oxide nanosheets. 13 ACS Paragon Plus Environment


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C CPMAS works due to polarization transfer to

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Page 14 of 22

C nuclei from its surrounding 1H.

1

The

2

Several studies report

3

this study are graphene oxide nanosheets and graphene oxide-carbon nanotubes. These materials

4

are prepared in such a way that the packing arrangement of

5

facilitating any polarization transfer and thus not producing any peaks in 13C CPMAS spectra.

6

When enzyme binds to glutaraldehyde treated surface it bonds with most free protons while

7

leaving some free 1H unbound with enzyme in the vicinity of the

8

phenomenon may have caused the

9

CNTs (in native as well as immobilized state) subjected to the 13C CPMAS experiments did not

10

yield a clear spectra. In native state, these may be having a abundance of highly mobile 1H or no

11

protons in the vicinity of

12

CNTs were treated with glutaraldehyde, there may have been free protons available for

13

polarization transfer. When enzyme binds to these protons, it leaves no free 1H for cross-

14

polarization to

15

CNTs are more efficient nanomaterials than that of GO nanosheets for immobilization of β-

16

amylase, which supports the finding in our previous section.

17

CONCLUSION

18

Solid-state NMR was employed to characterize the support (native and enzyme immobilized GO

19

and GO-CNT) used in immobilization of model biocatalyst β-amylase from peanuts. NMR

20

experiments were performed on 13C nuclei in natural abundance and these proved to be useful as

21

an indicator of the immobilization phenomenon. These methods could be used directly to the

22

dried samples without any need of

23

results suggest that these NMR spectra could be used to track the disturbances arising due to the

13

13

C CPMAS spectra for only graphene oxide, while the material used in

13

13

13

C nuclei and 1H nuclei are not

13

C atoms of GO. This

C CPMAS to work on these samples. Functionalized GO-

C atoms for cross-polarization to occur. When functionalized GO-

C, implying an increase in immobilization efficiency. This shows that GO-

13

C or

15

N labelling, thereby reducing cost and time. The


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1


interaction

of

the

enzyme

with

the

glutaraldehyde

activated

nanomatrices.

2 3

Acknowledgements

4

Ranjana Das acknowledges the scholarship from Indian Council of Medical Research (New

5

Delhi, India) in the form of JRF and SRF (Ref. No. 3/1/3/JRF-2012/HRD). Renuka Ranjan

6

acknowledges Senior Research fellowship from Council of Scientific & Industrial Research,

7

India (File No. 09/916(0085)/2015-EMR-I). Neeraj Sinha acknowledges financial support from

8

SERB India (grant no. EMR/2015/001758).

9

Conflict of interest

10

Authors declare no conflict of interest.

11

Supplementary Information: Additional figures are provided as supplementary information

12

13

References

14

1. Nehete, P.; Shah, N.; Ramamurthy, V.; Kothari, R., An Optimized Protocol for the Production

15

of High Purity Maltose. World Journal of Microbiology and Biotechnology 1992, 8, 446-450.

16

2. Ziegler, P., Cereal beta-Amylases. Journal of Cereal Science 1999, 29, 195-204.

17

3. Cerofolini, L.; Giuntini, S.; Louka, A.; Ravera, E.; Fragai, M.; Luchinat, C., High-Resolution

18

Solid-State NMR Characterization of Ligand Binding to a Protein Immobilized in a Silica

19

Matrix. The Journal of Physical Chemistry B 2017, 121, 8094-8101.

20

4. Küchler, A.; Yoshimoto, M.; Luginbühl, S.; Mavelli, F.; Walde, P., Enzymatic Reactions in

21

Confined Environments. Nature Nanotechnology 2016, 11, 409-420.



1

5. Das, R.; Mishra, H.; Srivastava, A.; Kayastha, A.M., Covalent Immobilization of β-Amylase

2

onto Functionalized Molybdenum Sulfide Nanosheets, its Kinetics and Stability Studies: A

3

Gateway to Boost Enzyme Application. Chemical Engineering Journal 2017, 328, 215–227.

4

6. Bommarius, A. S.; Riebel-Bommarius, B. R., Biocatalysis: Fundamentals and Applications;

5

John Wiley & Sons, 2004.

6

7. Sirisha, V.; Jain, A.; Jain, A., Enzyme Immobilization: An Overview on Methods, Support

7

Material, and Applications of Immobilized Enzymes. Advances in Food and Nutrition Research,

8

2016, 79, 179-211.

9

8. Faber, K.; Faber, K., Biotransformations in Organic Chemistry; Springer, 1992, 4, 31-313.

10

9. Jothiramalingam, R.; Wang, M. K., Review of Recent Developments in Solid Acid, Base, and

11

Enzyme Catalysts (Heterogeneous) for Biodiesel Production via Transesterification. Industrial &

12

Engineering Chemistry Research 2009, 48, 6162-6172.

13

10. Fauré, N. E.; Halling, P. J.; Wimperis, S., A Solid-State NMR Study of the Immobilization of

14

α-Chymotrypsin on Mesoporous Silica. The Journal of Physical Chemistry C 2013, 118, 1042-

15

1048.

16

11. Blanco, R. M.; Calvete, J. J.; Guisán, J., Immobilization-Stabilization of Enzymes; Variables

17

That Control the Intensity of the Trypsin (Amine)-Agarose (Aldehyde) Multipoint Attachment.

18

Enzyme and Microbial Technology 1989, 11, 353-359.

19

12. Tosa, T.; Mori, T.; Fuse, N.; Chibata, I., Studies on Continuous Enzyme Reactions. IV.

20

Preparation of a DEAE‐Sephadex–Aminoacylase Column and Continuous Optical Resolution of

21

Acyl‐DL‐Amino Acids. Biotechnology and Bioengineering 1967, 9, 603-615.

22

13. Mateo, C.; Fernández-Lorente, G.; Abian, O.; Fernández-Lafuente, R.; Guisán, J. M.,

23

Multifunctional Epoxy Supports: A New Tool to Improve the Covalent Immobilization of


Page 16 of 22

Page 17 of 22 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

Proteins. The Promotion of Physical Adsorptions of Proteins on the Supports before their

2

Covalent Linkage. Biomacromolecules 2000, 1, 739-745.

3

14. Chang, T. M.; MacIntosh, F.; Mason, S., Semipermeable Aqueous Microcapsules: I.

4

Preparation and Properties. Canadian Journal of Physiology and Pharmacology 1966, 44, 115-

5

128.

6

15. Singh, C.; Sinha, N., Mechanistic Insights into the Role of Water in Backbone Dynamics of

7

Native Collagen Protein by Natural Abundance 15N NMR Spectroscopy. The Journal of Physical

8

Chemistry C 2016, 120, 9393-9398.

9

16. Singh, C.; Rai, R. K.; Aussenac, F.; Sinha, N., Direct Evidence of Imino Acid–Aromatic

10

Interactions in Native Collagen Protein by DNP-Enhanced Solid-State NMR Spectroscopy. The

11

Journal of Physical Chemistry Letters 2014, 5, 4044-4048.

12

17. Rai, R. K.; Sinha, N., Dehydration-Induced Structural Changes in the Collagen–

13

Hydroxyapatite Interface in Bone by High-Resolution Solid-State NMR Spectroscopy. The

14

Journal of Physical Chemistry C 2011, 115, 14219-14227.

15

18. Rai, R. K.; Singh, C.; Sinha, N., Predominant Role of Water in Native Collagen Assembly

16

inside the Bone Matrix. The Journal of Physical Chemistry B 2014, 119, 201-211.

17

19. Gul-E-Noor, F.; Singh, C.; Papaioannou, A.; Sinha, N.; Boutis, G. S., Behavior of Water in

18

Collagen and Hydroxyapatite Sites of Cortical Bone: Fracture, Mechanical Wear, and Load

19

Bearing Studies. The Journal of Physical Chemistry C 2015, 119, 21528-21537.

20

20. Henzler-Wildman, K. A.; Lei, M.; Thai, V.; Kerns, S. J.; Karplus, M.; Kern, D., A Hierarchy

21

of Timescales in Protein Dynamics is Linked to Enzyme Catalysis. Nature 2007, 450, 913-916.

22

21. Palmer III, A. G., Probing Molecular Motion by NMR. Current Opinion in Structural

23

Biology 1997, 7, 732-737.



Page 18 of 22

1

22. Kay, L. E., Protein Dynamics from NMR. Biochemistry and Cell Biology 1998, 76, 145-152.

2

23. Casabianca, L. B.; Shaibat, M. A.; Cai, W. W.; Park, S.; Piner, R.; Ruoff, R. S.; Ishii, Y.,

3

NMR-Based Structural Modeling of Graphite Oxide Using Multidimensional

4

NMR and Ab Initio Chemical Shift Calculations. Journal of the American Chemical Society

5

2010, 132, 5672-5676.

6

24. Cai, W.; Piner, R. D.; Stadermann, F. J.; Park, S.; Shaibat, M. A.; Ishii, Y.; Yang, D.;

7

Velamakanni, A.; An, S. J.; Stoller, M., Synthesis and Solid-State NMR Structural

8

Characterization of

9

25. Abou-Hamad, E.; Babaa, M.-R.; Bouhrara, M.; Kim, Y.; Saih, Y.; Dennler, S.; Mauri, F.;

10

Basset, J.-M.; Goze-Bac, C.; Wågberg, T., Structural Properties of Carbon Nanotubes Derived

11

from 13C NMR. Physical Review B 2011, 84, 165417.

12

26. MacIntosh, A. R.; Harris, K. J.; Goward, G. R., Structure and Dynamics in Functionalized

13

Graphene Oxides through Solid-State NMR. Chemistry of Materials 2015, 28, 360-367.

14

27. Blumenfeld, A.; Muradyan, V.; Shumilova, I.; Parnes, Z.; Novikov, Y. N. Investigation of

15

Graphite Oxide by Means of

16

Forum, 1992, 91-93, 613-617.

17

28. Khandelwal, M.; Kumar, A., One-Step Chemically Controlled Wet Synthesis of Graphene

18

Nanoribbons from Graphene Oxide for High Performance Supercapacitor Applications. Journal

19

of Materials Chemistry A 2015, 3, 22975-22988.

20

29. Tang, X.-P.; Kleinhammes, A.; Shimoda, H.; Fleming, L.; Bennoune, K.; Sinha, S.; Bower,

21

C.; Zhou, O.; Wu, Y., Electronic Structures of Single-Walled Carbon Nanotubes Determined by

22

NMR. Science 2000, 288, 492-494.

13

13

C Solid-State

C-Labeled Graphite Oxide. Science 2008, 321, 1815-1817.

13

C NMR and 1H Spin-Lattice Relaxation. Materials Science


Page 19 of 22 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

30. Dreyer, D. R., Sungjin. Park, Christopher W. Bielawski, Rodney S. Ruoff, The Chemistry of

2

Graphene Oxide. Chemical Society Review 2010, 39, 228-240.

3

31. He, H.; Riedl, T.; Lerf, A.; Klinowski, J., Solid-State NMR Studies of the Structure of

4

Graphite Oxide. The Journal of Physical Chemistry 1996, 100, 19954-19958.

5

32. Mermoux, M.; Chabre, Y.; Rousseau, A., FTIR and

6

Carbon 1991, 29, 469-474.

7

33. Lerf, A.; He, H.; Riedl, T.; Forster, M.; Klinowski, J.,

8

Graphite Oxide and its Chemically Modified Derivatives. Solid State Ionics 1997, 101, 857-862.

9

34. Christiansen, S. C.; Hedin, N.; Epping, J. D.; Janicke, M. T.; del Amo, Y.; Demarest, M.;

10

Brzezinski, M.; Chmelka, B. F., Sensitivity Considerations in Polarization Transfer and Filtering

11

Using Dipole–Dipole Couplings: Implications for Biomineral Systems. Solid State Nuclear

12

Magnetic Resonance 2006, 29, 170-182.

13

35. Bower, P.; Louie, E.; Long, J.; Stayton, P.; Drobny, G., Solid-State NMR Structural Studies

14

of Peptides Immobilized on Gold Nanoparticles. Langmuir 2005, 21, 3002-3007.

15

36. Varghese, S.; Halling, P. J.; Häussinger, D.; Wimperis, S., High-Resolution Structural

16

Characterization of a Heterogeneous Biocatalyst Using Solid-State NMR. The Journal of

17

Physical Chemistry C 2016, 120, 28717-28726.

18

37. Liu, X.; Wei, S.; Chen, S.; Yuan, D.; Zhang, W., Graphene-Multiwall Carbon Nanotube-

19

Gold Nanocluster Composites Modified Electrode for the Simultaneous Determination of

20

Ascorbic Acid, Dopamine, and Uric Acid. Applied Biochemistry and Biotechnology 2014, 173,

21

1717-1726.

13

C NMR Study of Graphite Oxide.

13


C and 1H MAS NMR Studies of


1

38. Mani, V.; Chen, S.-M.; Lou, B.-S., Three Dimensional Graphene Oxide-Carbon Nanotubes

2

and Graphene-Carbon Nanotubes Hybrids. International Journal of Electrochemical Science

3

2013, 8, e60.

4

39. Das, R.; Talat, M.; Srivastava, O.; Kayastha, A. M., Covalent Immobilization of Peanut β-

5

Amylase for Producing Industrial Nano-Biocatalysts: A Comparative Study of Kinetics, Stability

6

and Reusability of the Immobilized Enzyme. Food Chemistry 2018, 245, 488-499.

7

40. Das, R.; Kayastha, A. M., An Antioxidant Rich Novel β-Amylase from Peanuts (Arachis

8

hypogaea): Its Purification, Biochemical Characterization and Potential Applications.

9

International Journal of Biological Macromolecules 2018, 111, 148-158.

10

41. Bernfeld, P., Amylases, α and β. Methods in Enzymology 1955, 1, 149-158.

11

42. Singh, C.; Purusottam, R. N.; Viswan, A.; Sinha, N., Molecular Level Understanding of

12

Biological Systems with High Motional Heterogeneity in its Absolute Native State. The Journal

13

of Physical Chemistry C 2016, 120, 21871-21878.

14

43. Yu, Z.; Lv, L.; Ma, Y.; Di, H.; He, Y., Covalent Modification of Graphene Oxide by

15

Metronidazole for Reinforce Anti-Corrosi on of Epoxy Coatings. RSC Advances 2016, 6, 18217-

16

18226.

17

44. Goze Bac, C.; Bernier, P.; Latil, S.; Jourdain, V.; Rubio, A.; Jhang, S. H.; Lee, S. W.; Park,

18

Y. W.; Holzinger, M.; Hirsch, A., 13C NMR Investigation of Carbon Nanotubes and Derivatives.

19

Current Applied Physics 2001, 1, 149-155.

20

45. Vacchi, I. A.; Raya, J.; Bianco, A.; Menard-Moyon, C., Controlled Derivatization of

21

Hydroxyl Groups of Graphene Oxide in Mild Conditions. 2D Materials 2018, 5, 3.


Page 20 of 22

Page 21 of 22 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

46. Thirupathi, R.; Reddy, Y. J.; Prabhakaran, E. N.; Atreya, H. S., Organic Fragments from

2

Graphene Oxide: Isolation, Characterization and Solvent Effects. Journal of Chemical Sciences

3

2014, 126, 541-545.

4

47. Abraham, A.; Mihaliuk, E.; Kumar, B.; Legleiter, J.; Gullion, T., Solid-State NMR Study of

5

Cysteine on Gold Nanoparticles. The Journal of Physical Chemistry C 2010, 114, 18109-18114.

6

48. Whipple, E. B.; Ruta, M., Structure of Aqueous Glutaraldehyde. The Journal of Organic

7

Chemistry 1974, 39, 1666-1668.

8

9

10

11

12

13

14

15

16

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