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Enzyme and Microbial Technology xxx (2016) xxxxxx

Contents lists available at ScienceDirect

Enzyme and Microbial Technology

journal homepage: www.elsevier.com/locate/emt

Biophysical characterization and activity analysis of nano-magnesium

supplemented cellulase obtained from a psychrobacterium following
graphene oxide immobilization
N. Dutta, S. Biswas, M.K. Saha
National Institute of Cholera and Enteric Diseases, West Bengal, India

a r t i c l e i n f o a b s t r a c t

Article history: Cellulase enzyme was puried from a psychrophilic strain of Bacillus subtilis obtained from east Himalayan
Received 2 February 2016 mountains. The native enzyme showed optimum activity at 15 C and pH 8.0.The Magnesium oxide
Received in revised form 13 April 2016 nanoparticle (MgN) supplemented enzyme when immobilized on graphene oxide nanosupport (GO),
Accepted 28 April 2016
via glutaraldehyde as cross linker, showed 2.98 folds increase in enzymatic activity at 8 C and more than
Available online xxx
3.5 folds activity increment at 90 C. The MgN-cel on graphene (GO-MgN-cel) showed a decrease in Km
by 6.7 folds at 8 C and 34 folds at 90 C. GO-MgN-cel showed 5 fold and 4.7 fold increase in Vmax at 8 C
Keywords:
and 90 C respectively than the untreated enzyme.When compared to native enzyme, GO-MgN-cel had
Psychrobacteria
Cellulase
t1/2 (half life) and Ed increased by 72.5 fold and 2.48 fold respectively at 90 C; and 41.6 fold and 2.19
Purication fold respectively at 8 C. Enzymatic activity of GO-MgN-cel was retained even after 12 repeated uses
Magnesium oxide nanoparticle and showed storage stability at 4 C for more than 120 days. This nanoparticle assisted immobilization
Graphene oxide technique can be utilized in bioprocessing industries which require functioning at these extreme ranges
Glutaraldehyde of temperature.
Kinetic parameters 2016 Elsevier Inc. All rights reserved.
Storage stability
Biophysical studies

1. Introduction
Cellulases are accountable for hydrolysis of the -1,4glucosidic
bonds in cellulose. This cellulolytic activity occurs by the synergis-
tic effect of three major components; endoglucanase (EC 3.2.1.4),
exoglucanase (EC 3.2.1.91) and glucosidase (EC 3.2.1.21) [1]. Cellu-
lases are used in different industrial applications, such as in textile
industry for bio-polishing for fabrics and producing stonewashed
look of denims, in animal feed industry for improving the nutri-
tional quality and digestibility, in food industry for processing of
fruit juices and in baking, in detergent industry for improving fab-
ric softness and brightness and removing the soil from cotton bres
[1]. Cold-active enzymes compared to mesophilic enzyme are more
attractive because of their high activity at low temperature which
is more preferred in application as they represent the lower limit of
Abbreviations: GO, graphene oxide nanosupport; cel, cellulase; MgN,
magnesium oxide nanoparticle; MgN-cel, cellulase activated by magnesium
oxide nanoparticle; GO-MgN-cel, magnesium oxide nanoparticlecoupled cellulase
nanocomposite immobilized on graphene oxide nanosupport.
Corresponding author.
E-mail address: sahamk@yahoo.com (M.K. Saha).
natural protein stability and are useful tool for studies in the eld of
protein folding [2]. In the textile industry, there is a need for novel
cellulases that are active at broad pH values. Cold active enzymes
are crucial in saving of energy in textile, detergent, fuel and etc.
industrial process [3].
Enzymes, owing to their selective nature catalyze specic
reactions with few by products. Thus they are environmentally
favourable alternative to conventional chemical synthesis, particu-
larly in the food and pharmaceutical industries where high reaction
selectivity is essential. Due to an absence of long term operational
stability and difculty in recovery and reuse of the enzyme, indus-
trial applications of enzymes are limited. These limitations can be
overcomed by immobilizing them on appropriate matrices. The
interaction between the enzyme and carrier governs the proper-
ties of immobilized enzyme, inuencing the chemical and other
thermodynamic properties. The property of immobilized enzyme
is also affected by particle mobility which is governed by particle
size and solution viscosity [4]. Nano-materials and nano structures
generally provide large surface area, low mass transfer resistance
and high enzyme loading capability. This aids in better interaction
with the enzyme, resulting in increased immobilization efciency,
recycling ability and long term storage of the enzyme. Nanomate-
rials can also be engineered to present multiple surface functional

http://dx.doi.org/10.1016/j.enzmictec.2016.04.012
0141-0229/ 2016 Elsevier Inc. All rights reserved.

Please cite this article in press as: N. Dutta, et al., Biophysical characterization and activity analysis of nano-magnesium supple-
mented cellulase obtained from a psychrobacterium following graphene oxide immobilization, Enzyme Microb Technol (2016),
http://dx.doi.org/10.1016/j.enzmictec.2016.04.012
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groups for interacting with biomolecules. A wide range of nano
materials has been studied and their positive effect on enzyme
was shown through immobilization process [57]. Graphene also
gets benetted by biological modication which improves its bio-
compatibility, solubility and selectivity. Proteins can exfoliate and
modify graphene due to presence of various functional groups,
through physical adsorption or chemical bonding [8]. Graphene
oxide (GO) has been studied as interesting nanosupport for a variety
of biologically active agents leading to novel biocatalysts, biosen-
sors, and drug delivery vehicles [9]. The morphology and large
accessible surface area of GO nanosheets along with the formation
of stable aqueous suspensions full the criteria for high enzyme
loading on support and thus for the development of catalysts for
biotechnological applications [10]. Horse-radish peroxidase and
oxalate oxidase have been successfully immobilized on GO sur-
faces and the extent of the electrostatic interaction between the
enzyme and nanomaterial surface was proved to change accord-
ing to the degree of the GOs reduction [11,12]. The introduction of
a glutaraldehyde spacer arm on the GO support enables tethering
of enzyme molecules to yield bio-conjugates with improved ther-
mostability, reusability and storage stability [13,14]. In this current
study, we investigated the immobilization efciency of Magne-
sium nanoparticle supplemented enzyme (cellulase) on graphene
oxide nanosupport in terms of enzyme activity and stability. Glu-
taraldehydyde mediated cross-linking of the enzyme was achieved
through simultaneous addition of the enzyme and glutaraldehyde
to GO. The enzyme-nanocomposite (GO-MgN-cel) could tolerate
both high (90 C, higher than its optimum temperatureof 15 C) and
cold temperatures (8 C) lower than its optimum temperature. It
was found that GO immobilized MgN-cel showed increased ther-
mal stability retaining up to 72.9% activity at 90 C after 240 min
and 83% activity at 8 after 210 min compared to the untreated
enzyme. Again GO-MgN-cel retained enzymatic activity after 12
repeated uses of the immobilized enzyme, thereby indicating an
efcient immobilization method which can be applied henceforth.
2. Materials and methods
2.1. Materials
The magnesium oxide nanoparticle water dispersion, (MgO,
99.95%, 50 nm, 20 wt% in water) (Stock #: US7018) was supplied
by US Research Nanomaterials, Inc. Single Layer Graphene Oxide
(SLGO) was purchased from mkNANO (80% carbon, 20% Oxy-
gen. Flake size: 0.55 m. Thickness: Single atomic layer >80%
Color: Bright yellow Purity: Carbon contents over 80%). Primers
for PCR (forward and reverse) and silver nitrate for silver stain-
ing were obtained from SigmaAldrich (Germany). -Cellulose,
agar powder and glutaraldehyde were purchased from Himedia,
India. All other reagents for bacterial culture media such as agar,
and salts were purchased from SRL (India) and were of analytical
grade. 20120 kDa protein markers were obtained from Fermentas
(Germany).
2.2. Methods
2.2.1. Identication and isolation of enzyme secreting bacterial
strains
Soil samples were collected from Gnathang valley of East Sikkim
(13,500 feet above sea level), India. Approximately 5 g of soil sample
was added to sterile 0.9% saline water. The sample saline mix was
incubated overnight at 20 C and the saline supernatant was used
as the bacterial source.Cellulolytic bacterial strains were isolated
from the soil by using enrichment and serial dilutions technique.
The medium used for isolation of cellulolytic bacteria contained
Please cite this article in press as: N. Dutta, et al., Biophysical characterization and activity analysis of nano-magnesium supple-
mented cellulase obtained from a psychrobacterium following graphene oxide immobilization, Enzyme Microb Technol (2016),
http://dx.doi.org/10.1016/j.enzmictec.2016.04.012
1.5% peptone, 1.2% carboxymethylcellulose (CMC), 0.2% K2 HPO4 ,
1.5% agar, 0.03% MgSO4 7H2 O, 0.25% (NH4 )2 SO4 and at pH 8.0 for
48 h of incubation at 20 C. Bacterial colonies were puried by
repeated streaking. Pure cultures of bacterial isolates were indi-
vidually transferred to CMC agar plates. The CMC agar plates were
incubated at 15 C for 5 days to allow for the secretion of cellulase.
At the end of the incubation, the agar medium was ooded with
a solution of Congo red (1% w/v) for 15 min. The Congo red solu-
tion was then poured off, and the plates were further treated by
ooding with 1 M NaCl for 15 min. The formation of a clear zone
of hydrolysis indicated cellulose degradation. The ratio of the clear
zone diameter to colony diameter was measured in order to select
for the highest cellulase activity producer. The largest ratio was
assumed to contain the highest activity.
2.2.2. Ribotyping studies
The cellulolytic strain with the highest activity was coined
as NCEL03.We performed 16S rDNA gene sequencing to iden-
tify the cellulase secreting bacterial strain. Genomic DNA was
prepared from NCEL03 by sodium dodecyl sulphate proteinase K-
cetyltrimethylammonium bromide (CTAB) method. The 16S rRNA
gene was partially amplied with the thermal cycler ABI 9700 (ABI,
Foster City, USA). The amplied and gel-eluted PCR fragments were
sequenced with an ABI 3100 Genetic Analyzer. Sequencing reaction
was performed by using the Big Dye terminator cycle sequencing
Kit V3.1 (Applied Biosystems, Foster City, USA). Then the nucleotide
sequence was deposited in GenBank.
2.2.3. Method of psychrophilic cellulase purication
Cellulase enzyme was puried from 100 ml culture of NCEL03.
Cell free supernatant was precipitated with 080% saturation
of ammonium sulphate followed by dialysis. All steps of the
purication procedure were performed at 4 C. The dialysed pro-
teins were loaded onto a CM-Sepharose column pre-equilibrated
with 10 mM sodium phosphate buffer (pH 8.0) and allowed to
equilibrate overnight. After washing the column with 10 mM
sodium phosphate buffer, a 60 ml increasing discontinuous gra-
dient (0200 mM) of NaCl dissolved in 10 mM sodium phosphate
buffer (pH 8.0) was applied to the column. Proteins were eluted
in fractions of 1 ml. The fractions showing cellulase activity were
concentrated using a Macrosep 10 K unit and loaded onto a glass
column packed with Sephadex G-100 (bed volume 30 ml) and
equilibrated with 10 mM sodium phosphate buffer. Elution of the
proteins was done using the same buffer. The collection of the
fractions and assay of enzyme activity were as described below.
Fractions were run on 12% SDS polyacrylamide using Bio-Rad elec-
trophoresis apparatus. The amount of protein that was loaded on
SDSPAGE gel lanes was 0.45 mg/ml. Protein markers and the pro-
tein bands were stained by silver staining.
2.2.4. Soluble enzyme assay for cellulase
Cellulase activity was assayed using dinitrosalisylic acid (DNS)
reagent to estimate reducing sugars released from CMC solubilised
in 50 mM sodium phosphate buffer (pH-8.0). Puried enzyme was
added to 0.5 ml of 1% CMC in 50 mM sodium phosphate buffer
and incubated at 20 C for 15 min. After incubation, reaction was
stopped by the addition of 1.5 ml of DNS reagent and the reac-
tion mixture was kept at 100 C in water bath for 10 min. Sugars
liberated were determined by measuring absorbance at 540 nm.
Cellulase activity was estimated by using glucose calibration curve.
One unit (U) of enzyme activity was expressed as the quantity of
enzyme that released 1 mol of glucose per min under standard
assay conditions.
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2.2.5. Circular dichronism studies
For the native cellulase enzyme at 15 C and 8 C, MgN-cel at
15 C and 8 C, and MgN at 8 C; CD spectra over the range of
190250 nm were obtained using a Chirascan spectropolarimeter
(Applied Photosystem, UK). Enzyme concentration for the puried
enzyme was maintained at 0.45 mg/ml for all observations.1 mm
pathlength cell and 1 nm bandwidth was maintained for every
observation while carrying out the spectral analyses with a scan
rate of 20 nm/min. A system of (10 mM sodium phosphate buffer,
pH 8.0) was used as blank.Data obtained from CD was analyzed
using the DICHROWEB website:http://www.dichroweb.cryst.bbk.
ac.
2.2.6. Measurement of Mg concentration by atomic absorption
spectra (AAS)
The Mg content of MgN and 1 mM MgCl2 , was measured using
AAS technique (Analyst 200, Perkin Elmer). Standard Mg ion solu-
tion was provided by Perkin Elmer. MgN of different dilutions
(0.005 M, 0.01 M, 0.015 M, 0.02 M,0.025 M, 0.03 M and 0.035 M)
were prepared and their concentration was determined by com-
parison of data with the standard solutions provided by Perkin
Elmer.
2.2.7. MgN effecting cellulase activity
Puried cellulase enzyme(100 l in volume with concentration
of 0.45 mg/ml) was incubated with MgN at nal concentra-
tions of 2.5 g/ml, 5.0 g/ml, 7.5 g/ml, 10.0 g/ml, 12.5 g/ml,
15.0 g/ml, and 17.5 g/ml. Enzyme activity for each fraction was
measured by the process described above.
2.2.8. Effect of temperature and pH on activity of MgN-cel and
untreated cellulase
The optimum temperature of MgN-cel and untreated cellulase
were determined by carrying out the standard enzyme assay in
sodium phosphate buffer at increasing temperatures i.e.8, 10, 15,
25, 37,50 and 60 C. The substrate was pre-incubated with the
enzyme at the said temperature for 25 min followed by the enzyme
assay for each observation. To observe the effect of MgN treat-
ment on enzyme activities with decreasing temperature, optimal
concentration of MgN was added to the enzyme. The control sys-
tem contained untreated enzyme. The effect of pH on MgN-cel was
studied in the pH range of 3.012.0 (citrate phosphate: pH 3.05.0,
sodium phosphate: 5.57.0, glycine buffer pH 8.012.0).
2.2.9. Stability of mgN-cel at lower and higher temperatures
To measure the retention of enzyme activity at lower and
higher temperatures, MgN treated cellulase was incubated for
0250 min at 8 Cand 90 C. The enzyme activities were determined
as described in the earlier section after incubation. The results
expressed were in comparison to the values of untreated (- MgN)
enzyme systems.
2.3. Enzyme immobilization on GO
Puried cellulase was coupled to the graphene oxide with the
help of glutaraldehyde as a cross linker. The optimum concentra-
tion of glutaraldehyde for cross-linking of cellulase was found out to
be 0.9(ml) and graphene oxide concentration required for enzyme
immobilization was found out to be 1.0 mg/ml (Supplementary Fig.
1). Graphene oxide dispersion was prepared by dissolving 1 mg/ml
of graphene in 25 mM sodium phosphate buffer (pH 8.0) followed
by treatment with glutaraldehyde (0.9 ml, 25% v/v in water) and
kept in dark for 4 h at room temperature. After treatment and buffer
wash, it was incubated with the MgN-cel under dark condition
for 22 h at 4 C. The immobilized enzyme was washed thoroughly
with phosphate buffer. Each step of immobilization was followed
Please cite this article in press as: N. Dutta, et al., Biophysical characterization and activity analysis of nano-magnesium supple-
mented cellulase obtained from a psychrobacterium following graphene oxide immobilization, Enzyme Microb Technol (2016),
http://dx.doi.org/10.1016/j.enzmictec.2016.04.012
3
by thorough washing with buffer and centrifugation at 8000 rpm
for 10 min at 4 C.
2.4. Immobilized enzyme assay
0.5 ml of 1% CMC in 50 mM sodium phosphate buffer (pH 8.0)
were added to graphene cross-linked with MgN-cel and was incu-
bated at 25 C for 30 min followed by centrifugation at 10000 rpm
for 3 min at 4 C. Supernatant was pipetted out in test tube and 1 ml
of 3,5 dinitrosalicylic acid was added, followed by placing it in boil-
ing water bath for 5 min. It was diluted with 10 ml of MilliQ water
before recording absorbance at 540 nm. One unit (U) of enzyme
activity was expressed as the quantity of enzyme that released
1 mol of glucose per min under standard assay conditions.
2.5. Stability and reusability of immobilized mgN-cel on GO
To observe the reusability of MgN-cel immobilized on GO, the
system (GO-MgN-cel) wastested for enzyme activity of 12 repeated
uses at a stretch with standard enzyme assay protocols. Storage
stability of GO-MgN-cel was tested over a period of 120 days with
15 days interval at 4 C.
2.6. Activationinactivation parameters and kinetics of cellulase
enzyme
The Km -Vmax , activation energy (Ea) and the activa-
tion/deactivation kinetics of both MgN treated and untreated
cellulase enzyme systems were studied using the standard reac-
tion mix and assay conditions as described in the earlier section.
The substrate (CMC) concentration used was from 0.015% to
1.25% to determine Km and Vmax . The assay temperature was case
specic(8 C or 90 C).The activation energies (Ea ) of GO-MgN-cel,
MgN-cel and cellulase were calculated for the temperature range of
825 C (277293 K) and 5090 C (323363 K) from the Arrhenius
plots. Inactivation parameters comprising half-life (t1/2 ), decay rate
constant (k), energy of deactivation (Ed ), enthalpy (H), entropy
(S) and free energy change (G) were obtained according to
Ortega et al., 2004 [15].
3. Results and discussion
3.1. Psychrophilic strain identication
The sequence of the partially amplied 16S rDNA fragments
from NCEL03 was found to be more than 98% similar to Bacillus
subtilis when compared to rDNA sequence database in GenBank.
The Genbank accession number was allocated as KT783470. A
cold-adapted cellulase CelG from the culture supernatant of the
Antarctic bacterium Pseudoalteromonas haloplanktis was isolated by
Garsoux et al., 2004 [16]. A Cold-Active cellulase from a novel psy-
chrotrophic isolate Bacillus sp. K-11 was isolated by Caf et al.,2014
[17].
3.2. Purication of cellulase from Bacillus subtilis strain NCEL03
60.72% recovery of cellulase was achieved after ion-exchange
chromatography. The recovery of enzyme activity was about
50.57% after gel-ltration. A single band was obtained in SDSPAGE
analysis indicating complete purication of the enzyme. Compared
to protein markers, the size of the puried enzyme was around
50 kDa (Fig. 1A). More than 7.83 fold purication was achieved with
a nal specic activity of 1665.8 units/mg (Table 1).
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Table 1
Purication of psychrophilic cellulase from Bacillus subtilis NCEL03.

Purication steps Total volume (ml) Total protein (mg) Total activity (unit) Specic activity (Unit/mg) Purication (fold) Yield (%)

Crude 100 48 10210 212.70 1.0 100

(080)% (NH4 )2 SO4 10 19 9659 508.36 2.39 94.60
CM-Sepharose 3.0 4.2 6200 1476.19 6.94 60.72
Gel Filtration (G-75) 1.5 3.1 5164 1665.8 7.83 50.57

catalytic site of the enzyme but it also brings about alteration in

intrinsic property of the enzyme. On investigating the effect of
different additives on enzyme systems of cel and GO-cel at 8 C
and 90 C it was found that though CaCl2 and MgCl2 somewhat
increased the activities of enzyme systems at both temperature
extremes, the presence of MgCl2 conferred optimum activity to
the enzyme systems (Supplementary Fig. 3). In our earlier studies
[19,30], we have demonstrated that if the metal co-factor (calcium)
of an enzyme is substituted by its nano-counterpart (Ca-NP), the
activity of the enzyme increases manifold. It was this rationale
which propelled us in trying out magnesium nanoparticle to inves-
tigate its effect on enzyme activity. Moreover on comparing the
enzyme activities at different concentrations of MgN and calcium
nanoparticles it was found that, at a concentration of 12.5 g/ml,
MgN nanoparticles triggered increased enzyme activity of 65% and
66.7% at 90 C and 8 C respectively against a similar concentration
of calcium nanoparticles (Supplementary Fig. 4).

3.6. Analysis of CD structures of puried cellulase

strands contribute towards imparting stability to the structure

Fig 1. SDS-PAGE (12%) showing purication of Cellulase from Bacillus subtilis
NCEL03.
Table 2
Magnesium concentration of MgN by Atomic Absorption Spectra.
System 1 mM MgCl2 MgN
Mg2+ concentration(g/l) 1.59 0.15 0.28 0.08
3.3. Mg content in MgN
The AAS absorbance studies showed that 1 mM MgCl2 contained
5.67 fold higher Mg2+ than MgN when considered on a per l basis.
(Table 2)
3.4. Determination of optimum temperature and pH of puried
cellulase
The optimal activity of cellulase was obtained at 15 C (Fig. 2A).
The activity was 54.07% higher than the activity at 8 C. The cellulase
was found to have pH optima of 8.0 (Fig. 2B). An isolated cold-active
cellulase from a novel psychrotrophic isolate Bacillus sp. K-11had
an optimum pH of 4.5 and its optimum temperature as 20 C [17]. A
novel endo-b-1,4-glucanase (named Cel5 M) was isolated from the
psychrophilic deep-sea bacteria Pseudomonas sp. MM15 showed
an optimum temperature of 30 C and pH of 4.5 [18].
3.5. Effect of MgN on activity of puried cellulase
The activity of puried cellulase increased with gradual increase
of MgN concentration when compared to control (-MgN). The opti-
mum concentration of MgN for cellulase activity was 12.5 g/ml
(Fig. 3A). There was a gradual fall in enzyme activity beyond this
concentration. Compared to cellulase (-MgN) at 8 C and 15 C, the
activity increased by 71.5% and 39.14% respectively for MgN-cel at
the optimum MgN concentration (Fig. 3A).This suggests that the
MgN not only increases enzyme activity upon interaction with the
of cellulase. (source:PDB http://www.rcsb.org/pdb/home/home.
do).The percentage of -strand content per 100 residues was 2.98at
the optimum temperature for enzyme activity of 15 C. The per-
centage of -strand content for the MgN-celsystem was 3.41 at the
same temperature. A nding of note was the increment in -strand
content by 34.07% for the MgN-cel system at 8 C compared to MgN-
cel system at 15 C. There was an increase of 53.76% of the-strand
content for the MgN-cel system at 8 C compared to the untreated
set at the same temperature. (Table 3) (Fig. 3B).
In a previous work by the author, CD spectroscopy of puri-
ed pectate lyase from Macrophomina phaseolina showed that the
enzyme falls in the all -secondary class [19]. The CD data suggests
that -strands are the most important contributor towards main-
taining the activity of pectate lyase and cellulase. It was observed
for the (-MgN) cellulase that at the temperature (8 C) lower than
(15 C) what supports its optimal activity decreased the -sheet
content by 0.89% with 43% loss in enzyme activity (Table 3).
3.7. Temperature prole for puried cellulase (MgN)
For cellulase (-MgN), the optimum temperature was 15 C which
became 8 C upon the addition of 12.5 g/ml MgN. The MgN-
cel activity increased by 66.97% 0.143 at 8 C compared to the
untreated enzyme at 8 C. An increment by 23.89% 1.03 at 12 C
and 28.11% 0.59 in enzymatic activity was observed at 15 C
with respect to the untreated enzyme set. Beyond 15 C, there
was gradual decrease of the MgN-cel activity. At 20 C, 30 C, and
50 C,MgN-cel activity decreased by 37.46% 0.98, 56.57% 1.002,
and 73.08% 0.014 respectively. The untreated set of enzyme reg-
istered decreased activity at and beyond 20 C (Fig. 2A). At higher
temperatures, both the enzyme systems suffered loss of activity
as per the nature of psychrophilic enzymes. The presence of MgN
boosted the enzyme activity at temperatures lower than the opti-
mum. This nding is important because this the rst time report of
MgN conferring stability to a psychrophilic cellulase at a tempera-
ture, lower than its optimum.

Please cite this article in press as: N. Dutta, et al., Biophysical characterization and activity analysis of nano-magnesium supple-
mented cellulase obtained from a psychrobacterium following graphene oxide immobilization, Enzyme Microb Technol (2016),
http://dx.doi.org/10.1016/j.enzmictec.2016.04.012
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Fig. 2. A. Comparison of cellulase activity as a function of temperature (860 C) in presence and absence of MgN. B. Comparison of cellulase activity as a function of pH
(312)in presence and absence of MgN. C. Activities of GO-MgN-cel, MgN-cel, GO + cel and cel at 8 C. D. Activities of GO-MgN-cel, MgN-cel, GO + cel and cel at 90 C.

Table 3
CD structure analysis of cellulase and MgN-cel at 15 C and 8 C.

Enzyme System () strand content (per 100 residues) at 15 C () strand content (per 100 residues) at 8 C change in () strand content (per 100 residues) at 8 C

cellulase 2.98 2.09 0.89

MgN-cel 3.41 4.52 1.11

3.8. Effect of pH on enzyme activity
On comparing with the enzyme activity at pH 8.0, MgN-cel sys-
tem retained enzyme activity by18.3%,30.05%, 56.69%, and 97.3%
respectively at pH 3,4, 6and 10.5. In comparison, cellulase (-MgN)
rapidly lost activity at pH values lower and higher than 8.0 (Fig. 2B).
MgN-cel retained 3560% of its optimal activity at pH values of 3and
8. In contrast, cellulase (-MgN) lost 4060% of its activity at the pH
values of 3 and 8.
3.9. Immobilization studies of cellulase on GO
The immobilization efciency of cellulase on GO was found out
to be 68.9%. (Supplementary Fig. 2).GO-MgN-cel showed increased
enzyme activity by 2.94 folds in comparison to native cellulase
at 8 C (Fig. 2C). Cellulase showed optimum activity at 15 C for
both immobilized and free enzyme(Fig. 2A). 60% increased enzyme
activity was observed for the MgN-cel at 8 C compared to cel (-
MgN). GO-MgN-cel showed more than 3.66 folds increased activity
than control set at 90 C (Fig. 2D). MgN-cel showed an activity
increase by 168.4% over control at this temperature. The high
activity of the GO-MgN-celsystem, as compared to the (-MgN)
cel and MgN-cel stems from the fact that the GO immobiliza-
tion increases conformational exibility of the enzyme increasing
enzyme rigidity. This is reected in improved stability of the
enzyme at high temperature which otherwise disrupts enzyme
structure and function [20]. The enhanced activity conferred to the
enzyme at temperature extremes by GO-MgN systems need fur-
ther investigation. It may be concluded that upon cross linking of
MgN-cel with GO, the system GO-MgN acts as a chaperone thereby
protecting the enzyme structure and integrity at temperature and
pH extremes. As the native structure of the enzyme remains intact
at extreme ranges of temperature and pH, it becomes extremely
efcient catalytic machinery. Enhancement in thermostability of
enzyme on being immobilized on nanomaterials has been shown
by others [21]. Earlier studies have characterised the mode of cross
linking of functionalized graphene by FTIR. The studies revealed
the presence of peaks corresponding to the C H stretch modes of
the cross linked molecule suggesting that cross linking between
functionalized graphene and glutaraldehyde has taken place. The

Please cite this article in press as: N. Dutta, et al., Biophysical characterization and activity analysis of nano-magnesium supple-
mented cellulase obtained from a psychrobacterium following graphene oxide immobilization, Enzyme Microb Technol (2016),
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Fig. 3. A.Cellulase activity as a function of MgN concentrations at 8 C and 15 C. B. Far-UV CD spectra (190250 nm) of the puried cellulase at 8 C and 15 C, MgN-cel at
8 C and 15 C and MgN.

hydroxyl groups can react with aldehyde through formation of
hemiacetal structure. The aldehyde functional groups present in
the molecule of glutaraldehyde, react with the sheets of func-
tionalized graphene as a cross-linker. Glutaraldehyde binds to the
hydroxyl group of the graphene oxide through its one arm contain-
ing aldehyde group and attaches to the enzyme via amino group
through its another aldehyde group. Glutaraldehyde is also known
to play an important role in the self-assembly of graphene oxide
nanosheets in aqueous solution, as the self-assembly of nano sheets
occur in more ordered fashion in presence of little amount of glu-
taraldehyde [22]. To validate our ndings, we performed similar
studies of immobilization on GO with xylanase enzyme obtained
from a mesophilic source (Genbank accession number:KF214094).
The trend of the data obtained were similar to the study in ques-
tion, as the mesophilic enzyme (optimum temperature 40 C) had
increased enzyme activity at 15 C and 95 C when immobilized on
GO. So, it is the process of immobilization on GO, supplemented
with nanoparticles which makes the enzyme psychrostable as well
as thermostable.
3.10. Reusability and storage stability of GO-MgN-cel
GO immobilized MgN-cel could be reused for 12 repeated times
retaining signicant activity at both high(61.69%; 90 C) and low
(64.4%; 8 C) temperatures whereas free enzymes lost their activ-
ity after single use (Fig. 4A,B). In sharp contrast, native cellulase
retained only 25% and 17% activity at 8 C and 90 C respectively.
(Fig. 4A,B). GO immobilization of MgN-cel via glutaraldehyde as
cross linker imparted exibility to the temperature range for the
enzyme activity by extending its functionality from psychrophilic
to thermophilic range. The immobilized MgN-cel on GO could be
stored at 4 C for more than 120 days with the enzyme retaining
more than 80% activity whereas the MgN-cel could only retain 45%
of its activity at the same temperature after 60 days (Fig. 4E). The
standing enzyme activity upon incubation of the enzyme systems
at 90 C showed that GO-MgN-cel could retain 72.9% enzyme activ-
ity after 240 min whereas the untreated set (-GO, MgN) lost half of
its initial activity after 60 min (Fig. 4D). At 8 C, GO-MgN-cel could
retain 83% enzyme activity after 210 min whereas the untreated set
lost one-fourth of its activity after 150 min (Fig. 4C). The observa-
tions underline the fact that the GO-MgN-cel is a supremely stable
system where the enzyme activity is enhanced and the integrity
of the catalytic sites of the enzyme are maintained even under
extreme conditions by the dual action of MgN and GO. This mode
of immobilization using MgN and GO in tandem is unique and a
nding of note. Reusability of a biocatalyst is an important fac-
tor that considerably reduces the processing cost of any industrial
merchandise. There are many reports which show improved sta-
bility of the enzymes through supported attachment via covalent
bonding [23,24]. Immobilized enzyme with good reusability and
prolonged stability provides an economical gain and can be used
constantly in both batch and continuous reactors. Improvement in
stability and reusability of enzymes following immobilization has
been well-documented [2527].

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Fig. 4. A. Analysis of reusability of cellulase as a function of number of use(s) at 8 C. B. Analysis of reusability of cellulase as a function of number of use(s) at 90 C. C. Retention
of cellulase activity as a function of time at 8 C. D. Retention of cellulase activity as a function of time at 90 C. E. Storage stability of GO-MgN-cel and cellulase(-MgN) at 4 C
as a function of number of days.

Table 4
Km , Vmax and Ea of cellulase and MgN-cel systems at 8 C and 90 C.

Activation kinetics parameters 8 C 90 C

cellulase MgN-cel GO-MgN-cel cellulase MgN-cel GO-MgN-cel

Km (mg/ml) 2.31 1.05 0.34 4.5 1.47 0.13

Vmax (Units/ml) 22.03 66 119.2 16.56 33.00 78.1
Ea (KJ/mol) 27.8 13.8 8.91 47.02 11.5 3.14
kcat /KM (mM1 s1 ) 2875.3 7247.1 8976.04 384.2 4598.03 7139.2

3.11. Kinetic parameters of cellulase systems
The kinetic parameters were evaluated for puried cellulase,
MgN-cel and GO-MgN-cel at 90 C and 8 C w.r.t the optimum
temperature of 15 C. The Km decreased with concomitant Vmax
increase for MgN-cel compared to cellulase (-MgN) at 8 C (Table 4).
This indicates that MgN increased afnity of the enzyme for the sub-
strate and rate of catalytic conversion compared to the untreated
enzyme. For cellulase (-MgN), Km and Vmax were 2.31 mM and
22.03 units respectively at 8 C. For MgN-cel, Km decreased by54.5%
(1.05 mM) and Vmax increased by 62% (66 Unit) compared to cel-
lulase (-MgN).For the GO-MgN-cel system, there was a decrease in

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Km by84.4% (0.34 mM) and an increase in Vmax by81% (119.2Unit)

60.667 1.076

163.54

177.72
187.17
192.46
170.95
compared tocellulase (-MgN)system (Table 4). Again at 90 C, the

40.838 0.13
Ed (KJ mol1 )
GO-MgN-cel had Km and Vmax values of 0.13 mM and 78.1 U respec-

(+)
tively. This value was 34.6 fold lower and 4.71 fold higher compared
to the Km (4.5 mM) and Vmax (16.56 U) of cellulase(-MgN). Arrhe-
nius plots of the enzyme at low temperature (825 C) showed that

288.7 0.46
cellulase (-MgN) had an Ea value of 27.8 kJ/mole, whereas MgN-cel
3.98 0.77
showed almost 2 fold decreased Ea value of 13.8 kJ/mole. At 8 C, the
life(t1/2 )
(min)

Ea value of 8.91 kJ/mole for GO-MgN-cel was 3.12 fold decreased

Half
363

S(J/mol/k) w.r.tto the cel (-MgN). At higher temperature range (5090 C), cel
GO + MgN
(-MgN) showed an Ea value of 47.02 kJ/mole, whereas GO-MgN-

467.62
454.72

436.44
475.09

445.07
cel showed almost 14.9 fold decreased Ea value of 3.14 kJ/mole
()
0.0024 0.67

(Table 4).Moreover, GO-MgN-cel showed an increase in the cat-

0.174 0.02
constant(k)

alytic efciency (kcat /Km ) by 18.5 fold at 90 C compared to the

(min1 )

untreated cellulase at the same temperature. Similarly at 8 C,

Decay

GO-MgN-cel system showed 3.12 fold increase in catalytic ef-

ciency compared to the untreated enzyme at the same temperature
223.5 0.35

(Table 4). It can be concluded from the ndings that the Go-MgN
6.53 1.2

system could catapult the enzyme activity at higher temperatures

life(t1/2 )

103.97
107.67
(min)

by increasing the afnity for the substrate while simultaneously

94.98
90.97

99.02
Half
353

(+)

increasing its conversion rate. The catalytic efciency prole of

the enzyme showed that GO-MgN composite provided increased
0.0031 0.52

substrate enzyme interaction at elevated temperatures compared

0.106 0.14
constant(k)

to the control set(-MgN). Again, the Km and Vmax values of GO-

(min1 )

MgN-cel and cel systems (at 8 C) indicates that the enzyme system
Decay

undergoes a conformational change making it more efcient in cat-

G(kJ/mol)

GO + MgN

alytic turnover. This effect is observed for the GO-MgN-cel system

161.6 1.21
11.74 0.42

92.291

93.517

alone and requires further investigation.The cellulase from NCEL03,

92.46

94.75
90.77
()

supplemented with MgN, could perform as an integrated enzyme

life(t1/2 )
(min)

system both at8 C and 90 C when immobilized on GO. The Km ,Vmax

Half
343

and Ea values for the enzyme indicate that substrate binding and
catalytic efciency were high at the temperature extremes. An ear-
0.0043 1.001

lier report of a fenugreek -amylase immobilized on functionalized

0.059 0.12
constant(k)

graphene showed better activity at higher temperatures as com-

(min1 )

pared to -amylase immobilized on phenyl boronate agarose [28]

Decay

37.896
38.052
38.062

and acrylic carriers [29]. Km value showed slight change from 1.56
38.14

37.81
Effect of variation in kinetic parameters for immobilized (MgN-cel) and cellulase(-MgN)(323K363 K).

to 1.82 mg/ml on being immobilized on functionalized graphene.

(+)

These differences can be related to the change in microenvironment

resulting from the enzymatic hydrolysis of substrate.
86.6 0.001
30.1 0.22
life(t1/2 )

3.12. Thermodynamic characteristics of immobilized and free

(min)
Half
333

enzyme
H(kJ/mol)

GO + MgN
0.023 0.992
0.0087 0.02

The thermodynamic parameters of thecellulase systems were

63.428
62.68

63.51
63.59
63.67
constant(k)

studied at a temperature range between (825 ) C (Table 6) and

()
(min1 )

(5090 ) C (Table 5) for activity analysis at temperature extremes.

Decay

The semi-logarithmic plots of enzyme activity versus time for MgN-

cel and cel(-MgN) systems exhibited linearity. The plots revealed
thatcel (-MgN) was cold inactivated following rst order kinetics
49.5 0.23
53.3 0.33

whereas MgN-cel was cold activated following rst order kinet-

life(t1/2 )
(min)

ics.For the cel(-MgN) system at both higher and lower temperature

Half
323

ranges, a trend of decrease in decay constant with increase in tem-

Enzymes

perature was observed. At 8 C; decay constant (k), half life (t1/2 )

0.014 0.027

and deactivation energy (Ed ) of cel (-MgN) were measured and

0.013 0.21
constant(k)

cel

gave values of 0.150 min1 , 4.62 min and 45.87 kJ/mole respec-
(min1 )
Decay

tively (Table 6). In comparison tocel (-MgN), MgN-cel had 35 fold

decreased k value of 0.0042 min1 while t1/2 (192 min) and Ed
(54.64 kJ/mole) were increased by 41.5 fold and 2.19 fold respec-
tively (Table 6). From these results, it was evident that MgN-cel
Go + MgN

system could resist deactivation at low temperatures unlike the

cellulase (-MgN) system. The cold and heat activation of GO immo-
Temperature (k)
()
(+)

bilized cellulase followed rst order kinetics.

It may be taken into account that a change in kinetic parame-
Enzymes

ters is brought about by immobilization process due to changes in

Table 5

the microenvironment of immobilized enzyme. At 90 C the decay

323
333
343
353
363
cel

constant (k), half life (t1/2 ) and deactivation energy (Ed ) of cellu-

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mented cellulase obtained from a psychrobacterium following graphene oxide immobilization, Enzyme Microb Technol (2016),
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Fig. 5. A. EAntropy-Enthalpy map of cellulase (-MgN) at (825) C (281298)k. B. Entropy-Enthalpy map of MgN-cel at (825) C (281298) k.

Table 6
Effect of variation in kinetic parameters for immobilized (MgN-cel) and cellulase(-MgN)(281K298 K).

281 288 293 298

Enzymes GO + MgN Decay Half Decay Half Decay Half Decay Half Ed (KJ mol1 )
constant(k) life(t1/2 ) constant(k) life(t1/2 ) constant(k) life(t1/2 ) constant(k) life(t1/2 )
(min1 ) (min) (min1 ) (min) (min1 ) (min) (min1 ) (min)
constant(k)

cel () 0.150 0.23 4.62 0.33 0.097 0.33 7.14 119 0.080 1.02 8.66 0.37 0.048 0.62 14.43 0.04 45.87 0.04
(+) 0.0042 0.005 192.5 0.99 0.0078 0.55 141.42 1.2 0.0130 0.78 77.02 1.03 0.025 0.43 22.35 1.12 54.64 0.97

Temperature (k) Enzymes H(kJ/mol) G(kJ/mol) S(J/mol/k)

GO + MgN GO + MgN GO + MgN

() (+) () (+) () (+)

281 cel 48.20 52.3 73.11 81.46 431.7 103.75

288 48.26 52.24 76.02 82.06 431.5 103.54
293 48.30 52.2 77.87 82.3 430.6 102.73
298 48.34 52.16 80.29 81.9 431.64 99.79

lase were found to be 0.174 min1 , 3.98 min and 60.66 kJ/mole increased t1/2 value of 288.7 min and 1.67 times increased Ed value
respectively (Table 5). As compared to these values GO-MgN- of 40.83 kJ/mole. These values indicated a lower rate of activity loss
cel had 72.5 times decreased k value of 0.0024 min1 , 72.53 fold at higher temperature coupled with an enhanced stability as evi-

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mented cellulase obtained from a psychrobacterium following graphene oxide immobilization, Enzyme Microb Technol (2016),
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Fig. 6. A. EAntropy-Enthalpy map of cellulase (-MgN) at (5090) C (323363)k. B. Entropy-Enthalpy map of MgN-cel at (5090) C (323363) k. All values are averages of
results from triplicate trials; error bars indicate the SD values.

dent from the Ed value(Table: 5).We have investigated the prospect
of immobilizing psychrophilic cellulase, coupled with MgN onto GO
via glutaraldehyde as crosslinker. The enhanced enzyme activity
at the upper and lower temperature extremes is a unique nding
from this study. The alteration in decay constant (k) with tem-
perature, high deactivation energy (Ed ) and the increase of t1/2
value with approaching temperature extremes of the GO-MgN-cel
implies that GO based immobilization conferred structural stability
to the enzyme at both lower and higher temperatures by guard-
ing it against harsh environments. Previous study by the author
[19,30] showed the effect of calcium nanoparticle in restoring and
enhancing the activities of bacterial cellulase, xylanase and fungal
pectate lyase. Herein, it has been shown that a psychrophilic cel-
lulase supplemented with MgN when immobilized on GO shows
improved activities at higher and lower temperature extremes. An
earlier study [31] with enzyme lipase from Rhizopus oryzae exhib-
ited exceptionally high thermal stability and high solvent tolerance
and even increased activity in acetone when immobilized onto a
graphene oxide (GO) nanosupport.
3.13. Entropy-Enthalpy analysis
The thermodynamic change associated with the enzymatic pro-
cess in the presence and absence of the nanoparticle is summarized
by the entropy-enthalpy map. When MgN was present in the sys-
tem, a compensatory behaviour was observed whereas the absence
of MgN triggered a critical change in the entropy-enthalpy pro-
le. The entropyenthalpy prole for the thermal inactivation
process of GO-MgN-cel at both higher and lower temperature
ranges showed that there was entropyenthalpy compensation
as the temperature approached the upper and lower temperature
extremes(Figs. 5 A,B and 6 A,B ). For MgN treated enzyme, the oppo-
site signs of H and S suggested a signicant entropy enthalpy
compensation. [32,33]. However this compensatory prole was not
observed for the untreated enzyme set as it was operative in nature
for the (-MgN) cellulase. The compensatory prole was lost, the
entropic and enthalpic contributions assumed the same sign (both
negative, showing poor compensation) for the untreated set as
the temperature approached the upper and lower temperature
extremes. The unfavourable entropic contribution perhaps indi-

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mented cellulase obtained from a psychrobacterium following graphene oxide immobilization, Enzyme Microb Technol (2016),
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cated a loss of structure (and enzymatic activity) as maintenance
of native structure normally is entropy driven process.
4. Conclusions
In this study, we have effectively optimized an enzyme immobi-
lization process on GO supplemented by MgN via glutaraldehyde as
cross linker. The immobilized enzyme system GO-MgN-Cel proved
to be an efcient machinery showing2.98 fold increase in enzyme
activity at 8 C and more than 3.5 folds activity increment at 90 C
compared to the untreated enzyme set at the same temperature.
The reusability and storage stability studies indicated durability
and functional efcacy of the immobilized enzyme. This method-
ology of immobilization was quick, effective and could be easily
implied upon similar studies for improved end products. This
nding brings to light an environment friendly bio processing of
enzymes without distorting the catalytic sites of the enzymes even
at extreme temperatures and pH.
Acknowledgements
The authors would like to acknowledge lab facility at National
institute of cholera and enteric diseases for the help and support.
The author would also like to thank technical ofcers Mr. S. Omesh
and Ms. M. Mallick of the ICMR institute for their sincere help in
media preparations.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.enzmictec.2016.
04.012.
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