Alpha amylase assisted synthesis of TiO2 nanoparticles: Structural characterization and application...

Accepted Manuscript

Title: Alpha amylase assisted Synthesis of TiO2

Nanoparticles: Structural Characterization and Application asAntibacterial Agents

Author: Razi Ahmad Mohd Mohsin Tokeer Ahmad MeryamSardar

PII: S0304-3894(14)00754-7DOI: http://dx.doi.org/doi:10.1016/j.jhazmat.2014.08.073Reference: HAZMAT 16264

To appear in: Journal of Hazardous Materials

Received date: 28-6-2014Revised date: 7-8-2014Accepted date: 26-8-2014

Please cite this article as: R. Ahmad, M. Mohsin, T. Ahmad, M. Sardar, Alphaamylase assisted Synthesis of TiO2 Nanoparticles: Structural Characterization andApplication as Antibacterial Agents, Journal of Hazardous Materials (2014),http://dx.doi.org/10.1016/j.jhazmat.2014.08.073

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http://dx.doi.org/doi:10.1016/j.jhazmat.2014.08.073

http://dx.doi.org/10.1016/j.jhazmat.2014.08.073

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Highlights

Green synthesis of TiO2 nanoparticles using an enzyme alpha amylase has been

described.

The morphology and shape depends upon the concentration of the alpha amylase

enzyme.

The biosynthesized nanoparticles show good bactericidal effect against both gram

positive and gram negative bacteria.

The bactericidal effect was further confirmed by Confocal microscopy and TEM.

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Alpha amylase assisted Synthesis of TiO2 Nanoparticles: Structural

Characterization and Application as Antibacterial Agents

Razi Ahmada, Mohd Mohsina, Tokeer Ahmadb and Meryam Sardara*aDepartment of Biosciences, Jamia Millia Islamia, New Delhi-110025, IndiabDepartment of Chemistry, Jamia Millia Islamia, New Delhi-110025, India

*Corresponding author.

Dept. of Biosciences, Jamia Millia Islamia

New Delhi-110025, India

E-mail address: [email protected]

Tel: 011-26981717

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Abstract

The enzyme alpha amylase was used as the sole reducing and capping agent for the synthesis

of TiO2 nanoparticles. The biosynthesized nanoparticles were characterized by X-ray

diffraction (XRD) and transmission electron microscopic (TEM) methods. The XRD data

confirms the monophasic crystalline nature of the nanoparticles formed. TEM data shows that

the morphology of nanoparticles depends upon the enzyme concentration used at the time of

synthesis. The presence of alpha amylase on TiO2 nanoparticles was confirmed by FTIR. The

nanoparticles were investigated for their antibacterial effect on Staphylococcus aureus and

Escherichia coli. The minimum inhibitory concentration value of the TiO2 nanoparticles was

found to be 62.50 µg/ml for both the bacterial strains. The inhibition was further confirmed

using disc diffusion assay. It is evident from the zone of inhibition that TiO2 nanoparticles

possess potent bactericidal activity. Further, growth curve study shows effect of inhibitory

concentration of TiO2 nanoparticles against S. aureus and E. coli. Confocal microscopy and

TEM investigation confirm that nanoparticles were disrupting the bacterial cell wall.

Keywords: TiO2 nanoparticles; TEM; Minimum inhibitory concentration; Antibacterial effect

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1. Introduction

In recent years the application of nanoparticles in various fields has expanded

considerably. The synthesis of titanium dioxide (TiO2) nanoparticles and nanostructures have

been a matter of interest due to their attractive material properties and applications in various

fields like optical devices, sensors, photocatalysis, organic pollutants and antibacterial

coatings[1-4]. TiO2 nanoparticles are considered to be amongst the best photocatalytic

materials due to their long-term thermodynamic stability, strong oxidizing power, and

relative non-toxicity [5, 6]. Presently there are chemical, physical and biological routes

available for the synthesis of metal oxide nanoparticles [7, 8]. It is well known that many

organisms can produce inorganic materials either intra or extracellular [9]. Biological

synthesis of metal oxide nanoparticles is hallmarked by ambient experimental conditions of

temperature, pH, and pressure [8, 10]. There is a need to develop environmentally safe

protocols for the synthesis of TiO2 nanoparticles. So far, only few reports are available on the

biosynthesis of TiO2 nanoparticles [8, 10-14]. Previously, nanoparticles has been synthesized

by using enzymes silicatein [15] , lysozymes [11] and urease [13]. Recently, our group

synthesized TiO2 nanoparticles using lactobacillus sp. [14]. We have also reported the

synthesis of silver [16] and gold [17] nanoparticles using alpha amylase from Aspergillus

oryzae. One of the major big challenges for researchers are facing is the occurrence of

antibiotic resistant bacterial strains, hence there is the need to develop the new drugs [18].

Therefore, the finding of new antimicrobial agents with novel mechanisms of action is

essential and extensively pursued in antibacterial drug discovery. In this paper, we describe

the biosynthesis of TiO2 nanoparticles using hydrolytic enzyme alpha amylase from

Aspergillus oryzae and their antibacterial effect against gram positive Staphylococcus aureus

(S. aureus) and gram negative bacteria Escherichia coli (E. coli).

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2. Experimental

2.1. Materials and Methods

Culture media Luria agar, Luria broth and Alpha amylase enzyme were purchased from

Hi-media (India). TiO2 powder and TiO2 nanoparticles (size < 25nm) were purchased from

Sigma Aldrich (USA). Bacterial strains Staphylococcus aureus (MTCC-3160) and

Escherichia coli (MTCC-405) were procured from Microbial Type Culture Collection

(MTCC), Institute of Microbial Technology (Chandigarh, India). All other chemicals and

solvents used were of analytical and biotechnology grade.

2.2. Biosynthesis of TiO2 nanoparticles by alpha amylase

Aqueous solution (25 ml) of 0.25 M TiO(OH)2 was added to 30 ml of alpha amylase

enzyme (2 mg/ml dissolved in 20 mM sodium acetate buffer pH 4.5) and it was incubated at

60 °C for 10 minutes. The solution was cooled and kept at 25°C with continuous shaking.

After 24 h the mixture was centrifuged at 3000 g for 10 minutes and the nanoparticles of

TiO2 obtained in the pellet were washed with distilled water for several times to remove the

unbound enzyme. The washed and air dried nanoparticles were used for further

characterization. In another set of experiment, enzyme concentration was increased to 15

mg/ml by keeping the other parameters constant.

2.3. XRD analysis

The XRD studies for the formation of metal oxide TiO2 nanoparticles was confirmed on

Bruker D8 advance diffractometer using Ni- filtered Cu-Kα X-rays of wavelength (λ)

=1.54056 Å over a wide range of Bragg angles (20°≤ 2θ ≤ 80°). The data was obtained at the

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scanning rate of 0.05º/s. The raw data obtained was subjected to the back ground correction

and Kα2 reflections were stripped off using normal stripping procedure.

2.4. Transmission electron microscope measurements of nanoparticles

Transmission electron microscope measurements were performed on a JEOL, F2100

instrument operated at an accelerating voltage at 200 kV for the size and morphological

studies of the nanoparticles. The TEM specimens were prepared by adding a drop of the

dispersed sample in distilled water followed by sonication on a porous carbon film supported

by a carbon coated copper grid, and dried in oven.

2.5. Fourier Transform infrared spectroscopy

Fourier transform infrared (FTIR) spectroscopy was recorded on a Bruker IR

Spectrophotometer. The spectra of the washed and purified TiO2 nanoparticles were recorded

from 4000-650 cm-1.

2.6. Minimum inhibitory concentration (MIC)

The microdilution method for estimation of MIC values was carried out to determine the

antimicrobial activity. The MIC values were determined on 96-well micro-dilution plates

according to the protocols developed previously [19].

2.7. Agar diffusion assay

The antibacterial activity of TiO2 nanoparticles was evaluated against S. aureus and E.

coli by agar diffusion method. Fresh cultures (0.2 ml) of S. aureus and E. coli were

inoculated into 5 ml of sterile Luria broth separately, and incubated for 3–5 h to standardize

the culture to McFarland standards (106 CFC/ml). Hundred µl of revived cultures from each

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was added on agar media and poured on three replicate plates for both cultures. Discs with

the size of 6 mm were placed on the agar plates and 10 µl of TiO2 nanoparticles (4 mg/ml

suspended in sterile water) loaded on one disc and on another disc 10 µl ampicillin (1 mg/ml)

was loaded as standard. On separate discs 10 µl alpha amylase (15 mg/ml) and 10 µl

TiO(OH)2 (4 mg/ml suspended in sterile water) were loaded as controls. The petriplates were

incubated at 37 ºC for 18 h for bacterial growth.

2.8. Growth curve study

S. aureus and E. coli were studied for their growth dynamics in the presence and absence

of TiO2 nanoparticles. Briefly, 100 µl of overnight fresh bacterial culture (~106 CFU/ml) was

inoculated in respective flasks containing 100 ml growth medium with and without TiO2

nanoparticles with varied concentration of nanoparticles from 15 to 250 µg/ml. The culture

flasks were incubated at 37 ºC and optical density (OD600 nm) was recorded at the interval of

2 h. The readings obtained were plotted and comparative studies were performed between

control and treated culture with TiO2 nanoparticles.

2.9. Confocal scanning laser microscopy

Confocal microscopy studies were performed on a confocal laser scanning microscope

Leica DMRE equipped with a confocal head TCS SPE (Leica, Wetzlar, Germany) and a 40X

water immersion objective with a laser of 532 nm wavelength. S. aureus and E. coli cultures

were labeled with propidium iodide (PI) dye. PI was used to determine the dead cells as PI

specifically stains only dead bacteria. The TiO2 (125 µg/ml) nanoparticles were added to the

overnight grown bacterial cultures and were incubated at 37 ºC for 30 minutes in the dark. 1

ml from both of these bacterial strain cultures were harvested by centrifuging at 2500 g for 5

min. The pellet was resuspended in 1 ml PBS buffer. Staining with PI was performed by

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adding 10 µl of PI to final concentration of 1 µg/ml. This solution was incubated for 3 h at 37

ºC. Likewise a control of both bacterial strains (not treated with TiO2 nanoparticles) was

grown under similar conditions. A drop of the above prepared samples were then placed on a

glass slide and mounted with the cover slip then cells were examined under a confocal

scanning microscope.

2.10. Transmission electron microscopy of bacterial cells

Test compounds at MIC were added to the Bacterial cell S. aureus and E. coli

suspensions in media (~1x106 c.f.u./ ml) and incubated for 16 h at 37 °C. All the cells were

fixed with 2% glutaraldehyde in 0.1 M phosphate buffer for 1 h at 20 °C [20]. Cells were

washed with 0.1 M phosphate buffer (pH 7.2) and post-fixed by using 1% osmium tetroxide

in 0.1 M phosphate buffer for 1 h at 4 °C. For ultrastructure studies, samples were dehydrated

with graded acetone, cleared with toluene and infiltrated with a toluene and araldite mixture

at room temperature then finally in pure araldite at 50 °C and embedded in an polypropylene

tube (1.5 ml) with pure araldite mixture at 60 °C. Samples were prepared using a sectioning

ultramicrotome (Lecia EM UC6) and were observed using TEM [20].

3. Results and discussion

The “green synthesis” approach has been proven to be a better method due to their

slower kinetics, better manipulation, control over crystal growth and stabilization. Recent

advancement in this area includes enzymatic method of synthesis suggesting the enzymes to

be responsible for the nanoparticle formation. Biomacromolecules have been used in the past

to synthesize metal oxides, including ZnO [21], Cu2O [22] and Ga2O3 [23]. Recently,

Jayaseelan et al. [24] reported the synthesis of TiO2 nanoparticles using Aeromonas

hydrophilla. The GC-MS analysis of the broth showed the major compound found in the

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Aeromonas hydrophilla is glycyl – proline and other compounds present in lesser amounts

are uric acid, glycl-glumatic acid, and Leucyl-leucine and compounds containing –COOH

and -C═O group. They concluded that glycil-L proline act as reducing agent for the synthesis

of TiO2 nanoparticles and the water soluble carboxylic acid compounds acts as stabilizing

agents [24]. In the present work we report the extracellular synthesis of TiO2 nanoparticles

using an enzyme alpha amylase from a non pathogenic fungal strain Aspergillus oryzae.

Alpha amylase from Aspergillus oryzae contains 478 amino acid residues out of which 21

residues are proline [25], 12 of them are exposed [26]. Here it is assumed that the amino

acid proline might have played an important role in the reduction of titanium hydroxide.

Moreover, alpha amylase from Aspergillus oryzae is an acidic enzyme with optimum pH of

4.5 and low isoelectric point (PI=4.2) [27, 28]. This enzyme has large percentage of carboxyl

groups which might be responsible for the stabilization of TiO2 nanoparticles. Fig. 1

represents the proposed mechanism for the synthesis of TiO2 nanoparticles with alpha

amylase having proline residues. Use of enzyme for nanoparticles synthesis has advantage

over other biological methods as they are available in pure form and their structure is also

well known. The mechanism of biosynthesis can be easily understood using well

characterized enzyme. So far lysozyme [11] and urease [13] has been reported in the

literature for the synthesis of TiO2 nanoparticles, compared to these enzymes alpha amylase is

a low cost enzyme.

The crystalline nature and phase purity of titania nanostructures were analyzed by X-ray

diffraction studies. Fig. 2 shows the XRD analysis of TiO2 nanoparticles synthesized with

different concentrations of alpha amylase enzyme. Both diffraction patterns could be indexed

to monophasic nanocrystalline structure of titanium dioxide and found to be in good

agreement with the available literature reports (PCPDF No. #84-1285). The observed

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reflections in Fig. 2a and b corroborates to the pure nanocrystalline TiO2 with anatase

structure. No peak corresponding to any impurity was present which indicates the high purity

of the samples. Fig. 3 showing the TEM images of the TiO2 nanoparticles synthesized using

different concentration of alpha amylase. The TEM analysis showed that the particle size and

morphology depends upon the concentration of enzyme used during synthesis. With the

alpha amylase concentration of 2 mg/ml, the grain size varies in the range of 30-70 nm with

an average grain size of 50 nm (Fig. 3a), however the grain size decreases significantly to 25

nm (Fig. 3b) by increasing the concentration of the enzyme upto 15 mg/ml. The morphology

of the nanoparticles formed in the enzymatic synthesis also exhibits the strong dependence on

enzyme concentration. The hexagonal along with spherical nanoparticles of TiO2 could be

seen at 2 mg/ml concentration of enzyme as shown in Fig. 3a. It is important to note that as

the enzyme concentration increased to 15 mg/ml, the nanorings were also observed in

addition to nanoparticles (Fig. 3b). To arrive at the definite response of nanoring formation,

the high resolution-TEM (HR-TEM) study has been carried out. HR-TEM image clearly

showed the formation of nanoring (Fig. 3c). This is clear from Fig. 3b and c that the average

ring outer and inner diameters are 55 nm and 33 nm respectively with the shell thickness of

13 nm. It has been reported that the nanoparticles of different sizes and shapes could be

synthesized using biological methods [8, 14, 16, 29]. Thus, by controlling the enzyme

concentration one can achieve the controlled shape and size of the nanoparticles. Further,

research on the synthesis of TiO2 nanoparticles at different enzyme concentration is under

investigation which will give some insight on this aspect. FTIR spectra of alpha amylase and

biosynthesized TiO2 nanoparticles using amylase at two different concentrations were

recorded. The FTIR of nanoparticles with different enzyme concentrations was found similar

(Fig. 4a and b). The spectral bands of enzyme at 1625 cm-1 could be assigned to amide I and

absorption signal at 1520 cm-1 indicate a characteristic amide II band [30, 31]. The

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characteristic amide I and amide II bands of alpha amylase was observed in the FTIR spectra

(Fig. 4c). FTIR spectroscopy clearly indicating that alpha amylase is responsible for the

synthesis of nanoparticles and in their stabilization.

The antibacterial effect of enzyme assisted TiO2 nanoparticles was evaluated against

gram positive and gram negative bacterial strains. Standard antibiotic ampicillin was used as

a control. The MIC value for the TiO2 nanoparticles was found to be 62.5 µg/ml for both

strain S. aureus and E. coli (Table 1). The MIC value of the biosynthesized nanoparticles was

compared with the chemically synthesized nanoparticles (procured from sigma). The

chemically synthesized nanoparticles show greater MIC values for both the strains as shown

in Table 1. Further, the stability of the biosynthesized nanoparticles was also studied in terms

of antibacterial effect. To check the stability of the enzyme assisted TiO2 nanoparticles, they

were stored at 4°C for six months and used to calculate the MIC value against bacterial

strains. The nanoparticles show good stability as no change in the MIC results was obtained.

The Inhibition was further confirmed using disc diffusion assay as shown in Fig. 5. It is

evident from the zone of inhibition that TiO2 nanoparticles possess potent bactericidal

activity. The bactericidal effect of TiO2 nanoparticles has been attributed to the

decomposition of bacterial outer membranes by reactive oxygen species (ROS), primarily

hydroxyl radicals (OH), which leads to phospholipid peroxidation and ultimately cell death

[32, 33]. It was proposed that nanomaterials that can physically attach to a cell can be

bactericidal if they come in contact with cell [34]. The growth curve study of S. aureus and E.

coli in the presence and absence of TiO2 nanoparticles showed the effect of inhibitory

concentration of TiO2 nanoparticles against S. aureus and E. coli till 20 h of incubation (Fig.

6). In control cell lag phase ends upto 4-6 h after which log phase starts which ends upto 16-

18 h. However when cells were treated with TiO2 nanoparticles, then delay in the lag phase

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occurred. At MIC concentration lag phase ends upto 12 h. There occurred delay in lag phase

on exposure to different concentration of TiO2 nanoparticles and thus a prominent decrease in

growth of S. aureus and E. coli was observed. To prove the effect of TiO2 nanoparticles on

the viability of S. aureus and E. coli strain, we performed confocal laser scanning microscopy

(CLSM) in the presence of PI. Bacterial cells were grown and stained with PI as described

above. PI penetrates only cells with severe membrane lesions; the entire bacterial cells appear

red (Fig. 7). It should be noted that PI can only stain the cells in which the cell membrane is

disrupted since it intercalates into the double-stranded nucleic acids [35]. Results presented

here showing that PI penetrates the bacterial cell. S. aureus and E. coli were taken for the

study. A control stained with PI without the TiO2 nanoparticles (Fig. 7a) and samples with

the nanoparticles (Fig 7b and c) were tested for the invasiveness of the nanoparticles inside

the cells. The confocal studies showed that control is not showing any fluorescence while the

sample with TiO2 nanoparticles showing the good fluorescence proving the concept of

invading the cells. Since the PI is binding to the double stranded DNA it is confirmed that the

TiO2 nanoparticles damage the bacterial cell wall. The nanoparticles disrupt the bacterial cell

walls whether it is gram positive or gram negative bacterial strain. TEM images of ultrathin

sections of treated cells showing the damaged cell wall and cell membrane while in untreated

cells the cell membrane structures are intact (Fig. 8a). Treated cells are showed the leakage of

the intracellular contents (Fig. 8b). The focus of this study was to examine the antibacterial

activity of the biologically synthesized nanoparticles and also the effect of their particle size.

In this study size effect is not observed as seen by the MIC data, confocal and TEM studies.

4. Conclusion

Here, the biosynthesis of TiO2 nanoparticles using the enzyme alpha amylase has been

described. The present studies suggest that the proline residues present in the enzyme play an

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important role in the reduction of titanium hydroxide. The biosynthesized nanoparticles were

exploited for their antibacterial activity. The disc diffusion and growth curve confirms their

antibacterial effect and indicates that TiO2 nanoparticles can be considered as potent

antibacterial compound. As the synthesis is eco-friendly, the antibacterial properties of these

nanoparticles can be further explored in future on other bacterial strains, for their use in

various industrial and medical applications.

Acknowledgments

First author RA acknowledge the Indian Council of Medical Research, Govt. of India,

for providing the financial support in the form of Senior Research Fellowship.

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Legends to Figures

Fig. 1. Possible mechanism for the synthesis of TiO2 nanoparticles.

Fig. 2. X-ray diffraction pattern of TiO2 nanoparticles synthesized using alpha amylase (a)

2mg/ml and (b) 15mg/ml.

Fig. 3. TEM images of TiO2 nanoparticles using (a) 2mg/ml and (b) 15mg/ml alpha amylase

concentration (c) is the HRTEM image of the nanoring.

Fig. 4. FTIR spectra of TiO2 nanoparticles: (a) alpha amylase. (b) Nanoparticles synthesized

using 2mg/ml alpha amylase. (c). Nanoparticles synthesized using 15mg/ml alpha amylase.

Fig. 5. Zone of Inhibition of TiO2 Nanoparticles against S. aureus and E.coli: (a) Ampicillin

(b) TiO2 nanoparticles (Av. size 50 nm) (c) TiO2 nanoparticles (Av. size 25 nm) (d)

TiO(OH)2 (e) alpha amylase.

Fig. 6. Growth curve study of bacterial strain S. aureus and E.coli: (a) S. aureus with

different concentration of TiO2 nanoparticles (Av. size 50 nm) (b) S. aureus with different

concentration of TiO2 nanoparticles (Av. size 25 nm) (c) E. coli with different concentration

of nanoparticles (Av. size 50nm) (d) E. coli with different concentration of TiO2

nanoparticles (Av. size 25 nm). Each data point represents the mean ± standard deviation of

three independent observations.

Fig. 7. Laser confocal images of S. aureus and E.coli: cells with membrane damage were

stained with PI (red signals) (a) untreated control cells showing no fluorescence (b) cells

treated with 125µg/ml of TiO2 nanoparticles (Av. size 50 nm) showing the red fluorescence

(c) cells treated with 125µg/ml of TiO2 nanoparticles (Av. size 25 nm) showing the red

fluorescence.

Fig. 8. TEM of S. aureus and E. coli: (a) untreated control cells (b) cells treated with

62.5µg/ml of TiO2 nanoparticles (Av. size 50 nm) (c) cells treated with 62.5µg/ml of TiO2

nanoparticles (Av. size 25 nm).

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Table 1Zone of Inhibition (mm) and MIC (µg/ml) of alpha amylase and chemically synthesized TiO2

nanoparticles against S. aureus and E. coli.

Particle size (Av. Size 50nm)

Particle size (Av. Size 25nm)

Chemically synthesized NPS

Bacterial strain

MIC (µg/ml)a

Inhibition Zone(mm)

MIC (µg/ml)a

Inhibition Zone(mm)

MIC (µg/ml)

S. aureus (MTCC-3160) 62.5 15 62.5 16 200

E. coli (MTCC-405) 62.5 11 62.5 13 200

aParticle size is not affecting the MIC of nanoparticles.

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Fig. 8

Alpha amylase assisted synthesis of TiO2 nanoparticles: Structural characterization and application...

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