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#-Amylase@magnetite: magnetic nanocomposites with enhanced thermal stability for starch hydrolysis Bazhena V. Astafyeva, Olga E. Shapovalova, Andrey S. Drozdov, and Vladimir V. Vinogradov J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01298 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 6, 2018

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Journal of Agricultural and Food Chemistry

α-Amylase@ferria: magnetic nanocomposites with enhanced thermal stability for starch hydrolysis Bazhena V. Astafyeva, Olga E. Shapovalova, Andrey S. Drozdov,∗ and Vladimir V. Vinogradov Laboratory of Solution Chemistry of Advanced Materials and Technologies, ITMO University, St. Petersburg, 191002, Russian Federation E-mail: [email protected]

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Abstract

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The present study is devoted to the development of a new class recyclable magnetic

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catalytic nanocomposites for starch hydrolysis. Alpha-amylase was entrapped within

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magnetite derived xerogel matrix in a course of a room-temperature sol-gel transition,

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leading to enzyme immobilization within the pores of a rigid magnetic matrix. For

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hybrid organo-inorganic composites with enzyme mass fractions less than 10 wt. %

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no enzyme leaching was observed. At 80◦ C amylase@ferria composite demonstrates

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catalytic activity on the level of 10 U/mg and starch hydrolysis rate comparable to

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free enzyme, while at 90◦ C the activity of amylase@ferria at least twice higher that

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of free amylase due to higher thermal stability of the composite. Entrapped amylase

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showed excellent stability and lost only 9 % of its activity after 21 days of storage in a

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buffer solution, while free enzyme was totally inactivated after 17 days. The material

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can be used either a magnetically-separable reusable catalyst or can be applied as a

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catalytic ceramic coating with at least 10 cycles of use.

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ACS Paragon Plus Environment


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Introduction

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Advance in nanomaterials synthesis and nanotechnology led to active implementation of

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nanostructured materials in different areas of industry as adsorbents, catalysts or construc-

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tion materials. 1–4 When it comes to food industry, the main attention is paid to enzyme

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immobilization, as it can significantly reduce costs of enzymatically catalyzed reactions by

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thermal and chemical stabilization of enzymes, simplifying separation of enzymes from reac-

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tion mixtures and offering the possibility to make recyclable enzymatic catalysts. 5–8 Selection

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of a support material can have a critical effect on the stability of enzymes and the efficiency

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of enzyme immobilization, although it is difficult to predict in advance which support will be

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the most suitable for a particular enzyme. The support must be insoluble in water, should

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have high enzyme loading capacity, be mechanically stable and must not have deleterious

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effect on the catalytic activity of the enzyme. The variety of nanostructured materials for

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immobilization of enzymes includes both organic and inorganic polymers and materials, such

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as: porous polymeric matrixes, 9,10 polystyrene particles, 8,11,12 phospholipid liposomes, 13–15

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zeolites, 16,17 metal oxides, 18,19 silica matrixes 20–22 and ect. Among all enzymatic nanocom-

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posites significant attention is paid to ones with magnetic properties. 23,24 Magnetic field

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susceptibility revealed a mechanism for efficient recovery of the enzyme complex thereby

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preventing the contamination of the final product by enzymatic catalyst. Magnetic enzy-

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matic catalysts mainly presented by either core-shell type structures or polymeric matrixes

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with encapsulated magnetic nanoparticles and are known to be effective systems with good

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application results. 25–30 Despite the effectiveness, magnetic composites shares one common

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disadvantage of their complex structure that involves a magnetic core, an enzyme and a

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linking agent. Such multicomponent structure requires multi-step synthesis procedures with

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intermediate purification steps resulting in higher production costs that can negate the prac-

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tical advantage of such systems. In this article, we describe magnetic nanocomposite with

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amylase activity consisting of only two components: amylase and magnetite ceramic matrix.

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The material is produced by direct entrapment of the enzyme within magnetite xerogel in a 2


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course of the room-temperature sol-gel transition. Entrapped amylase not only demonstrated

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the ability to perform catalytic hydrolysis of starch solutions with the rate comparable to that

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of the free enzyme, but also showed excellent thermal stability and extended temperature

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of activity window and prolonged period of storage. Composites demonstrated leach-proof

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behavior and can be magnetically separated and reused up to 10 times with only 5% decrease

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of its initial activity. Alternatively the composite can be applied in a form of ceramic coating

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to ensure effective application in batch or flow reactors.

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Materials and methods

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Chemicals: Iron (II) chloride tetrahydrate, iron (III) chloride hexahydrate, ammonia 25%

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solution, α-amylase from porcine pancreas (100 units/mg) and soluble starch, all obtained

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from Sigma-Aldrich.

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Preparation of a ferria hydrosol: Pure ferria hydrosol was prepared from iron (II) chloride

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tetrahydrate and iron (III) chloride hexahydrate as described in the Ref. 31. Briefly, 2.5

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g FeCl2 *4H2 O and 5 g FeCl3 *6H2 O were dissolved in 100 mL of deionized water under

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constant stirring (500 rpm). Then, 12 mL of aqueous ammonia solution was added at room

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temperature. Using a magnet, the formed magnetite precipitate was collected and washed

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with deionized water until neutral pH. The washed black precipitate was mixed with 100 mL

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of deionized water and subjected to ultrasonic treatment (37 kHz, 110 W) under constant

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stirring (300 rpm) for 120 minutes. The mass fraction of magnetite nanoparticles in the

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resulting sol was 2 wt. %.

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Entrapment of α-amylase in a ferria matrix: 20 mL of freshly prepared ferria sol was

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mixed with 0 to 1250 µL of α-amylase solution (4000 U/mL). After stirring, the contents

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were dried in a vacuum desiccator to yield composites with enzyme mass fractions ranging

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from 0 to 12.5 wt. % (0-11 U/mg). The composite materials were milled in an agate mortar

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and used in experiments. To evaluate the release profile of the enzyme, 20 mg of the crushed

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composite in a quartz cuvette was treated with 2 mL of the saline solution and the absorption

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spectrum at 37◦ was measured over time at 210 nm.

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Amylase@ferria coatings: In order to prepare the catalytic coating mixture of 5 mL of

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freshly prepared ferria sol with 1 mL of α-amylase solution (4000 U/mL) was applied on

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the walls of a glass and condensed on air. The glass was continuously rotated to ensure the

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homogeneity of the coating.

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Preparation of the starch solution: 2 g of soluble starch were placed in 98 mL of water

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preheated to 55◦ and thoroughly stirred. The temperature was gradually elevated to 80◦ with

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continuous stirring using a magnetic stirrer for an hour. After incubation the temperature

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of the solution was decreased to 60◦ , it was stirred for another 30 minutes and used in the

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experiments.

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Evaluation of amylase@ferria particles enzymatic activity: Activity of the amylase@ferria

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composite was evaluated by measuring the viscosity of the starch solution. 7 mL of the starch

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solution was placed into a thermostated rotary viscometer sleeve and, after adding 100 mg

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of the 10 wt. % amylase@ferria composite (enzymatic activity 10 U/mg), the viscosity

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was measured in the kinetic mode at different temperatures ranging from 60 to 90◦ . For

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comparison, 7 mL of the starch solution was incubated with 25 µL of amylase solution (4000

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U/mL) and subsequently tested in the same manner.

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Evaluation of amylase@ferria particles reusability: In order to test the reusability of the

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composite magnetic catalyst 100 mg of the 10 wt. % amylase@ferria composite (enzymatic

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activity 10 U/mg) was added to 7 mL of the starch solution and stirred for 20 minutes while

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monitoring the process by measuring the viscosity of the solution. After that, the composite

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material was separated by applying magnetic field and added to a new portion of the starch

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solution for another catalytic cycle.

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Catalytic activity of amylase@ferria coatings: Into a model reactor with the total volume

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1 L, coated with amylase@ferria nanocomposite, 100 mL of the starch solution was added

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and mixed at 80◦ for 15 minutes. The process was monitored by measuring the viscosity.

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After the incubation, the solution was changed with a new portion of the starch solution and

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another catalytic cycle was performed.

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Characterization methods: The crystalline phase of the samples was measured by X-

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ray diffraction (Bruker D8 Advance) using Cu-Kα radiation (λ = 1.54 ˚ A); the samples

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were scanned at a rate of 0.5 degrees per minute. To analyze the samples with a high-

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resolution scanning electron microscopy (SEM), the obtained ground xerogel was deposited

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on a metal tip and investigated without additional spraying using a Magellan 400L ultra-high

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resolution electron microscope. The samples for a transmission electron microscopy (TEM)

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were obtained by dispersing a small probe in ethanol to form a homogeneous suspension.

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Then, a suspension drop was coated on a copper mesh covered with carbon for a TEM

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analysis (FEI TECNAI G2 F20, at an operating voltage of 200 kV). Specific surface area,

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pore volume and pore size distribution were investigated using Quantachrome Nova 1200e by

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nitrogen adsorption at 77 K and analyzed by the BET and BJH equations. Prior to analysis,

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all samples were degassed at room temperature for 48 hours. Hydrodynamic diameter was

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measured by the DLS technique on Photocor Compact Z. The Raman spectra were recorded

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using the 633 nm He-Ne laser line on a Horiba Jobin Yvon Micro Raman 300 spectrometer.

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The laser power on the samples employed was 0.03 mW and 0.344 mW, with 300 s and 120

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s exposition per diffraction window, respectively. In all the measurements, 50x Olympus

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lens, hole of 500mm, slit of 100mm and a diffraction grid with 1800 grooves/mm were

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employed. The IR measurements were performed in transmission and ATR modes. Viscosity

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was determined by a Fungilab rotational viscometer.

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Results and discussion

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Synthesis of amylase@ferria composites

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In order to produce amylase@magnetite composites, the stable magnetite hydrosol (desig-

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nated as ferria) was used that was described in details in our previous works. 31 The hydrosol 5



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was synthesized by ultrasound-assisted coprecipitation procedure using non-stoichiometric

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ratios of iron(II) and iron(III) salts with the subsequent ultrasonic treatment of the resulting

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sol in deionized water. The final hydrosol consists of magnetite nanoparticles with an aver-

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age hydrodynamic diameter of 30 nm dispersed in water at neutral pH level (pH 7). Surface

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of the particles is densely covered with the hydroxyl groups both of magnetite and Fe(OH)2

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nature as proven by Raman spectroscopy (Fig. 1a). Due to this fact, the isoelectric point

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of the magnetite NPs is shifted from pH = 6.8, which is common for magnetite, to a higher

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pH value of 8.3 (Fig. 1b). As a result, the magnetite NPs showed high zeta-potential values

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(+36 mV at neutral pH level) and excellent colloidal stability, forming stable hydrosols which

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are behaves as typical magnetic fluids (Fig. 1c).

Figure 1: Ferria hydrosol. The ATR spectra demonstrates the presence of the hydroxyl groups of both Fe(OH)2 and Fe3 O4 nature (a); in contrast to magnetite, the isoelectric point of the ferria nanoparticles is shifted to pH = 8.3 (b); due to the high zeta potential values the ferria nanoparticles form a stable hydrosol with a magnetic fluid-like behavior at a neutral pH level (c).

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Amylase@ferria composites were produced by the condensation of the ferria hydrosol

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with α-amylase solution under reduced pressure at room temperature. Upon removal of

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the solvent, a sol-gel transition occured leading to the formation of the mesoporous xerogel

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matrix. The matrix was composed of the 10 nm magnetite nanoparticles with a truncated

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octahedron morphology according to SEM and TEM images (Fig. 2a and 2b). The XRD

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pattern of the material demonstrated the lines typical for magnetite (compared to JCPDS

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file no. 19-0629) (Fig. 2c). The main peak at 35.45◦ attributed to the crystalline plane with 6


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Miller indices of (3 1 1) is clearly seen. Other distinctive peaks at 18.52◦ - (0 2 0), 30.10◦ -

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(2 2 0), 43.08◦ - (4 0 0), 53.45◦ - (4 2 2), 56.98◦ - (5 1 1), 62.57◦ - (4 4 0), 70.99◦ - (5 3 3),

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and 74.02◦ - (4 4 4) (Fig. 2f) matched those of the standard magnetite diffraction pattern. 32

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Another proof that the ferria xerogel matrix consisted of the magnetite nanoparticles came

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from Raman spectroscopy. The Raman spectra of the sample showed the characteristic

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broad band of magnetite at 667 cm−1 (Fig. 2d, line a) when the spectra was recorded with

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a 633 nm He-Ne laser (0.030 mW laser beam power and an acquisition time of 300 sec per

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diffraction window). 33,34 Further confirmation came from observations of the known phase

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transformations of magnetite under the Raman-laser measurement conditions: when the

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power was increased to 0.344 and 1.35 mW (acquisition times of 90 and 30 sec, respectively),

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then for the former the characteristic maghemite bands at 711, 492, and 372 cm−1 are seen

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(Fig. 2d, line b), and for the latter full oxidation to α-hematite is observed (Fig. 2d, line c)

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by the characteristic bands at 591, 385, 273, and 213 cm−1 . 33,34

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The formed matrix possesses well-developed mesoporous nanoarchitecture (Fig. 2a,b)

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with a high specific surface area (110 m2 /g for 10 wt. % amylase@ferria and 119 m2 /g

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for pure ferria xerogel) and an average pore diameter of 8 nm (Fig. 1S(a,b)). While the

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molecule of amylase has the size of 7x6x5 nm, 35 it is complementary to the pores diameter

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of ferria matrix, and it is entrapped in the course of a sol-gel transition. The mechanism

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of the entrapment can be briefly described as follows: at the first step, negatively charged

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amylase molecules (isoelectric point of amylase is at pH = 5.5) 35 electrostatically interacts

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with positively charged magnetite nanoparticles (isoelectric point of magnetite sol is at pH

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= 8, Fig. 1b). Upon drying of the resulting mixture, the rigid magnetite xerogel matrix is

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formed (Fig. 2a,b), in which enzyme molecules may be buried inside of the matrix or located

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in the surface layer. According to our previous observations, such structural organization

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of a material allows the entrapped enzyme to interact with macromolecular substrates, such

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as polysaccharides and to perform enzymatic transformations without being released from

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a xerogel matrix. 36,37 Due to the formation of interparticle Fe-O-Fe bonds in the process of

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Figure 2: The ferria xerogel matrix. The ferria matrix consists of nanoparticles with a truncated tetrahedron morphology. Interplanar spacing is shown in the insert (a); SEM image of the ferria xerogel demonstrates porous structure of the material (b); XRD patternt of the ferria xerogel shows peaks typical for the magnetite crystal phase (c); Raman spectra recorded at a 0.03 mW laser beam power demonstrates spectra typical for magnetite (line a), after increasing the laser power to 0.344 mW magnetite partially converts into maghemite (line b), at 1.35 mW complete conversion of magnetite into hematite is observed (line c). All lines are obtained from a single sample (d); the magnetization curve of ferria and amylase@ferria. Absence of hysteresis can be observed in the insert (e); the material is readily attracts to the magnet (f). 163

sol-gel transition, the formed composite matrix is not prone to spontaneous re-suspendeition

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in water media and can be used as a magnetically-sensitive catalytic composite. 38 The mate-

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rial demonstrated the typical superparamagnetic behavior: its magnetization curve showed

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absence of the hysteresis loop and no remnant magnetization in absence of magnetic field

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(Fig. 2e). The magnetic moment of the composite at 6000 Oe amounted to 61 emu/g for

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10% amylase@ferria, that is 25% lower than for the pure ferria matrix. Nevertheless, a high

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magnetic susceptibility allowed to separate the amylase@ferria composite using an external

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magnet (Fig. 2f). The entrapment of amylase may be either full or there may be partial

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release from the composite depending on the mass fraction of the enzyme. According to 8


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release studies, at mass fractions of entrapped amylase below 10 wt. %, only a minor release

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of approximately 1% of the total entrapped enzyme amount occurred, while at higher mass

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fractions release was increased up to 21% of the total amount due to matrix enzyme loading

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capacity overload (Fig. 2S). 36

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Catalytic activity of amylase@ferria

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To evaluate the enzymatic activity of the synthetized biocomposites, the enzymatic starch

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cleavage reaction was selected. Amylase acts on α-1,4-glycosidic bonds of starch, cleav-

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ing polysaccharides inside the chain and leading to formation of low-molecular hydrolysis

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products, normal α-dextrins. Further hydrolysis gives maltose, maltotriose, and glucose. 39

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Since the hydrolysis process results in biopolymer cleaving to smaller monomers, the viscos-

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ity of the system decreases, and this phenomenon serves as a convenient analytical signal

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to describe the catalytic process. Amylase is a thermally stable enzyme and has an opti-

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mal temperature (Topt ) valued 70◦ C. To evaluate the catalytic activity of the composites,

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amylase@ferra with 10 wt. % was selected and incubated with the starch solution at tem-

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peratures varied from 60 to 90◦ C. For comparison, the process was performed using equal

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amounts of free enzyme (Fig. 3). Evaluation of the process at 60◦ C demonstrated that the

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amylase@ferria composite demonstrates catalytic activity and is able to hydrolyze the starch

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solution. Compared to free amylase, the composite material showed approximately 1.5-fold

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lower activity under these conditions, hydrolyzing starch in 34 minutes, while free enzyme

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performed this process in 22 minutes (Fig. 3a). With elevation of the temperature to 70◦ C

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accelerated the reaction rate of the both systems, but also makes the difference between free

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enzyme and amylase@ferria even more pronounced: while free enzyme catalyzes the process

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in 5 minutes, for the amylase@ferria composite the rate of the reaction was 2.5-folds lower

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and required 13 minutes for hydrolysis of the same amount of the substrate (fig. 3b). The

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difference between the reaction rates can be easily explained by the fact that in case of the

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free amylase the reaction takes place in a homogeneous solution, while for the immobilized 9



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enzyme the system is heterogeneous and diffusion processes limits the reaction. While amy-

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lase has an optimal temperature of 70◦ C, further elevation of the temperature to 80◦ C led to

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its denaturation and inactivation processes. This is true for the free enzyme, and a notable

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decrease in the reaction rate is observed, with prolongation of the hydrolysis time from 5

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min at 70◦ C to 14 min at 80◦ C. In contrast, entrapped amylase showed the catalytic activity

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almost identical to that at 70◦ C and close to the activity of the free enzyme (Fig. 3c). Fur-

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ther increase of the reaction temperature to 90◦ C made the difference in the behavior of the

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catalysts more obvious. While in the case of free amylase the starch hydrolysis was halted

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due to the denaturation, the composite material demonstrated a high degree of catalytic

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activity compared to the activity measured at 70 and 80◦ C (Fig. 3d). Preservation of the

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catalytic activity by entrapped amylase at elevated temperatures is in good correlation with

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our previous observations. 40 This phenomenon can be explained by tight interactions of the

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enzyme’s tertiary structure with the inorganic matrix pore walls, resulting in stabilization

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of the protein structure and its stabilization against harsh conditions.

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One of the features of the amylase@ferria composite material is its magnetic responsive-

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ness. The composite material can be separated from the reaction mixture by an external

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magnetic field and be reused for another cycle of the catalytic process. Experiments showed

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that the activity of the composite material remains essentially at the same level at least after

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ten cycles of use (Fig. 3e), decreasing by only 4% in total and revealing a high stability of

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the composite material and a prolonged profile of action, characteristic for the entrapped

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enzymatic systems, when the activity is not related to the release of the enzyme. Another

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important parameter of enzymatic formulations and composite materials is their storage sta-

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bility. By their nature, enzymes are unstable in solutions and deteriorate over time. One of

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the goals that can be achieved though enzyme immobilization is an increase of its stability

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and prolongation of the storage period. 22,41 In order to evaluate the effect of entrapment

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within sol-gel magnetite in amylase long-term stability the material was stored in phosphate

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buffer solution (pH = 7.4) at 4◦ C for a period of 21 days and the activity was compared

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to one measured for free enzyme (Fig. 3f). Entrapped amylase demonstrated significantly

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higher stability at storage conditions compared to free enzyme. While free amylase lost all

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its activity within 17 days, composite material demonstrates 91% of its initial activity after

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21 days of storage with only 0.5%/day degradation rate. When compared to results reported

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earlier it can be seen that immobilization of amylase by entrapment results in higher stability

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compared to covalent bonding or adsorption. 22,42–45 This fact can be explained by tighter

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three dimensional interaction of the enzyme with inorganic matrix and absence of covalent

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bonds between the supporting material and the enzyme that can affect the conformation of

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enzyme. 40

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Amylase@magnetite ceramic catalytic coatings

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As an alternative to application as a magnetically separable catalyst, amylase@magnetite

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composite can be applied as a ceramic coating on a surface of a reactor. For this purpose,

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mixture of amylase and stable magnetite hydrosol was applied on the vessel surface before

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solvent removal by spraying, dip-coating or by using the Mayer rod. Upon condensation,

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irreversible sol-gel transition occurred resulted in the formation of the enzymatically-doped

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ceramic magnetite coating on the surface of the substrate. The thickness of the formed

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ceramic layer depends on the amount of the applied colloid solution by the factor of 24,

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(that is, application of the 5 micrometer layer of hydrosol mixture resulted in the formation

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of the composite ceramic coating with the thickness of ≈210 nm) and can be precisely

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regulated in that way. The formed coating showed a high degree of the homogeneity (Fig.

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4a).

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The composite bioceramic coating demonstrated high thermal stability similar to the

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one observed on the magnetic particles (Fig. 4b). The relative activity of the composite

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coating was stable at elevated temperatures without notable degradation at 70◦ C, Topt of

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α-amylase, and only slight degradation of the material for 4% after incubation at 80◦ C after

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90 min of incubation was observed. At 90◦ the enzymatic activity linearly decreased over 11



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the time, loosing 0.37% of its relative activity per minute, but still demonstrated higher

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thermal stability than free enzyme, which was fully inactivated in 5 minutes at this tem-

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perature. The performance of the model reactor was tested by evaluating the rate of starch

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hydrolysis in successive catalytic cycles. For this purpose, the starch solution was subjected

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to hydrolysis in batch experiments in the reactor at 80◦ C, the process was monitored by

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measuring the viscosity of the solution every 5 minutes (Fig. 4c). When the viscosity of

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solution reached the constant value, the starch solution was changed with a new portion and

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the hydrolysis dynamic was measured again (for details see Experimental section). Viscosity

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measurements in the set of experiments demonstrated the excellent stability of the model re-

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actor performance in time. Starch solution hydrolysis demonstrated almost equivalent rates

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in 10 successive experiments and took 20 minutes at 80◦ C with only minor degradation by

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4%, which demonstrated the high stability of the hybrid amylase@ferria coating catalytic

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properties. It can be concluded that sych and makes it promising material for application

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in design of new types of advanced batch and flow reactors.

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Acknowledgement

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This work was supported by Russian Foundation for Basic Research, grant 18-33-01170

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mol a. The authors declare no conflict of interests.

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Figure 3: Catalytic hydrolysis of starch solution by free amylase and amylase@ferria kinetic curves. Hydrolysis process kinetics at 60◦ C (a), 70◦ C (b), 80◦ C (c), and 90◦ C (d); relative activity of the amylase@ferria composite reused in ten successive experiments (e); long term stability of amylase@ferria and free amylase stored at 4◦ C in phosphate buffer solution (f). The results presented is represented by average values of 3 measurements for each of the curves.

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Figure 4: Enzymatically-doped ceramic coating amylase@ferria on the surface of the glass vessel. SEM cross section of the coating is presented (a); the dynamic of starch hydrolysis in the model reactor in successive cycles. Catalytic coating in model experiment have demonstrated a good stability against thermal inactivation and peeling, and showed high degree of reusability of the system with only 6% degradation after 10 cycles of starch hydrolysis.

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199x268mm (300 x 300 DPI)


Î±-Amylase@ferria - American Chemical Society

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