Advanced Cellulose Fibers for Efficient Immobilization of Enzymes

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ADVANCED CELLULOSE FIBERS FOR EFFICIENT IMMOBILIZATION OF ENZYMES Iris Beatriz Vega Erramuspe, Elnaz Fazeli, Tuomas Näreoja, Jani Trygg, Pekka E. Hänninen, Thomas Heinze, and Pedro Fardim Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00865 • Publication Date (Web): 30 Aug 2016 Downloaded from http://pubs.acs.org on August 31, 2016

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Biomacromolecules

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ADVANCED CELLULOSE FIBERS FOR

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EFFICIENT IMMOBILIZATION OF ENZYMES

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Iris Beatriz Vega Erramuspe,* Elnaz Fazeli,¥ Tuomas Näreoja,¥,‡ Jani Trygg,* Pekka Hänninen,¥

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Thomas Heinze,§ Pedro Fardim.†,*

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*

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Akademi University, Porthansgatan 3, FI 20500 Åbo, Finland

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¥

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Turku, Tykistökatu 6A, 5th Floor, FI 20520 Turku, Finland

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‡

Fibre and Cellulose Technology Laboratory, Faculty of Science and Engineering, Åbo

Laboratory of Biophysics, Cell Biology and Anatomy, Institute of Biomedicine, University of

Department of Neuroscience, Karolinska Institutet, von Eulers väg 3, SE 17177 Stockholm,

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Sweden

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§

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Macromolecular Chemistry, Friedrich Schiller University of Jena, Humboldtstraße 10, 07743

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Jena, Germany

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†

Center of Excellence for Polysaccharide Research at Institute of Organic Chemistry and

Department of Chemical Engineering (CIT), Celestijnenlaan 200 F, 3001 Leuven, Belgium.

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ACS Paragon Plus Environment

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KEYWORDS. Glucose oxidase (EC 1.1.3.4), Horseradish peroxidase (EC 1.11.1.7),

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Immobilized enzymes, Bleached kraft pulp fibers, Amino groups containing fibres, STED, XPS,

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Advanced functional materials.

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ABSTRACT. Biocatalytic pulp fibres were prepared using surface functionalization of bleached

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kraft pulp with amino groups (F) and further immobilization of a cross-linked glucose oxidase

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(G*) from Aspergillus niger. The cross-linked enzymes (G*) were characterized using X-ray

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spectroscopy, Fourier transform infrared spectroscopy, dynamic scanning calorimetry and

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dynamic light scattering. According to standard assays, the G* content on the resulting fibers

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(FG*) was of 11 mg/g of fiber, and enzyme activity was of 215 U/g. The results from confocal-

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and stimulated emission depletion microscopy techniques demonstrated that glucose oxidase do

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not penetrate the interlayers of fibers. The benefit of pulp fiber functionalization was evident in

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the present case, as the introduction of amino groups allowed the immobilization of larger

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amount of enzymes and rendered more efficient systems. Using the approach described on this

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paper, several advanced materials from wood pulp fibers and new bioprocesses might be

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developed by selecting the correct enzyme for the target applications.

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INTRODUCTION

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In the drive towards efficient and safe industrial processes with low ecological footprint,

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enzymatic catalysis has been considered as one attractive tool for the synthesis of specific

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chemical compounds. Currently, enzymes are used in an enormous variety of products such as

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laundry products, textiles, food, pulp and paper, diagnostic tests, pharmaceutical products, and

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leather production, among others.1 Thanks to the development of enzyme immobilization

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methodologies, the lack of long-term stability and difficulties on recovery and re-use showed by


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the free-enzymes was overcome, and the use of selective and efficient enzymes at the industrial

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scale has become a reality.1,2 For example, immobilized enzyme systems are very important for

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the food industry in the case of fructose synthesis from glucose, using glucose isomerase

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(EC 5.3.1.5) with the high catalyst productivity of 11.000 kg of product per kg of enzyme. Also,

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in the conversion of lactose into glucose and galactose (lactose-free milk and whey) catalyzed by

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β-Galactosidase (EC 3.2.1.23).3,4 There are several types of enzyme immobilization methods,

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divided in three major groups: binding to a solid support of carrier, entrapment (encapsulation),

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and cross-linking.4,5,6,7 When using a solid support, the enzymes can be bound to the surface

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thanks to weak physical interactions, or due to strong ionic- or covalent forces. In the synthesis

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of the pre-fabricated carriers, it has been used different biopolymers (e.g. cellulose, starch,

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agarose, chitosan, poly-N-isopropylacrylamide, or acrylates-derived copolymers), hydrogels (e.g.

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polyvinyl alcohol hydrogels) and inorganic polymers (e.g. nanosilica or zeolites). The cost of the

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carrier might be a limitation in the case of strong support binding, because the carrier often

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renders unusable, once the enzyme in not active anymore. Entrapment involves the synthesis of

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the polymeric network in the presence of the enzyme that needs a physical restraint. With this

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methodology, additional covalent binding is often required. Although the immobilization

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methods using support binding or entrapment show many advantages, some drawbacks of these

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methods are important and should not be ignored. For example, immobilization of enzymes on a

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solid carrier is usually detrimental for the enzyme activity, principally at high enzyme loadings.

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Moreover, mass transfer limitations can play a significant role in the performance of the system.

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As an alternative of support bonding, the carrier-free immobilization methods such as cross-

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linking are available.


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The carrier-free cross-linking of enzymes using bifunctional cross-linker reagents such as

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glutaraldehyde, offers many advantages in comparison with the immobilization of enzymes in

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solid materials. For instance, it avoids the inevitable ‘dilution of activity’ observed in the final

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material prepared via support binding or encapsulation methods, because of the low fraction of

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enzyme immobilized in the inert matrix. In comparison with these methods, cross-linking is

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usually simpler, show high enzyme activity, and is less expensive. To avoid low activity

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retention, poor reproducibility, low mechanical stability and difficulties in handling the

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cross-linked enzymes, several strategies such as the preparation of cross-linked enzyme

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aggregates (CLEA®), combining purification and cross-linking, have been successfully

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developed in the latest decades. More recently, Combi-CLEAs® involving catalytic cascade

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processes, or enzymes cross-linked to a polymer membrane such as polytetrafluoroethylene

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(PTFE) have been attracted much interest.4

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Glutaraldehyde (GA) is frequently used to bind enzymes to each other in order to create a

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enzymatic matrix (crosslinkage-), and has been found to be very effective for enzyme

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immobilization as it generates thermally and chemically stable cross-links.8,9,10,11 According to

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literature, commercial GA solutions contains a mixture of monomeric structures such as the

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aldehyde group or the cyclic hemiacetal form, and polymeric structures such as the cyclic

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hemiacetal oligomer form.12 Under acidic or neutral conditions, the reaction between GA and

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proteins is expected to yield monomeric cyclic acetals or its multimeric forms.13 The covalent

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bonds created after the chemical reaction between GA and the commercial enzymes yield cross-

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linked enzymes. The enzyme activity, thermal stability, resistance to pH changes, and the

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hydrodynamic volume distribution of the macromolecules in solution are usually modified after

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cross-linking.


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In the search for bio-based alternatives to traditional carriers, natural fibers such as the

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obtained from agricultural resources have been studied for immobilization of enzymes with

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positive preliminary results.14 The possibility of using agricultural residues for the preparation of

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new systems is widely argued to be more beneficial for the environment. However, the

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sustainability of the bio-based systems still remains a challenge in many cases. For example, the

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heterogeneous and highly variable composition of natural fibers represents an important

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drawback. Due to that and because of the strong dependence of the enzymes behavior on its

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micro-environment, it is difficult achieving reproducible and efficient systems with natural

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fibers. In this context, bleached kraft wood pulp fibers may offer many advantages. They have a

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similar composition independently of the production batch and are already available on the

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market on the large scale from the pulp mill. The bio-based material with very large surface area

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is insoluble in water but highly hydrophilic, which offers the favorable environment for the

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immobilization of most of the enzymes except for lipases.2 In addition, it is possible improve the

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poor reactivity of pulp fibers by introducing functional groups such as amino groups on the

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surface of these fibers, while preserving their excellent mechanical properties and large surface

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area.15 Bleached kraft wood pulp fibers have been intensively studied and its surface anionic

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charge and excellent mechanical properties are determined. Some dimensions related to native

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softwood fibers and the corresponding bleached kraft pulp fibres are reported in the

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literature.16,17,18 This information is highly valuable when studying pulp fibers as a solid carrier

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for the immobilization of enzymes. Moreover, the accessibility of pulp fibers to macromolecules

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such as glucose oxidase enzyme, which hydrodynamic particle size of about 8 nm has been

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reported by Talbert et al.19, can be assessed using fluorescence microscopy.20

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In previous works it has been studied the modification of cellulose fibers using multifunctional

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polysaccharide derivatives.15,21,22 This innovative approach provides an easy way to introduce

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functional groups on bleached kraft wood pulp fibers (BKPP) in aqueous media, at room

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temperature. The method is simple, reproducible, and allows fiber functionalization without

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losing the excellent mechanical properties of the original fibers.22 For example, using this

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method BKPP can be modified with (3-carboxypropyl)trimethylammoniumchloride ester of

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6-deoxyazidocellulose, yielding functional fibers decorated with azide groups (Scheme 1). As it

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was demonstrated, the azide groups introduced to the fibers are available for catalyzed alkyne-

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azide Huisgen cycloaddition reaction (CuAAc) with alkyne group-containing molecules.15 The

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reaction of the azide functional groups-containing fibers with propargylamine allows the

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preparation of amino functional groups-containing fibers F. In the present work, we are

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demonstrating that cross-linked glucose oxidase enzymes (G*), which are produced from a

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commercial glucose oxidase enzyme (G), can be covalently bonded to the amino functional

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groups-containing fibers F using a known procedure, yielding biocatalytic fibers (FG*). The

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differences between a G and G* have been studied. The amount of enzymes on the fibers FG*

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was determined, and the enzyme activity in these fibers have been measured. In addition, the

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distribution of G* on the fiber surfaces was studied using high-resolution fluorescence

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microscopy techniques. The performance of the system FG* was tested at different conditions

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(pH, T). The information gathered on this paper is a proof of concept that enzymes can be

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immobilized on functional fibers obtained from bleached kraft pulp fibers.

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((Scheme 1 here))


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MATERIALS AND METHODS

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Materials

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Bradford reagent, methanol (anhydrous, 99.8 %), sodium chloride (BioXtra, ≥ 99.5 %), and

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sodium phosphate dibasic heptahydrate (ACS reagent), were purchased from Sigma. Sodium

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phosphate monobasic dihydrate (puriss p.a., crystallized ≥ 99 %), glutaraldehyde (grade II, 25 %

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in H2O), Tris, paraformaldehyde (PFA), dimethyl sulfoxide (DMSO), and mowiol 4-88 were

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purchased from Sigma-Aldrich ( all with ACS reagent). Glucose oxidase (type VII prepared from

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Aspergillus niger, ≥ 100 U.mg-1), HRP Peroxidase (type II prepared from horseradish,

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150-250 U.mg-1), 2,2’-Azino-di-(3-ethyl-benzothiazoline-6-sulphonic acid), and glucose oxidase

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activity assay kit MAK097, were also purchased from Sigma-Aldrich. ACS grade sodium

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hydrogen carbonate was purchased from J.T. Baker. ACS reagent phosphate buffered saline was

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purchased from Gibco (ThermoFisher Scientific). Abberior® STAR 635 (NHS ester) was

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purchased from Abberior. Never dried BKPP prepared from Pinus sylvestris L., with brightness

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of 86 from kraft cooking process followed by the bleaching sequence DO-EOP-D1-P was

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supplied by Metsä Fiber (Rauma Mill, Finland). Amino functional groups-containing fibers F

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were prepared from BKPP as described in our previous work.15 Both BKPP and F, were stored

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at -20 °C and taken out of the freezer thawed before use. The chemicals were used without

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further purification. Unless specified otherwise in this paper, all the results from weight

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measurements related to wood pulp fibres are expressed on oven-dry weight basis.

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Methods


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Cross-linking of glucose oxidase (G*)

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Cross-linked glucose oxidase (G*) were prepared following a similar procedure as described

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by Solomon et al.23 Briefly, 125 mg of G was dissolved at room temperature (rt) in 0.1 M

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phosphate buffer solution (PBS) with pH 6.2, and the mixture was brought to a final volume of

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25 ml with the same buffer solution. Subsequently, 500 µl of GA solution (25 % in water) was

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slowly added to the enzyme solution and the mixture was stirred at 200 rpm on an orbital shaker

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(SKF2050, Lab. companion), at rt. After 12 h of reaction, the solution was dialyzed against 5 l of

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0.1 M PBS pH 6.2 twice, and then four times against the same volume of deionized water. The

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dialyzed solution was freeze dried, and the cross-linked enzyme was aliquoted and stored in the

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freezer at -20 °C, until further use.

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Preparation of the biocatalytic fibers (FG*)

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Crosslinked enzymes G* were dissolved in 0.1 M PBS pH 6.2, so that the concentration of the

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resulting solution S1 was 1.6 mg/ml. Subsequently, 2.0 ml of S1 solution was mixed with

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100 mg of amino functional groups-containing fibers F in 15 ml Falcon test tube

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(17 mm × 120 mm, polypropylene). The mixture was agitated on the orbital shaker for 2 h, at rt

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and 170 rpm. After that, the supernatant S2 was carefully removed with a pipette and 10 ml of

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0.1 M PBS pH 6.2 and 1.0 ml of GA were added to the Falcon tube (Scheme 2). The suspension

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was agitated for 5 min, at rt and 170 rpm. Finally, the supernatant S3 with excess of GA was

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removed and the treated fibers were thoroughly washed three times with 10 ml of 0.1 M PBS pH

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6.2, yielding biocatalytic fibers (FG*).

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Biomacromolecules

((Scheme 2 here))

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Characterization of the enzymes G and G*

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Differential scanning calorimetry (DSC). Differential scanning calorimetry measurements

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were performed using a TA Instruments' Q1000 Tzero DSC. The experiments were carried out

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using nitrogen gas as a sweep fluid at a flow rate of 50 ml/min. The heating rate was 2 °C/min,

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the cooling rate was 50 °C/min. The initial, upper, and lower temperatures were 20 °C, 80 °C,

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and 0 °C, respectively. The mass of analyzed G was equal to 4.40 mg, while the mass of G* was

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equal to 1.12 mg.

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Hydrodynamic particle size measurements. The measurements of the hydrodynamic particle

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size of the enzymes in 0.1 M PBS pH 6.2 have been performed using dynamic light scattering

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(Zetasizer Nano ZS, Malvern Instruments, Malvern, UK). The commercial enzyme (G) and the

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crosslinked enzyme (G*) were dissolved to a concentration of 3 mg/ml in 0.1 M PBS pH 6.2.

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After 10 min sonication, the samples were transferred to plastic cuvettes (sample level of 1.0 ml).

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The measurements were done in triplicate, and average value was calculated from the individual

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results. Poly dispersive index (PDI) was used as a measure of the size heterogeneity

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hydrodynamic size of the macromolecules in the studied solutions.

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ATR-FTIR instrumentation. The FTIR spectra were obtained using a Thermo Scientific™

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Nicolet™ iS™50 FTIR spectrometer equipped with deuterated tri glycine sulfate (DTGS)

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detector and a built in Thermo Scientific™ Nicolet™ iS™50 ATR module (diamond crystal).

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All spectra were recorded over the spectral range from 4000 cm-1 to 400 cm-1 at rt, with 4 cm-1


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resolution. 64 scans were collected for each spectrum, using autogain function, and aperture of

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100.

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X-ray spectroscopy. X-ray photoelectron spectra (XPS) of solid enzymes were obtained with a

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Physical Electronics PHI Quantum 2000 ESCA instrument equipped with a monochromatic Al

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Kα X-ray source of 1486.6 eV at 25.0 W and a combination of electron flood gun and ion

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bombarding for charge compensation. The take-off angle was 45° in relation to the sample

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surface. At least ten spots were analyzed on each sample with a beam diameter of 100 µm

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(analyzer mode FAT). The wide-scan was measured using 117.40 eV pass energy in 5.5 min

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(sampling depth up to 10 nm). The chemical states distribution of the different elements found in

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the sample were studied using pass energy of 46.95 eV and the following times: 10.77 min for

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C1s, 5.39 min for O1s and 10.77 min for N1s, yielding high resolution spectra.

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Determination of enzyme activity of G and G* in solution. For the determination of the activity

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of the cross-linked enzymes G* in 0.1 M PBS pH 6.2 solution at 37 °C, a 1.7 µg/ml solution of

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enzyme G* in 0.1 M PBS pH 6.2 was prepared, and the same was done in the case of G. Then,

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20 µl of the diluted enzyme solution was mixed with the two components ABTS-Peroxidase

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substrate system (400 µl of component A: 0.16 M glucose and HRP in O2-saturated 0.1 M PBS

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pH 6.2; 400 µl of component B: 0.9 µg.ml-1 of ABTS in O2-saturated 0.1 M PBS pH 6.2). After

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exactly 6 minutes of reaction, the absorbance at 415 nm (A415) was measured. The measurements

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were done by triplicate, and each individual value A415 was corrected by the absorbance of the

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sample blank (sample without enzyme). The mean value A’415 was obtained from the individual

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corrected values, for each sample. The amount of hydrogen peroxide, which is generated


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together with D-glucono-δ-lactone during the enzymatic conversion of glucose24, was calculated

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calibration curve A’415 vs nmol of H2O2 The calibration curve was prepared with a set of

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standard solutions of H2O2 in 0.1 M PBS pH 6.2, and the two component ABTS-Peroxidase

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system using the same procedure described above, for the enzyme solutions with unknown

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activity.

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Quantitation of the enzyme in solution

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A procedure similar to the standard protein assay described in Bradford (1976)25 was used for

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the quantitation of the enzyme in solution. Briefly, enzyme solutions containing 27-160 µg of

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enzyme in a volume up to 0.1 ml of 0.1 M PBS pH 6.2 were mixed by inversion with 3 ml of

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Bradford reagent in 15 ml Falcon test tubes (17 mm × 120 mm, propylene). Additionally, a

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sample referred as ‘reagent blank’ was prepared in a similar way by mixing 0.1 ml of PBS pH

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6.2 with 3 ml of the Bradford reagent. The absorbance was measured at 595 nm after 2 min and

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before 1 h in 3 ml polystyrene cuvettes against a reagent blank, using UV-2600

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spectrophotometer from Shimadzu. The concentration of the enzyme in solution was plotted

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against the corresponding absorbance, and the resulting standard curve was used for the

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determination of the enzyme solution concentration in unknown samples.

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Enzymes labelling

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Enzyme-labelling with Abberior® STAR 635 (NHS ester) was carried out following main

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guidelines described in the Abberior® protocol.26 Briefly, the procedure consisted in the

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following three steps. Step 1: preparation of the dye solution. Less than 1 mg of Abberior®


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STAR 635 (NHS ester) was dissolved in 50 µl of dry DMSO, yielding a concentrated dye

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solution. According to the literature, a solution of the fluorescent dye in methanol shows the

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absorption maximum at 639 nm with an extinction coefficient (ε) of 63000 M-1cm-1.26 Thus, the

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concentration of the prepared dye solution was determined using UV-Vis spectroscopy by

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measuring the absorbance at 639 nm (A639), using a dilution of 1:500 in methanol. Step 2:

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preparation of the enzyme solutions. 1 mg of solid free enzyme G was dissolved in 50 µl of

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100 mM NaHCO3 buffer (pH 8.0-8.5) solution. Similarly, 1 mg of cross-linked enzyme G* was

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dissolved in NaHCO3. Step 3: enzyme labelling. The exact volume of concentrated dye solution

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that is necessary for a 10-fold molar excess of dye over the free enzyme G (or 6-fold molar

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excess of dye over G*) was added slowly to the corresponding stirred enzyme solution. The

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mixture was gently stirred in the dark at rt, and after 2 h, 1 ml of 10 mM Tris buffered saline

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(TBS) with pH 7.4 was added to the system to stop the reaction. The unbound dye was removed

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and the dye-labeled enzymes were washed three times with 1X phosphate-buffered saline

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solution using 30 KDa Amicon® Ultra-4 centrifugal filters (Merck Millipore Ltd., Ireland) at

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3000 rpm for 12 min. The dye-labeled enzymes were suspended in 100 µl of 1X phosphate-

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buffered saline solution, and transferred from the Amicon Pro device into a safe-lock micro-

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centrifugal tube using a pipette. The dye/enzyme ratio (D/E ratio) was determined by measuring

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the absorbance at 635 nm (A635) and 280 nm (A280) of the dye-labeled enzyme in phosphate-

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buffered saline solution pH 6. The prepared dye labeled enzyme solutions were named as LG and

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LG* in the case of free enzyme G and G*, respectively.

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Immobilization of dye-labelled enzymes on amino-groups containing fibres

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2 µl of dye-labeled enzyme solution, LG or LG*, was mixed with few wet amino functional

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groups-containing fibers F, and the system was incubated in the dark for 2 h. A 100 µl of 3-4 %


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paraformaldehyde (PFA) was added into the system, and after 15 min the supernatant was

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removed by centrifugation at 10000 rpm during 3 min. The pellet was washed two times with

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1X phosphate-buffered saline and one time with milliQ water, with 3 min of centrifugation at

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10000 rpm between each washing step. The fibers bearing LG enzymes were named as FLG and

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the fibers bearing LG* enzymes were named as FLG*. A set of each type of fibers was mounted

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on clean glass microscope slides, embedded in Mowiol mounting medium and kept at +4 °C

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until further measurements. Similar experiments using bleached kraft pine pulp fibers with LG*

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enzymes were also performed as a BLANK sample. The resulting fibers were labeled as

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BKPP-LG*.

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Characterization of fibers

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Quantitation of the enzyme in the biocatalytic fibers FG*. In order to determine enzyme

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activity, it is essential to know the amount of enzyme present in the sample. For the present case

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the amount of enzymes on FG* fibres was determined indirectly. During the experiment

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performed for the immobilization of the enzyme G* on surface of the amino functional groups-

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containing fibers F, several liquid samples were collected and named as S2, S3, and washing

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solutions. The total amount of enzyme that was recovered in these unknown samples was

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determined by performing the Bradford protein assay described above. The quantity of

274

immobilized enzyme (QIE) in the biocatalytic fibers was expressed in mg of immobilized

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enzyme per gram of fibers. The QIE value represents the maximum amount of immobilized

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enzyme in the cellulose fibers and it was estimated using the following equation:

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QIE (mg/g) = (Eo - Elosses ).(1/m)


(Eq. 4)

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were Eo (mg) is the amount of enzyme in the solution S1, Elosses(mg) represents the total

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amount of enzyme that is not immobilized in the fibers, and m (g) is the mass of amino

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functional groups-containing fibers F used in the experiment. The Elosses value includes the total

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amount of enzyme that is collected in the unknown samples (i.e., S2, S3, and washing solutions)

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and the amount of protein that is bound to the Falcon tube surface. The amount of the enzyme

283

that is bound to the surface of the Falcon tube was estimated separately. For that purpose, 2 ml of

284

S1 were transferred to an empty Falcon tube and stirred for 2 h, at rt. The amount of enzyme

285

bound to the Falcon tubes was estimated from the difference in the amount of enzyme in the

286

liquid solutions before and after contact with the tube.

287

Enzymatic assay of the biocatalytic fibers. The enzyme activity is usually determined using

288

enzyme assays. Results are expressed in enzyme unit (U), which is defined as the amount of

289

enzyme that produces a certain enzymatic activity. In the case of glucose oxidase, 1 U

290

corresponds to the amount of enzyme that generates 1 µmol of H2O2 per minute at 37 °C (1 U

291

corresponds to 16.67 nkat).27,28 The enzyme activity in the biocatalytic fibers (in mU/g) was

292

measured with the glucose oxidase activity assay kit MAK097. The measurements were

293

performed following the instructions briefly described as following: (1) Preparation of the

294

standard solutions. A sufficient amount of glucose oxidase buffer for preparing 150 µl of H2O2

295

standard solutions was added to 0, 4, 8, 12, 16, and 20 µl of the 50 µM H2O2 solutions, which

296

were obtained with the 0.88 mM H2O2 solution provided in the kit. (2) Preparation of reaction

297

mixture. Each 50 µl of reaction mixture contained 36 µl of glucose oxidase assay buffer, 2 µl of

298

glucose oxidase developer, 2 µl of fluorescent peroxidase substrate, and 10 µl of glucose oxidase

299

substrate. (3) Preparation of reaction mixture for the blank samples. Each 50 µl of reaction

300

mixture for the blank sample contained 46 µl of glucose oxidase assay buffer, 2 µl of glucose


14

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301

oxidase developer, and 2 µl of fluorescent peroxidase substrate. Preparation of the samples: A

302

150 µl of glucose oxidase buffer was added to 1.5 ml polypropylene Eppendorf containing c.a.

303

0.2 mg of wet biocatalytic fibers and kept at room temperature for one hour before the assay.

304

Standard curve: A 50 µl or reaction mixture was added to 150 µl of H2O2 standard solutions

305

having a concentration in the range of 0 mM (blank) to 3.33 mM. These H2O2 standard solutions

306

were prepared by dilution of the 88 mM H2O2 with the glucose oxidase buffer solution provided

307

with the kit. (4) Enzymatic assay: A 50 µl of the reaction mixture described above was added to

308

the suspension containing the wet fibers. The mixture was shaken and incubated at different time

309

intervals (each 2 min, max. 10 min) at 37 °C. Upon the reaction time was accomplished, the

310

suspension was removed from the incubator, and 150 µl of PBS was added. The system was

311

quickly filtered through 0.2 µm nylon membrane (13 mm Acrodisc® syringe filter) and the

312

enzyme activity was quantified using UV-Vis analysis (λ=570 nm). The final measurement of

313

the absorbance at 570 nm (A570) was obtained using a UV-Vis 2600 spectrophotometer from

314

Shimadzu.

315

Studies on the effect of pH and temperature on the biocatalytic fibers. The performance of the

316

system FG* was studied at four different pHs (3.5; 5.5; 6.2; and 7.5), and three different

317

temperatures (32 °C, 37 °C, and 52 °C). The control of pH was achieved using 0.1 M sodium

318

formate buffer pH 3.5, 0.1 M sodium acetate buffer pH 5.5, 0.1 M potassium phosphate buffer

319

pH 6.2, or 0.1 M sodium phosphate buffer pH 7.5. About 5 mg of biocatalytic fibers FG* were

320

mixed with 5 ml of 0.16 M glucose in the selected buffer solution and incubated under stirring,

321

over a period of time. After that, the samples were easily filtered through a 0.2 µm nylon

322

membrane (13 mm Acrodisc® syringe filter). Immediately after, the amount of H2O2 produced

323

during the incubation time and recovered in the buffer solution was measured with the two


15

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Page 16 of 45

324

components ABTS-HRP system, similarly to the method described for the determination of

325

enzyme activity of G and G* in solution. Two parallel samples were incubated for each condition

326

of pH and T, and the amount of hydrogen peroxide produced in each sample was determined by

327

triplicate yielding individual values. Mean values were calculated from the individual values, for

328

each sample.

329

In addition, the effect of immobilization using glutaraldehyde was also evaluated. For that

330

purpose, FG fibers were prepared following the same procedure as in the case of FG* (see

331

Scheme 2), using the fibers F, the enzyme G, but without using glutaraldehyde. The effect of pH

332

and temperature was studied with fibers FG and FG*, in parallel, and the results are expressed as

333

the percentage of the maximum value observed from the set of experiments.

334 335

Fluorescence imaging. Images were acquired using Leica SP5 STED microscope. Sample was

336

excited using 635 nm pulsed laser and fluorescence was collected with an avalanche photo diode

337

(APD) detector at 665705 nm range. During stimulated emission depletion (STED) imaging

338

excited sample was depleted at 760 nm. Images were obtained with an oil immersion objective

339

(N.A.1.4 100x Oil, Leica) while confocal pinhole was set to 151.6 µm equal to one airy unit,

340

with a line-scan speed of 600 Hz and a line averaging of 8. Pixel-size was according the Nyquist

341

sampling requirement, about 25 nm for STED and 100 nm for confocal microscopy.

342

Field Emission Scanning Electron Microscopy (FE-SEM) measurements. The amino functional

343

groups-containing fibers F and the fibers treated with the immobilized enzymes (FG*) were

344

mechanically separated in aqueous suspension, smeared on small glass slides, and sputtered with

345

carbon in a Tamcarb TB500 sputter carbon equipped with a rotating base. FE-SEM images with


16

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346

magnification orders of 500X, 1000X, and 2500X were obtained in a FE-SEM LEO 1530

347

Gemini. The dimensions in the FE-SEM pictures were assigned with ImageJ software.29

348 349

RESULTS AND DISCUSSION

350

Characterization of the enzymes.

351

DSC was used as a method for assessing the differences of thermodynamic properties between

352

the commercial glucose oxidase (G), and the cross-linked enzyme (G*) obtained after the

353

reaction between G and GA. Due to the covalent bonds created after the chemical reaction

354

between glutaraldehyde and the commercial enzyme, the thermal stability of the immobilized

355

enzyme is usually changed and these changes might be visible using DSC analysis. The DSC

356

thermograms of G and G* are shown in Figure 1. The values of enthalpy (∆H), the maximum

357

heat capacity of the phase transition (Tpeak), and the temperature at which the tangent in the

358

inflection point crosses the baseline (Tonset) were calculated from each thermogram. The

359

increment on the ∆H value, evidences the increment on organization and stability of the

360

immobilized enzyme G* in comparison with the free enzyme G.30 Another indication for the

361

better organization and stabilization of the system is the decreasing on peak width, which is

362

usually calculated as the difference between Tpeak and Tonset. In addition, the tailing of the

363

endothermic peak showed in Figure 1 in the case of G* can be also considered as an indication

364

that cross linking took place. Similar results were observed in the third cycle (Figure S1in

365

supporting information).

366


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367

Page 18 of 45

((Figure 1 here))

368 369

The ‘hydrodynamic particle size’ or ‘size’ of the enzymes G* (or G) in 0.1 M PBS pH 6.2

370

solution was measured with ZetaSizer Nano ZS, at rt. The size of ca. 7.7 nm for the native

371

glucose oxidase enzyme, which was reported by Talbert (2014), was used as a reference value.19

372

According to the results showed in Table 1, the sample containing G* in PBS solution consist of

373

several distinct particle populations, with size of about ca. 850.9 nm (60.9 %I), 65.7 nm (9.6 %I),

374

and 13.5 nm (29.5 %I), respectively. On the other hand, the sample containing G in PBS solution

375

seems to be predominantly composed of free enzymes with a size of ca. 8.8 nm (81.9 %I). The

376

increment in the size of the particles suggests that the cross-linking took place. However, free

377

enzymes are also present in the sample containing cross-linked enzymes, indicating that the

378

cross-linking reaction was not complete or aggregates were partially broken during sonication. It

379

is necessary to state that, even though the results obtained with Zetasizer Nano ZS are in line

380

with the results observed with DSC and above, they should be not used as reference values, and

381

further studies will be needed to determine more precisely the size of G* (or G) in PBS solution.

382

((Table 1 here))

383 384

The IR spectra of the free glucose oxidase G and that of the cross-linked glucose oxidase G*

385

exhibit the expected characteristic absorption bands amide A, amide B, and amides I-VII

386

associated with the vibrational modes of the secondary amide groups of proteins (Figure 2).31

387

The IR absorption bands labelled as amide A (ca. 3300 cm-1) and amide B (ca. 3100 cm-1)


18

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388

originate from the secondary amide N-H stretching vibrations and the secondary amide II

389

overtone. The amide I band (ca. 1642 cm-1) is usually associated with C=O stretching coupled to

390

C-N stretching and N-H bending vibrations. The band amide II (at about 1535 cm-1) is usually

391

associated with N-H bending vibration coupled with C-N stretching vibrations. The bands of

392

amide I and II were the strongest absorption bands of the infrared spectra, being frequently the

393

most important bands for the analysis of the secondary structure of proteins. Particularly in the

394

case of amide I band, the exact wavenumber of the associated vibrations depend on the nature of

395

hydrogen bonding involving amide groups, which are determined by the particular secondary

396

structure adopted by the protein. Therefore, the similarities between the amide I band of the free

397

enzyme G and the amide I band of the immobilized enzyme G* evidence the similarities on the

398

secondary structure of these enzymes. In fact, it is claimed that these can be taken as an

399

indication that the enzyme activity is preserved after the immobilization.32 The absorption band

400

at 1300 cm-1 is associated to the amide III band, which is due to C-N str, N-H bending, C=O

401

stretching, O=C-N bending, and also non identified vibrations. The week IR band in the range

402

from 900 cm-1 to 800 cm-1 is assigned to symmetrical C-N-C stretching vibrations. Despite of the

403

similarities between the G and G* spectra, the comparison of the spectra reveals an increment in

404

the relative intensity of the bands at 2824 cm-1 and 1465 cm-1, which are associated with CH2

405

deformation vibrations. At the same time, the bands in the range of wavenumber from

406

1342-1359 cm-1, which can be assigned to CH deformation vibrations, showed stronger

407

absorbance in the case of G*. This absorption increments support the hypothesis that the

408

chemical reaction between G and GA render a compound with larger number of methylene

409

groups as suggested by Migneault et al.11 Unlike in the case of the spectrum of G*, the bands at

410

1144 cm-1, 1105 cm-1, 1062 cm-1, 963 cm-1, and 842 cm-1 are not observed in the spectrum of G.


19

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Page 20 of 45

411

These bands were assigned to deformation vibrations from acetals and NH deformation

412

vibrations.11

413 414

((Figure 2 here))

415 416

At least ten different spots from G and from G* were analyzed using X-ray spectroscopy

417

(XPS). According to the results obtained from the analysis of low-resolution spectra, the atomic

418

composition of G was very uniform among the analyzed spots. Carbon and oxygen were the

419

major atoms in that sample (Table 2). On the contrary, the atomic composition of G* was not

420

uniform among the different analyzed spots, and the values can be pooled in two main groups

421

referred as G*A and G*B. In comparison with G, the group G*B showed a similar carbon atomic

422

percentage, larger atomic percentage of oxygen, and lower atomic percentage of nitrogen. The

423

differences between G*B and G become even more evident when the elemental ratios O/C, N/C,

424

and O/N are compared. Thus, the group G*B shows lower N/C, and larger O/N ratios. These

425

observations are in agreement with the hypothesis that G* resulted enriched with carbon and

426

oxygen in comparison with G, due to the chemical reaction between G and GA.11 Comparing the

427

high-resolution spectra of G and G*B, the differences in chemical composition are also evident

428

(Figure 3). Although an appropriate curve fitting of the C1s spectra was not possible due to the

429

complexity of the samples (i.e., multiplicity of the carbon chemical states), the C1s spectrum of

430

G*B shows the presence of a peak at about 287 eV, which is not present in the case of C1s

431

spectrum of G. On the other hand, the XPS results from G and the group G*A did not show

432

significant differences.


20

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433

Although lack of uniformity in the XPS results in the case of G* opens a debate whether XPS

434

analysis is suitable technique for the study of this sample, there is a clear difference between the

435

chemical composition of G and G*. Moreover, the XPS results are agreement with the results

436

obtained from DSC and DLS, where free and cross-linked enzymes were detected in the sample

437

G*.

438 439

((Figure 3 here))

440

((Table 2 here))

441 442

The activity of G and G* in 0.1 M PBS pH 6.2 solution was measured at 37 °C using the two

443

component ABTS-HRP system, and measuring the H2O2 that is produced over exact period of

444

time with the help of the calibration curve (standard curve range from 0-10 nmol) showed in

445

Figure S2 (in supporting information). As expected, the immobilization leads to the loss of

446

catalyst productivity (kg of product/kg of enzyme), and the cross-linked enzymes G* in solution

447

showed an enzyme activity of about 34 % of the enzyme activity showed by the original

448

enzymes G in solution.

449

Enzyme quantitation. The concentration of the enzyme in solution was plotted against the

450

corresponding absorbance, yielding the Bradford enzyme concentration standard curve (Figure

451

S3 in supporting information). The enzyme standard curve was used for the determination of the

452

enzyme concentration in unknown samples.


21

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Page 22 of 45

453 454

Characterization of fibers.

455

Quantitation of the enzyme in the treated fibers. The QIE value, which represents the

456

maximum amount of enzyme G* immobilized in F, was calculated from the Equation 4.

457

According to the results, QIE value was equal to 11 mg/g (or 2.6 mg of G* per gram of wet F,

458

dry content of 24 %). No enzyme was detected in the supernatants S2 or in the washing solutions

459

represented in Scheme 2. The amount of enzyme G* that is bound to the Falcon tube is very

460

small, about 1.2 % of the original amount of enzyme utilized in the experiments.

461

Enzymatic assay of biocatalytic fibers. The enzyme activity was determined by a coupled

462

enzyme array from enzyme assay kit MAK097, in which the target enzyme oxidizes D-glucose,

463

generating H2O2. Because of the chemical reaction between H2O2 and a probe, the formation of a

464

colorimetric compound (λmax = 570 nm) took place. The amount of H2O2 in glucose oxidase

465

assay in buffer solution was plotted against the corresponding absorbance at 570 nm (A570),

466

yielding the H2O2 standard curve (Figure S4 in supporting information). The amount of H2O2

467

(nmol) that was generated per gram of FG* was calculated from the H2O2 standard curve, and

468

used for plotting the amount of H2O2 versus time (Figure 4). The glucose oxidase activity of the

469

enzymes immobilized in the fibers F (nmol of H2O2/min per gram of FG*) was determined using

470

Figure 4. According to the results and based on the definition of enzyme unit, the immobilized

471

enzymes G* in the prepared fibers FG* showed an activity of 215 U/g of fiber (or 906 U/g of

472

wet fiber, dry content of 24 %). In other words, it was possible to convert 215 µmol of glucose

473

per minute, per gram of dry biocatalytic fiber.

474


22

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475

Biomacromolecules

((Figure 4 here))

476 477

Effect of pH and temperature on the biocatalytic fibers. According to the results, the

478

performance of the system FG* (expressed in terms of hydrogen peroxide/kg of fiber) was higher

479

than in the case of FG, under all the tested conditions (Figure 5). Evidently, the enzyme cross-

480

linking and subsequent immobilization of the cross-linked enzymes on the cellulose fibers using

481

glutaraldehyde is beneficial. As it will be shown later in the fluorescence images, the retention of

482

the enzyme on the solid carrier surface is very poor without the covalent bonds between fibers

483

and enzymes. Therefore, the ‘dilution of activity’ that is mentioned for example in Sheldon

484

(2007)4 is observed and because of that the system FG is less efficient than FG*.

485

Comparing the performance of the systems (FG* or FG) at the different tested conditions, the

486

highest yield was observed at 32 °C and pH of 5.5, whereas no enzyme activity was observed at

487

52 °C. Surprisingly, the enzymes on FG* are very active under pH of 3.5, at 37°C. It is well

488

known that the stability of the enzymes is affected at high temperatures, and therefore, it is not a

489

surprise the drop of enzyme activity observed at 52 °C. However, further studies are needed in

490

order to explain the observations at lower temperatures and to optimize the performance of the

491

system.

492 493

((Figure 5 here))

494


23

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Page 24 of 45

495

Imaging of enzymes on fibre surfaces. The cross-linked enzymes G* were labelled with a

496

fluorescent dye and immobilized in the amino functional groups-containing fibers F. The

497

resulting material FLG* was studied with confocal microscopy and STED microscopy. The

498

picture showing the orthogonal optical sections of a three dimensional confocal image created

499

from a z-stack of xy-scans of a fiber FLG* revealed a dense and uniform distribution of the

500

fluorophore groups on the surface of the fibers but not to the interlayers of the fibers (Figure 6a).

501

The used STED microscope has about 100 nm lateral resolution, which represents a 4-5 fold

502

increase of lateral resolution as compared to the used confocal microscopy (the comparison is

503

valid in ideal case of transparent samples). Thus, the use of STED microscopy allowed an

504

enhanced visualization of the fluorophore groups on the surface of the treated fibers. Small fiber

505

components with nanoscale dimensions, such as the fine cellulosic strands extending to the

506

periphery of the pit chamber, can be more clearly distinguished on STED images in comparison

507

with confocal images (Figures 5c and 5b, respectively). The fibers FLG, which contains dye-

508

labeled G, were also studied using confocal- and STED microscopy (Figure 6). The STED

509

figures and the set of pictures from confocal microscope that are compiled in the video (see

510

video in supporting information) permits to distinguish the LG enzymes located only at the

511

surface of the fibers FLG. In other words, as in the case of LG*, the LG enzymes cannot

512

penetrate into the inter layers of the cellulosic fibers, even though the enzymes LG possess

513

smaller size than the enzymes LG*. The images obtained with the confocal or STED microscope

514

provide evidence on the large area available for immobilization of enzymes, and show the

515

complex morphology of the solid carrier. Unlike FLG and FLG*, the fibers named as BKPP-

516

LG* did not show significant fluorescence under confocal microscope or STED microscope


24

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Biomacromolecules

517

(Figure S5 in supporting information). The remarkable difference demonstrates the benefits of

518

amino-functionalization of pulp fiber surface for the immobilization of enzymes.

519 520

((Figure 6 here))

521

((Figure 7 here))

522 523

FE-SEM images from FG* clearly show the presence of the enzymes at the surface of the

524

fibers (Figure 8). Evidently, the immobilized enzyme is not evenly distributed on the surface of

525

biocatalytic fibers. Crystallization occurs at the surface in many areas (Figure 8-d), whereas

526

smaller foreign structures are observed around the pits (Figure 8-c). The heterogeneous

527

distribution on particle size of the enzymes G* is evidenced by FE-SEM measurements in the

528

case of the sample containing biocatalytic fibers FG*, and from the measurements using

529

Zetasizer Nano ZS in the case of the sample with G* in solution. However, the values for particle

530

size obtained with these techniques are not comparable. In the case of FE-SEM results, the

531

values are estimated from pictures obtained from solid samples under high-vacuum and after

532

coating with carbon, whereas the hydrodynamic particle size obtained with ZetaSizer Nano ZS is

533

obtained from enzymes G* in solution.

534 535

((Figure 8 here))

536


25

Biomacromolecules

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Page 26 of 45

537

CONCLUSIONS

538

A new method for efficient immobilization of glucose oxidase from Aspergillus niger onto

539

pulp fibre surfaces leading to biocatalytic fibres was developed. The method is based on adding

540

amino functional groups to pulp fibre surfaces prior to covalent anchoring enzymes to fibres with

541

glutaraldehyde. According to the results from SEM, confocal- and STED microscopy, the

542

advanced cellulose fibres containing amino groups are suitable for anchoring of enzymes both in

543

original or cross-linked forms. In addition, the high resolution and sensitivity of the fluorescence

544

techniques have shown that neither original nor cross-linked enzymes penetrate the interlayers of

545

the functional fibers. In addition, fluorescence microscopy evidenced that the unmodified pulp

546

fibers can load very low amount of enzymes in comparison with advanced cellulose fibers. Using

547

the approach discussed in this paper, different enzymes could be immobilized to the amino

548

functional groups-containing fibers. Because of the specificity relative to the reaction that each

549

enzyme can catalyze, the properties of the biocatalytic fibres can be tuned by selecting the

550

correct enzyme. Thus, the results discussed could lay the groundwork for the utilization of pulp

551

fibers as a carrier for the immobilization of enzymes with potential applications in very different

552

areas.

553

ASSOCIATED CONTENT

554

Supporting Information. Figure S1: Differential scanning calorimetry (DSC) of the free

555

enzyme (G) and the immobilized enzyme (G*) showing the corresponding endothermic peaks

556

after the second heating named as cycle 3. Figure S2. H2O2 standard curve showing the

557

absorbance at 415 nm (A’415) of the stable product from ABTS oxidation versus the amount of

558

H2O2 in solution. Figure S3: Bradford protein concentration standard curve showing the


26

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Biomacromolecules

559

absorbance at 595 nm (A595) of enzyme-dye complex system versus the concentration of

560

immobilized enzyme in phosphate buffered solution pH 6.2. Figure S4. H2O2 standard curve

561

showing the absorbance at 570 nm (A570) of protein-dye complex system versus the amount of

562

H2O2 in solution. Figure S5: Confocal microscope picture of a bleached kraft pine pulp fiber

563

section decorated with immobilized enzymes labeled with Abberior® STAR 635 (NHS ester)

564

(a), and the corresponding transmission image (b). Video: set of pictures obtained with confocal

565

microscope showing fluorescence from dye-labeled free enzymes immobilized on amino

566

functional groups containing-fibers.

567 568

AUTHOR INFORMATION

569

Corresponding Author

570

†,*Pedro Fardim, Tel.: +32 16 32 09 70, E-mail: [email protected]

571

§

Thomas Heinze, Tel.: +49 173 58 95 373. E-mail: [email protected]

572 573

Present Addresses

574

* Fibre and Cellulose Technology Laboratory, Faculty of Science and Engineering, Åbo

575

Akademi University, Porthansgatan 3, FI 20500 Åbo, Finland

576

†

Department of Chemical Engineering (CIT), Celestijnenlaan 200 F, 3001 Leuven, Belgium.

577 578

Author Contributions


27

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Page 28 of 45

579

The manuscript was written through contributions of all authors. All authors have given approval

580

to the final version of the manuscript.

581 582

Funding Sources

583

The authors would like to thanks the Finnish Funding Agency for Technology and Innovation

584

(Tekes) and Åbo Akademi Research Grant for the financial support.

585 586

ACKNOWLEDGMENTS

587

The authors would like to thanks Linus Silvander for carrying out the SEM images, Mohammad

588

Khajeheian for his support with the DSC measurements, Peter Holmlund for his support with

589

DSC and XPS measurements, Björn Törngren for carrying out the measurements with the

590

Zetasizer Nano, Jyrki Juhanoja for his support with regard to the analysis of XPS results, and

591

Carl Lange for providing the bleached kraft pine pulp. The authors would also like to thanks

592

Samuel Fernandez for his valuable contribution during the previous work, which provided the

593

basis for the present paper.

594 595

ABBREVIATIONS

596

∆H, enthalpy; BKPP, bleached kraft pine pulp fibers; BKPP-LG, bleached kraft pine pulp fibers

597

bearing dye-labeled glucose oxidase; DSC, dynamic scanning calorimetry; DLS, dynamic light

598

scattering; FG*, biocatalytic fibers bearing immobilized glucose oxidase; FLG*, biocatalytic

599

fibers bearing dye-labeled immobilized glucose oxidase; ATR-FTIR, Fourier transform infrared


28

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Biomacromolecules

600

spectroscopy with attenuated total reflection accessory; Glutaraldehyde, GA; G, free glucose

601

oxidase; G*, immobilized glucose oxidase enzyme;; LG, dye-labeled enzyme; LG*, dye-labeled

602

immobilized enzyme; PBS, phosphate buffered solution; PDI, poly dispersive index; QIE,

603

quantity of immobilized enzyme; RT, room temperature; FE-SEM, Field Emission Scanning

604

Electron Microscopy; STED, Stimulated emission depletion; U, enzyme unit; UV-Vis,

605

ultraviolet-visible; XPS, X-ray photoelectron spectroscopy.

606 607 608

REFERENCES (1)

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DiCosimo, R.; McAuliffe, J.; Poulose, A. J.; Bohlmann, G. Chem. Soc. Rev. 2013, 42, 6437.

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Sheldon, R. A. Advanced Synthesis and Catalysis. 2007, pp 1289–1307.

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Cao, L.; van Langen, L.; Sheldon, R. A. Current Opinion in Biotechnology. 2003, pp 387–

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Vega, B.; Wondraczek, H.; Bretschneider, L.; Näreoja, T.; Fardim, P.; Heinze, T. Carboh. Polym. 2015, 132, 261–273.

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Talbert, J. N.; He, F.; Seto, K.; Nugen, S. R.; Goddard, J. M. Enzyme Microb. Technol.


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Grigoray, O.; Wondraczek, H.; Fardim, P.; Heinze, T. Macromol. Mater. Eng. 2015, 300, 277–282.

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Solomon B., Lotan N., K.-K. E. Biopolymers 1977, 16, 1837–1851.

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Leskovac, V.; Trivić, S.; Wohlfahrt, G.; Kandrač, J.; Peričin, D. Int. J. Biochem. Cell Biol.

647

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Bradford, M. Anal. Biochem. 1976, 72, 248–254.

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Young, D. S. Ann. Clin. Lab. Sci. 1977, 7, 93–98.

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Rzepka, K.; Norman, M.; Klapiszewski, Ł.; Bartczak, P.; Kaczorek, E.; Jesionowski, T.

659

Open Chem. 2015, 13, 138–148.

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Biomacromolecules

661

Figure 1. Differential scanning calorimetry (DSC) of the free enzyme (G) and the cross-linked

662

enzyme (G*), showing the corresponding endothermic peaks after the first heating named as

663

cycle 1.

664 665


33

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 45

666

Figure 2. ATR-FTIR of the glucose oxidase enzyme (G) and the cross-linked enzyme (G*),

667

which is obtained after reaction of G with glutaraldehyde followed by dialysis.

668 669


34

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

670

Figure 3. XPS high-resolution spectra of the commercial glucose oxidase G, and the cross-

671

linked enzyme G* (results from G* are grouped as G*A and G*B).

672


35

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

673

Page 36 of 45

Figure 4. Enzyme activity showed by biocatalytic fibers FG*.

674


36

Page 37 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

675

Figure 5. Relative performance of the biocatalytic fibers (in %), which is defined as the amount

676

of hydrogen peroxide that is produced per kg of biocatalytic fibers, over the same period of time.

677

For the comparison, the maximum value observed was considered equal to 100 %.

678 679


37

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 45

680

Figure 6. Orthogonal sectioning (xy, xz, and yz) from the confocal microscope pictures of a

681

biocatalytic fiber FG* (a), confocal microscope picture with focus on the cellulosic strands of the

682

same pit chamber exhibited in picture a (b), and the STED microscope picture of the same

683

section than in picture b (c). The fluorescence observed in these pictures derived exclusively

684

from the fluorophore Abberior® STAR 635 (NHS ester), which is covalently bonded to the

685

enzymes (Dye/Enzyme ratio of 1.6). Picture dimensions: size-width and sizeheight of 75 µm;

686

stepdepth of 15.6 µm and stepsize of 0.25 µm (a); 10.73 µm × 6.62 µm (b,c).

687 688


38

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

689

Figure 7. STED image of a biocatalytic fiber cross-section, bearing free enzymes labeled with

690

Abberior® STAR 635 (a), and the corresponding transmission image (b). Dye/Enzyme ratio of

691

1.4. Picture dimensions: 32.53 µm × 32.53 µm (a,b).

692


39

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 45

693

Figure 8. Field emission scanning electron microscope (FESEM) pictures of the reference fibers

694

(a,b), and the fibers obtained after chemical reaction between the functional fibers (F), the

695

immobilized enzymes (G*), and GA (c,d), all pictures at 25Kx magnification.

696


40

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

697 698

Biomacromolecules

Scheme 1. Chemical structure of the cellulose derivative (3-carboxypropyl)trimethylammonium chloride ester of 6 deoxyazidocellulose.

699


41

Biomacromolecules

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Page 42 of 45

700

Scheme 2. Preparation of the biocatalytic fibers (FG*) from amino-groups.containing fibers (F).

701

S1, enzyme solution; PBS, phosphate buffered solution; Glutaraldehyde, GA; S2 and S3,

702

supernatants; rt, room temperature.

703 704 705


42

Page 43 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

706

Biomacromolecules

Table 1. Results obtained with Zetasizer Nano ZS at room temperature. Sample

707 708

Size (S) and percentage of intensity (%I)

G

Peak 1 Size %I (nm) 8.8 81.9

Peak 2 Size %I (nm) 300 15.8

Peak 3 Size %I (nm) 5093 2.2

G*

850.9

13.5

65.7

60.9

29.5

9.6

PDI Average size (nm) 22.5

0.26

51.1

1.00

G, commercial enzyme; G*, immobilized enzyme; PBS, phosphate buffered saline; PDI, poly dispersity index.


43

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

709

Table 2. X-ray spectroscopy (XPS) results Sample

Atomic percentage C (±σ)

710 711

Page 44 of 45

N (±σ)

Atomic percentage ratios O (±σ)

O/C (±σ)

N/C (±σ)

O/N (±σ)

G*A

64.9

0.7

11

3

24

4

0.40

0.1

0.17

0.05

3

2

G*B

65.9

0.5

1.2

0.4

33

1

0.50

0.01

0.02

0.01

30

9

G

66.4

0.6

7

1

27

1

0.40

0.02

0.10

0.02

4

1

G, commercial enzyme; G*A and G*B, immobilized enzymes grouped in groups A and B according to atomic percentage ratios.

712


44

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

713

Biomacromolecules

Table of Contents Graphic

714


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Advanced Cellulose Fibers for Efficient Immobilization of Enzymes

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