Laccase Complex with Polyvinylamine Bearing Grafted TEMPO is a

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Laccase Complex with Polyvinylamine Bearing Grafted TEMPO is a Cellulose Adhesion Primer Jieyi Liu, Robert Pelton, Jaclyn M. Obermeyer, and Anton Esser Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm4009827 • Publication Date (Web): 10 Jul 2013 Downloaded from http://pubs.acs.org on July 15, 2013

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Laccase Complex with Polyvinylamine Bearing Grafted TEMPO is a Cellulose Adhesion Primer Jieyi Liu1, Robert Pelton1*, Jaclyn M. Obermeyer1, and Anton Esser2 1

Department of Chemical Engineering, McMaster University, Main St. West, Hamilton, Ontario,

Canada, L8S 4L7 2

BASF AG, ZKM/D 15425, 67056 Ludwigshafen, Germany

KEYWORDS cellulose oxidation, wet strength, TEMPO, hydrogel, adhesion, laccase ABSTRACT Polyelectrolyte complexes formed between laccase and polyvinylamine with grafted TEMPO moieties, PVAm-T, adsorb onto cellulose causing oxidation. All evidence supports the view that aldehyde groups on oxidized cellulose condense with primary amine groups giving a grafted layer of PVAm-T complexed with laccase. The grafted PVAm-T serves as a primer layer promoting wet cellulose-to-cellulose adhesion in the presence of PVAm adhesive. The cellulose modification occurs at ambient temperatures and pH 5. The adhesion improvements with mixtures of PVAm-T and laccase are remarkable because both components are macromolecular, which should inhibit the ability of the TEMPO to act as a shuttle between the enzyme and the primary hydroxyl groups on cellulose. It is proposed that PVAm-bound oxoammonium ions exchange with neighboring TEMPO moieties, providing a mechanism for the transfer of oxidation activity from immobilized enzyme to the cellulose surfaces.

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INTRODUCTION One of the limitations of paper packaging and other lignocellulosic materials is sensitivity to water, resulting in poor mechanical properties when wet. We have shown that the adhesion between wet regenerated surfaces can be greatly enhanced first by mild TEMPO mediated oxidation with bleach (NaClO), followed by the application of polyvinylamine (PVAm). 1 Based on substantial but indirect evidence, we 2 and others3, 4 have argued that increased wet adhesion results from aminal and imine bonds formed by the reaction of PVAm with aldehyde groups on oxidized cellulose.

Covalent bond formation in adhesive joints is a common attribute of

polymers that enhance wet cellulose adhesion. 5 The newly formed bonds can serve as crosslinks in the strength-enhancing polymer and as grafts between the polymer and cellulose substrate, giving water resistant joints.

1, 5

Although, there are many unanswered scientific details

concerning the role of covalent bond formation in the interactions of PVAm with oxidized cellulose, the focus of work described herein was to solve two significant technological problems.

Specifically, TEMPO/bleach/PVAm treatment has little commercial potential in

papermaking because of the requirements for high pH (~10) and because of the cost and environmental impact of high TEMPO concentrations. This paper presents a new approach to circumvent these problems by grafting TEMPO onto PVAm together with the use of laccase/oxygen as the primary oxidant. Recently we described the preparation and characterization of polyvinylamine (PVAm) derivatized with 4-carboxyTEMPO giving PVAm-T, a cationic, water-soluble polymer with pendant TEMPO moieties.6 PVAm-T adsorbed onto cellulose surfaces in water and, in the presence of bleach (NaClO) at high pH, catalyzed the oxidation of cellulose, yielding aldehyde groups that subsequently formed covalent bonds with primary amine groups on the PVAm.


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Thus, PVAm-T performed a dual role as a cellulose oxidation mediator and as a reactive adhesive promoting cellulose-cellulose wet adhesion. Compared to our original approach based on free TEMPO, the PVAm-grafted TEMPO offers the advantage that much less TEMPO is required, and that TEMPO is fixed to the solid cellulose, avoiding water contamination problems. Herein we describe replacing bleach at high pH with laccase/oxygen at pH 5 together with PVAm-T.

TEMPO is a mediator for the oxidation that must be activated to form the

corresponding oxoammonium ion by a primary oxidant such as bleach – see Scheme 1. It is the oxoammonium ion that oxidizes the primary alcohols in cellulose and hemicellulose.

7, 8

The

9

The

enzyme laccase in the presence of oxygen is an alternative primary oxidant to bleach.

advantage of O2-laccase as a primary oxidant is that the TEMPO reaction occurs under mild conditions at neutral pH, conditions more appropriate for conventional papermaking. There have been a number of studies on the O2-laccase-TEMPO system including mechanistic aspects,

10-13

and on the influence of oxidation wet cellulose-to-cellulose adhesion in paper products. 14-16 In this paper, we describe the specific conditions under which O2-laccase can activate PVAmT for cellulose oxidation and give enhanced adhesion. There are significant challenges because of the polymeric nature of both the enzyme and the immobilized TEMPO. Laccase is a 62 kDa polymer bearing a net negative charge under reaction conditions (pKa 4.3-4.5

17

) whereas our

PVAm-T is a highly cationic, 45 kDa linear polymer. We anticipated that laccase and PVAm-T would form strong, irreversible polyelectrolyte complexes and they did. In addition, we were concerned that the limited mobility of the polymer-bound TEMPO would prevent TEMPO from entering the laccase catalytic domain. Finally we were concerned that any oxoammonium ions formed would have insufficient mobility to come in contact with the cellulose surfaces. The


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results herein show that there are conditions under which laccase can activate PVAm-T for cellulose oxidation and enhanced adhesion at neutral pH. EXPERIMENTAL SECTION Materials. Polyvinylamine (PVAm) with a number-average molecular weight of 45 kDa, and a degree of hydrolysis of 75% was obtained from BASF, Ludwigshafen (Lupamin® 5095). The polymers were further purified by dialysis against water, and freeze-dried for storage. Regenerated cellulose dialysis tubing (Spectra/Por® 2, 12-1400 Da MWCO) was purchased from Spectrum Laboratories. Tubing was cut into rectangles of two sizes: top membranes (2 cm×6 cm) and bottom membranes (3 cm × 6 cm), and then were boiled in deionized water to remove plasticizer. Laccase from Trametes versicolor (EC 1.10.3.2), TEMPO, 4-carboxy-TEMPO, NHydroxysulfosuccinimide

sodium

salt

(sulfo-NHS),

1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide (EDC), 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were purchased from Sigma-Aldrich. Other salts for buffer preparation were purchased from Caledon Laboratories Ltd. Water was purified with a Nanopure purification system to a specific resistance of at least 18MΩ cm−1. PVAm-T Preparation and Characterization.

Polyvinylamine with grafted TEMPO

moieties (PVAm-T) was synthesized by EDC/sulfo-NHS-mediated conjugation of 4-carboxyTEMPO to PVAm as described previously.6 Table 1 summarizes the grafting conditions and the properties of the polymers used in the experiments herein. The grafted polymers were dialysed for two weeks, freeze-dried and stored in a desiccator. Conductometric titration and Electron Paramagnetic Resonance (EPR) spectroscopy were performed to determine the TEMPO contents of PVAm-T.


The conductometric titrations,

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performed with a PC Titrate (Man-Tech Associates), determined the primary amine contents in lyophilized PVAm-T samples.

Comparison with the starting material yielded the amine

contents. EPR spectroscopy was performed on a Bruker ELEXSYS E580 spectrometer at 140K in an EPR tube. Typically, 10 g/L PVAm-T solutions were prepared for EPR spectroscopy. 120 µL solutions in EPR tubes were frozen by liquid nitrogen then thawed in a vacuum for degasification. TEMPO concentrations in PVAm-T solutions were determined by double integration of the EPR signal using 8.7 mM TEMPO solution as a standard. Laccase Activity Assay Typically, 0.04 mL of enzyme solution were mixed with 3 mL 0.5 mM ABTS in 0.05 M sodium acetate pH 5 buffer at 25oC. Laccase activity was determined by monitoring the oxidation rate of ABTS at 420 nm (ε420=36 000 M-1 cm-1). 18 One unit of laccase activity is defined as the amount of enzyme required to oxidize 1 µmol of ABTS/min at 25oC. Oxidation of Cellulose Membranes.

The oxidation agents were PVAm-T, or TEMPO,

laccase and oxygen. Three methods were used to expose these agents to cellulose – sequential treatment, pre-made PVAm-T/laccase complexes, and layer-by-layer (LbL) adsorption. In a typical sequential experiment, 20 mg PVAm-T was dissolved in 130 mL of sodium acetate buffer (50mM, pH 5) and four pairs of cellulose membranes were immersed in the solution for 30 min. Then, 20 mL of 1 mg/mL laccase solution (190 Units), filtered by syringe filter (0.45 µm), was added to initiate the oxidation at room temperature. The final oxidation mixture was 150 mL solution consisting of 133 mg/L PVAm-T and 133 mg/L laccase. The oxidation solution was stirred under oxygen purging (1 bubble/second) for 24 h. After the oxidation, membranes were immersed in sodium acetate buffer (50 mM, pH 5) for 5 min and rinsed with buffer three times to remove excess unabsorbed polymer.


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Various control experiments were performed in which components of the “typical experiment” were either not included or replaced – see Table 2.

In experiment 8 in Table 2, the mediator

PVAm-T was physically separated from the laccase to show whether or not physical contact between the mediator and the enzyme was required. For this, 20 mL of enzyme solution in buffer was place inside a 12-14 kDa cut-off dialysis tubing that was suspended in the PVAm-T solution with the cellulose membrane strips. Cellulose treatments with pre-made PVAm-T/laccase complexes were conducted in the following manner. In a typical experiment, 10 mL of 1g/L laccase solution was dropped into 140 mL of PVAm-T14 solution consisting of 10 mg PVAm-TEMPO to form PVAm-T/laccase complex. The final oxidation mixture was 150 mL solution consisting of 67 mg/L PVAm-T14 and 67 mg/L laccase. Four pairs of cellulose membranes were oxidised by immersing in this oxidation mixture for 24 hours with oxygen purging. For the results in Figure 8, four pairs of cellulose membranes were immersed in the 150 mL pre-made PVAm-T14/laccase complex solution consisting of 67 mg/L PVAm-T14 and 67 mg/L laccase. After 1 hour the first set of membranes was removed and rinsed with buffer, and a second set of membranes was immersed in the same oxidation mixture. The procedure was repeated over 9 sets of membranes Finally in the LbL cellulose treatment, cellulose membranes were dipped in 1 g/L PVAm-T6 solution for 30 min and then rinsed with pH 5, 50 mM sodium acetate solution for 15 min. After removing surface water, the membranes were dipped in 1 g/L laccase solution for 30 min, followed by rinsing with pH 5, 50 mM sodium acetate solution for 15 min. The same procedures were repeated for multilayer fabrication. After LbL treatment, membranes were soaked in 150


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mL pH 5, 50 mM sodium acetate buffer for 24 hours with oxygen purging, followed by directly applying extra PVAm solution to give a layer coverage of 15 mg/m2. Laminate Preparation and Delamination Wet cellulose adhesion test laminates consisting of two cellulose membranes were prepared using direct application methods. In this method, the bottom cellulose membrane was placed on a polished TAPPI standard stainless-steel drying plate. The excess surface water was removed by gently dabbing with Kimwipes. To give two cellulose membrane tails for attachment to Instron clamps and a uniform crack in the laminate, a piece of Teflon tape (40×12.7 mm, G.F. Thompson, TWB480P) was placed across the top edge of the bottom membrane. Then a 15 µL drop of PVAm solution (1g/L) was applied using a 20 µL micropipette (eppendorf) onto the bottom membrane. The top membrane was progressively placed over the bottom membrane carefully so that the polymer solution between membranes could uniformly spread with negligible loss of polymer solution. Then the laminate was pressed (89 kN) between two TAPPI standard blotters for 30 min in a Carver press and dried at 23oC and 50% humidity for 24 h. The delamination tests and data analysis were conducted as described previously. 6 Confocal Laser Scanning Microscopy (CLSM) The distribution of aldehyde groups through cellulose membranes was measured by soaking the membranes in 0.5 g/L of fluorescein-5thiosemicarbazide overnight at pH 8. The excess fluorescein probe was removed by soaking and rinsing the membranes in pH 8 Na phosphate buffer 10 times. The membranes were cut with Cryostat (CM3050S, Leica) and the cross-section of cellulose membranes was observed under a laser beam at the wavelength of 488 nm. The images of PVAm-T treated, soluble TEMPO treated and un-oxidized cellulose membranes were taken under the same parameter settings of CLSM.


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Quartz Crystal Microbalance (QCM-D) Estimates of the mass coverage PVAm-T6 and laccase resulting from LbL assembly on cellulose were based on QCM-D measurements using a Q-Sense quartz crystal microbalance (E4 model from Q-Sense, Sweden) fitted QSX 334 cellulose sensors. 50 mM pH 5 sodium acetate buffer was employed as the solvent for the polymer and laccase solutions as well as the rinse solutions. The flow rates were 0.150 mL/min and the concentrations of polymer and enzyme solutions were 0.1 g/L. QCM-D data are given in the supporting information.

RESULTS A series of seven PVAm-T polymers was prepared by EDC/S-NHS catalyzed coupling of 4carboxy-TEMPO to 45 kDa PVAm.

Table 1 summarizes the grafting conditions and the

TEMPO contents of the polymers. The grafting extents ranged from 0.007 to 0.164 TEMPO moieties per mole amine group, corresponding to TEMPO mass fractions of 1.2% to 15.8%. The PVAm-T polymers were used as oxidation mediators for wet regenerated cellulose membranes. The following paragraphs present evidence for the formation of aldehydes on the cellulose film surfaces and the subsequent formation of covalent bonds between cellulose and PVAm-T. Fluorescein-5-thiosemicarbazide, an aldehyde reactive fluorescent dye, was used to visualize the aldehyde distribution through the cellulose film whereas adhesion measurements were used as an indication of a covalent network linking wet laminated cellulose films.

19

The

adhesion measurements consisted of 90-degree peel delamination measurements for wet regenerated cellulose strips laminated with a very thin layer of PVAm adhesive.


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Grafting conditions and TEMPO contents of PVAm(45 kDa)-TX oxidation

Table 1

mediators. The TEMPO contents are expressed as the mole fraction of amine groups bearing a TEMPO.

PVAm -TX

PVAm (mg)

4-carboxyTEMPO (mg)

EDC (mM)

Sulfo-NHS (mM)

TEMPO Content (mole %)

Grafting Yield (mole %)

T4

200

120.8

32.21

5.00

9.6

29

T6

200

241.8

32.21

5.00

16.4

25

T9

100

17.8

16.10

1.87

4.2

43

T10

100

8.9

8.05

0.94

0.7

15

T11

100

3.6

64.42

0.27

0.9

45

T14

200

149.0

32.21

5.00

10.8

26

T16

200

150.0

32.21

5.00

6.1

15

Table 2 compares adhesion results corresponding to various cellulose oxidation conditions using the sequential procedure for introducing PVAm-T and laccase. Without mediator (Exp. 2) or without laccase (Exp. 3) the adhesion was very low. Using TEMPO instead of PVAm-T, with (Exp. 5) and without (Exp. 4) PVAm, gave less than one half of the base case adhesion (Exp. 1). It will be shown that laccase forms a colloidal polyelectrolyte complex with PVAm-T, so it could be argued that laccase is simply acting as a strength enhancing polymers. A laccase sample was denatured by boiling before use and the results (Exp. 7 in Table 2) confirm that denatured laccase does not give strong adhesion. Exp. 8 in Table 2 shows the results for an experiment in which the laccase was isolated from the PVAm-T by dialysis membrane during the oxidation experiment, preventing direct contact


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between the mediator and the enzyme. The resulting adhesion was very low, suggesting that TEMPO and enzyme must be capable of direct contact for oxidation.

Table 2

The influence of cellulose oxidation conditions on the wet delamination forces.

PVAm-T6 and laccase were added by the sequential method. Oxidations were performed in 50 mM acetate buffer at pH 5 for 24 hours.

PVAm (45 kDa) was used as an adhesive. Delamination force (N/m)

133

PVAm adhesive (mg/m2) 15

0

133

15

1.2 ±0.2

Blank

0

0

15

1.9 +0.5

4

TEMPO

20 TEMPO

133

15

14.9 ±0.8

5

TEMPO/ PVAm

113 PVAm+20 T

133

15

15.3 ±1.3

6

No PVAm

133 PVAm-T6

133

0

5.1 ±1.2

7

Denatured Laccase

133 PVAm-T6

133*

15

4.4 ±1.4

8

Separated

133 PVAm-T6

133

15

1.0 ±0.4

Exp. # 1

Description

Mediator (mg/L)

Laccase (mg/L)

Base case

133 PVAm-T6

2

No mediator

3

35.5 ±2.1

*boiled to denature laccase Our original goal was to develop a polymer, PVAm-T, to act as both an oxidation catalyst and as a wet adhesive for the laminated cellulose films. However both our original work using bleach as a primary oxidant,

6

and the current work with laccase shows that additional PVAm

adhesive was required after the cellulose strips were oxidized to achieve high wet adhesion – compare the results for Exp. 1 with those for Exp. 6 in Table 2. The distribution of aldehyde groups through the thickness of the cellulose membranes was characterized by conjugating a fluorescent dye to the aldehyde groups, followed by imaging the fluorescence distribution through the thickness of cellulose membranes with confocal


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microscopy. Figure 1 shows three cellulose membrane cross sections. Image (a) was unoxidized cellulose that gave very little signal. Image (b) was oxidized using molecular TEMPO as a mediator. Although laccase, the primary oxidant, would have restricted mobility within the membrane, the low molecular weight TEMPO facilitated oxidation throughout the thickness. By contrast, the high molecular weight PVAm-T mediator was restricted to external surfaces, giving surface-localized oxidation. This characteristic is important for cellulose fibers where it is desirable to functionalize the external surfaces while minimizing the weakening of the fiber mechanical properties. The role of immobilized TEMPO moieties was further illustrated by measuring the wet adhesion as a function of the TEMPO content in PVAm-T. The results are shown in Figure 2. For comparison, also shown a set of results with NaClO as the primary oxidant. With no TEMPO (i.e. pure PVAm), the wet adhesion was low with both primary oxidants. With laccase as the primary oxidant, a degree of TEMPO substitution of 10% or greater was required for high adhesion. By contrast, with NaClO, the lowest TEMPO content, less than 1 percent gave high adhesion. Figure 3 shows the influence of oxidation time on the strength of wet cellulose laminates. About one hour was required to achieve maximum strength. There was slight degradation of strength at longer times. PVAm-T/Laccase Complex Formation. PVAm-T is a cationic polyelectrolyte and at neutral pH approximately one half of the primary amine groups are ionized. experimental conditions laccase is an anionic protein.

20

By contrast, under our

Therefore we anticipated that the

oppositely charged, water-soluble polymers should form polyelectrolyte complexes. 21 Solutions of laccase and PVAm-T were mixed and the absorbance of a PVAm-T/laccase (1:2 w/w) as a


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function of time is shown in Figure 4. The absorbance increased over the first two hours, indicating the presence of dispersed PVAm-T/laccase complex particles. A major factor influencing the polyelectrolyte complex properties is the mixing ratio. For example, in a detailed study of the phase behaviour of PVAm complex with carboxymethyl cellulose, we showed that if either polymer is in stoichiometric excess, a water-soluble complex is formed.

21

By contrast, when mixing the polymers under conditions near charge balance, a

macroscopic precipitate forms. At intermediate mixing ratios, colloidal complexes form. The sign of the charge on the colloidal complexes is determined by the charge of polymer in excess. PVAm-T was mixed in various ratios with laccase in the cellulose oxidation.

The

electrophoretic mobilities of the resulting complexes were measured and are show in Figure 5, together with the corresponding delamination force measurements. When laccase was in excess, the electrophoretic mobility was low and negative, reflecting the protein net charge density. By contrast, excess PVAm-T gave a positive mobility, reflecting an excess of PVAm-T on the corona of the complex. The highest two adhesion measurements corresponded to a positively charged complex. Influence of PVAm and PVAm-T on Laccase Activity. One could imagine the PVAm-T or PVAm polymer chains forming a polyelectrolyte complex with laccase could wrap around the enzyme and block active sites. Two types of experiments were performed to explore this possibility. The oxidation of TEMPO to the oxoammonium ion (Scheme 1) was measured with and without PVAm and the results are summarized in Figure 6. The presence of PVAm lowered the oxidation rate and decreased the maximum conversion from 100 to 50%. In this experiment the low molecular weight TEMPO should be highly mobile, facilitating interaction with the


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active sites on laccase. By contrast, with PVAm-T the mobility of TEMPO moieties is inhibited by the polymeric nature of PVAm-T. The catalytic activity of laccase present as PVAm-T/laccase complex was evaluated by the ABTS assay and the results are shown in Figure 7. It took about ten hours to halve the apparent enzyme activity. The PVAm-T/laccase complexes in these experiments were positively charged and can promote cellulose wet adhesion (see Figure 5). In conventional papermaking, the water removed during paper formation (white water) is reused within a hour to dilute concentrated pulp to make more paper. Thus the extended complex lifetime shown in Figure 7 suggests that unadsorbed complex in the papermachine white water may subsequently adsorb and thus be active.

To demonstrate this effect, a

suspension of PVAm-T/laccase complex was prepared and a series of cellulose strips was sequentially mixed in the suspension for 60 minutes, and then laminated. The adhesion results in Figure 8 show that only after five repeated uses of the suspension, did the wet adhesion start to decrease. We propose that the drop in adhesion after 9 treatments reflects the loss in complex activity with time (Figure 7). We considered that the results in Figure 8 could be explained by the depletion of PVAm-T/laccase complex with repeated experiments. However, we estimated that the depletion in PVAm-T/laccase complex concentration was less than 1% in these experiments. Layer-by-Layer Mediator/laccase Assembly. Layer-by-layer (LbL) assembly was evaluated as an alternative to depositing PVAm-T/laccase complexes onto wet cellulose since LbL assembly has been shown to be an effective way of putting adhesive layers onto cellulose.

22, 23

In addition, LbL assembly has been used to immobilize poly(diallyldimethyl ammonium chloride) and laccase layers on cellulose fiber surfaces and maintain enzyme activity.


24

In our

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work, PVAm-T was adsorbed first onto wet cellulose, followed by laccase. As with our other adhesion experiments, a final 15 mg/m2 layer of PVAm was placed between the two cellulose films during the lamination step. Figure 9 shows the delamination force as a function of the number of pairs PVAm-T followed by laccase layers placed on the cellulose films. For all experiments in Figure 9, the adhesion forces were very low.

It seems that by adsorbing the

PVAm-T before exposure to laccase, the immobilized TEMPO moieties do not have sufficient mobility to access both the active sites on laccase and then the C6 hydroxyls on cellulose. To gain insight into the LbL structures with PVAm-T and laccase, consecutive adsorption and rinsing experiments were performed on cellulose-coated quartz crystal microbalance (QCM-D) sensor surfaces. The Sauerbrey adsorbed layer amounts are summarized in Table 3, and raw QCM-D data are given in the supporting information file.

The estimated coverages are

consistent with consecutive adsorbed monolayers of PVAm-T and laccase.

∆f/n (Hz)

Sauerbrey Adsorbed Mass (mg/m2)

PVAm-T6 on cellulose

-6.97

1.23

Laccase on PVAm-T6

-13.27

2.35

PVAm on cellulose

-5.91

1.05

Laccase on PVAm

-18.47

3.27

Laccase on cellulose

-7.84

1.39

Layer

Table 3

Summary of adsorption coverages on cellulose measured by QCM-D.

Measurements were performed in 50 mM acetate buffer at pH 5. The QCM plots are available in the supporting information.


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DISCUSSION The current view of TEMPO mediated oxidation mechanisms is that TEMPO acts a shuttle. The TEMPO is oxidized to the oxoammonium ion (see Scheme 1) by laccase and then that oxoammonium ion diffuses (shuttles) to contact a primary alcohol on cellulose, forming an adduct that decomposes to TEMPO and an aldehyde group. How can this mechanism possibly function in the PVAm-T + laccase system? The TEMPO moieties in our work have greatly reduced mobility because they are covalently bonded to PVAm. Furthermore, the PVAm-T is immobilized as a polyelectrolyte complex, further reducing the ability of the TEMPO moieties on PVAm-T to function as a shuttle. The influence of the TEMPO content on oxidation (Figure 2) points to one possible explanation. With laccase/oxygen as the primary oxidant, the minimum TEMPO content required to achieve high wet adhesion was 10 mole percent. By contrast, with mobile NaClO/NaBr as the primary oxidant, the lowest TEMPO content of less than 1 percent gave high adhesion. Based on these observations, we propose that the TEMPO moieties are relatively immobile in the PVAm-T/laccase complexes and that the oxoammonium ions generated on TEMPOs near the laccase active sites exchange oxidations states with neighboring TEMPO moieties. Therefore the oxoammonium ions move along and between PVAm-T chains until they contact cellulose – see Figure 10. The oxidative transfer between TEMPO moieties requires short distances between neighboring groups and hence the requirement for high degrees of TEMPO substitution. By contrast, the mobile ClO- ions, in the case of the NaClO/NaBr system, can easily access all the TEMPO moieties and those near cellulose will induce oxidation. We recognize that the concept of oxidative activity moving along a PVAm-T in the form of oxoammonium ions is speculative, nevertheless it is a hypothesis that explains the current


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results. Furthermore, a similar mechanism has been used to explain electron transport along nonconjugated polymers. 25 The role of topochemical effects is further illustrated by the differences in the adhesion between surfaces treated with PVAm-T/laccase complexes and those treated with LbL deposition of PVAm-T followed by laccase. Although there are other adhesive systems described in the literature where LbL structures give stronger adhesion,

22

herein treatment with pre-made

complexes (see Figure 5) gave much stronger laminates than those treated with LbL sequential adsorption (see Figure 9).

Perhaps the LbL structure is simply too inflexible to promote

substrate/enzyme complex formation or the transfer of oxoammonium ions between neighbouring TEMPO moieties. The laccase performs two roles. First, with oxygen it acts as the primary oxidant that activates TEMPO moieties to give reactive oxoammonium ions.

Second, it remains as part of the

adhesive or surface treatment layer. Indeed, with care it may be possible to retain catalytic activity in the PVAm-T/laccase hydrogel surface layer on the cellulose surfaces. The importance of the catalytic role for the laccase was demonstrated by comparing active versus denatured enzyme (compare experiment 1 with experiment 7 in Table 2). Although denatured laccase forms polyelectrolyte complexes with PVAm-T, the resulting adhesion values were very low. From a technology perspective, the high laccase dosage requirement is a negative aspect. On the other hand, the reactions occur at neutral pH and ambient temperature. Furthermore, the binding efficiency (retention) of cationic PVAm-T/laccase onto anionic cellulose should be very high, avoiding water pollution issues. For example, high residual TEMPO concentrations could be an issue in the approach described by the recent work of Aracri et al. who reported paper wet strength improvement with laccase + TEMPO treatments.

14, 15


Their work did not involve the

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use of any polymers. The laccase dosages were comparable to those herein, whereas Aracri’s TEMPO concentrations were extraordinarily high (2-8% wt based on dry cellulose) – at least ten times higher than the equivalent TEMPO contents in our PVAm-T/laccase experiments. As described in our early work, 6 immobilizing the TEMPO on PVAm greatly lowers the required dosage of TEMPO because the polymer retains the TEMPO on the cellulose surfaces. Years ago we showed that if cellulose film is oxidized with TEMPO and then a monolayer of PVAm is adsorbed, strong wet adhesion is observed when two such surfaces are laminated.

1

Our original aim for the current work was combine the oxidation and PVAm adhesive addition steps by having a single polymer, PVAm-T, serve as both the oxidation mediator and as the sole wet adhesive.

As shown in Table 2, this approach failed (compare Exp. 1 with Exp. 6).

However, additional PVAm adhesive was required during the lamination step to get good adhesion. Although we could speculate, we do not have a definitive explanation as to why PVAm-T/laccase complex is a poor adhesive, nor why it functions as an excellent “primer” when additional PVAm adhesive is employed. In summary, the PVAm-T/laccase complex approach described in this paper offers four significant advantages over small molecule TEMPO (soluble TEMPO) mediators, and over using bleach as the primary oxidant. First, the oxidation of porous fibers or films is restricted to the exterior surfaces only, as PVAm-T has a high molecular weight, which avoids the excessive oxidation and weakening of interior surfaces. Second, TEMPO is concentrated on cellulose surfaces by being tethered to PVAm. Compared with water-soluble TEMPO, the total dose of TEMPO required to oxidize fibers is much less than that required by soluble TEMPO. Third, laccase catalysis occurs at neutral pH under mild conditions. It not only avoids the harmful halide reagents, but also improves the chemical stability of PVAm in oxidation solution because


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of the high substrate selectivity of the enzyme. Fourth, polymer-bound TEMPO stays with the solid phase, limiting environmental impacts. CONCLUSIONS In conclusion, laccase forms polyelectrolyte complexes with PVAm-T that oxidize and graft to cellulose surfaces under ambient conditions at pH 5. The cellulose grafted PVAm-T complex acts as primer, enhancing the ability of PVAm to increase the wet adhesion between cellulose surfaces. Furthermore, O2-laccase activated PVAm-T is potentially an effluent-free, very highyield cellulose surface treatment to give a polyvinylamine grafted surface layer without degrading the interior cellulose chains. The current results support the hypothesis that in spite of the TEMPO moieties being immobilized, the reactive oxoammonium ions are effectively mobile within PVAm-T/laccase complexes due to the exchange of oxidation states between neighboring grafted TEMPO moieties. Finally, PVAm or PVAm-T slowly deactivates laccase presumably by blocking active sites, the PVAm-T complex is oxidatively active for many hours. ASSOCIATED CONTENT Supporting Information. The SI file includes: the comparison of laccase activity, before and after purification; the influence of PVAm on laccase activity; and, QCM-D data corresponding to the results in Table 3.

This material is available free of charge via the Internet at

http://pubs.acs.org. ACKNOWLEDGEMENTS The authors acknowledge Professor Lars Wågberg for suggesting laccase/oxygen as a primary oxidant and Prof. Art van der Est from Brock University for performing the ESR measurements. In addition, we thank Profs Dominic Rochefort, University of Montreal, and Raja Ghosh,


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McMaster University for advice on the purification and quantitation of the enzyme. Finally the authors thank BASF Canada and NSERC for research funding. RP holds a Tier 1 Canada research chair in Interfacial Technologies.


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FIGURES AND TABLES

Scheme 1

TEMPO mediated oxidation of cellulose with laccase + oxygen based on the

mechanism proposed by Fabbrini et al. 9


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Figure 1

Distribution of fluorescein labeled aldehyde groups in cellulose membrane cross

sections. Aldehydes were labeled with green fluorescein-5-thiosemicarbazide (excited at 488 nm). The black background on the original laser scanning confocal images was inverted to white. The cellulose films were oxidized by the sequential addition method using PVAm-T6.


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Wet Delamination Force (N/m)

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Lamination: PVAm 45 kDa 15 mg/m2 PVAm-T

30

20

10

PVAm-T14/NaClO/NaBr

PVAm-T14/laccase 1/2 Sequential Addition

0 0% 5% 10% 15% 20% Mole Percentage Amines with Grafted TEMPO

Figure 2

Influence of TEMPO grafting extent on cellulose wet adhesion, comparing two

primary oxidants. NaClO mediated oxidation experiments were performed in solutions containing 50 mg/L of NaBr and 20 mg/L of PVAm-T and 6.8 mmol/L NaClO at pH 10.5. Laccase mediated oxidation experiments were conducted using the sequential treatment method in pH 5 50 mM sodium acetate buffer solutions consisting of 66.7 g/L PVAm-T 14 and 133.3 mg/L laccase. The oxidation time was 24 hours.


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Figure 3

Influence of oxidation time on cellulose wet adhesion. Cellulose films were

oxidized with 67 mg/L PVAm-T14 in acetate buffer at pH 5 for 30 min followed by 133 mg/L laccase solution. Oxidized films were laminated with 15 mg/m2 PVAm 45 kDa.

40 Wet Delamination Force (N/m)

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Oxidation: PVAm-T14/laccase 1/2 sequential addition Lamination: PVAm 15 mg/m2

30

20

10

0

0 0.1

II 1

10 100 Oxidation Time (min)


1000

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0.3 Absorbance at 500 nm

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67 mg/L PVAm-T14 133 mg/L Laccase 0.2

0.1

0 0 1

Figure 4

II

10 100 1000 Oxidation Time (min)

10000

The absorbance of mixture of PVAm-T14 (66.7 mg/L) and laccase (133 mg/L) as

a function of oxidation time at pH 5 (50mM Na acetate buffer) and 25°C.


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0.6 0.4

30 0.2 20

0 -0.2

10 Preformed PVAm-T Complex with 133 mg/L Laccase 0

-0.4

Electrophoretic Mobility 10-8m2Vs-1

40

-0.6 0

50 100 PVAm-T6 Concentration (mg/L)

150

40 1

30

20 0 10 Preformed Laccase Complex with 67 mg/L PVAm-T14 0

-1 0

Figure 5

Electrophoretic Mobility 10-8m2Vs-1


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50 100 Laccase Concentration (mg/L)

150

The influence of PVAm-T/laccase dosing ratios on complex electrophoretic

mobility and on adhesion. Experiments were conducted in pH 5 in 50 mM acetate.


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100% TEMPO Conversion

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Laccase

80%

Laccase + 0.3 g/L PVAm

60% 40% 20% 0% 0

100

200

300

Time (min)

Figure 6

Influence of PVAm (200 mg/L) on laccase (40 mg/L) activation of TEMPO (10

mM) at pH 5 in 50 mM buffer. The conversion of TEMPO to an oxoammonium ion was measured by UV-Vis spectroscopy.


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100%

Laccase (67 mg/L)/PVAm-T14 (67 mg/L) Complex ABTS Oxidation Assay

80% Residual Activity

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60% 40% 20% 0% 0

Figure 7

5

10

15 Time (h)

20

25

30

The catalytic activity of PVAm-T14/laccase complex, measured by ABTS

oxidation, as a function of time. Complexes were formed and aged in 50 mM acetate buffer at pH 5.


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30 Oxidation 150 mL pH 5 buffer 67 mg/L PVAm-T14 67 mg/L Laccase 1 hour Lamination PVAm 15 mg/m2

25 20 15 10 5 0

0 2 4 6 8 10 Sets of Cellulose Strips Oxidized Sequentially

Figure 8

Evaluating how many sets of cellulose membranes can be oxidized by the same

PVAm-T14/laccase complex dispersion.


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Figure 9

Wet adhesion of laminates prepared by layer-by-layer assembly of PVAm-T6 and

laccase on cellulose. The coverage of the PVAm 45 kDa adhesive layer was 15 mg/m2.

40

30

Cellulose

Laccase PVAm-T

PVAm

20

PVAm-T Laccase

Laminate Structure

Cellulose

Delamination Force (N/m)

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10

0 0

1 2 3 Number of Laccase Layers/Cellulose Film


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Figure 10

We propose that the oxoammonium ion (i.e. activated TEMPO), formed by

laccase, exchanges with neighbouring TEMPO moieties, resulting in the transport of oxidation activity to the cellulose surface in spite of the immobilized state of the TEMPO moities.

T to T Electron Transfer Along PVAm-T T T

T

T Laccase

T Laccas e

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T

T T T

T

T

T T

T T

Cellulose


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Reference 1. DiFlavio, J. L.; Bertoia, R.; Pelton, R.; Leduc, M., The Mechanism of Polyvinylamine Wet-Strengthening. In Advances in Paper Science and Technology: Transactions of the 13th Fundamental Research Symposium, SJ, I. A., Ed. Pulp & Paper Fundamental Research Society: Cambridge, UK, 2005; Vol. 1, pp 1293-1316. 2. DiFlavio, J. L.; Pelton, R.; Leduc, M.; Champ, S.; Essig, M.; Frechen, T., Cellulose 2007, 14, 257-268. 3. Saito, T.; Isogai, A., Ind. Eng. Chem. Res. 2007, 46, 773-780. 4. Lai, T.-w.; Pinschmidt Jr, R. K., Amine Functional Polymers Containing Aminal Groups. EP Patent 0,461,399: 1995. 5. Espy, H. H., Tappi J. 1995, 78, 90-99. 6. Pelton, R.; Ren, P. R.; Liu, J.; Mijolovic, D., Biomacromolecules 2011, 12, 942–948. 7. Anelli, P. L.; Biffi, C.; Montanari, F.; Quici, S., J. Org. Chem. 1987, 52, 2559-2562. 8. Kitaoka, T.; Isogai, A.; Onabe, F., Nordic Pulp & Paper Res. J. 1999, 14, 279-284. 9. Fabbrini, M.; Galli, C.; Gentili, P.; Macchitella, D., Tetrahedron Lett. 2001, 42, 75517553. 10. Arends, I. W. C. E.; Li, Y.-X.; Ausan, R.; Sheldon, R. A., Tetrahedron 2006, 62, 66596665. 11. Kulys, J.; Vidziunaite, R., Journal of Molecular Catalysis B-Enzymatic 2005, 37, 79-83. 12. Barreca, A. M.; Sjögren, B.; Fabbrini, M.; Galli, C.; Gentili, P., Biocatalysis and Biotransformation 2004, 22, 105-112. 13. Lee, S.-K.; George, S. D.; Antholine, W. E.; Hedman, B.; Hodgson, K. O.; Solomon, E. I., J. Am. Chem. Soc. 2002, 124, 6180-6193. 14. Aracri, E.; Vidal, T.; Ragauskas, A. J., Carbohydr. Polym. 2011, 84, 1384–1390. 15. Aracri, E.; Valls, C.; Vidal, T., Carbohydr. Polym. 2012, 88, 830-837. 16. Lund, M.; Felby, C., Enzyme Microb. Technol. 2001, 28, 760-765. 17. Zouari-Mechichi, H.; Mechichi, T.; Dhouib, A.; Sayadi, S.; Martínez, A. T.; Martínez, M. J., Enzyme Microb. Technol. 2006, 39, 141-148. 18. Johannes, C.; Majcherczyk, A., J. Biotechnol. 2000, 78, 193-199. 19. Kurosu, K.; Pelton, R., J. Pulp Paper Sci. 2004, 30, 228-232. 20. Katchalsky, A.; Mazur, J.; Spitnik, P., J. Polym. Sci. 1957, 23, 513-30. 21. Feng, X.; Pelton, R.; Leduc, M.; Champ, S., Langmuir 2007, 23, 2970-2976. 22. Wagberg, L.; Forsberg, S.; Johansson, A.; Juntti, P., J. Pulp Paper Sci. 2002, 28, 222228. 23. Feng, X.; Zhang, D.; Pelton, R., Holzforschung 2009, 63, 28–32. 24. Xing, Q.; Eadula, S. R.; Lvov, Y. M., Biomacromolecules 2007, 8, 1987-1991. 25. Heller, A., Phys. Chem. Chem. Phys. 2004, 6, 209-216.


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TOC Graphic


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Laccase Complex with Polyvinylamine Bearing Grafted TEMPO is a

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