Optimization of process variables by central composite design for the immobilization of urease...

ORIGINAL PAPER

Optimization of process variables by central composite designfor the immobilization of urease enzyme on functionalized goldnanoparticles for various applications

Mahe Talat • Ashwani Kumar Singh •

O. N. Srivastava

Received: 27 October 2010 / Accepted: 10 January 2011 / Published online: 26 January 2011

� Springer-Verlag 2011

Abstract In the present study, enzyme urease has been

immobilized on amine-functionalized gold nanoparticles

(AuNPs). AuNPs were synthesized using natural precursor,

i.e., clove extract and amine functionalized through

0.004 M L-cysteine. Enzyme (urease) was extracted and

purified from the vegetable waste, i.e., seeds of pumpkin to

apparent homogeneity (sp. activity 353 U/mg protein).

FTIR spectroscopy and transmission electron microscopy

was used to characterize the immobilized enzyme. The

immobilized enzyme exhibited enhanced activity as com-

pared with the enzyme in the solution, especially, at lower

enzyme concentration. Based on the evaluation of activity

assay of the immobilized enzyme, it was found that the

immobilized enzyme was quite stable for about a month

and could successfully be used even after eight cycles

having enzyme activity of about 47%. In addition to this

central composite design (CCD) with the help of MINI-

TAB� version 15 Software was utilized to optimize the

process variables viz., pH and temperature affecting

the enzyme activity upon immobilization on AuNPs. The

results predicted by the design were found in good agree-

ment (R2 = 96.38%) with the experimental results indi-

cating the applicability of proposed model. The multiple

regression analysis and ANOVA showed the individual and

cumulative effect of pH and temperature on enzyme

activity indicating that the activity increased with the

increase of pH up to 7.5 and temperature 75 �C. The effects

of each variables represented by main effect plot, 3D sur-

face plot, isoresponse contour plot and optimized plot were

helpful in predicting results by performing a limited set of

experiments.

Keywords Au nanoparticles � Central composite design �Clove extract � Functionalization � Process variables �Urease

Introduction

With the application of nanotechnology in biology and

medicine, the need for nano-bioconjugate has significantly

increased. Gold nanoparticles (AuNPs) have attracted

enormous amount of attention in the recent years, especially,

in the biological and chemical researches because of their

good biocompatibility [1]. AuNPs which have high-affinity

for biomolecules, have been used in biosensors, [2] immu-

noassays, [3] therapeutic agents, [4] and vectors for drug

delivery [5]. The conjugation of AuNPs and biomolecules

has become a major area of research for advancing the use of

nanotechnology in applications covering sensors [6]. Pro-

teins, enzymes, DNA, and oligonucleotides and several other

biosystems have all been immobilized onto AuNPs [7].

Before immobilization, nanoparticles have to be surface

modified to make them stable and compatible for prepa-

ration of bioconjugate. Various methods have been devel-

oped till now for the synthesis, functionalization, and

protection of the gold nanoparticles using amino acids like

tryptophan, glutamic acid and histidine [8, 9]. Functional

groups, such as thiol (–SH) and amino (–NH2) groups, are

known to have high-affinity for gold and therefore, an

amino acid, containing both functional groups, such as

L-cysteine, is a promising compound to be used for the

biofunctionalization of gold nanoparticles which could be

used for coupling of biomolecules such as enzymes and

M. Talat � A. K. Singh � O. N. Srivastava (&)

Department of Physics, Nanoscience and Nanotechnology Unit,

Banaras Hindu University, Varanasi 221005, India

e-mail: [email protected]

123

Bioprocess Biosyst Eng (2011) 34:647–657

DOI 10.1007/s00449-011-0514-2

protiens. Compared with free enzyme in solution, the

immobilized enzyme is more stable and resistant to various

environmental changes [10]. Among all the enzymes, ur-

eases are the most appropriate in immobilized form for a

number of medical and analytical applications. Ureases

(EC 3.5.1.5, urea amidohydrolase) are nickel-dependent

metalloenzymes that catalyze the hydrolysis of urea to

ammonia and carbon dioxide. Urease is ubiquitously

present in plants, fungi and bacteria. In plants, urease is the

only enzyme that is able to recapture nitrogen from urea

[11]. This enzyme is utilized in direct urea detection in

biomedical samples as the content of urea in blood or in

urine is an important substance in the diagnosis of renal

and liver diseases. Urease is also used as medicine for the

removal of urea from blood or dialysate in treatment of

uremia and in analytical determinations of urea [12]. The

most effective way of removing urea from aqueous solu-

tions is the utilization of immobilized urease. However,

high-cost and limited availability of immobilized enzyme

preparations are two important limitations in the wider

applications of enzymes. Most of the studies have utilized

urease obtained from expensive sources like jack bean,

pigeon pea, etc. Therefore, still there exists a need to obtain

and immobilize urease from a non-conventional, unutilized

and economically viable source. In the present study, gold

nanoparticles were amine functionalized through L-cysteine

which was further utilized to immobilize urease obtained

from the dehusked seeds of pumpkin which is a vegetable

waste [13]. The characteristics of enzymes such as kinetic

constants, activation energy, temperature, pH, storage sta-

bility and reusability, etc. determine the usability and

productivity of enzymes, therefore, immobilized enzyme

was further characterized and different parameters were

studied with respect to the soluble enzyme. Recently, the

response surface methodology (RSM) has been widely

employed for the optimization of enzymatic processes as

well as in other catalytic studies [14]. It is a collection of

mathematical and statistical techniques useful for analyz-

ing the effects of several independent variables [15].

Application of RSM for conventional method is advanta-

geous, because there is a variation of only one parameter at

a time, keeping the other parameters constant, and thus, the

cumulative effect of all the affecting parameters at a time

cannot be studied [16]. However, in RSM, the interactions

of two or more variables can be studied simultaneously. In

addition to this, it results in higher percentage yields,

reduced process variability, closer confirmation of the

output response to nominal and target achievement, and

less treatment time with minimum costs [17]. In this report,

we demonstrate the use of the statistical method to change

the factor units in a quadratic central composite response

surface frame to obtain an improved experimental design

frame that encloses the optimal responses. This study also

investigates the feasibility of using urease immobilized on

functionalized gold nanoparticles as a potential urea bio-

sensing device and for environmental monitoring as well.

Experimental

Chemicals

Chloroauric acid (AuCl4), L-cystiene were purchased from

Sigma-Aldrich. All chemical reagents were of AR grade.

Pumpkin seeds were procured from the local market and de-

husked just before soaking. Bovine serum albumin was

obtained from Sigma Chemical Co., USA. Sephadex G-200

was from Pharmacia Fine Chemicals, Uppsala, Sweden.

Nessler’s and Folin–Ciocalteau reagents were from Quali-

gens Fine Chemicals, Mumbai. All other reagents were ana-

lytical grade chemicals either from BDH or E. Merck, India.

Synthesis of Au nanoparticles

Au nanoparticles have been obtained by reduction of per-

chlorate ions for which, clove extract has been used as

reducing agent in the ratio of 1:1 [18]. The details of syn-

thesis route of rapid green synthesis route and mechanism of

formation of AuNPs have been described by us [18]. For the

synthesis of gold nanoparticles using clove extract, different

volumes of clove extract (5–50 ml) were added to 50 ml of

0.004 M AuCl4 solution (aqueous) with rigorous stirring at

200 rpm. After reduction, the solution was centrifuged and

nanoparticles were collected and redispersed in water. The

centrifugation process was repeated for several times so that

other impurities may get washed out. Effect of the amount

of clove extract on the synthesis of gold nanoparticles was

monitored by observing the product formed, using UV-

visible spectroscopy and transmission electron microscopy

(TEM) measurement. These synthesized nanoparticles are

highly monodispersed having circular shape. Size of the

particles ranges between 90 and 120 nm.

For the synthesis of –NH2 functionalized Au nanopar-

ticles; 5 ml of clove extract was added to 50 ml of 0.004 M

solution of AuCl4. After 5 min of reaction, 50 ml of

0.004 M of L-cysteine was added to the reaction mixture

during stirring. This amine functionalized gold nanoparti-

cles was further centrifuged and washed. For the immobi-

lization, enzyme (urease) were extracted and purified from

the vegetable waste, i.e., seeds of pumpkin to apparent

homogeneity (sp. activity 353 U/mg protein). Immobili-

zation of urease was performed by incubating amine-

functionalized gold nanoparticles with the enzyme

overnight at 4 �C. The enzyme-immobilized gold nano-

particles were thoroughly washed with double-distilled

water by centrifugation at 5,000 rpm for 5 min at 25 �C

before the activity assay.

648 Bioprocess Biosyst Eng (2011) 34:647–657

123

Characterization of nanoparticles

Synthesized AuNPs obtained after reduction was washed

out twice with double-distilled water and then character-

ized by employing transmission electron microscope

(TEM), XRD infrared spectroscopy by X’Pert Pro X-ray

diffractometer (PANalytical BV, the Netherlands). FTIR

spectra were recorded corresponding to different steps of

urease immobilization (compared with the previous data)

onto AuNPs viz., AuNPs, AuNPs–cysteine, AuNPs–cys-

teine–urease using Perkin Elmer Spectrum 100 instrument.

To obtain a good signal-to-noise ratio, 50 scans of the film

were taken in the range 450–4,000 cm-1.

Enzyme and protein assay

Urease from dehusked seeds of pumpkin was isolated and

purified to electrophoretic homogeneity as described earlier

[13]. The most important factor in the enzyme immobili-

zation procedure is the complete retention of biocatalytic

activity of the bioconjugate. The biocatalytic activity of

urease was done in the solution form and in the immobi-

lized state (on amine-functionalized AuNPs) by following

a somewhat modified form of the technique employed by

Prakash et al. [19], for calcium alginate-immobilized ure-

ase obtained from water melon seeds.

One unit of urease activity is defined as the amount of

enzyme, which liberates 1.0 lmol NH3 from urea per min

at 35 �C at pH 7.0.

Protein was estimated by Folin–Ciocalteu reagent cali-

brated with crystalline bovine serum albumin as described

by Lowry et al. [20]. The quantity of protein bound on the

AuNPs was calculated by subtracting the protein recovered

in the supernatant after washing the enzyme-immobilized

AuNPs with the double-distilled water.

Effect of temperature on the activity of free

and immobilized urease

The temperature activity profile of soluble and immobi-

lized urease was determined in the range 10–85 �C.

Effect of pH on the activity of free and immobilized urease

Urease activity as a function of pH was determined at

35 �C in 50 mM phosphate buffer of 50 mM concentration

in the pH range of 5.5–8.5.

Operational stability and storage stability of free


The operational and storage stability of the immobilized

urease was studied by detecting the residual activity. The

immobilized urease on AuNPs was thoroughly washed to

remove any residual substrate on immobilized urease sur-

face after completion of each reaction cycle. They were

then reintroduced into fresh reaction medium and urease

activities were detected under standard assay conditions.

Freshly immobilized enzyme was taken as control for each

assay. The storage stability was evaluated by storing the

soluble and immobilized enzyme at 4 �C for 1 month and

the urease activity was measured every second day ini-

tially, then after 5 days. The total duration of the mea-

surements corresponded to 30 days.

Design of experiments

A two-level two-factor (22) full factorial design has been used

to observe the effect of the variables influencing the activity

of immobilized urease enzyme. As a result the two variables

viz., pH (low 5.5 and high 8.5 mM) and temperature

(30–85 �C) have been considered. A variety of factorial

designs are available to achieve this task. The most successful

and best among them is the central composite design (CCD)

which is accomplished by adding two experimental points

along each coordinate axis at opposite sides of the origin and

at a distance equal to the semi-diagonal of the hyper cube of

the factorial design, and new extreme values (low and high)

for each factor added in this design. This design has also been

used earlier for the successful optimization of the production

of inulase from Cryptococcus aureus and also for diosgenin

with Trichoderma reesei [21, 22].

This response surface model was used to predict the

result by isoresponse contour plots and three dimensional

surface plots. Contour plot is the projection of the response

surface as a two dimensional plane, whereas 3D surface

plots is the projection of the response surface in a three

dimensional plane. For optimization of enzyme activity at

variables, pH and temperature, a 22 factorial central com-

posite design with four axial (a = 1.414) points, four cube

points and six central points resulting in a total 13 or 14

experimental points will be used in a single block.

Results and discussion

Characterization

AuNPs

The XRD characterization, typical XRD pattern of as-

synthesized particle corresponds to that of Au lattice

structure. TEM characterizations were performed on

TECHNI 20 G2-electron microscope operated at an accel-

erating voltage of 200 kV. The samples of gold nanopar-

ticles synthesized using AuCl4 and clove extract as

Bioprocess Biosyst Eng (2011) 34:647–657 649

123

reducing agent were prepared by placing a drop of reaction

mixture over copper grid and allowing water to evaporate

and dry before loading into the TEM machine.

Synthesized AuNPs completely matches with the known

Au cubic lattice structure. This analysis was further con-

firmed by extensive TEM investigation employing both

imaging and diffraction modes. Figure 1a shows repre-

sentative bright field TEM image of the AuNPs together

with the formation of mostly spherical AuNPs. Inset in the

Fig. 1a shows the typical selected area electron diffraction

pattern (SAD) of the Au precipitates. Figure 1b shows

EDAX data suggesting the presence of Au alone in the as-

synthesized nanoparticles. All these observations and

analyses of the as-synthesized deposits corresponded to

that of Au confirms that the deposited particles are AuNPs.

AuNPs–urease

The microstructural characterization of AuNP-immobilized

urease was carried out through TEM. Figure 2a shows a

typical TEM micrograph of enzyme-immobilized Au

nanoparticles which show that the AuNPs are encapsulated

within the enzyme urease. TEM Micrographs also show

dispersion of gold nanoparticles with random orientations.

Fig. 1 a TEM micrographs of

Au nanoparticles and b energy-

dispersive spectrum (EDAX) for

the elemental analysis of AuNPs


123

TEM images of gold nanoparticles (dark spots in clusters)

decorating the surface of the urease enzyme can clearly be

seen. This is illustrated quite clearly in Fig. 2b where the

interface between enzyme matrix is seen with a dense

population of gold nanoparticles. This clearly indicates that

the gold nanoparticles are bound to the enzyme urease at a

very high concentration. The clusters are united in a more

complex network and a bridge-like structure is formed by

the AuNPs, because of crosslinking of the enzyme.

Apparently the microstructure suggests the presence of

AuNPs within the enzyme. It was further investigated

employing FTIR as discussed below. The FTIR spectra, as

shown below, confirms the covalent linkages between

AuNPs embedded in the enzyme matrix. In Fig. 2a, it can

be seen that, the morphology of configuration is nearly

spherical with somewhat irregular periphery. The particles

are fairly spherical, with slightly irregular edges. It is clear

that on immobilization the various AuNPs get tagged with

urease via amino group of cysteine residues. Because

linkage involves several radicals, which are set throughout

the periphery, the shape of the AuNp–urease configurations

become irregular.

FTIR analysis (AuNPs, AuNPs–cysteine,

AuNPs–cysteine–urease)

The sequential steps of functionalization and immobiliza-

tion were examined by FTIR. At every step of character-

ization for the FTIR analysis, as-synthesized gold

nanoparticles were washed and dried. While washing, Au

nanoparticles were centrifuged at a speed of 15,000 rpm

for 20 min in double-distilled water and the purified pre-

cipitates of gold nanoparticles were dried separately to

remove the water completely. FTIR spectra of (AuNPs,

AuNPs–cysteine, AuNPs–cysteine–urease scans of the film

were taken in the range 450–4,000 cm-1) gold nanoparti-

cles in powder form were recorded using Perkin Elmer

Spectrum 100 instrument. The powder was then mixed

with KBr to make a pellet to measure its FTIR spectrum.

During the first step of immobilization, AuNPs were

functionalized with L-cysteine [18]. In order to check the

successful functionalization, FTIR spectra of the func-

tionalized Au nanoparticles were taken. The FTIR spectra

confirmed that functionalization of Au nanoparticles with

amine group has occurred.

FTIR of pumpkin urease immobilized on gold nanoparticles

As is well known, the details of the peaks in the FTIR

spectra of the infrared absorption bands of amide I and

amide II can provide detailed information on the secondary

structure of proteins. Amide I band at 1,610–1,690 cm-1

results from the C=O stretching vibration of peptide link-

ages in the protein backbone. The amide II band around

1,500–1,600 cm-1 is due to the combination of N–H

bending and C–N stretching [23]. Figure 3 exhibits FTIR

of urease-immobilized AuNPs, where two bands are seen at

1,669 and 1,460 cm-1. These bands are assigned to the

amide I and II bands of proteins, respectively. The band

present at 1,669 cm-1 (C=O stretching of amide group) is

due to the attachment of urease enzyme through the peptide

linkage and confirms the covalent immobilization on

AuNPs. This peak is also indicative of the formation of

Schiff’s base as a result of the reaction between carbonyl

group of urease and amine group of cysteine. It is well

known that proteins can bind to gold nanoparticles either

through free amine groups or cysteine residues in the

proteins, and therefore, stabilization of the gold nanopar-

ticles by surface-bound proteins is a possibility [24–26].

These observations are indicative of the binding of protein

with Au nanoparticles through free carboxylate group [27].

This is so, since, from the previous reported work, it has

been established that amide I and amide II will significantly

diminish, if the enzyme was denatured [28]. However, in

Fig. 2 TEM micrographs of

urease immobilized on

amine-functionalized gold

nanoparticles (AuNPs marked

with arrows)


123

our FTIR both the bands are very intense and visible which

supports that upon immobilization of enzyme (urease), its

native structure is not denatured. This was further con-

firmed, when the immobilized enzyme exhibited its

enzyme activity with its substrate. The stretch vibrations

of –CO are also found at 1,157 cm-1. The band at

3,417 cm-1 corresponds to stretching vibration of –OH and

–NH2. It occurs due to amine group stretching vibrations

superimposed on the side of hydroxyl group band. This

absorption band indicates the presence of residual amino

groups on the Cys–Au nanoparticles that are used to

immobilize the urease. The –CH stretching vibration is

manifested through strong peaks at 2,923–2,838 cm-1

(C–H stretching of –CH2 groups). The band visible at

1,080 cm-1 corresponds to bonded sulfide group, and

the stretch present at 2,500 cm-1, probably is a S–H

stretch. Urease being a sulfahydryl enzyme the appearance

of these bands further supports the covalent immobilization

of enzyme on AuNPs. The band appeared at 1,320 cm-1

in spectrum is attributed to the C–N bonds of amide group.

Effect of temperature on the activity of free


The chemical reactions generally proceed through an

energy rich activated state. A requisite amount of energy is

required to bring the reactants to this state. This feature

leads to the recognition of the profound influence of tem-

perature on the rate of a chemical reaction. The rate of the

enzymatic reaction increases usually with the temperature.

At higher temperatures, beyond a certain limit, the coun-

teracting force of protein denaturation becomes prevalent,

which leads to severe decrease in the activity.

In order to examine the effect of temperature the cata-

lytic activity of the free and immobilized urease was studied

over a temperature range of 10–85 �C. The effect of tem-

perature on the relative activity of the free and immobilized

urease is shown by the representative Fig. 4. From this it is

clear that at comparatively lower temperatures (up to 45 �C)

there is no significant difference in the activity of free and

immobilized enzyme. Since the stability of free urease is

strongly dependent on temperature and a sharp optimum

was obtained at 70 �C and above this temperature, it loses

its activity very rapidly, whereas the urease immobilized on

AuNPs was observed to be thermally stable up to 75 �C.

The increase in optimum temperature may be because of the

change in the enzyme’s conformational structure upon

immobilization which largely affects the observed changes

in enzyme activity. This improved stability of the immo-

bilized urease can be attributed to the covalent binding of

the enzyme to the matrix. This linkage between the gold

nanoparticles and urease would, therefore, lead to reduced

conformational freedom with respect to effects such as high

temperatures. This feature is in keeping with other enzyme

immobilization protocols [12].

Effect of pH on the activity of free and immobilized urease

A change in optimum pH generally occurs upon immobi-

lization depending on nature of the immobilizing matrix

used. This type of change is very useful in understanding

the structure–function relationship of enzyme protein.

Therefore, it is very useful to compare the activity of the

free and immobilized enzyme as a function of pH. Changes

in the enzyme structure result into change in the pH

activity of the enzyme, due to the change taking place in

the microenvironment. However in the present study of pH

dependence of activity of soluble and immobilized urease,

it was observed that the AuNPs-immobilized urease has the

Fig. 3 FTIR spectra of urease immobilized on amine-functionalized

gold nanoparticles

Fig. 4 Effect of temperature on the activity of soluble and immo-

bilized urease


123

optimum pH, i.e., 7.5 which is the same as that of the

soluble enzyme, but the pH activity profile is considerably

widened due to diffusional limitations.

It can be seen from the data (Fig. 5) that immobilized

urease displayed a broadened pH profile particularly toward

the acidic side, around pH 6.5–5.0. The shift to acidic

optimal pH upon immobilization could be because of the

urease-catalyzed degradation of urea into ammonia and

carbon dioxide. Released ammonia results in an increase in

the local pH, and due to the diffusional constraint of the

support retaining a higher concentration of enzyme product,

in the vicinity of the support that adsorbed enzyme favors

the shift in optimum pH toward alkaline side. Several

reports are available with the similar observations upon

immobilization of urease and other enzymes [12, 29]. Thus,

the microenvironment around the enzyme was more alka-

line than that of the bulk solution. In general, the immobi-

lized enzymes like lipase have either a broader or the same

pH range of high activity as that of the free enzyme [30, 31].

Operational and storage stability of free and immobilized

urease

In order to investigate the commercial applicability of the

immobilized urease, the operational and storage stability of

immobilized urease are important parameters. Also, the

stability of immobilized urease is very essential in

the continuous hydrolysis reactions. The stability of the

enzymes might be expected to either increase or decrease

upon immobilization, depending on whether the carrier

provides a microenvironment capable of denaturing the

enzymic protein or of stabilizing it. Storage stability of the

soluble and reusability of AuNPs-immobilized urease was

studied at 4 �C. The stability of AuNPs-immobilized ure-

ase was much more than the soluble enzyme. However, the

results exhibited a gradual loss in the enzyme activity and

the soluble enzyme showed 60% loss on the tenth day of

storage which increased to about 78% on the 15th day

(Fig. 6a). On the other hand, the AuNPs-immobilized

enzyme retained a considerable amount of activity even

after 1 month (Fig. 6b). Also, there was practically no

leaching of enzyme over a period of 2 weeks.

The reusability was also checked for the AuNPs-

immobilized enzyme. It was recorded that 47% activity of

urease was retained even after eight-repeated cycles. On

the other hand, reports available suggest only poor reus-

ability of the alginate-immobilized pumpkin urease [13].

Pumpkin urease immobilized in alginate beads retained

\25% of the initial activity after fourth reuse even after

storing at -4 �C. This improved retention of enzyme

activity for the pumpkin urease on AuNPs-immobilized

enzyme as compared with the alginate immobilization

could be explained by the Zulu effect. It explains that as the

surface area of the immobilizing matrix increases, the local

concentration of enzyme will also increase; because of this

effect, a significant enzyme activity is observed over

number of washes.

Response surface factorial design for the optimization

of the process

The individual and cumulative effect of the variables, i.e.,

pH and temperature, which were affecting the activity of

immobilized urease, were evaluated by using a statistical

Fig. 5 pH activity profile of soluble and immobilized urease

0

20

40

60

80

100

0 2 4 6 8Cycle Number

% R

esid

ual

Act

ivit

y

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4(a) (b)

2 6 10 14 18 22 26 30 34

Days

Ab

sorb

ance

at

405

nm

Soluble Enzyme AuNPs Immobilized

Fig. 6 a Operational and

Storage stability of free and

immobilized urease and

b reusability of the AuNPs-

immobilized enzyme


123

and graphical technique known as Response Surface

Methodology (RSM). For this purpose 22 full factorial,

central composite design (CCD), which is a part of RSM, is

used with the help of software MINITAB� Release

15(Demo version). Thus 14 experimental values were

required for the two-level two-full factorial Central Com-

posite Design (CCD), as per requirement of the design.

Experiments were performed accordingly, where the pH

was changed from 5.5 to 8.5 and temperature from 30 to

85 �C (Table 1). Significant changes in enzyme activity

were observed for all the combinations, implying that both

these variables were significantly affecting the enzyme

activity.

(a) Interpretation of the regression analysis: The response

surface regression analysis was performed to deter-

mine the significance of the regression coefficient of

each variable and the result thus obtained are: the SE

coefficient, T value and the P value along with the

constant and coefficient (estimated using coded

values) given in Table 2. It can be observed from

the results given in Table 2 that the value of constant

was 0.6862 with the T value of 38.993 and P value of

0.000. Thus the value of constant is significant,

because of high T value and low P value. It also

represented that the value of constant does not depend

on linear term, square term as well as interaction term

of the variables. It can also be observed from the

Table 2 that linear term ‘‘pH’’ was significant because

of the low (P \ 0.005) and high value, i.e.,

T = 6.790. However, the quadratic term of pH was

also noticed to be significant (P = 0.000) with

T = 9.330, because T value in case of quadratic term

was higher than the linear term of pH. This indicated

that there was a curve relationship between pH and

enzyme activity. Thus, initially the enzyme activity

will increase with the increase in pH and after

attaining the maximum, the enzyme activity decreases

with the further increase of pH. Similarly the linear

term ‘‘temperature’’ was also found significant,

because low P value of 0.000 with a very high

T value of 22.406, where as quadratic term of

temperature is also significant as P value is 0.000,

but T value is only 5.508 and less is than the linear

term, where T value is 22.406. This indicated that the

linear term temperature is more significant over

quadratic term of temperature. Thus, there is a linear

relationship between temperature and enzyme activity

that means there will be an increase in the activity

with an increase in the temperature. In addition to

this, it was also observed that both the linear term

temperature and pH were having the positive value of

regression coefficient that meant because of the

synergistic effect of pH and temperature enzyme

activity increases initially with the increase of tem-

perature and pH. Whereas quadratic term of pH and

temperature are having the negative value of regres-

sion coefficient, this indicates antagonistic effect of

square of pH and temperature on activity which

means at higher pH and temperature, i.e., with the

further increase of pH and temperature, enzyme

activity will decrease.

On the basis of results of regression analysis, the second

order quadratic model was proposed representing the

empirical relationship between the enzyme activity and the

variables pH and temperature is given below:

Enzyme activity yð Þ ¼ 0:6862þ 0:1010 pH

þ 0:2629 temperature

� 0:2272 pH2

� 0:1464 temperature2: ð1Þ

The goodness of the fit of the model was checked by the

multiple correlation coefficient (R2). The predicted value of

Table 1 Predicted and experimental data showing the enzyme

activity of urease at different pH and temperature (8C)

pH Temperature

(8C)

Experimental

value

Predicted

value

Residual

5.5 35 0.083 0.053969 0.0290306

6.0 35 0.186 0.213860 -0.0278605

6.5 35 0.292 0.323255 -0.0312554

7.0 35 0.368 0.382154 -0.0141544

7.5 35 0.399 0.390557 0.0084426

8.0 35 0.334 0.348464 -0.0144644

8.5 35 0.241 0.255875 -0.0148755

7.5 30 0.280 0.285287 0.0477132

7.5 40 0.488 0.484118 0.0038818

7.5 50 0.638 0.636110 0.0018900

7.5 60 0.752 0.741262 0.0107378

7.5 70 0.803 0.799575 0.0034252

7.5 75 0.830 0.811166 0.0188338

7.5 80 0.790 0.811048 -0.0210478

Table 2 Estimated regression coefficients of enzyme activity

(absorbance at 405 nm) of urease versus pH and temperature in coded

units

Term Coef. SE coef. T P

Constant 0.6862 0.01760 38.993 0.000

pH 0.1010 0.01487 6.790 0.000

Temperature 0.2629 0.01173 22.406 0.000

pH 9 pH -0.2272 0.02436 -9.330 0.000

Temperature 9 temperature -0.1464 0.02657 -5.508 0.000

R2 = 99.20%, R2(pred.) = 96.38%, R2(adj.) = 98.85%


123

the response are shown in Table 2 and it was found that

predicted response were close to the experimental results.

(b) Interpretation of the analysis of variance (ANOVA):

The analysis of variance was performed to check the

adequacy and significance of the quadratic model in

terms of larger F and low P value. The result of

ANOVA is predicted in Table 3. It was found from

the results that the P value for all the variables were

lower than 0.05 which shows the significant correla-

tion of the regression equation with the response

variable in the interpretation of regression analysis.

Significant high value of F, i.e., 280.16 indicated that

the second order polynomial model response ratio in

Eq. 1 was adequate to represent the actual relation-

ship between the response, i.e., enzyme activity and

model process variables namely pH and temperature.

(c) Interpretation of residual analysis: The analysis of the

residuals was done with the help of residual plots in

order to determine whether the model meets the

assumption of the analysis. The normal probability

plot of residual value of enzyme activity is shown in

Fig. 7. It was observed that the residual points on the

plot fall fairly close to the straight line which

represented a normal distribution of the residual [32].

(d) Main effect plot: In order to visualize the main effects

of each variables viz., pH and temperature, the main

effect plot was drawn and shown in Fig. 8a for pH

and in Fig. 8b for temperature. Figure 8a shows that

the response was greater than the average value at pH

7.5, while it was less than average for rest of the pH,

i.e., 5.5, 6.0, 6.5, 7.0, 8.0 and 8.5. This was further

noted that the mean enzyme activity was observed to

be maximum affected by low pH levels of 5.5 in

comparison with high pH, i.e., 8.0 and 8.5. It was

further noted that the maximum value was observed

at pH 7.5, whereas Fig. 8b shows that the enzyme

activity increases with the increase in the tempera-

ture. At the initial temperature such as 30 and 35 �C

the response was below the mean value, whereas for

the rest of the temperature 40–80 �C, response was

above the mean value. The mean response was

observed to be maximum affected by higher temper-

atures 70, 75 and 80 �C and the maximum achieved at

75 �C. Further, it is to be noted that the average

response was least affected at 40 �C. This type of

main effect plot using statistical design has been

shown for the effect of two parameters, i.e., force and

speed in the output voltage of nanogenerators [33].

(e) Response surface 3D and contour plot: The three

dimensional response surface plot, which is a 3D

graphic representation of the regression equation

showing the individual and cumulative affect of the

variable, and the mutual interaction between the

variable (Fig. 9). This figure indicates the quadratic

nature of the surface with the maxima and the minima

of the response and the significance of the coefficient

of the canonical equation. Whereas isoresponse con-

tour plot is a projection of the response surface on a 2D

plane. Contour plot gives better understanding about

the influence of variable and their interaction on the

response as compared with the 3D surface plot [34].

Table 3 Analysis of variance of enzyme activity (absorbance at

405 nm) of urease versus pH and temperature in coded units

Source DF Seq. SS Adj. SS Adj. MS F P

Regression 4 0.811571 0.811571 0.202893 280.16 0.000

Linear 2 0.718781 0.449176 0.224588 310.11 0.000

Square 2 0.092790 0.092790 0.046395 64.06 0.000

Residual error 9 0.006518 0.006518 0.000724

Total 13 0.818089

Fig. 7 Normal probability plot of the difference between the

observed and the predicted value

Fig. 8 Effect of pH and temperature on the mean enzyme activity

represented by main effect plot


123

Contour plot representing the combined effect of pH and

temperature on the enzyme activity showed as absorbance

was represented in Fig. 10 which shows that the absor-

bance increased with the increase in temperature, whereas

in case of pH with the increase in pH, the enzyme activity

was noticed to first increase from 5.5 to 7.5 and thereafter

decreased with further increases of pH from 8.0 to 8.5. The

maximum enzyme activity was noticed to be at 7.5 pH and

75 �C temperature.

(f) Optimization plot: A response optimization plot was

drawn in order to identify the factor settings that

optimize a single response or a set of responses. It is

useful in determining the operating conditions that

will result in a desirable response. In the present

study, the objective was to obtain a value at or near

the target value of absorbance 0.85 at which maxi-

mum enzyme activity was observed. The optimum

condition, which is defined as the best combination of

factor settings for achieving the optimum response,

was found to be pH 7.5080 and temperature 75.4668

for a predicted response of absorbance 0.8108 which

is close to the target value of 0.85 with a desirability

score of 0.95339 (Fig. 11). There are many other

advantages of optimization plot, i.e., to achieve

predicted response with higher desirability score,

lower cost factor settings with near optimal properties,

to study the sensitivity of response variables, to

change in the factor settings, and to get required

response for factor setting of interest.

Conclusion

The present study showed that the properties and kinetics

of pumpkin urease were improved after immobilization on

amine-functionalized AuNPs. The immobilized enzyme is

more stable thermally (it is stable up to 75 �C). Storage

stability of immobilized urease as compared with soluble

enzyme is improved and can be reused up to eight cycles

continuously, having a residual activity of 47% (Fig. 6a,

b). The excellent storage stability, thermal stability, reus-

ability and operational stability of the immobilized urease

demonstrates the superior potential of immobilized forms.

These may found various applications in biotechnology,

biomedicine, agriculture and environment. The influence of

parameters, such as pH and temperature, upon immobili-

zation of enzyme was also investigated by response surface

methodology. Thus, a two-level two factor (22) central

composite design (CCD) was applied with help of statis-

tical and quadratical software, Minitab� version 15. The

results predicted by the design were found in good agree-

ment (R2 = 96.38%) with the experimental results indi-

cating the applicability of proposed model. The multiple

regression analysis and ANOVA showed the individual and

Fig. 9 3D plot of the cumulative effect of pH and temperature on the

enzyme activity of urease

Fig. 10 Contour plot of the cumulative effect of pH and temperature

on the enzyme activity of urease

Fig. 11 Plot for the optimization of variables (pH and temperature)

for maximum immobilization of enzyme urease on AuNPs


123

cumulative effect of pH and temperature on enzyme

activity.

Acknowledgments The authors acknowledge with gratitude the

financial support from DST: UNANST, CSIR, UGC and Dr. Kalpana

Awasthi for recording FTIR.

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