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A SIMPLE ONE POT PURIFICATION OF BACTERIALAMYLASE FROM FERMENTED BROTH BASED ON AFFINITYTOWARDS STARCH FUNCTIONALIZED MAGNETICNANOPARTICLETanima Paul a , Saptarshi Chatterjee a , Arghya Bandyopadhyay a , Dwiptirtha Chattopadhyaya , Semanti Basu a & Keka Sarkar aa Department of Microbiology , University of Kalyani , Nadia , West Bengal , IndiaAccepted author version posted online: 19 May 2014.

To cite this article: Tanima Paul , Saptarshi Chatterjee , Arghya Bandyopadhyay , Dwiptirtha Chattopadhyay , SemantiBasu & Keka Sarkar (2014): A SIMPLE ONE POT PURIFICATION OF BACTERIAL AMYLASE FROM FERMENTED BROTH BASED ONAFFINITY TOWARDS STARCH FUNCTIONALIZED MAGNETIC NANOPARTICLE, Preparative Biochemistry and Biotechnology, DOI:10.1080/10826068.2014.923454

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A SIMPLE ONE POT PURIFICATION OF BACTERIAL AMYLASE FROM FERMENTED BROTH BASED ON AFFINITY TOWARDS STARCH

FUNCTIONALIZED MAGNETIC NANOPARTICLE

Tanima Paul1, Saptarshi Chatterjee1, Arghya Bandyopadhyay1, Dwiptirtha Chattopadhyay1, Semanti Basu1, Keka Sarkar1

1Department of Microbiology, University of Kalyani, Nadia, West Bengal, India

Corresponding author: Keka Sarkar,, . Fax No. – 03325828282, Tel No. - 09432191495

E-mail: drkekasarkar@yahoo.com

Abstract

Surface functionalized adsorbant particles in combination with magnetic separation

techniques have received considerable attention in recent years. Selective manipulation

on such magnetic nanoparticles permits separation with high affinity in the presence of

other suspended solids. Amylase is used extensively in food and allied industries.

Purification of amylase from bacterial source is a matter of concern because most of the

industrial need of amylase is met by microbial source. Here we report a simple, cost

effective, one pot purification technique of bacterial amylase directly from fermented

broth of Bacillus megaterium utilizing starch coated Superparamagnetic iron oxide

nanoparticle (SPION). SPION was prepared by co-precipitation method and then

functionalized by starch coating. The synthesized nanoparticles were characterized by

TEM, SQUID, zeta potential, UV-vis and FTIR spectroscopy. The starch coated

nanoparticles efficiently purified amylase from bacterial fermented broth with 93.22%

recovery and 12.57 fold purification. SDS-PAGE revealed that the molecular weight of

the purified amylase was 67 kDa and native gel showed the retention of amylase activity

even after purification. Optimum pH and temperature of the purified amylase was 7 and

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50 ºC respectively and was stable over a range of 20 ºC to 50 ºC. Hence, an improved

one pot bacterial amylase purification method was developed using starch coated SPION.

KEYWORDS: Superparamagnetic iron oxide nanoparticle (SPION), bacterial amylase,

enzyme-purification, magnetic carrier technology

INTRODUCTION

Nanotechnology offers exciting opportunities to intermix with various other fields of

biosciences which have set in motion the development of an emerging research area

called nanobiotechnology [1]. In the past years, the synthesis of superparamagnetic

nanoparticles has been intensively developed not only for its fundamental scientific

interest but also for many technological applications that includes magnetic storage

media[2], biosensing applications[3], targeted drug delivery[4,5], detection of

antiphospholipid antibodies using magnetoliposomes [6,7] and contrast agents in magnetic

resonance imaging (MRI) [8–14].

Isolation, separation and purification of various types of proteins and peptides, are

important in almost all branches of biosciences [15, 16]. New separation techniques which

are capable of isolating even minute amount of target molecules from the presence of vast

amounts of accompanying compounds or particulate matter are necessary. These

separation techniques are also applicable in lab scale as well as small and large scale

process. In the area of biosciences, the isolation of proteins and peptides is usually

performed using variety of chromatography, electrophoresis, ultrafiltration, precipitation

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and other procedures [17]. However, the disadvantage of all standard column liquid

chromatography procedures is the impossibility of the standard column systems to cope

with the samples containing particulate material; so, they are not suitable for work in

early stages of the isolation or purification process where suspended solid and fouling

components are present in the sample. Thus the basic requirement is a simple, targeted

protein extraction process. Here magnetic separation processes, have shown their

usefulness.

The principle underlying magnetic carrier technology is the use of nonmagnetic particles

as components of a sample matrix to attach to magnetic nanoparticles [18,19]. Magnetic

carriers in the form of nanoparticles are surface modified to increase the affinity towards

target compound [20, 21]. Such nanoparticles are mixed with a sample containing target

compound as well as other substances. Following an incubation period when the target

compound binds to the magnetic particles the whole magnetic complex is easily removed

from the sample using an appropriate magnetic separator. After washing out the

contaminants, the isolated target compound is eluted and used for further work.

Magnetic separation techniques have several advantages in comparison with standard

separation procedures. It can separate the target enzyme from the reaction media by

applying magnetic field and due to its larger surface area large amounts of enzyme can be

captured. This process is simple, with a few handling steps. All the steps of the

purification procedure can take place in a single vessel hence termed as one-pot

purification. This eliminates the need for expensive liquid chromatography systems,

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centrifuges, filters or other equipments. The separation process can be performed directly

from crude samples containing suspended solid materials [22].

Enzymes are very essential and vital for our daily life, industrial processes and in nature

for degradation and renewal of energies. But purification of enzymes or proteins from

nature or various other resources is tough, time taking and tedious. Downstream

processing for the production of pure enzymes can generally constitute a major

percentage of overall production cost, especially if end purity requirements are stringent.

Most enzymes are purified by chromatographic techniques after crude isolation by

precipitation and membrane separations [23]. Therefore there is requirement of cost

effective technique that would have less steps yet would be faster, efficient and

economic. Here, we are concerned with the development of a novel protocol for

successful extraction of enzymes taking bacterial amylase as a model. Amylases are one

of the most important industrial enzymes that account for about 30 % of the world’s

enzyme production [24] and the need has anticipated by microbial origin.

The present work focus on purification of bacterial amylase directly from bacterial

fermented broth using starch coated Super Paramagnetic Iron Oxide Nanoparticle

(SPION). The enzyme activity has also been compared before and after purification.

EXPERIMENTAL

Preparation Of Superparamagnetic Iron Oxide Nanoparticles (SPION)

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Superparamagnetic iron oxide nanoparticles (SPION) were prepared by chemical co-

precipitation of Fe2+ and Fe3+ ions in an alkaline solution [25]. For this 2.7 g FeSO4, 7H2O

and 5.7 g FeCl3 were dissolved separately in 10 ml nano pure distilled water. Both the

solutions were mixed thoroughly and added to double volume 10 M ammonium

hydroxide with constant stirring at 25ºC. Thus obtained Fe3O4 particles were heated at

80ºC in a water bath for 30 min. Impurities were removed by washing the particles

several times with nano pure water. Particles were dispersed in 20 ml nanopure water and

sonicated for 10 min at 60 MHz. Particles thus obtained have magnetic property.

Nanoparticles were dried at 60ºC which were stable at room temperature.

Preparation Of Starch-Coated SPION

For the purification of bacterial amylase surface functionalization of SPION was carried

out by starch coating. Modification of SPION was done by mixing 1 g of prepared

magnetic nanoparticle with 10 ml of distilled water to which 0.1g/ml of soluble starch

solution was added under stirring condition for 2 hours at 60ºC.The precipitate was

washed with distilled water several times and separated by applying magnetic field.

Characterization Of Superparamagnetic Iron Oxide Nanoparticles And Starch-

SPION

Characterization of SPION was carried out using various means. Transmission electron

microscopy (TEM; Tecnai S-Twin, FEI, Hillsboro, OR, USA) revealed the size of the

nanoparticle. Superconducting Quantum Interference Device (SQUID; MPMS, Quantum

Design Inc., San Diego, CA, USA) was used for measuring the magnetic momentum of

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the synthesized SPION. Dynamic light scattering (Zetasizer, Malvern Instruments Ltd,

Malvern, UK) was used for measuring the zeta- potential of the synthesized SPION. The

starch coated SPION was characterized using FTIR study. FTIR spectra (Perkin Elmer,

USA) were measured in the 450–4000 cm−1 region with samples dispersed in KBr

medium at room temperature.

Production Of Bacterial Amylase

Bacillus megaterium was used for the production of bacterial amylase. Production of

bacterial amylase was carried out using submerged fermentation in Erlenmeyer flask by

taking 100 ml of amylase production medium [26]; containing Peptone (6.0g/L), MgSO4

(0.5g/L), KCl (0.5g/L), Starch (1g/L) and kept in rotary incubator at 37 ºC for 48 hours.

Fermented broth was centrifuged at 7000 rpm for 15 min and the supernatant was

concentrated in a rotary vacuum evaporator at 45ºC.The resulting liquid was used as the

bacterial amylase source for the purification of amylase using starch-coated SPION.

Purification Of Bacterial Amylase Using Starch-Coated SPION

Fermented broth containing bacterial amylase was mixed with starch-SPION for 15 min

in the ratio 5:1 (v/v) at room temperature. The immobilized enzymes on starch-SPION

were isolated by applying magnetic field, and the supernatant was discarded. After

washing the complex (enzymes-starch-SPION) with distilled water, bound amylase was

eluted out from the starch-SPION by adding acetonitrile buffer (0.01% formic acid and

5% acetonitrile) of pH 8 at shaking condition for 15 min. Magnetic field was applied to

separate the starch-SPION from the buffer containing purified amylase. Recovered

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supernatant contains the purified bacterial amylase. Thus the entire procedure of

purification of amylase by using starch-SPION has been depicted in Scheme 1.

Determination Of Amylase Activity

For determining the amylase activity 0.1 ml aliquot of the enzyme preparation was mixed

with 1 ml soluble starch solution (2% w/v) as a substrate and allowed to react at 37ºC for

10 min. The reaction was stopped by adding 1 ml of 3, 5-dinitrosalicylic acid [27].

Reaction mixture was heated in boiling water bath for 10 min and then cooled rapidly in

cold water. Absorbance of 0.1 ml reaction mixture diluted with 0.9 ml distilled water was

measured at 546 nm using maltose as the standard curve. One unit of activity was defined

as the amount of enzyme that is able to produce 1 g of maltose per minute at 37ºC [28].

Determination Of Molecular Weight

Molecular weight of the purified protein was determined by SDS-PAGE taking 12.5% as

resolving gel and 3.75% as stacking gel. The running buffer was Tris-glycine (pH 8.3)

and electrophoresis was carried out at 150 volts for 2 hours. Protein ladder from 14.4 kD

to 94.0 kD was used.

Determination Of Retention Of Enzyme Activity After Purification

Native polyacrylamide gel electrophoresis [29] was used to determine the retention of

enzyme activity even after purification. Native gel was preferred so that SDS could not

denature the protein and the enzyme may loss its activity. After electrophoresis in the

above described parameters, gel was soaked in 1% soluble starch solution for 30 min and

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then stained with Lugols iodine to ensure the activity of amylase even after the treatment

with starch-SPION [30].

Effect Of Ph On Amylase Activity

The pH optimum of the amylase activity was determined in the pH range from 2 to 12 by

using glycine–HCl buffer for pH 2.0–3.0, phosphate citrate buffer for pH 4.0–7.0, tris-

HCl buffer for pH 8.0 and 9.0, NaHCO3-NaOH buffer for pH 10.0 and NaOH-HCl buffer

for pH 11.0 – 12.0 [31]. The molarity of each solution was 0.2 M and the reaction was run

at 37ºC for 10 min.

Effect Of Temperature On Amylase Activity

The amylase activity was determined in the temperature range from 20ºC to 100ºC by

using 0.1 M phosphate buffer having a pH of 7.0 and the reaction was run for 10 min [32].

Thermal Stability Of Amylase

The enzyme was first incubated in the temperature range from 20ºC to 100ºC for 30 min,

and the amylase enzyme activity was then determined in 0.1 M phosphate buffer having a

pH of 7.0 at 37ºC for 10 min [33].

RESULTS AND DISCUSSION

Characterization Of The Superparamagnetic Iron Oxide Nanoparticles (SPION)

To explore the large surface area /unit volume for adsorption and magnetic property for

easy separation, SPION was prepared by co-precipitation method. The resulting

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nanoparticles were characterized for its size, magnetic property, and charge distribution.

The image of SPION under TEM revealed the size of the nanoparticle to be 8 nm (Fig.

1a). Fig. 1b represents M-H curve [magnetization (emu/g) verses applied field (Oe)]

obtained from SQUID data. The absence of hysteresis loop confirmed the lack of

magnetic remanence which indicated the superparamagnetic nature of the synthesized

iron oxide nanoparticles. Zeta potential was found to be -15.04 mV (Fig. 1c). The large

negative value suggested the small particle size as well as the stability of nanoparticles

without agglomeration.

Characterization Of Starch Coated SPION

The goal of our research was to prepare a simple enzyme purification system using

SPION. To that end, we planned to develop starch-coated SPION that specifically bind to

amylase with high affinity. A simple novel adsorption technique has been utilized to

prepare starch coated SPION without using epichlorohydrin as attempted in earlier cases

[34–36] for such modification. Our method is simpler, has less chemical involvement, thus

reducing chance for interference during protein purification.

The starch coating onto the SPION was confirmed by FTIR. Fig. 2 shows the FTIR

spectra of SPION and starch coated SPION. The IR spectrum of SPION exhibit strong

band at 3127.27, 2005.67, 1619.64, 1401.40, 1121.17, 771.81 and 587.49 cm-1 whereas

additional bands on 2923.51, 1179.09, 1023.54, 859.27 and 609.90 cm-1 signifies that a

layer of starch was coated over the SPION.

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Purification Of Bacterial Amylase From Fermented Broth Using Starch-SPION

Though similar attempts were made to isolate amylase from human saliva [34], soybean

[35] and tilapia fish gut [36], we for the first time made an attempt to use the magnetic

carrier technology in the form of Superparamagnetic iron oxide nanoparticle and its

modification with starch by our novel method for the isolation of bacterial amylase

because microbial amylases meet most of the industrial demand. The major advantage of

using microorganisms for production of amylases is in economical bulk production

capacity and easy manipulation to obtain enzymes of desired characteristics [37]. In our

study the bacterial fermented broth was directly subjected to starch-SPION purification

process. This method is simpler than the existing ones since our method does not require

ammonium sulfate precipitation and also eliminates the requirement of dialysis and

centrifugation.

Total activity of both starch-SPION purified amylase and fermented broth containing

amylase were measured by DNS method. Analysis of these parameters indicated that

over 93.22 % of the amylase in fermented broth was recovered with the starch-SPION

resulting in 12.57 fold purification (Table 1).

Characterization Of Starch-SPION Purified Bacterial Amylase From Fermented

Broth

To determine the molecular weight of the starch-SPION purified bacterial amylase SDS-

PAGE was carried out. Fermented broth containing amylase and starch-SPION purified

amylase were run in two different lanes (Fig. 3). A number of bands were found in the

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lane 2 (fermented broth containing amylase) indicating the presence of several proteins

along with the band corresponding to the purified amylase in lane 3 (starch-SPION

purified amylase). Lane 3 showed one major band corresponding to the band of protein

ladder with molecular weight 67 kDa. Thus the molecular of the purified amylase was 67

kDa. Lighter band was found in lane 2 as compared to lane 3 indicating lesser amylase

content in unpurified product than starch-SPION purified product.

To investigate whether the purified enzyme has retained its activity even after

purification or not, native gel was run so that SDS could not interfere the enzyme

activity. Native gel was soaked in soluble starch solution which when stained with iodine

gives blue colour. Clear zone surrounding the band of fermented broth containing

amylase and starch-SPION purified amylase were found indicating that the amylase was

active even after purification. Amylase in the band degrades the surrounding starch so the

area remains unstained. Clear zone of starch-SPION purified amylase was much more

than fermented broth containing amylase because Starch-SPION purified process

contains 12.57 fold purified enzyme (Fig. 4).

Effect of pH and temperature on amylase activity and thermal stability were determined

for analyzing the biochemical characteristics before and after purification. Effect of pH

was determined with pH range 2–12 at 37ºC for 10 min. Fig. 5 shows the optimum pH of

enzyme activity for both fermented broth containing amylase and starch-SPION purified

amylase. The pH range of starch-SPION purified enzyme activity was narrower than that

of unpurified enzyme activity. The unpurified enzyme exhibited higher activity over a

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broad pH range from 4.5 to 9, while the activity of the starch-SPION purified enzyme

showed its maximum at pH 7. The optimum temperature of enzyme activity for both

unpurified amylase and starch-SPION purified amylase was shown in (Fig. 6). Optimum

enzyme activity of the starch-SPION purified enzyme was at 50ºC. The activity of the

unpurified enzyme was over 85% between 20ºC and 60ºC but decreased sharply at 70ºC.

The temperature range of starch-SPION purified enzyme activity was narrower than that

of unpurified enzyme activity. Regarding thermal stability, both unpurified amylase and

starch-SPION purified amylase was found to be stable between 20ºC to 55ºC (Fig. 7).

The activity of the fermented broth containing amylase decreased sharply at 50ºC and

became negligible above 70ºC. The starch-SPION purified amylase was stable over a

range of 20ºC to 40ºC and gradually decreased its activity upto 60ºC and became

negligible above 70ºC.

Thus the robustness and simplicity of our method makes it applicable even in the

industrial level because a bacterial source of amylase is paramount.

CONCLUSIONS

The present study demonstrates that the magnetic carrier technology could be used as

carrier support to adsorb and purify amylase, and this technology can serve as model for

protein purification. By using magnetic separation in this way, several stages of sample

pretreatment (especially by centrifugation, filtration and membrane separation) that are

normally necessary to condition an extract before its application on packed bed

chromatography columns, may be eliminated. Few attempts to scale up magnetic

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operations in biotechnology have been reported so far. The application of magnetic

carrier technology to purify enzymes involves functionalizing an inert solid support

which has a magnetic property with an appropriate substrate. In this study,

superparamagnetic iron oxide nanoparticle (SPION) was employed as a support for the

preparation of starch functionalized superparamagnetic iron oxide nanoparticles (starch-

SPION) to purify bacterial amylase. The results showed that the starch-SPION had high

adsorption specificity for bacterial amylase.

Compared to the traditional methods used in the purification of proteins, our novel starch-

SPION based purification technology is fast, scalable and easy to handle. Moreover the

process does not require sophisticated instruments leading to cost savings and easy

recovery of enzymes. Amylase is an industrially and economically important enzyme

used for clarification of fruit juices, in coffee industry, textile industry, pulp industry,

production of ethanol, treatment of human bezoars, in syrups and various other purposes.

So, in our study we have considered amylase as a model enzyme for purification. The

enzyme purified by superparamagnetic nanoparticle is practically feasible in the

laboratory scale, opening up the scope of utilizing magnetic career technology in the field

of separation science. Thus, further research and optimization should be carried out to

scale up the magnetic separation technique for industrial production scale.

ACKNOWLEDGEMENTS

This research work has been carried out with the financial support of Dept. of Science &

Technology, Govt. of India (Project-Nanomission: SR/NM/NS-48/2009) and University

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of Kalyani, Nadia, West Bengal. We also acknowledge the help of Prof. T Basu (Dept. of

Biochemistry and Biophysics, University of Kalyani) and Dr. P Roy (SINP) for providing

dynamic light scattering and TEM facilities respectively.

AUTHOR’S CONTRIBUTION

TP carried out the actual purification steps whereas SC and AB were involved in the

synthesis and characterization of the nanoparticle. SB and DC were also involved in the

study and optimization. KS supervised the work along with critical interpretations during

manuscript preparation.

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TABLE 1 Purification of bacterial amylase by starch-SPION

Sample Total

activity (U)

Total protein

(mg)

Specific activity

(U/mg)

Yield (%) Purification

fold

Crude

enzyme

1019 6.02 302.15 100 1

Starch-

SPION

purified

950 0.25 3800 93.22 12.57

Yield = (Total activity of purified enzyme/total activity of crude enzyme) ×100

Purification fold = Specific activity of purified enzyme/specific activity of crude

enzyme

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Scheme 1 Procedure of amylase purification from bacterial broth using Starch coated

SPION.

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FIGURE 1 Characterization of SPION: 1a) Transmission Electron Microscopy (TEM) of

SPION showing the size of the nanoparticle to be 8 nm; 1b) SQUID data of SPION

showing M-H curve (lack of hysteresis loop confirmed superparamagnetic property); 1c)

Zeta potential of SPION.

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FIGURE 2 Characterization of starch-SPION by FTIR spectrophotometric study.

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FIGURE 3 SDS-PAGE of Starch-SPION purified bacterial amylase (Lane1: Marker

protein, Lane 2: Fermented broth containing amylase, Lane 3: Starch-SPION purified

amylase).

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FIGURE 4 Native gel electrophoresis of Starch-SPION purified amylase soaked in starch

solution and stained with iodine (Lane 1: Fermented broth containing amylase, Lane 2:

Starch-SPION purified amylase).

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FIGURE 5 Effect of pH on Starch-SPION purified amylase and Fermented broth

containing amylase.

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FIGURE 6 Effect of temperature on Starch-SPION purified amylase and Fermented broth

containing amylase.

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FIGURE 7 Thermal stability of Starch-SPION purified amylase and Fermented broth

containing amylase

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