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. 2024 Jun 25;25(7):4428–4439. doi: 10.1021/acs.biomac.4c00457

Enzymatically Oxidized Carbohydrates As Dicarbonyl Biobased Cross-Linkers for Polyamines

Owen M Mototsune †, Sung Hwa Hong †, Hani E Naguib ‡, Emma R Master †,§,*
PMCID: PMC11238324  PMID: 38917058

Abstract

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Carbonyl cross-linkers are used to modify textiles and form resins, and are produced annually in megatonne volumes. Due to their toxicity toward the environment and human health, however, less harmful biobased alternatives are needed. This study introduces carbonyl groups to lactose and galactose using galactose oxidase from Fusarium graminearum (FgrGalOx) and pyranose dehydrogenase from Agaricus bisporus (AbPDH1) to produce four cross-linkers. Differential scanning calorimetry was used to compare cross-linker reactivity, most notably resulting in a 34 °C decrease in reaction peak temperature (72 °C) for FgrGalOx-oxidized galactose compared to unmodified galactose. Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), and proton nuclear magnetic resonance (1H NMR) spectroscopy were used to verify imine formation and amine and aldehyde depletion. Cross-linkers were shown to form gels when mixed with polyallylamine, with FgrGalOx-oxidized lactose forming gels more effectively than all other cross-linkers, including glutaraldehyde. Further development of carbohydrate cross-linker technologies could lead to their adoption in various applications, including in adhesives, resins, and textiles.

Introduction

Conventional carbonyl cross-linkers, such as formaldehyde, glyoxal, and glutaraldehyde, are derived from petroleum and have adverse environmental and/or human health effects.1−5 This project aims to produce alternatives to conventional cross-linkers that are biobased (i.e., derived from biomass streams), made using bioprocesses (i.e., converted to cross-linkers using enzymatic reactions), and permit the controlled assembly of polymers. It is anticipated that the generated carbohydrate cross-linkers will be relevant to applications in textile reuse/recycling technologies and in biobased wood adhesives for fully biobased engineered wood building materials.6,7

Lactose, β-D-galactopyranosyl-(1 → 4)-d-glucose, is a biomass side-stream that is largely underused and undervalued due to its low sweetness, its relatively high cost, the inability of industrially relevant microbes to use it as a carbon source, and the prevalence of lactose intolerance in many people.8 These factors make lactose a strong candidate for use as a feedstock for biobased cross-linker production, which could address challenges with low value (i.e., bioethanol, electricity) and low volume (i.e., pharmaceuticals, cosmetics) applications for lactose.8−11 To replicate the carbonyl chemistry of conventional cross-linkers in carbohydrates, enzymatic processes are preferred over chemical processes like TEMPO and periodate oxidation.12−15 Compared to chemical methods, enzyme technologies permit regiospecific modifications to carbohydrates without requiring complex protection protocols that reduce product yield, avoid resource- and energy-intensive conditions (e.g., high temperature, pressure), and use water as a solvent instead of hazardous, petroleum-based solvents.13,16−19

Many enzymes acting on carbohydrates are curated in the carbohydrate-active enzyme (CAZyme) database (CAZy.org). Among those, the auxiliary activity (AA) family of CAZymes is of interest as they perform redox reactions that modify carbohydrate chemistry.20,21 Oxidized carbohydrates have been investigated for many applications, including in paper, hydrogels, lubricants, cosmetics, gelling agents, and nutraceuticals.22 In this work, enzymes from families AA3 and AA5, specifically pyranose dehydrogenase from Agaricus bisporus (AbPDH1, AA3_2, EC 1.1.99.29) and galactose oxidase from Fusarium graminearum (FgrGalOx, AA5_2, EC 1.1.3.9) were used to modify galactose G0 and lactose L0 to produce dicarbonyl cross-linkers (Schemes 1, S1).

Scheme 1. Enzymatic Oxidation of Galactose by FgrGalOx and AbPDH1 To Produce Crosslinkers (a), Crosslinker Ring-Opening Reactions in Water To Reveal Reactive Groups (b), and Crosslinking Reactions with HMDA (c).

Scheme 1

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AbPDH1 (E.C.1.1.99.29) is a flavin-dependent oxidoreductase, containing a flavin adenine dinucleotide (FAD) binding domain and a covalently linked FAD cofactor, with a molecular weight of approximately 75 kDa.23,24 It catalyzes the oxidation of the C-1 hydroxyl and secondary hydroxyls of several pyranose monosaccharides and oligosaccharides while reducing quinones, such as benzoquinone and 2,6-dichlorophenolindophenol, or organometallic ions, such as ferricenium.18,25−27 When applied to galactose G0, AbPDH1 has been reported to exclusively oxidize the C-2 alcohol (Scheme 1a) to 2-ketogalactose G2, as the C-4 axial hydroxyl prevents proper orientation for C-1, C-3, or C-4 oxidation.18,25,26,28,29AbPDH1 has been reported to oxidize the C-2 secondary hydroxyl of lactose L0 to 2-ketolactose L2, as well as the C-1 hemiacetal to the corresponding lactone, lactobionolactone L3 (Scheme S1).30 Previous studies have shown AbPDH1 can oxidize the terminal pyranose units at the reducing and/or nonreducing ends of xylooligosaccharides performing multiple oxidations on the same substrates.18AbPDH1 and other pyranose dehydrogenases have been used to modify sugars to produce tagalose (an alternative sweetener) and lactulose (a laxative).26,27 Studies have also investigated its use in enzymatic biofuel cells and biosensors.26,27

FgrGalOx (E.C.1.1.3.9) is a single-copper metalloenzyme with a molecular weight of approximately 65 kDa.22 It catalyzes the oxidation of the C-6 primary alcohol in D-galactose G0 to the corresponding aldehyde, galacto-hexodialdose G1 (or D-galacto-hexodialdo-1,5-pyranoside), while catalyzing the reduction of molecular oxygen to hydrogen peroxide (Scheme 1a).22,31FgrGalOx also oxidizes the galactopyranosyl subunits of oligosaccharides such as lactose L0, melibiose, and raffinose, as well as polysaccharides such as galactomannan, galactoglucomannan, and galactoxylomannan.32,33FgrGalOx has been used in various biotechnological applications, including for labeling glycoproteins, functionalizing polysaccharides, creating aerogels, and in biosensors. FgrGalOx can also be used in enzyme cascades to make furan dicarboxylic acid (biobased plastic monomer), naftifine (antifungal agent), aminated sugars and lactams (precursors to biobased plastic monomers), and islatravir (an HIV drug).19,34−36

Typically, FgrGalOx and AbPDH1 oxidize one single hydroxyl group on a monosaccharide to a carbonyl or one single anomeric hemiacetal to the corresponding lactone. To cross-link molecules, a carbohydrate cross-linker requires a second reactive carbonyl group, which can be exposed to reducing sugars by dissolving them in water. Reducing sugars (e.g., galactose G0) contain C-1 hemiacetals and undergo ring openings in water to expose C-1 aldehydes G4 (Scheme 1b). Therefore, a reducing sugar oxidized at a different location (like G1 or G2) can form an open chain dicarbonyl (like G5 or G6, respectively), and can then act as a cross-linker, reacting with two amine groups (Scheme 1c) to produce two imine cross-links (G9 and 10, respectively).

Reducing sugars that have been oxidized at the C-1 hemiacetal to the corresponding lactone (e.g., galactolactone G3) are readily hydrated to form open-chain carboxylic acids (e.g., galactonic acid G7). Unlike carbonyls, these carboxylic acids do not undergo typical covalent-bond forming reactions with amines (i.e., imine cross-linking reactions) and instead undergo an acid–base proton exchange (Scheme 1c), producing the conjugate base (e.g., galactonate G11). Therefore, these C-1 oxidized compounds are not expected to act as cross-linkers for amines. Reactions with lactose L0 corresponding to the reactions described here are shown in Scheme S1.

The current study investigates the potential of FgrGalOx and AbPDH1 to synthesize carbohydrate-derived cross-linkers from galactose and lactose, thereby creating renewable alternatives to petroleum-based cross-linking molecules (e.g., formaldehyde, glutaraldehyde, glyoxal). Whereas the impact of enzymatic oxidation on the reactivity of the carbohydrates was quantified using differential scanning calorimetry (DSC), the formation of expected cross-links was followed by attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), and proton nuclear magnetic resonance spectroscopy (1H NMR). This study also investigates the ability of cross-linkers to induce gelation in polyallylamine solutions.

Experimental Methods

Materials

Galactose, lactose, hexamethylenediamine (HMDA), benzoquinone (BQ), sodium acetate, n-butylamine (n-BA), and 2,3-dichloronitrobenzene (2,3-DCNB) were of analytical grade and purchased from Millipore-Sigma. 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was purchased from Biobasic. Polyallylamine (Mw ∼ 65 kDa) was purchased from Millipore-Sigma as a 10% (w/w) solution in water. Catalase (E.C. 1.11.1.6, from bovine liver, Sigma-Aldrich), laccase (E.C. 1.10.3.2, Novozym 51003, Novozymes), and horseradish peroxidase (E.C. 1.11.1.7, from horseradish, Millipore-Sigma) were used. Deuterated dimethyl sulfoxide (DMSO-d6) with 99.9% deuterium was purchased from Millipore-Sigma.

Enzyme Production and Purification

FgrGalOx and AbPDH1 were heterologously produced in Pichia pastoris KM71H as previously described.19 Proteins were produced using an Infors Minifors bioreactor (5 L) with pH and dissolved oxygen sensors (Hamilton Company) and controlled using Iris NT version 5.01, following a modified version of the Invitrogen Pichia Fermentation Process Guidelines protocol.37 Briefly, 200 mL precultures in BMGY were first inoculated from streaked YPD plates and grown overnight at 30 °C shaking at 180 rpm, as previously described.19 Cells were pelleted by centrifugation at 3000 × g for 5 min, resuspended in 50 mL of water, and then transferred into the bioreactor containing 2 L of pH 5 fermentation basal salts medium with PTM1 trace salts at 30 °C. When the glycerol had been depleted (∼20 h), a fed-batch protein-expression phase was initiated whereby 50% glycerol with 1.2% PTM1 trace salts was fed into the bioreactor at 5–35 mL/h for 4–6 h or until OD = 180–220 g/mL. This was followed by the methanol-fed batch phase where 100% methanol with 1.2% PTM1 trace salts was fed into the reactor at 5–10 mL/h for 5 days, keeping the reactor at 15 °C.

Following induction, the pH was adjusted to 6.0 (AbPDH1) or 7.5 (FgrGalOx), and cells and salts were removed by two cycles of centrifugation at 18,000 × g for 45 min. Ammonium sulfate was added to 1.0–2.0 M and supernatants were filtered using 0.45 μm capsule filters (Cytiva Whatman Polycap TC). The filtrate was then loaded onto a 20 mL HiPrep Phenyl Sepharose Fast Flow (High Sub) 16/10 hydrophobic interaction chromatography (HIC) column (Cytiva). Proteins were eluted from the column using a linear gradient over 15 column volumes to 0 M ammonium sulfate. Fractions with protein were pooled and buffer-exchanged to 50 mM pH 7.5 sodium/phosphate buffer with 300 mM sodium chloride and 20 mM imidazole using 10 kDa centrifugal filters. The protein mixture was then loaded onto a Ni-NTA-agarose affinity chromatography column, washed with loading buffer (50 mM pH 7.5 sodium/phosphate, 300 mM sodium chloride, 20 mM imidazole) for 10 column volumes, and then eluted using a step elution from 20 to 250 mM imidazole. Fractions with protein were pooled and buffer-exchanged with 10 kDa centrifugal filters to remove imidazole. Protein identity and purity were verified using SDS-PAGE. FgrGalOx activity was measured using an ABTS and horseradish peroxidase (HRP) assay with the following reaction parameters: 200 μL total volume, 30 mM MOPS buffer (pH 7.5), 150 mM galactose (KM = 42 mM), 7.5 U/mL HRP, 2.0 mM ABTS, measured at 420 nm over 30 min at 30 °C.19,38 One FgrGalOx activity unit (U) is equal to 1 μmol of H2O2 produced per minute (=2 μmol of ABTS oxidized to 2 μmol of ABTS•+ per minute). AbPDH1 activity was measured using the benzoquinone activity assay with the following reaction parameters: 200 μL total volume, 50 mM sodium acetate buffer (pH 5.5), 3 mM benzoquinone, 25 mM galactose (KM = 4.2 mM), measured at 290 nm over 30 min at 30 °C.25,39 One AbPDH1 activity unit (U) is equal to 1 μmol of benzoquinone reduced to 1 μmol of hydroquinone per minute.

Enzymatic Preparation of Oxidize Galactose and Lactose for Cross-Linking Reactions

To prepare enough cross-linkers for cross-linking studies, reaction volumes were increased to accommodate 1.42 mmol of carbohydrate (0.25 g of galactose or 0.50 g of lactose). Oxidations of galactose by FgrGalOx (referred to below as FgrGalOx-Gal) were performed for 6 h in 30 mM MOPS buffer (pH 7.5) with vigorous shaking at 30 °C and comprised 150 mM galactose, 200 U/mL (0.17 mg/mL) FgrGalOx, 27 U/mL (18 μg/mL) HRP, and 5400 U/mL (0.17 mg/mL) catalase. Oxidations of lactose by FgrGalOx (to produce FgrGalOx-Lac) replaced galactose with 150 mM lactose and comprised 600 U/mL FgrGalOx, 81 U/mL HRP and 16,200 U/mL catalase. Oxidations by AbPDH1 on galactose (to produce AbPDH1-Gal) were performed for 24 h with vigorous shaking at 30 °C and comprised: 150 mM galactose, 10 mM benzoquinone, 18 U/mL (1.3 mg/mL) AbPDH1, and 18 U/mL (72 μg/mL) laccase. Oxidations of lactose by AbPDH1 (to produce AbPDH1-Lac) replaced galactose with 75 mM lactose and used 22 U/mL (1.6 mg/mL) of AbPDH1 and 22 U/mL (90 μg/mL) laccase and 100 mM sodium acetate buffer (pH 5.5). At the end of the reaction, 50 μL aliquots of reaction solutions were filtered through 10 kDa membrane filters to remove enzymes and saved for analysis by HPAEC-PAD and LC-MS. The rest of the product solutions were frozen and freeze-dried.

HPAEC-PAD and HILIC-ESI-MS Analysis of Enzymatically Produced Carbohydrate Cross-Linkers

The extent of enzymatic oxidation was measured by carbohydrate substrate depletion using a high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using a Dionex ICS-5000 HPLC system with a CarboPac PA1 (2 × 250 mm) analytical column and guard column (2 × 50 mm) (Thermo Fisher Scientific). The solutions were diluted in water to a carbohydrate concentration of 0.5 mM and filtered through 0.22 μm PES membranes before being injected (12.5 μL) onto the column. Injections were eluted at 0.25 mL/min with a constant concentration of NaOH (0.1 M) and a linear gradient of sodium acetate (from 0 to 1 M) over 55 min. A modified elution protocol was used for quantifying galactonic acid and lactobionic acid in PDH oxidation products: for 10 min, a linear increase in NaOH concentration from 1 to 100 mM and sodium acetate concentration from 0 to 500 mM, then a step increase to 1 M sodium acetate (remaining at 100 mM NaOH) for 5 min. Data were analyzed using the Chromeleon Chromatography Data System version 7.2 (Thermo Fisher Scientific).

Hydrophobic interaction liquid chromatography coupled with electrospray ionization mass spectrometry (HILIC-ESI-MS) was used to identify products lacking standards. In this case, reaction samples were dissolved in Milli-Q water and filtered through 0.22 μm filters, then 10-μL aliquots were injected at 0.4 mL/min into a Thermo Scientific Ultimate 3000 UHPLC system with a Waters Acquity BEH HILIC (3.0 mm × 150 mm, 1.7 μm) equipped with guard column. Injections were 10 μL, the flow rate was 0.4 mL/min, and the column temperature was 40 °C. The elution program consisted of 1 min at 50% eluent A (20 mM ammonium acetate 20 mM ammonium hydroxide in water) and 50% eluent B (acetonitrile), then an increase to 100% eluent B over 1 min, hold for 9 min at 100% B, then reduction to 50% B over 0.5 min, and hold at 50% B for 3.5 min. A Thermo Scientific Q-Exactive equipped with a HESI-II probe (spray voltage: 3.5 kV, capillary temperature: 320 °C) was used for mass spectroscopy. Full MS was performed over a m/z range of 70–1000 with a resolution of 140,000 and AGC target of 3 × 106 in both positive and negative polarity mode. Data-dependent MS2 was performed with an isolation window of 0.4 m/z, with a resolution of 17,500, and an AGC target of 1 × 105.

Differential Scanning Calorimetry To Measure Cross-Linker Reactivity

Equimolar amounts of FgrGalOx-Gal, FgrGalOx-Lac, AbPDH1-Gal, or AbPDH1-Lac and hexamethylenediamine (HMDA) were dissolved/suspended at 1.32 M in water. Total reaction mixtures had total masses of 5–20 mg. Reaction mixtures were sealed in high-pressure capsules. A TA Instruments Q2000 DSC was used to first equilibrate the capsule at 10 °C for 10 min and then to heat the samples to 200 °C at a ramp rate of 5 °C/min. The products of the DSC reactions were collected at the end of the run and freeze-dried to prepare them for analysis by FTIR and XPS.

Raw data were analyzed in TA Universal Analysis version 4.5.A. Smoothing over ranges of 1–2 °C was applied to reduce noise. Enthalpies of reaction were calculated by peak integration with a sigmoidal tangent baseline. Peak temperature is defined as the temperature at which the curve deviates most from the baseline drawn under the peak. Onset/offset points are defined as the intersection of an initial tangent line with the tangent line after the on/offset.

Cross-Linker Reactivity Testing at Static Temperatures

Equimolar amounts of cross-linker and HMDA were dissolved in water in 200 μL PCR tubes at concentrations of 1.32 M in 10–70 mg total reaction masses. Reactions were heated to 30, 65, and 80 °C for 30 min, then cooled on ice, frozen, and freeze-dried.

ATR-FTIR and N 1s XPS To Characterize Cross-Linkers and Products of Cross-Linking Reactions

Cross-linker and HMDA reaction products (from DSC and static temperature reactions) were analyzed using a Bruker Alpha ATR-FTIR and data were analyzed using OPUS version 7.2. Solid samples (∼5 mg) are loaded directly into the instrument after calibrating using air as the background. Spectra are produced by averaging 24 scans from 400–4000 cm–1.

Cross-linker and HMDA reaction products were analyzed using a K-Alpha XPS system (Thermo Fisher Scientific, East Grinstead (UK)) with a monochromatized Al K-Alpha X-ray source and a 400 μm spot size (2:1 ellipse, with the major axis being the noted spot size). A low-energy electron/ion (Ar) flood source was used for charge neutralization. First, to identify all detectable surface species, a survey spectrum was acquired at a pass energy of 200 eV and a point density of 1 eV. Next, regional scans were performed at high energy resolution (lower pass energy (50 eV), and with a correspondingly higher point density of 0.1 eV. The dwell time for the acquisition of these spectra was 50 ms. Regional spectra were used for quantification. Thermo Fisher’s Avantage software version 5.982 was used to process the data. Surface elemental compositions were calculated from background-subtracted (smart background) peak areas derived from transmission function-corrected regional spectra. Sensitivity factors used to calculate the relative atomic percentages were provided by the instrument manufacturer. Spectra were charge shifted to align the lowest binding energy C 1s peak (C–C) to 284.8 eV. A simplex peak fitting algorithm (Gaussian–Lorentzian product function) with the following parameters was used: 1 × 105 maximum iterations, <0.01 convergence, and a 25–35% Lorentzian–Gaussian ratio (L/G). Full width at half maximums (fwhm) were constrained to 0.2 eV ranges between 1.2 and 1.75 eV and peak energies were constrained to at most ±0.1 eV for reported peaks.

1H NMR To Measure Cross-Link Formation

Proton (1H) NMR was used to track the production of imines and depletion of aldehydes from reactions between a constant amount of FgrGalOx-Gal (12.5 μmol) and a varying amount of n-butylamine (0, 1, 2, 3, 5, 6.25, 12.5, 25, and 50 μmol) in 500 μL of DMSO-d6. Proton signals were further analyzed by correlated spectroscopy (COSY) to annotate protons by identifying their cross peaks. Reactions were performed in microcentrifuge tubes and run for 1.5 h at 60 °C, before cooling and adding 1.25 μmol of 2,3-DCNB as an internal standard. Reactions were transferred to 3 mm NMR tubes and analyzed using a 600 MHz Agilent DD2 NMR spectrometer. Spectra were analyzed using MestReNova version 14.3.0. Raw spectra were processed by calibrating the chemical shift by referencing the DMSO peak at 2.50 ppm, correcting the phase to produce a flat baseline, and correcting the baseline using a multipoint, cubic spline algorithm. Integrals were selected manually and calculated relative to the internal standard (1.25 μmol of 2,3-DCNB).

Polyallylamine Cross-Linking Using Carbohydrate Cross-Linkers

In brief, 3–6 mg of dry carbohydrate cross-linkers were dissolved in 100 μL aliquots of polyallylamine (PAA) solutions (Sigma, 65,000 kDa) at 10% (i.e., 100 mg/mL), 5, and 2.5% in 200 μL PCR tubes. Glutaraldehyde was used as a positive control, and lactobionic acid L7 was used as a negative control. Native galactose and lactose were also tested. Gelling was observed qualitatively by eye and by touch and associated with the cross-linkers reacting with amine groups to form imine cross-links (Scheme 2). Pictures of samples were taken in front of a blue lab notebook (Winnable SKU# WL-191-BE) using a OnePlus 5T smartphone with default camera settings.

Scheme 2. PAA Crosslinking with Dicarbonyl Crosslinker Galacto-Hexodialdose G1.

Scheme 2

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Results

Verification and Identification of FgrGalOx and AbPDH1 Oxidation Products by HPAEC-PAD and LC–MS

FgrGalOx and AbPDH1 were produced with volumetric yields of 31 and 60 mg/L, respectively, and had activities of 250 and 15 U/mg on galactose, respectively. Enzymes were then used to oxidize galactose G0 and lactose L0 to produce cross-linkers. Conversions to oxidized products were calculated by substrate depletion, as determined by HPAEC-PAD. FgrGalOx reactions resulted in conversions of 90 and 100% for galactose and lactose, respectively, and AbPDH1 showed conversions of 76 and 98% for galactose and lactose, respectively (Figure 1a,b). Higher enzyme loadings and longer reaction times did not result in greater galactose depletion (Figure S1). In addition to substrate depletion, the HPAEC-PAD chromatograms revealed product peaks resulting from the enzymatic treatments (Figure S2). The identification and quantification of reaction products by HPAEC-PAD could not be completed due to a lack of reference standards for galacto-hexodialdose G1, 2-ketogalactose G2, lacto-hexodialdose L1, and 2-ketolactose L2. Nevertheless, given the commercial availability of galactonic acid G7 and lactobionic acid L7, a modified HPAEC-PAD elution protocol was used to determine the extent of C-1 oxidation of galactose and lactose by AbPDH1 (Figure 1c,d). This same analysis confirmed neither FgrGalOx cross-linkers contained galactonic acid G7 or lactobionic acid L7.

Figure 1.

Figure 1

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HPAEC-PAD chromatograms showing galactose (a) and lactose (b) depletion after oxidation by FgrGalOx and AbPDH1 and galactonic acid (c) and lactobionic acid (d) production after AbPDH1 oxidations (n = 2).

Further product identification was enabled by HILIC-ESI-MS, which was used to identify likely aldehyde and ketone modifications to galactose and lactose by FgrGalOx and AbPDH1 (Table 1, Figure S3). FgrGalOx-Gal contained galactose with the C-6 hydroxyl converted to an aldehyde, galacto-hexodialdose G1 (C6H10O6, m/z = 177.0405), as well as the double oxidation to a carboxylic acid, galacturonic acid G12 (C6H10O7, m/z = 193.0354). FgrGalOx-Lac contained lactose with a C6 hydroxyl converted to an aldehyde, lacto-hexodialdose L1 (C12H20O11, m/z = 339.0933) and to a lesser extent converted to a carboxylic acid, lactobiuronic acid L12 (C12H20O12, m/z = 355.0864). In both cases, FgrGalOx produced mostly single-oxidized C6 aldehyde–containing products, and some C6 carboxylic acid–containing products, reflecting the consensus from previous studies.22

Table 1. Product Identification Was Carried Out by LC-MS and HPAEC-PADa.

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a

‡ Identified by LC-MS. † Identified by HPAEC-PAD.

For AbPDH1-Gal, galactose with a ketone at one of the secondary alcohol sites (C6H10O6, m/z = 177.0405), most likely, 2-ketogalactose G2, was observed. Galactonic acid G7 (C6H12O7, m/z = 195.051), was also detected, agreeing with observations from HPAEC-PAD. The double-oxidized (most likely 2,3-diketogalactose G13, C6H8O6, m/z = 175.0248) galactose was also observed. AbPDH1-Gal also contained the combination of these oxidations, 2-ketogalactonic acid G14 (C6H10O7, m/z = 193.0354). The product of AbPDH1-oxidation of lactose (AbPDH1-Lac) contained the C-2/C-2′-oxidized lactose, 2/2′-ketolactose L2 (C12H20O11, m/z = 339.0933), and lactobionic acid L7 (C12H22O12, m/z = 357.1038). The combination of the C-1 and C-2 oxidations was also observed, likely corresponding to 2/2′-ketolactobionic acid L13, (C12H20O12, m/z = 355.0864). Broadly, AbPDH1 produced a mixture of ketone-containing and carboxylic acid–containing products, in agreement with previous observations.26,29,30

Assessing Reactivity of Enzymatically Oxidized Galactose and Lactose in Cross-Linking Reactions with HMDA Using DSC

Cross-linkers were then tested for reactivity toward HMDA using DSC, with a lower reaction temperature corresponding to a higher reactivity (Figure 2). Reaction temperature measurements were reproducible within 3 °C for most cross-linkers (Figures 2, S4). Unmodified galactose and lactose both showed reactivity with HMDA, reacting at 106.2 and 112.4 °C, respectively, as both are reducing sugars that can react with amines through the C-1 aldehyde when the sugar is in the open-chain form (G4, G8 in Scheme 1). While this open-chain form accounts for only a small fraction of the sugar (about 0.006%), when the aldehyde form is consumed the equilibrium shifts to the open-chain aldehyde form, allowing the reaction to proceed.40,41

Figure 2.

Figure 2

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DSC heat flow profiles for galactose (a) and lactose (b) cross-linker + HMDA reactions with summarized reaction quantities, showing reactivity toward amines. Values are expressed as means and standard deviations of triplicates, shown in Figure S4.

FgrGalOx-Gal showed the greatest improvement in reactivity (Figure 2a), where the reaction peak temperature decreased by 34 °C; by comparison, AbPDH1-Gal showed only a slight increase in reactivity where the peak temperature decreased by 4 °C. This result is consistent with the expectation that aldehydes (the new functional group of FgrGalOx-Gal) will be more reactive than ketones (the new functional group of AbPDH1-Gal) toward amines. The impact of FgrGalOx on lactose reactivity toward HMDA was less dramatic, with FgrGalOx-Lac reacting at 110.7 °C, a reduction of less than 2 °C (Figure 2b). Reactions comprising AbPDH1-Lac and HMDA led to a comparatively small change in heat flow at a temperature close to other lactose cross-linkers (107.9 °C). Notably, a small peak at this temperature was also observed in reactions containing AbPDH1-Lac alone, suggesting reactivity with the molecule (Figure S5). The lack of AbPDH1-Lac reactivity toward HMDA is likely due to lactobionic acid L7 being the major product of lactose oxidation by AbPDH1. HMDA is not expected to react with lactobionic acid but instead undergoes an acid–base proton exchange that occurs spontaneously once regents are dissolved in water and is not detectable by this DSC protocol. Based on the DSC analyses, FgrGalOx-Gal was identified as the strongest candidate for use as a cross-linker of polyamines.

Confirmation of Imine Cross-Link Formation by XPS and ATR-FTIR

To verify imine cross-link formation following reactions between cross-linkers and HMDA, reactions were performed at 30, 65, 80, and 200 °C, and products were analyzed using XPS and FTIR. XPS and FTIR were selected as they are techniques that do not require solubilizing the products in water and so prevent the hydrolysis of the imine bonds formed during the reactions. Based on DSC experiments, the cross-linking reaction was the only reaction detected below 200 °C (Figures 2, S5); therefore, it is reasonable to assume the cross-linked products are stable up to at least 200 °C under the reaction conditions tested.

XPS was used to investigate the formation of imine bonds. Three main peaks can be confidently separated in N 1s spectra: the low binding peak at 399.0 eV is attributed to primary amines, the medium energy peak at 400.0 eV is attributed to imines and amides, and the high binding energy peak at around 401.1 eV is attributed to hydrogen-bonded and protonated primary amine bonds.42,43 Therefore, as imine cross-links are formed, the 400.0 eV peak is expected to increase, and correspondingly as amines are consumed, the 399.0 and 401.1 eV peaks are expected to decrease. XPS peak integrals correspond to atomic composition, so by normalizing across all measured peaks for the elements detected (in this case, carbon, nitrogen, and oxygen), one can calculate the atomic percent related to a specific atomic environment.

As seen with DSC, FgrGalOx increased the reactivity of galactose, with the FgrGalOx-Gal and HMDA reaction products showing a considerable increase in imine bond formation at 65 °C compared to that at 30 °C (Figure 3b); for unmodified galactose, there was no increase from the baseline 400.0 eV peak at 30 °C until the reaction temperature was increased to 200 °C (Figure 3a). For lactose, however, FgrGalOx had little effect. Like its unmodified counterpart, FgrGalOx-Lac only showed an increase in the 400.0 eV peak at 200 °C (Figure 3d,e). Similarly, AbPDH1 had different effects on galactose and lactose. In reactions with HMDA and AbPDH1-Gal, an increase in the 400.0 eV peak was observed at 80 °C, while for unmodified galactose the increase was only observed at 200 °C (Figure 3c,a). By contrast, in reactions with HMDA and AbPDH1-Lac, only a small change in the 400.0 eV peak was observed, even at 200 °C, demonstrating a loss of reactivity after the enzyme oxidation. Critically, the XPS results agreed with the reactivity trends observed by DSC and verified the reactions observed by DSC corresponded to imine bond formation.

Figure 3.

Figure 3

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XPS N 1s spectra with atomic percent for 400.0 eV peak noted for products of reactions of HMDA and cross-linkers Gal (a), FgrGalOx-Gal (b), AbPDH1-Gal (c), Lac (d), FgrGalOx-Lac (e), and AbPDH1-Lac (f) at 30, 65, 80, and 200 °C. Peaks fitted to data at 401.1 eV (protonated and H-bonded amines) are shown in blue, at 400.0 eV (imines and amides) are shown in purple, and at 399.0 eV (neutral amines) are shown in green. Raw data are reported in Table S1.

Similar to XPS analyses, ATR-FTIR analysis of reactions performed at 30, 65, 80, and 200 °C indicated temperature-dependent formation of imines, shown by the formation of the peak at 1561 cm–1 and the broad shoulder in the 1690–1650 cm–1 range, and the depletion of amines, shown by decreases in peaks at 3328, 3225, 3165, 1605, and 1043 cm–1, (Figure 4).44,45 Other peaks not necessarily related to imine bond formation are shown in Figure S7. In particular, and consistent with the XPS and DSC analyses, the highest reactivity with HMDA was observed using the FgrGalOx-Gal cross-linker, where the amine peaks at 1605 and 1043 cm–1 decreased and imine peaks at 1650 and 1561 cm–1 increased in reactions at 65 and 80 °C compared to reactions at 30 °C (Figure 4 b). Like unmodified lactose, FgrGalOx-Lac spectra did not show evidence of imine formation until the temperature reached 200 °C (Figure 4d,e). Reactions comprising HMDA and AbPDH1-Gal revealed an increase in imine peaks and a decrease in amine peaks at 80 °C compared to 30 and 65 °C, showing an earlier onset temperature compared to unmodified galactose while confirming the lower reactivity of AbPDH1-Gal compared to FgrGalOx-Gal (Figure 4a,c). Consistent with DSC and XPS analyses, ATR-FTIR analyses revealed a lack of reactivity between AbPDH1-Lac and HMDA at any tested temperature.

Figure 4.

Figure 4

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FTIR spectra for products of HMDA reactions with cross-linkers Gal (a), FgrGalOx-Gal (b), AbPDH1-Gal (c), Lac (d), FgrGalOx-Lac (e), and AbPDH1-Lac (f) performed at 30 °C (orange), 65 °C (yellow), 80 °C (green), and 200 °C (blue), with wavenumbers associated with amines and imines shown with green dotted lines (3328, 3225, 3165, 1605, and 1043 cm–1) and purple dashed lines (1650, 1561 cm–1), respectively.

Confirmation of Aldehyde Depletion and Imine Formation by 1H NMR

1H NMR was used to simultaneously investigate carbonyl depletion and imine formation in reactions with FgrGalOx-Gal, which was selected for its high reactivity as measured by DSC, XPS, and ATR-FTIR. Unlike the FTIR and XPS, NMR could not be conducted using the corresponding DSC products due to their low solubility in solvents for 1H NMR (i.e., D2O, DMSO-d6, and CDCl3). Instead, NMR reactions were conducted with n-butyl amine (n-BA); this compound was selected for having only one amine group to produce low molecular weight products soluble in DMSO-d6 (Figure 5).46−48 DMSO-d6 was used as a solvent as aldehydes can be hydrated in D2O to form gem-diols, making it difficult to observe aldehydes by NMR. Water (and D2O) can also hydrolyze imine bonds under acidic conditions, so DMSO-d6 was preferred to stabilize the imine bonds formed. Additionally, using DMSO-d6 as the solvent stabilizes the closed-ring conformation of the FgrGalOx-Gal products, preventing the C-1 aldehyde from forming and reacting with n-BA.

Figure 5.

Figure 5

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Reaction between galacto-hexodialdose G1 (major component of FgrGalOx-Gal) and n-BA with aldehyde protons H-6 in purple and to imine protons H-6’ in green. 1H NMR (600 MHz, C2D6OS) of reactions of 12.5 μmol of FgrGalOx-Gal with increasing μmol of n-BA, as indicated on the numbers on the left. 2,3-dichloronitrobenzene (peaks at 8.06, 8.01, and 7.64 ppm) was used as an internal standard (shown as IS).

The 1H NMR spectrum of FgrGalOx-Gal showed aldehyde H-6 singlet peaks at 9.49–9.63 ppm (Figure 5), in the range reported previously as aldehyde protons in galacto-hexodialdose G1.47 Adding increasing amounts (0–50 μmol) of n-BA to 12.5 μmol of FgrGalOx-Gal resulted in decreases in aldehyde H-6 peaks from 3 to 0 μmol and the introduction of an imine H-6’ peak at 8.48 ppm, which increased to 0.5 μmol with increasing n-BA. Although the setup of this reaction is different than those previously discussed, it shows simultaneous depletion of carbonyl groups and production of imines.

Proof of Concept Application: Carbohydrate-Based Cross-Linkers Assemble Polyamines

Different cross-linkers displayed varying ability to gel 5% (i.e., 50 mg/mL) solutions of polyallylamine (PAA) (Figure 6). The positive control, glutaraldehyde, immediately reacted with PAA solutions but did not generate a uniform gel product. The negative control, lactobionic acid, did not form a gel with PAA at any tested concentration, consistent with the hypothesis that carboxylic acids do not react with amines under the tested conditions to form covalent cross-links (Scheme 1 c).

Figure 6.

Figure 6

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Gelation of 5% PAA solutions with carbohydrate cross-linkers. The first instance of full gelation is boxed in blue.

FgrGalOx-Lac was most effective at gelling 5% PAA, where gels formed in 10 min. Although FgrGalOx-Gal also induced PAA gelation, the gel took several hours to form. Given that DSC cross-linking experiments identified FgrGalOx-Gal as most reactive toward HMDA, the faster gelation of PAA using FgrGalOx-Lac over FgrGalOx-Gal was unexpected. It is possible that the larger molecular weight of lactose encouraged interpolymer cross-link formation. Notably, the lower reactivity of carbohydrate cross-linkers compared to glutaraldehyde allowed them to be fully mixed into PAA solutions before reacting, generating more uniform gels.

Neither AbPDH1 cross-linkers formed gels with 5% PAA even after 6 days of incubation at room temperature. The lack of gelation by these cross-linkers can be attributed to the lower carbonyl content of these molecules (AbPDH1-Gal is 25% galactonic acid G7; AbPDH1-Lac is 62% lactobionic acid L7) and the lower reactivity of keto-groups in AbPDH1 cross-linkers compared to aldehydes present in FgrGalOx cross-linkers. As expected, untreated galactose and lactose did not gel at 5% PAA under the conditions tested. Notably, when increasing PAA concentration to 10% w/v, gelation of PAA by AbPDH1 cross-linkers was observed after 4.5 h (Figure S11). Gelation of 10% PAA by untreated galactose and lactose was also observed, but only after 6 days; this can be attributed to Maillard reactions whereby the C-1 lactone reacts with amine groups to form dicarbonyls that undergo a pair of imine-forming cross-linking reactions.49

Conclusions

The enzymatic conversion of underused carbohydrates to cross-linking molecules is especially relevant to food, biomedical, and sustainable packaging applications where fully biobased components are preferred. In this study, four carbohydrate cross-linkers were produced using FgrGalOx and AbPDH1. Most notably, FgrGalOx-Gal reduced the peak reaction temperature with HMDA by 34 °C compared to untreated galactose, demonstrating the beneficial impact of enzymatic oxidation on the cross-linking activity of carbohydrates with polyamines. FTIR and XPS analysis of corresponding reaction products confirmed imine bond formation and amine depletion in reactions performed at 65 and 80 °C. Although FgrGalOx-Gal cross-linkers showed the highest reactivity with HMDA, the gelation time of 5% PAA was the lowest when using FgrGalOx-Lac cross-linkers. The dependency of cross-linker performance on substrate and experimental method underscores the importance of including substrates with different molecular weights and degrees of substitution, as well as employing both analytical and physical techniques when evaluating the suitability of carbohydrate cross-linkers in new biobased materials. In addition to replacing PAA with bioderived polyamines and polyols, future studies will extend the enzyme application to create cross-linking molecules from other underused biomass fractions, including hemicelluloses from plant fiber.

Acknowledgments

The authors would like to acknowledge the contributions of the team of brilliant scientists they have had the pleasure of working with: Dr. Olanrewaju Raji, for running HPAEC-PAD samples; Dr. Robert Flick, for running HILIC-ESI-MS samples; Dr. Peter Brodersen, for running XPS samples; Dr. Thu Vuong, for assistance and instruction on protein production and purification; Spencer Imbrogno, whose research this study is built upon; Dr. Johanna Karppi, for providing the Pichia pastoris transformants to produce AbPDH1 and FgrGalOx; Professor Maija Tenkanen, for fruitful discussions and sound direction on AbPDH1 and FgrGalOx.

Glossary

Abbreviations

1H NMR

proton nuclear magnetic resonance spectroscopy

2,3-DCNB

2,3-dichloronitrobenzene

AbPDH1

pyranose dehydrogenase from Agaricus bisporus

ABTS

2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt

ATR-FTIR

attenuated total reflectance Fourier-transform infrared spectroscopy

BEH HILIC

bridged ethylene hybrid hydrophobic interaction liquid chromatography

BMGY

buffered glycerol complex media

BQ

benzoquinone

DSC

differential scanning calorimetry

FgrGalOx

galactose oxidase from Fusarium graminearum

HMDA

hexamethylenediamine

HPAEC-PAD

high-performance anion-exchange chromatography with pulsed amperometric detection

HPLC

high-performance liquid chromatography

n-BA

n-butylamine

PAA

polyallylamine

SDS-PAGE

sodium dodecyl sulfate polyacrylamide gel electrophoresis

YPD

yeast peptone dextrose media

HILIC-ESI-MS

hydrophobic interaction chromatography coupled with electrospray ionization mass spectroscopy

XPS

X-ray photoelectron spectroscopy

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biomac.4c00457.

  • Lactose reaction schemes and supplemental data from HPAEC-PAD, HILIC-ESI-MS, DSC, XPS, 1H NMR, COSY 1H NMR, and PAA gelation experiments (PDF)

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. E.R.M. conceived and coordinated the study. O.M.M. designed the experiments and performed the protein production and purification; the enzymatic oxidations of galactose and lactose; the analysis of enzyme-oxidized products; the cross-linker reactivity testing with polyallylamine; the preparation of NMR, XPS, and FTIR samples; and the data analysis. O.M.M. and S.H.H. performed the DSC and FTIR. S.H.H. performed the NMR analysis.

This project was supported by the Genome Canada and Ontario Genomics project Synbiomics (Project Number 10405), the Natural Sciences and Engineering Research Council of Canada (NSERC) CREATE for BioZone (Project Number 528163), the NSERC Alliance program (BioMax Project Number 570676), and the Faculty of Applied Science and Engineering (University of Toronto) Dean Strategic Funding to the Low Carbon Renewable Materials Centre.

The authors declare no competing financial interest.

Supplementary Material

bm4c00457_si_001.pdf (6.2MB, pdf)

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bm4c00457_si_001.pdf (6.2MB, pdf)

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