Journal of Bioscience and Bioengineering

VOL. xx No. xx, 1e8, 2017

www.elsevier.com/locate/jbiosc

Nano-magnesium aided activity enhancement and biophysical characterization of

a psychrophilic a-amylase immobilized on graphene oxide nanosupport

Nalok Dutta, Subrata Biswas, and Malay Kumar Saha*

National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road, Scheme e XM, Beliaghata, Kolkata, 70001 West Bengal, India

Received 25 October 2016; accepted 1 February 2017

Available online xxx
In the current literature we have devised an immobilization technique for conferring psychrostability to a cold
active a-amylase (amy) enzyme by the use of magnesium nanoparticle (MgNP) and graphene oxide (GO). The GO-
MgNP-amy nanocomposite showed enhanced enzymatic activity and thermostability at both upper (90
C) and lower
(8
C) temperature extremes. The GO-MgNP-amy showed increased afnity towards substrate, reected in the decrease
in its Km by 2.35 and 14.9-fold at 8
C and 90
C, respectively, than the untreated enzyme. GO-MgNP-amy showed 2.34-
fold and 4.29-fold increase in Vmax at 8
C and 90
C, respectively, than the untreated enzyme. When compared to native
enzyme at 90
C, GO-MgNP-amy had t1/2 (half life) increased by 44-fold with simultaneous increase in Ed by 1.9-fold.
Again at 8
C, GO-MgNP-amy had t1/2 increased by 6.48-fold with simultaneous increase in Ed by 2.21-fold when
compared to the native enzyme. The enzymatic activity of GO-MgNP-amy was retained even after 12 repeated uses and
showed storage stability at 4
C for more than 120 days. The ability of GO-MgNP to sustain and aggravate enzyme ac-
tivity and stability at temperatures beyond the optimal range can be utilized in bioprocessing industries which re-
quires functioning at these extreme ranges of temperature.
2017, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Psychrobacteria; a-Amylase; Purication; Magnesium oxide nanoparticle; Graphene oxide; Glutaraldehyde; Kinetic parameters;
Storage stability; Biophysical studies; Reusability]

Psychrophilic enzymes offer vast opportunities in economic and
applicatory aspects as compared to thermophilic enzymes (1). a-
Amylases are endo-1,4-a-D-glucan glucanohydrolases (EC 3.2.1.1)
which cleave the 1,4-a-D-glucosidic linkages between the adjacent
glucose units in linear amylase and amylopectin chain of starch. The
a-amylases belong to family GH-13 (glycoside hydrolase group of
enzymes) and require metal ions for enzyme activity (2). Amylases
have multifaceted role as biocatalysts with wide usage in industrial
applications, such as additives in processed food industries, additives
in detergents, waste-water treatment, biopulping, bioremediations
and in molecular biology. These enzymes account for about 30% of
the worlds enzyme production (3). Applications of industrial en-
zymes can be increased manifold by the method of immobilization of
the enzymes on suitable carriers or matrices. Immobilization tech-
nology promotes reusability and operational stability of an enzyme
thereby altering its thermodynamic characteristics (4). Nanomaterial
supports are being widely used nowadays as they provide larger
surface area for the substrate to interact with the enzyme coupled
with increased loading capability. It is the higher surface to volume
ratio of the nanomaterials which increase immobilization efciency,
storage and reusability of the enzyme. Of late, a number of nano-
support materials have been extensively studied and effectively
utilized for facilitating bioprocesses based on enzyme-
immobilization (5e7). Bio-engineering involving graphene has
gained headway in recent past with promising results with respect to
* Corresponding author. Tel.: 91 33 2363 3856/91 9433 081013; fax: 91 33
2363 2398/91 2370 5066.
E-mail address: sahamk@yahoo.com (M.K. Saha).
its stereoselectivity and biosolubility (8). Graphene oxide (GO),
owing to its varied exibility in biological applications as nano-
support has found its use in production of biocatalysts, biosensors,
and drug delivery vehicles (9,10). Recent studies show that enzymes
in the form of oxalate oxidase and horseradish peroxidase have been
immobilized on graphene with satisfactory outcome (11,12). The use
of glutaraldehyde as a cross linker for enzyme immobilization on
graphene was found to confer increased structural stability to GO-
enzyme complex which reected in its increased activity and mul-
tiple usages (13,14).
In this current study, we investigated the immobilization ef-
ciency of Magnesium nanoparticle supplemented enzyme (a-
amylase) on graphene oxide nanosupport in terms of enzyme ac-
tivity and stability. Glutaraldehyde mediated cross-linking of the
enzyme was achieved through simultaneous addition of glutaral-
dehyde and the enzyme to GO. Upon immobilization, the enzyme-
nanocomposite (magnesium oxide nanoparticle coupled a-amylase
nanocomposite immobilized on graphene oxide nanosupport,
GO-MgNP-amy) tolerated temperatures higher (90
C) and lower
(8
C) than its optimum temperature of 18
C. In our earlier studies
(15,26), 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 enzymes (cellulase, xylanase and pectate lyase)
increased manifold. It was this rationale which propelled us in trying
out MgNP to investigate its effect on enzyme activity. As no previous
study has been conducted with MgNP to confer increased activity
and thermostability to an enzyme, the current study represents the
rst hand report in this respect.

1389-1723/$ e see front matter 2017, The Society for Biotechnology, Japan. All rights reserved.
http://dx.doi.org/10.1016/j.jbiosc.2017.02.002

Please cite this article in press as: Dutta, N., et al., Nano-magnesium aided activity enhancement and biophysical characterization of a psy-
chrophilic a-amylase immobilized on graphene oxide nanosupport, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.02.002
2 DUTTA ET AL.
MATERIALS AND METHODS
The magnesium oxide nanoparticle water dispersion (MgNP, 99.95%, 50 nm, 20
wt% in water) (stock no. US7018) was supplied by US Research Nanomaterials, Inc.
Single Layer Graphene Oxide (SLGO) was purchased from mkNANO (80% carbon, 20%
oxygen; ake size: 0.5e5 m; thickness: single atomic layer >80%; colour: bright
yellow; purity: carbon contents over 80%). Sigma Aldrich supplied agar powder,
glutaraldehyde and all other reagents required for subsequent media preparation.
Protein markers were obtained from Fermentas, a Germany based organization.
Identication, isolation and optimization studies of enzyme secreting
bacterial strains Collection of soil samples were done physically from Gna-
thang valley of East Sikkim (approximately 4120 m above sea level), India. To sterile
saline water approximately 5e6 g of soil sample was added. The sample saline mix
was incubated overnight at 15
C and the saline supernatant was used as the bacterial
source. Amylolytic bacterial strains were isolated from the soil by using enrichment
and serial dilutions technique. The medium used for isolation of Amylolytic bacteria
contained (0.5% peptone, 0.3% yeast extract, 1% soluble starch, 0.3% NaCl, 0.1% K2HPO4
and 0.02% MgSO4$7H2O) and at pH 8.5 for 48 h of incubation at 20
C. Bacterial col-
onies were puried by repeated streaking. Pure cultures of bacterial isolates were
individually transferred to starch-nutrient agar plates. The starch-nutrient agar plates
were incubated at 15
C for 5 days to allow for the secretion of amylase. Single
colonies which formed clear halos with Grams iodine were identied as starch
utilizing strains. The halo diameters of selected single colonies were measured to
determine the halo diameter to colony diameter ratio. Selected single colonies
were puried by repeated streaking and transferred to starch-nutrient agar slant.
16S rRNA gene sequencing We performed 16S rRNA gene sequencing as
previously described by the author (15). Finally, the nucleotide sequence was
deposited in GenBank. The Genbank accession number was allocated as KT783469.
Methods of psychrophilic a-amylase purication a-Amylase enzyme was
puried from an initial (100 ml) culture of NAMY01. Cell free supernatant was
precipitated with 0e30; 30e80% saturation of ammonium sulphate followed by
dialysis. All steps of the purication procedure were performed at 4
C. The
dialysed proteins were loaded onto a DEAE-Sephadex A-50 column (20 mm
diameter  60 mm long) column that had been pre-equilibrated with 25 mM
sodium phosphate buffer (pH 8.5) and allowed to equilibrate overnight. After
washing the column with 25 mM sodium phosphate buffer, a 60 ml increasing
discontinuous gradient (0e200 mM) of NaCl dissolved in 25 mM sodium
phosphate buffer (pH 8.5) was applied to the column. Proteins were eluted in
fractions of 1 ml and were checked for a-amylase activity. The fractions showing
activity were concentrated further and loaded onto Sephadex G-100 (bed volume
30 ml) glass column and equilibrated with 25 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 according to Swain and Ross (16) using Bio-Rad
electrophoresis apparatus. The amount of protein that was loaded on SDSePAGE
gel lanes was 0.50 mg/ml.
a-Amylase enzyme assay a-Amylase enzyme activity was estimated as
described by Okolo et al. (17). The assay mixture containing 250 ml of 25 mM
TriseHCl buffer (pH 8.5), 250 ml of 1% soluble starch (substrate, 0.25% of the assay
mixture composition), and 500 ml of appropriately diluted enzyme solution and
the mixture was incubated at (4e25)
C for 30 min. The reaction was stopped by
adding 3 ml of DNS reagent and maintained in boiling water for 5 min and 1 ml
of Rochelle salt solution was added nally. Absorbance of the reaction mixture
was measured at 540 nm. Absorbance values were plotted in a standard graph
prepared with different concentration of D-glucose. One unit of enzymatic activity
was dened as the amount of enzyme required to produce 1 mmol of glucose/min
under the assay condition.
Circular dichroism studies For the native amylase enzyme at 18
C and 8
C,
MgNP-amy at 18
C and 8
C, and MgNP at 8
C; CD spectra over the range of
190e250 nm were obtained using spectropolarimeter as per the procedure
described by Dutta et al. (26). The enzyme concentration for the puried enzyme
was maintained at 0.50 mg/ml for all observations.
Measurement of Mg concentration The Mg content of MgNP and 1 mM
MgCl2, was measured by Atomic absorption spectra. Standard Mg ion solution was
provided by Perkin Elmer. MgNP of different dilutions over the range
(0.001e0.040 M) were prepared and their concentration was determined by com-
parison of data with the standard solutions as provided.
MgNP effecting a-amylase activity Puried amylase enzyme (100 ml in
volume with concentration of 0.50 mg/ml) was incubated with MgNP at nal con-
centrations of (2.5e17.5 mg/ml). Enzyme activity for each fraction was measured by
the process described above.
Effect of temperature and pH on activity of MgNP-amy and untreated a-
amylase The optimum temperature of MgNP-amy and untreated amylase were
determined by carrying out the standard enzyme assay in sodium phosphate
buffer at increasing temperatures, i.e., 4
C, 6
C, 8
C, 10
C, 15
C, 25
C, 37
C, 50
C
and 60
C. The enzyme was pre-incubated at these said temperatures for 25 min
followed by the enzyme assay. The control system contained only untreated
enzyme (-MgNP). The effect of pH on MgNP-amy was studied in the pH range of
Please cite this article in press as: Dutta, N., et al., Nano-magnesium aided activity enhancement and biophysical characterization of a psy-
chrophilic a-amylase immobilized on graphene oxide nanosupport, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.02.002
J. BIOSCI. BIOENG.,
3.0e12.0; citrate phosphate buffer: pH 3.0e5.0; sodium phosphate buffer: pH
5.5e7.0; and glycine buffer: pH 8.0e12.0.
Stability of MgNP-amy at lower and higher temperatures To measure the
retention of enzyme activity at lower and higher temperatures, MgNP treated
amylase was incubated for 5e250 min at 8
C and 90
C. The enzyme activities were
determined after incubation as described in the earlier section. The results
expressed were in comparison to the values of untreated (-MgNP) enzyme systems.
The enzyme activity of both MgNP treated and untreated sets were determined
under same conditions.
GO-MgNP-amy immobilization For optimum functionality of amylase, we
envisaged the method of crosslinking the enzyme with GO by means of glutaral-
dehyde. The optimum concentration of glutaraldehyde for cross-linking of amylase
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 (Table S1). GO dispersion was
prepared by dissolving 1 mg/ml of graphene in 25 mM sodium phosphate buffer
(pH 8.5) followed by treatment with glutaraldehyde (25% v/v in water) and kept
in darkness for 14 h at room temperature. After this treatment and buffer wash,
this system was incubated with the MgNP-amy for 22 h at 4
C in darkness. The
immobilized enzyme was thoroughly washed with phosphate buffer.
Assay for GO-MgNP-amy To GO-MgNP-amy, 250 ml of 1% soluble starch in
25 mM sodium phosphate buffer (pH 8.5) were added and was incubated at
4e25
C for 30 min followed by centrifugation at 9300 g for 3 min at 4
C. The
assay mixture contained 250 ml buffer (pH 8.5), 250 ml of 1% soluble starch
(substrate, 0.25% of the assay mixture composition), and 500 ml of GO-MgNP-amy
amounting to 1 ml in volume. The supernatant was pipetted out in test tube and
1 ml of 3,5 dinitrosalicylic acid was added, followed by placing it in boiling water
bath for 5 min. It was diluted with 10 ml of Milli Q water before recording
absorbance at 540 nm. One unit (U) of enzyme activity was expressed as the
quantity of enzyme that released 1 mmol of glucose per min under standard assay
conditions.
Stability and reusability of immobilized MgNP-amy on GO To observe the
reusability of MgNP-amy immobilized on GO, the system (GO-MgNP-amy) was
tested for enzyme activity of 10 repeated uses at a stretch with standard enzyme
assay protocols. Storage stability of GO-MgNP-amy was tested over a period of
120 days with 15 days interval at 4
C.
Activationeinactivation parameters and kinetics of amylase The
KmeVmax, activation energy (Ea) and the activation/deactivation kinetics of both
MgNP treated and untreated amylase enzyme systems were studied using the
standard reaction mix and assay conditions as described in the earlier section. The
substrate (starch) 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-MgNP-amy, MgNP-amy and amylase were calculated for the temperature
range of 8e25
C (277e293 K) and 50e90
C (323e363 K) from the Arrhenius plots.
Inactivation parameters comprising half-life (t1/2), decay rate constant (k), energy of
deactivation (Ed), enthalpy (DH), entropy (DS) and free energy change (DG) were
obtained according to Ortega et al. (18).
RESULTS AND DISCUSSION
Psychrophilic strain identication The sequence of the
amplied partial 16S rDNA fragments from NAMY01 was found to
be more than 98% similar to Bacillus cereus when compared to rDNA
sequence database in GenBank. A psychro-halo-tolerant B. cereus
GA6 was isolated from soil of Gangotri glacier, Western Himalaya
that produced maximum cold-active a-amylase at 20  1
C after
96 h of incubation in alkaline medium by using glycerol and
ammonium acetate as a substrate (19). Wang et al. (20) puried
and characterized of cold active a-amylase excreted by a strain of
marine cold adaptive Penicillia sp.
Purication of a-amylase enzyme from Bacillus cereus strain
NAMY01 After ion-exchange chromatography, 43.9% recovery of
a-amylase was achieved. The recovery of enzyme activity was
about 35.3% after gel-ltration. A single band was obtained in
SDSePAGE analysis indicating complete purication of the
enzyme. Compared to protein markers, the size of the puried
enzyme was around 40 kDa (Fig. 1A). Approximately, 60-fold
purication was achieved with a nal specic activity of
3875 units/mg (Table 1).
Determination of optimum temperature and pH of puried
a-amylase The native a-amylase enzyme exhibited optimal ac-
tivity at a temperature of 18
C and pH 8.5 (Fig. 2A,B). When
VOL. xx, 2017
FIG. 1. SDS-PAGE (12%) showing purication of a-amylase from Bacillus cereus NAMY01.
compared to the activity of this enzyme at 8
C, the enzyme activity
at 18
C was found to be 47.36% higher.
In general, bacterial a-amylases are produced over a wide range
of temperature. Literature survey showed that Bacillus amylolique-
faciens, Bacillus subtilis, Bacillus licheniformis and Bacillus stear-
othermophilus are among the most commonly used Bacillus sp.
reported to produce a-amylase at temperatures 37e60
C (21,22). A
cold active a-amylase from Antarctic psychrophile Alteromonas
haloplanktis was reported to exhibit maximum a-amylase produc-
tion at 4
C (23). pH is known to affect the synthesis and secretion of
a-amylase just like its stability. Earlier studies have revealed that
fungi required slightly acidic pH and bacteria required neutral pH
for optimum growth. Bacterial cultures such as B. subtilis,
B. licheniformis and B. amyloliquefaciens required an initial pH of 7.0
(24,25).
Mg content in MgNP The AAS absorbance analysis revealed
that 1 mM MgCl2 contained 5.67-fold higher Mg2 than MgNP
when comparative studies of both systems were analysed at ml
concentrations.
Effect of MgNP on activity of puried a-amylase The ac-
tivity of puried a-amylase increased with gradual increase of MgNP
concentration when compared to control. The optimum concentra-
tion of MgNP for a-amylase activity was 12.5 mg/ml. There was a
gradual fall in enzyme activity beyond this concentration (Fig. 3A).
Compared to a-amylase at 8
C and 18
C, the activity increased by
3.02-fold and 1.36-fold, respectively, for MgNP-amy at the
optimum MgNP concentration (Fig. 3A). For investigating the effect
TABLE 1. Purication prole of psychrophilic a-amylase from Bacillus cereus NAMY01.
Purication step Total volume (ml) Total protein (mg)
Crude 200 152
0e30; 30e80% (NH4)2 SO4 10 34.2
DEAE-Sephadex 5 2.9
Gel ltration (G-100) 4 0.8
Please cite this article in press as: Dutta, N., et al., Nano-magnesium aided activity enhancement and biophysical characterization of a psy-
chrophilic a-amylase immobilized on graphene oxide nanosupport, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.02.002
NANO-MG INCREASE ACTIVITY OF GO IMMOBILIZED a-AMYLASE 3
of different additives on enzyme activity; enzyme systems of (i)
Amy and (ii) GO-amy were assayed at 8
C and 90
C in presence of
5 mM of various metal ions and additives (Fig. S2). It was found
that at 8
C, MgCl2 increased amylase activity by 73.1% whereas the
activity of amylase immobilized on graphene increased by 2.07
folds. Similar trends were observed for enzyme activities at 90
C
with 11.6% and 63% increase in enzyme activity of amylase and GO-
amy systems in presence of MgCl2. Though the presence CaCl2 and
MnCl2 somewhat increased the activities of enzyme systems at
both temperature extremes, the presence of MgCl2 conferred
optimum activity to the enzyme systems. On comparing the
enzyme activities at different concentrations of MgNP and
calcium nanoparticles it was found that, at a concentration of
12.5 mg/ml, MgNP nanoparticles triggered increased enzyme
activity of 59.2% and 43.2% at 90
C and 8
C, respectively, against a
similar concentration of calcium nanoparticles (Fig. S3).
This suggests that the MgNP not only increases enzyme activity
upon interaction with the catalytic site of the enzyme but it also
brings about alteration in intrinsic property of the enzyme.
Analysis of CD structures of puried a-amylase Analysis of
the CD structures by dichroweb software revealed that at the op-
timum temperature of 18
C for a-amylase, the b-strand % was 58.9.
The b-strand % for the MgNP-amy conjugate was 62.23 at the same
temperature. A nding of note was increase in b-strand by 8.3% for
the MgNP-amy system at 8
C compared to MgNP-amy system at
18
C. Also, an increase in b-strand by 5.65% was observed for the
MgNP-amy system at 8
C compared to the control set at the same
temperature. It was seen that the a-helix % of MgNP-amy system
at 8
C decreased by 5.13% compared to the MgNP-amy system at
18
C (Table 2) (Fig. 3B). This decrease in a-helix % did not affect
the activity of a-amylase. In a previous work by the author, CD
spectroscopy of puried pectate lyase from Macrophomina
phaseolina showed that the enzyme falls in the all b-secondary
class (26). The CD data conrms that b-strands are important
contributors towards maintaining the activity of pectate lyase and
a-amylase. It was observed that at 8
C, a temperature
considerably lower than what supports native enzyme activity
(18
C), the b-strand % decreased by 0.19 with 2.5-fold loss in
activity of enzyme.
Temperature prole for puried a-amylase (MgNP) The
optimum temperature for a-amylase activity was found to be 18
C.
Upon addition of 12.5 mg/ml MgNP it changed to 8
C. When MgNP-
amy was immobilized on GO, the activity increased considerably at
8
C. Both MgNP-amy and GO-MgNP-amy enzyme systems showed
activity optima at 8
C with 3.05-fold and 3.98-fold increased
activity at 8
C, respectively, compared to the untreated enzyme
set at 8
C. Increment in enzyme activity by 1.35-fold and 2.50-
fold was observed for MgNP-amy and GO-MgNP-amy,
respectively, at 18
C with respect to the untreated enzyme set
(Fig. 2A). At 4
C and 6
C there was respectively 3.93-fold and
4.09-fold increase in activity for GO-MgNP-amy compared to
untreated enzyme, whereas an activity increase by 2.21-fold and
2.43-fold was observed in case of MgNP-amy compared to
untreated enzyme set at the said temperature. Beyond 8
C, there
was gradual decrease of the MgNP-amy activity. At 50
C, MgNP-
amy activity decreased by 5.24-fold. However, the GO-MgNP-amy
activity retained 74% of its activity at 50
C compared to its
Total activity (unit) Specic activity (unit/mg) Purication (fold) Yield (%)
8764 57.65 1.0 100
6543 191.31 3.31 74.6
3856 1329.65 23.06 43.99
3100 3875 67.21 35.37
4 DUTTA ET AL. J. BIOSCI. BIOENG.,

FIG. 2. (A) Comparison of enzyme activities of a-amylase, GO-MgNP-amy, and MgNP-amy as a function of temperature (4e50
C). Comparison of a-amylase activity as a function of
pH 3e12 in presence and absence of MgNP. (B) Activities of GO-MgNP-amy, MgNP-amy, GOamy and a-amylase at 8
C and 90
C.

activity optima at 8
C. The untreated set of enzyme registered
decreased activity at and beyond 18
C (Fig. 2A).
Pseudoalteromonas arctica GS230, a cold-adapted amylase
producing bacterium isolated from cold seawater showed optimal
enzyme activity at 30
C, while the activity was highly decreased
at temperatures above 40
C. Ca2 improved the thermostability
of the enzyme signicantly (27). A cold-adapted amylase
produced by Nocardiopsis sp. 7326, showed optimal activity at
35
C and the activity decreased signicantly at temperatures
above 45
C (28). At higher temperatures, both the enzyme
systems suffered loss of activity as the nature of psychrophilic
enzymes suggest. The presence of MgNP boosted the enzyme
activity at temperatures lower than the optimum. This nding is
important because this the rst time report of MgNP conferring
stability to a psychrophilic a-amylase at a temperature, lower
than its optimum.
pH prole for puried a-amylase (MgNP) On comparing
with the enzyme activitiy at pH 8.5, MgNP-amy system was found
to retain 15.1%, 28.79%, 34.45%, 94.6% and 91.5% activity at pH 3, 4, 6,
9 and 10, respectively (Fig. 2A). In comparison, a-amylase rapidly
lost activity at pH values lower and higher than 8.0 (Fig. 2A). At
pH 8.5 and 12.0 MgNP-amy showed 2.2-fold and 2.9-fold
increased enzyme activity compared to the native enzyme. A
cold-adapted amylase produced by Nocardiopsis sp. 7326, showed
stability in pH range between 5 and 10 with the optimum pH at
8. Ca2, Mn2, Mg2, Cu2, and Co2 enhanced enzyme activity
while Rb2, Hg2, and EDTA inhibited the activity (28).
Immobilization studies of a-amylase on GO The immobi-
lization efciency of a-amylase on GO was found out to be 76.9%
(Fig. S1). For the MgNP-amy at 8
C, a 3-fold increase in enzyme
activity was observed compared to amy. GO-MgNP-amy showed
more than 4.79-fold increased activity than control set at 90
C
(Fig. 2B). MgNP-amy retained 27.5% of its optimum activity at
90
C. At 8
C and 90
C, the GO-amy system showed an activity
increase by 1.33 and 1.56-fold, respectively, than the untreated
enzyme at the said temperatures. The high activity of the GO-
MgNP-amy system, as compared to the amy and MgNP-amy
stems from the fact that the GO immobilization increases
conformational exibility of the enzyme which is reected in
improved stability of the enzyme at high temperature which
otherwise disrupts enzyme structure and function (29). It is
noteworthy that GO alone cannot confer same magnitude of
activity to the native enzyme. The MgNP mediated retention of
enzyme activity at low temperature implies that MgNP interacts

Please cite this article in press as: Dutta, N., et al., Nano-magnesium aided activity enhancement and biophysical characterization of a psy-
chrophilic a-amylase immobilized on graphene oxide nanosupport, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.02.002
VOL. xx, 2017 NANO-MG INCREASE ACTIVITY OF GO IMMOBILIZED a-AMYLASE 5

FIG. 3. (A) a-Amylase activity as a function of MgNP concentrations at 8
C and 18
C. (B) Far-UV CD spectra (190e250 nm) of the puried a-amylase at 8
C and 18
C, MgNP-amy at
8
C and 18
C and MgNP.

TABLE 2. Analysis of CD structure of a-amylase and MgNP-amy at 18
C and 8
C.

Enzyme system b strand % at 18 C

a helix % at 18
C b strand % at 8
C a helix % at 8
C Change in b strand % at 8
C Change in a helix % at 8
C

a-Amylase 58.9 13.92 52.1 8.78 6.8 5.14

MgNP-amy 62.23 9.29 67.45 6.42 5.22 2.87

with the active site of the enzyme conferring psychrostability
and thermostability. The reason behind the effectiveness of
nanoparticle formulations of magnesium in conjunction with GO
in stabilizing the enzyme activity as compared to Ca-NP needs
further investigation (Fig. S4A and B). Immobilization on GO
enhances the performance of this concerted enzyme system.
Enhancement in thermostability of enzyme on being immobilized
on nano materials has been shown by other studies (30).
Reusability and storage stability of GO-MgNP-amy The
standing enzyme activity upon incubation of the enzyme systems
at 90
C showed that GO-MgNP-amy could retain 73.4% enzyme
activity after 240 min whereas the untreated set lost half of its
initial activity after 60 min (Fig. 4B). At 8
C, GO-MgNP-amy could
retain 77.2% enzyme activity after 210 min whereas the untreated
set lost one-third of its activity after 150 min (Fig. 4A). GO-MgNP-
amy could be reused for 12 repeated times retaining signicant
activity at both high (64.35%; 90
C) and low (71.17%; 8
C)
temperatures whereas the free enzyme lost activity after single
use. In sharp contrast, native amylase retained only 11.1% and
9.8% activity at 8
C and 90
C, respectively. GO-MgNP-amy
imparted exibility to the temperature range for the enzyme
activity by extending its functionality from psychrophilic to
thermophilic range. The GO-MgNP-amy could be stored at 4
C for

Please cite this article in press as: Dutta, N., et al., Nano-magnesium aided activity enhancement and biophysical characterization of a psy-
chrophilic a-amylase immobilized on graphene oxide nanosupport, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.02.002
6 DUTTA ET AL. J. BIOSCI. BIOENG.,

FIG. 4. (A) Retention of a-amylase activity as a function of time at 8
C. (B) Retention of a-amylase activity as a function of time at 90
C. (C) Storage stability of GO-MgNP-amy and
amylase at 4
C as a function of number of days. All values are averages of results from triplicate trials; error bars indicate the SD values.

more than 120 days with the enzyme retaining more than 78.8%
activity whereas the amylase could only retain 50% of its activity
at the same temperature after 60 days (Fig. 4C). The observations
underline the fact that the GO-MgNP-amy 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 MgNP and GO. This
mode of immobilization using MgNP and GO in tandem is unique
and distinguished nding.
Kinetic parameters of a-amylase systems The kinetic pa-
rameters were evaluated for puried a-amylase, MgNP-amy and
GO-MgNP-amy at 90
C and 8
C with respect to the optimum
temperature of 18
C. The Km decreased with concomitant Vmax
increase for MgNP-amy compared to a-amylase at 8
C (Table 3).
This indicates that MgNP increased afnity of the enzyme for the
substrate and rate of catalytic conversion compared to the
untreated enzyme. The Km and Vmax for a-amylase, were
0.283 mM and 3.98 IU ml1, respectively, at 8
C. The Km
(0.19 mM) and Vmax (6.82 IU ml1) decreased and increased by
32.8% and 42%, respectively, for MgNP-amy as compared to
a-amylase. The Km (0.12 mM) decreased by 42.4% for GO-MgNP-
amy and Vmax (9.35 IU ml1) increased by 234% compared to
a-amylase system. At 90
C, the GO-MgNP-amy had Km and Vmax
values of 0.23 mM and 13.82 IU ml1, respectively, which was
14.95-fold lower and 4.29-fold higher, respectively, compared to
the Km (3.44 mM) and Vmax (3.22 IU ml1) of a-amylase.
Arrhenius plots of the enzyme at low temperature (8e25
C)
showed that a-amylase had an Ea value of 59.7 kJ/mol, whereas
MgNP-amy showed 4.29-fold decrease in Ea value of 13.8 kJ/mol.
At 8
C, the Ea value of 8.95 kJ/mol for GO-MgNP-amy was 6.67-
fold decreased with respect to a-amylase. At higher temperature
range (50e90
C), a-amylase showed an Ea value of 106.32 kJ/mol,
whereas GO-MgNP-amy showed almost 6-fold decreased Ea value
of 17.7 kJ/mol (Table 3).
The Km and Vmax values of GO-MgNP-amy and a-amylase sys-
tems (at 8
C) indicates that the enzyme system undergoes a
conformational change making it more efcient in catalytic turn-
over. This effect is observed for the GO-MgNP-amy system alone
and requires further investigation. The a-amylase from NAMY01,
supplemented with MgNP, could perform as an integrated enzyme
system, both at 8
C and 90
C when immobilized on GO. The
Km,Vmax and Ea values for the enzyme indicate that substrate
binding and catalytic efciency were high at the temperature
extremes.

Please cite this article in press as: Dutta, N., et al., Nano-magnesium aided activity enhancement and biophysical characterization of a psy-
chrophilic a-amylase immobilized on graphene oxide nanosupport, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.02.002
VOL. xx, 2017
TABLE 3. Km, Vmax and Ea of a-amylase and MgNP-amy systems at 8
C and 90
C.
Activation kinetics parameters Temperature 8
C
Amy MgNP-amy
Km (mg/ml) 0.283 0.19
Vmax (mmol min1 ml1) (IU ml1) 3.98 6.82
Ea (KJmol1) 59.72 13.95
Thermodynamic characteristics of immobilized and free
enzyme The thermodynamic parameters of the a-amylase sys-
tems were studied at temperature ranges between 8e25
C;
(Table S2) and 50e90
C; (Table S3) for activity analysis at
temperature extremes. The semi-logarithmic plots of enzyme
activity versus time for GO-MgNP-amy and a-amylase systems
exhibited linearity. For the a-amylase system at both higher and
lower temperature ranges, a trend of decrease in decay constant
with increase in temperature was observed. At 8
C; decay constant
(k), half life (t1/2) and deactivation energy (Ed) of a-amylase were
measured and gave values of 0.147 min1, 23.8 min and 70.076 kJ/
mol, respectively (Table S2). In comparison to a-amylase, GO-
MgNP-amy had 36.7-fold decreased k value of 0.004 min1 while
t1/2 (155 min) and Ed (64.94 kJ/mol) were increased by 6.5-fold and
1.92-fold, respectively (Table S2). At 90
C the decay constant (k),
half life (t1/2) and deactivation energy (Ed) of a-amylase were found
to be 0.011 min1, 6.3 min and 53.6 kJ/mol, respectively
(Table S3). As compared to these values GO-MgNP-amy had 97.7%
decreased k value of 0.0025 min1, 44-fold increased t1/2 value of
277.2 min and 192.7% increased Ed value of 49.78 kJ/mol. These
values indicated a lower rate of activity loss at the higher
temperature coupled with an enhanced stability as evident from
the Ed value (Table S3). The alteration in decay constant (k) with
temperature, high deactivation energy (Ed) and the increase of t1/2
value with approaching temperature extremes of the GO-MgNP-
amy implies that GO based immobilization conferred structural
stability to the enzyme at both lower and higher temperatures by
guarding it against unfavourable environments.
Entropy and enthalpy analysis The entropy and enthalpy
prole for the thermal inactivation process of GO-MgNP-amy at both
higher and lower temperature ranges showed that there was entropy
and enthalpy compensation as the temperature approached the
upper and lower temperature extremes (Fig. S4A,B; Fig. S5A,B). For
MgNP treated enzyme, the opposite signs of DH and DS suggested
signicant entropy enthalpy compensation (31,32). However this
compensatory prole was not observed for the untreated enzyme
set as it was operative in nature for the amylase (Tables S4 and
S5). The loss of compensatory prole, negative signs of entropic
and enthalpic contributions (a marker for feeble compensation)
were salient features of the untreated set as the temperature
approached the upper and lower temperature extremes (Tables S4
and S5). This entropy and enthalpy prole behaved similarly to
that of pectate lyase in presence of Calcium nanoparticle at higher
temperatures that otherwise disintegrates its native form (26). In
the current study, we have effectively optimized an enzyme
immobilization process on GO supplemented by MgNP via
glutaraldehyde as cross linker. It was found that GO-MgNP-amy
showed increased thermal 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-MgNP-amy retained enzymatic
activity after 12 repeated uses of the immobilized enzyme, thereby
indicating an efcient immobilization method which can be
applied henceforth to other bioprocesses.
The retention in activity of GO-MgNP-amy enzyme system at
90
C is indeed a unique observation. Supplementation with NP
enhanced the psychrophilicity of amylase. When this was followed
by GO immobilization, the enzyme exhibited thermostability as
Please cite this article in press as: Dutta, N., et al., Nano-magnesium aided activity enhancement and biophysical characterization of a psy-
chrophilic a-amylase immobilized on graphene oxide nanosupport, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.02.002
NANO-MG INCREASE ACTIVITY OF GO IMMOBILIZED a-AMYLASE 7
Temperature 90
C
GO-MgNP- amy Amy MgNP-amy GO-MgNP- amy
0.12 3.44 1.2 0.23
9.35 3.22 7.21 13.82
8.91 106.32 71.5 17.7
well as psychrostability for several hours. The enhanced activity
conferred to the NP-enzyme at temperature extremes by GO needs
further investigation. Earlier studies show that a lipase immobi-
lized on functionalized graphene showed better activity at higher
temperatures as compared to free enzyme (33). The GOelipase
showed improved catalytic performance (93.4%) with respect to the
free lipase, and the reusability after 10 times was 69.9%. This
approach could be utilized for enzyme recycling without affecting
the integrity of the catalytic sites of the enzymes, even at extreme
temperatures. The reusability and storage stability studies indi-
cated durability and functional efcacy of the immobilized enzyme.
This methodology of immobilization was quick, effective and could
be easily implied upon similar studies for improved stable 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.
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.jbiosc.2017.02.002.
ACKNOWLEDGMENTS
The author would like to acknowledge the ICMR 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.
Sudhir Omesh and Ms. Maosam Mallick of the ICMR institute for
their sincere help in media preparations.
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