Water-soluble chitosan derivatives and pH-responsive hydrogels by selective C-6 oxidation mediated by TEMPO-laccase redox system

Chitosan is a polysaccharide with recognized antioxidant, antimicrobial and wound healing activities. However, this polymer is soluble only in dilute acidic solutions, which restricts much of its applications.

Carbohydrate Polymers 186 (2018) 299–309 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Water-soluble chitosan derivatives and pH-responsive hydrogels by selective C-6 oxidation mediated by TEMPO-laccase redox system T ⁎ Suse Botelho da Silvaa,b, , Malgorzata Krolickaa,c, Lambertus A.M van den Broeka, ⁎ August E Frissena, Carmen Gabriela Boeriua, a Wageningen Food & Biobased Research, Department Biobased Products, Bornse Weilanden 9, 6708 WG Wageningen, The Netherlands Polytechnic School, UNISINOS University, Av Unisinos 950, 93022-000 São Leopoldo, RS, Brazil c Wageningen University, Bioprocess Engineering Group, Droevendaalsesteeg 1, 6708 PD, Wageningen, The Netherlands b A R T I C L E I N F O A B S T R A C T Keywords: Chitosan Oxidation 2,2,6,6-Tetramethylpiperidinoxyl radical (TEMPO) Laccase pH-responsive hydrogel Soluble chitosan Chitosan is a polysaccharide with recognized antioxidant, antimicrobial and wound healing activities However, this polymer is soluble only in dilute acidic solutions, which restricts much of its applications A usual strategy for improving the functionality of polysaccharides is the selective oxidation mediated by 2,2,6,6-tetra-methyl-1piperidinidyloxy (TEMPO) using laccase as a co-oxidant In this work, the TEMPO-laccase redox system was used for the first time to selectively oxidize chitosan in order to produce tailored derivatives The reaction was performed at pH 4.5 under continuous air supply and the oxidized products were characterized structurally and functionally The TEMPO-laccase oxidation successfully added aldehyde and carboxylate groups to chitosan structure resulting in derivatives with oxidation between and 7% These derivatives showed increased solubility and decreased viscosity in solution If chitosan is dissolved in diluted hydrochloric acid prior to TEMPOlaccase oxidation, a crosslinked chitosan derivative was produced, which was able to form a pH-responsive hydrogel Introduction Polysaccharides obtained from renewable sources or agro-industrial waste streams play an important role in the context of a biobased and sustainable economy, or bio-economy (Persin et al., 2011) Chitin is the most abundant polysaccharide in nature after cellulose, it is found mainly in the exoskeleton of crustaceans and insects, but also in fungi and algae Industrially, chitin is obtained from crab and shrimp processing waste in countries such as USA, Japan and India (Kardas et al., 2013), and also Brazil The partial or fully deacetylation of N-acetylglucosamine moieties of chitin leads to formation of chitosan, also considered as a natural biopolymer (Luo & Wang, 2013) Therefore, chitin and chitosan are copolymers with similar structure, but in chitin predominates 2-acetamido-2-deoxy-D-glucopyranose units and in chitosan, 2-amino-2-deoxy-D-glucopyranose units are more frequent Chitin is insoluble in most common solvents, while chitosan has a limited solubility, being soluble for example in acidic aqueous solutions (Mourya, Inamdar, & Choudhari, 2011) Chitosan is recognized as a nontoxic, biodegradable and biocompatible polymer, whose properties are being exploited in industrial and technological applications since the last century These applications ⁎ cover areas such as food, pharmaceutical, textile, packaging, cosmetics and agriculture, and are often justified by the antimicrobial activity of this polymer, but also for its antioxidant activity (Kong, Chen, Xing, & Park, 2010; Luo & Wang, 2013) The poor solubility of chitosan, however, restricts many of its potential applications One of the strategies to improve the solubility of chitosan and other polysaccharides is to modify the structure of the molecule by the addition of hydrophilic functional groups Chitosan is a versatile molecule that can be modified by different methods mainly due to its structure, which contains several hydroxyls (primary hydroxyl at C-6 and secondary hydroxyl at C-3), and highly reactive amino groups (C-2) (Luo & Wang, 2013; Prashanth & Tharanathan, 2007) Direct oxidation of the hydroxyl groups is possible by the introduction of carboxyl and carbonyl groups at C-6 (Bragd, Besemer, & van Bekkum, 2000) In addition to increasing solubility, the insertion of these new functional groups could also convert chitosan into a potential crosslinking structure, which newly inserted aldehyde groups would hypothetically crosslink with existing animo groups Tan, Chu, Payne, and Marra (2009) reported a development of a composite hydrogel derived from succinyl-chitosan and oxidized hyaluronic acid stabilized with this kind of crosslinks In the last decades, the catalytic oxidation of carbohydrates using Corresponding authors at: Wageningen Food & Biobased Research, Bornse Weilanden 9, 6708 WG Wageningen, The Netherlands E-mail addresses: susebs@unisinos.br (S Botelho da Silva), carmen.boeriu@wur.nl (C.G Boeriu) https://doi.org/10.1016/j.carbpol.2018.01.050 Received 18 August 2017; Received in revised form January 2018; Accepted 16 January 2018 Available online 03 February 2018 0144-8617/ © 2018 The Authors Published by Elsevier Ltd This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/) Carbohydrate Polymers 186 (2018) 299–309 S Botelho da Silva et al Table Synthesis conditions of modified chitosan products, deacetylation and oxidation degree, and aldehyde and carboxylate contents Sample Acid1 Product P1 P2 P3 P4 HAc2 HAc HAc HCl Product Product Product Product TEMPO (g/100 g chitosan) Laccase (U/g chitosan) Ratio Laccase/ TEMPO (U/g) DD%3 10 10 10 70 7 700 700 70 70 83.6 79.8 83.4 82.7 a a a a OD%4 1.1 7.0 4.6 4.0 a b c c Aldehyde content (mmol/kg chitosan) Carboxylate content (mmol/kg chitosan) 50.9 a 385 b 264 c 227 c 13.5 28.0 10.4 10.8 a b a a a, b, c: The same letter in each column are not significantly different at the 5% level (Tukey’s test) 0.1 M acid diluted solution used to dissolve chitosan prior to TEMPO-laccase oxidation HAc is acetic acid DD is deacetylation degree OD is oxidation degree using the Mark–Houwink–Sakurada equation (Kasaai, 2007) and 1H NMR spectroscopy (Hirai, Odani, & Nakajima, 1991), respectively Laccase from Trametes versicolor, 2,2,6,6-tetramethylpiperidinoxyl radical (TEMPO), 3-ethylbenzothiazoline-6-sulphonic acid (ABTS), 2,5dihydroxybenzoic acid (DHB) and other chemicals were purchased from Sigma-Aldrich Endochitinase from Myceliophthora thermophila C1 was a kind gift from DuPont Industrial Biosciences (Wageningen, the Netherlands) and was purified and characterized in our lab (Krolicka et al., 2018) the stable nitroxyl radical 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) became one of the most promising methods for the conversion processes of polysaccharides into polyuronic acids (Coseri et al., 2013) This method is appropriate for the selective oxidation of primary alcohols to aldehydes or carboxylic acids groups It is very effective for the functionalization of high molecular weight polysaccharides, providing a material with increased solubility (Bragd, van Bekkum, & Besemer, 2004) In classical TEMPO-mediated oxidations, well discussed by Bragd et al (2004), sodium hypochlorite and bromide are used as TEMPO regenerating agents in situ The oxidation of TEMPO by primary oxidants (NaOCl/NaBr) generates the nitrosonium ion, which is the actual oxidation agent responsible for the conversion of alcohols in aldehydes or carboxyl groups Although this process is widely applicable, there are some concerns about the environmental impact because of the use of halogenated reagents (NaOCl and NaBr) and organic (co)-solvents (Sheldon & Arends, 2004) An alternative would be the use of oxidative enzymes to regenerate TEMPO, such as peroxidases and laccases, instead of NaOCl and NaBr In particular, laccases have been identified as promising ecofriendly oxidants, because they catalyze the oxidation of TEMPO and other mediators via reduction of O2 producing only H2O (DíazRodríguez, Martínez-Montero, Lavandera, Gotor, & Gotor-Fernández, 2014) The chemo-enzymatic approach of TEMPO oxidation has been previously used for cellulose and starch (Jaušovec, Vogrinčič, & Kokol, 2015; Kierulff, 2000; Mathew & Adlercreutz, 2009; Patel, Ludwig, Haltrich, Rosenau, & Potthast, 2011; Xu, Song, Qian, & Shen, 2013) Only recently, Pei, Yin, Shen, Bu, and Zhang (2016) used the same approach to oxidize a chitooligomer, but to the best of our knowledge, so far the TEMPO-laccase system had never been reported for the oxidation of the chitosan polymer Our hypothesis is that the TEMPO-laccase system can be used to promote the regioselective oxidation of chitosan in order to produce tailored derivatives with new enhanced properties Therefore, the objective of this work was to evaluate the TEMPO-laccase oxidation applied to chitosan polymers and investigate the structural and functional characteristics of the synthesized chitosan derivatives produced These derivatives were structurally characterized by FT-IR and NMR spectroscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis Furthermore, solubility, rheological behavior, thermal stability, and gel morphology of chitosan derivatives were also investigated in order to provide insights into the reaction mechanism and to demonstrate the potential for new applications 2.2 Laccase activity assay Laccase activity was determined using ABTS as substrate at 20 °C The assay reaction contained 200 μL enzyme solution, 100 μL mM ABTS and 700 μL 100 mM sodium acetate buffer pH 4.5 The oxidation of ABTS was monitored by the increase in absorbance at 436 nm (ε436 = 2.9 × 104 M−1 cm−1) (Niku-Paavola, Karhunen, Salola, & Raunio, 1988) The enzyme activity was expressed in Units (U), defined as μmol of ABTS oxidized per under the above conditions 2.3 TEMPO-laccase oxidation of chitosan Chitosan was dissolved overnight in 0.1 M acetic acid or 0.1 N HCl under stirring at room temperature After complete dissolution, the pH was adjusted to 4.5 using a NaOH solution Modification of chitosan was performed using a procedure based on the TEMPO-laccase oxidation of alcohols (Arends, Li, & Sheldon, 2006) and cellulose (Mathew & Adlercreutz, 2009; Xu et al., 2013) TEMPO was added into 0.1 M sodium acetate buffer (pH 4.5) and the mixture was stirred for a few minutes until it was dissolved The chitosan solution was added, and compressed air was applied by a fritted glass sparger placed inside the mixture Finally, laccase in 0.1 M sodium acetate buffer (pH 4.5) was added to the air-saturated mixture The reaction mixture containing 1.5% (w/v) chitosan was treated with different TEMPO-laccase concentrations, namely TEMPO 1–10% (w/w of chitosan), and laccase U–70 U/g chitosan, in different combinations (Table 1) The experiments were carried out at 20 °C for 18 h, under stirring at 750 rpm and continuous air flow The dissolved oxygen (DO) in the reaction mixture was continuously measured by a DO Meter (WTW InoLab, Oxi 7310, Germany) Initial reaction rates were calculated in terms of initial consumption rate of dissolved oxygen, by linear regression of dissolved oxygen data On completion of the reaction time, the pH of the reaction mixture was adjusted to 7.0 with M NaOH, and the modified chitosan product was precipitated by adding ethanol (5:1 v/v) ratio to the volume of the solution (Huang et al., 2013) The precipitated solid material was recovered by centrifugation (4000 × g, min) and washed three times with ethanol Finally, the product was dried under vacuum at 40 °C for 48 h Material and methods 2.1 Materials Commercial chitosan (Flonac C) was obtained from Nippon Suisan Kaisha, Ltd (Japan) The viscosity-average molecular weight (Mv) of 93 kDa and the deacetylation degree (DD) of 82.8 % were determined 300 Carbohydrate Polymers 186 (2018) 299–309 S Botelho da Silva et al of absolute viscosity are the average of three replicate experiments The viscosity-average molecular weight (Mv) of untreated chitosan was determined from the Mark–Houwink–Sakurada (MHS) Eq (1) using the rheological data of chitosan in solution The intrinsic viscosity (η) was obtained from the initial slope of the dependence between the natural logarithm of relative viscosity and polymer concentration, following the approach developed by Wolf (2007) The constants of the equation, k and a, were calculated considering the degree of acetylation of chitosan, pH, and ionic strength of buffer solution according Kasaai (2007) 2.4 Characterization of oxidized chitosan derivatives 2.4.1 FT-IR spectroscopy Fourier Transform Infrared (FT-IR) spectroscopy of the native and modified chitosan was carried out in attenuated total reflectance (ATR) mode on a Varian Scimitar 1000 FT-IR spectrometer Spectra were collected over the range 4000–650 cm−1 with a resolution of cm−1 and with 128 co-added scans 2.4.2 NMR spectroscopy NMR spectra of the native and modified chitosan were recorded on a Bruker Avance II 400 MHz spectrometer For 1H and 13C NMR spectra, samples were dissolved in 0.1 M DCl The deacetylation degree of samples was determined by 1H NMR spectroscopy according to Hirai et al (1991) η = kMv a (1) 2.4.6 Aqueous solubility The aqueous solubility was determined at pH 7.4 and therefore mL 0.1 M sodium phosphate buffer (pH 7.4) was added to 10 mg sample The same procedure as described by Azevedo, Santhana Mariappan, and Kumar (2012) was followed The suspension was stirred for 48 h at room temperature and subsequently, undissolved solids were separated by centrifugation Aliquots (0.8 mL) of the supernatant were dried under vacuum at 50 °C for 24 h The solubility was calculated considering the mass of solids in solution minus the mass of buffer solids determined in the control 2.4.3 Determination of carboxyl and aldehyde content and degree of oxidation The content of the aldehyde groups of the samples was determined using the spectrophotometric method described by Jaušovec et al (2015) Briefly, chitosan samples reacted with 2,3,5-triphenyltetrazoliumchloride in the presence of KOH for at 80 °C Methanol was added to the reaction mixture to extract the reddish-crystals of triphenyltetrazolium formazan that was recorded spectrophotometrically at 482 nm The amount of aldehyde groups was calculated from the calibration curve prepared with D-glucosamine as substrate The method for determination of uronic acids by Filisetti-Cozzi and Carpita (1991) was used to quantify the amount of carboxyl groups In this procedure, samples were mixed with sulfamic/K-sulfamate and hereafter treated with H2SO4 and sodium tetraborate, in order to generate products that react with m-hydroxybiphenyl The absorbance was measured at 525 nm and a calibration curve of galacturonic acid was used as a standard to calculate the amount of carboxyl groups The oxidation degree (OD) was calculated from the sum of the number of moles of aldehyde and carboxylate groups 2.4.7 Hydrogel morphology The hydrogel morphology was characterized by scanning electron microscopy (SEM) Initially, the samples were prepared by dissolving appropriate amounts of oxidized dried chitosan in 0.05 M potassium biphthalate buffer (pH 4.0) To induce gel formation, the pH of the solution was increased up to 7.0 using 0.025 M sodium bicarbonate/ carbonate buffer (pH 10.0), and the volume was adjusted with 0.04 M sodium-potassium phosphate buffer (pH 7.0) to reach a concentration of 3% (w/v) Hydrogels were frozen at −80 °C and subsequently lyophilized for 24 h Samples were gold-coated prior to viewing at 15.00 KV in a Zeiss EVO MA15 scanning electron microscope (Carl Zeiss SMT, Germany) 2.4.8 Thermal analysis Thermal analysis (TGA-DTG) of untreated chitosan and modified products was performed using a thermogravimetric analyzer model TGA Discovery (TA Instruments, USA) The measurements were performed under nitrogen atmosphere from 25 to 600 °C at a heating rate of 10 °C/min 2.4.4 Fragment analysis from enzymatically depolymerized (modified) chitosan using MALDI-TOF MS To obtain low molecular mass fragments from (modified) chitosans, the polymers were enzymatically hydrolyzed using an endo-chitinase with chitosanase activity (Krolicka et al., 2018) Samples were incubated at 50 °C for 24 h under gentle stirring The reaction was terminated by heating at 96 °C for 10 min, and samples were stored at °C Fragments were identified using MALDI-TOF-MS The mass spectra were recorded on a Bruker UltraFlextreme (Bruker Daltonics, Germany) in the reflective mode and positive ions were examined The instrument was calibrated using maltodextrins with known molecular masses and the matrix solution consisted of 10 mg mL−1 DHB in 50% (v/v) acetonitrile in milliQ water Prior to analysis, samples were desalted by adding a small amount of Dowex AG50W-X8 resin in the hydrogen form (Bio-Rad, Hercules, CA, USA) to 50 μL sample solution Hereafter, the suspension was centrifuged and μL of the supernatant was transferred to μL matrix solution 0.5 μL of the mixture was added to the matrix plate and dried under a stream of dry air The lowest laser intensity required to obtain a good quality spectrum was used and 10 times 50 laser shots randomly obtained from the sample were accumulated Measurements were performed in the m/z 300–3000 range 2.5 Statistical analysis A one-way analysis of variance (ANOVA) was performed on experimental data Significant differences between means were determined by Tukey’s test at 5% probability level Results and discussion 3.1 Oxidation of chitosan using the TEMPO-laccase catalytic redox system In order to perform the oxidation of chitosan, we used the TEMPOlaccase catalytic redox system, which is an efficient catalytic system for oxidation of aliphatic and aromatic alcohols (Arends et al., 2006) and polyhydroxy polymers like cellulose and starch (Jaušovec et al., 2015; Mathew & Adlercreutz, 2009) In this process (Scheme 1), the primary hydroxyl of C-6 chitosan (R-CH2-OH) is selectively oxidized by TEMPO radicals, leading mainly to the formation of carbonyl groups, but also to the formation of carboxyl groups TEMPO is continuously regenerated in situ by laccase to form the oxoammonium ion, which oxidizes the alcohol groups to aldehyde and is reduced by generating the N-hydroxy TEMPO derivative Finally, the reduced TEMPO is reoxidized by laccase due to the reduction of O2 in H2O The alcohol oxidation mediated by 2.4.5 Rheology and viscosity-average molecular weight Rheological measurements of untreated and modified chitosan in 0.3 M CH3COOH/0.2 M CH3COONa aqueous solution (pH = 4.6) were carried out at 25 ± 0.1 °C using a rotational rheometer in couette geometry (Brookfield Model DV II+ Pro and Rheocalc software, Brookfield Engineering Laboratories, Inc., USA) Rheological parameters were calculated from shear stress versus shear rate data Values 301 Carbohydrate Polymers 186 (2018) 299–309 S Botelho da Silva et al Scheme Oxidation of chitosan mediated by TEMPO-laccase catalytic redox system in presence of oxygen, adapted from Bragd et al (2004) TEMPO-laccase During the initial phase of the reaction, a fast decrease in dissolved oxygen (DO) was observed, and consequently, the aldehyde content rapidly increased in the first minutes (Fig 1b) Hereafter, the oxidation rate slightly decreased for the next h until it reached a plateau (Fig 1a) The initial rate of oxygen consumption (measured for the first min) was 0.85 mmol kg−1 min−1 The decrease in DO is directly related to the demand of oxygen for TEMPO regeneration As more TEMPO is reduced for chitosan oxidation, laccase needs more oxygen for the reoxidation of TEMPO (Scheme 1) The point of inflection in the curve (minimum DO) occurs around 45 and is related to the maximum number of active sites for the regeneration of TEMPO (maximum oxygen demand) The subsequent increase in DO results from a decrease in TEMPO regeneration We hypothesize that the fast replacement of the hydroxyl groups by carbonyl groups and the eventual formation of hemiacetals make the structure less accessible to TEMPO, thereby reducing the formation rate of aldehydes In addition, it is possible that the DO can be decreased due to the degradation of laccase in presence of oxoammonium salts, as reported by Arends et al (2006) the TEMPO-laccase system is discussed by Arends et al (2006) In our design, chitosan was dissolved overnight in diluted acetic acid or hydrochloric acid Reactions were performed at different ratios of TEMPO-chitosan and TEMPO-laccase, in order to optimise the conditions and to obtain products with different degrees of modification In Table 1, an overview of the different conditions studied is shown The combined use of laccase and TEMPO with continuously air supply successfully catalyzed the oxidation of chitosan at 20 °C and pH 4.5 Fig shows an example of the consumption of oxygen and formation of aldehyde groups during the time course of oxidation mediated by 3.2 Structure and chemical characterization of oxidized chitosan derivatives 3.2.1 Spectroscopic characterisation FT-IR spectra from untreated chitosan and derived products are shown in Fig The untreated chitosan reveals characteristics bands at 1645 cm−1 and 1583 cm−1 (Kumirska et al., 2010) for the stretching and bending vibrations of amide I and amide II, respectively The spectrum of Product (low TEMPO and low laccase) is quite similar to the FT-IR spectrum of untreated chitosan, as expected due to the very low level of substitution of this product The band around 2873 cm−1 (CeH stretching, in eCH2 groups) was present for untreated chitosan and was decreased for derivative products, particularly for Product This indicates that C-6 primary hydroxyls were converted to C-6 carbonyl and carboxylate groups The FT-IR spectrum of Product indicates the presence of carboxyl groups, as two new absorption bands appeared at 1593 cm−1 and 1404 cm−1, corresponding to symmetrical and asymmetrical stretching vibration of COO− A similar pattern was also reported by Pierre et al (2013) for chitosan oxidation using the TEMPO/NaOCl- NaBr system Fig Time course of dissolved oxygen (blue line) and formation of aldehyde groups (red squares) during oxidation of chitosan (Product 3) mediated by the TEMPO-laccase system a) is full time course; b) Initial time course (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 302 Carbohydrate Polymers 186 (2018) 299–309 S Botelho da Silva et al Fig FT-IR spectra of (a) chitosan, (b) Product 1, (c) Product 3, (d) Product and (e) Product H NMR results In Fig 3b, the resonance due to the aldehyde proton (–CHO) appears around 9.1 ppm and the imine proton (eCH]Ne) from Schiff’s base is clearly detected at 8.08 in the 1H NMR of Product Azevedo et al (2012) also reported the formation of Schiff’s base detected by 1H NMR in aldehyde-functionalized chitosan obtained by oxidation with nitrogen oxides To the best of our knowledge, this is the first time that the aldehydeand carboxylate-functionalization of chitosan by TEMPO-laccase oxidation is demonstrated Previously, only the chemical approach using TEMPO with halogenated reagents as primary oxidants has been reported (Bordenave, Grelier, & Coma, 2008; Kato, Kaminaga, Matsuo, & Isogai, 2004; Pierre et al., 2013) For Products 1, and these bands were hardly visible due to the low concentration of COO− groups These new bands can be also attributed to acetates (CH3COO−) bonded to protonated amino groups, which comes from the acetic acid used to dissolve chitosan prior to the oxidation reaction to obtain Products 1, and This hypothesis is supported by Mikhailov, Tuchkov, Lazarev, and Kulish (2014) and also by Nunthanid et al (2004) that reported a strong peak around 1550–1600 cm−1 and a weak peak near 1400 cm−1 in the FTIR spectrum of chitosan acetate The typical signal of aldehydic carbonyl groups around 1720 cm−1 was not detected since the aldehydes interact with neighboring alcohol and amino groups The new band at 791 cm−1 in Product can be attributed to hemiacetals formed by the interaction between aldehydes and alcohol groups, while some changes in the wavenumber region of 1550–1650 cm−1 mainly present in Products and are related to the formation of imine bonds between aldehyde and amino groups (Schiff bases) DiFlavio et al (2007) also reported the difficulty to detect the aldehyde band in FT-IR spectra of regenerated cellulose treated with TEMPO–NaBr–NaClO and cross-linked with polyvinylamine This observation was explained by the interaction between aldehydes and alcohols and aldehydes and amines Further characterization of chitosan derivatives was performed by H and 13C NMR (Fig 3) The 13C NMR spectra of untreated chitosan as a control (Fig 3a) showed the typical signals of both GlcN and GlcNAc residues, i.e C2 (55.7 ppm), C6 (59.9 ppm), C3 (70.0 ppm), C5 (74.7 ppm), C4 (76.23 ppm), C1 (97.4 ppm) and the N-acetyl group of GlcNAc (22.0 ppm), as reported previously by (Kumirska et al., 2010) The spectrum of Product (Fig 3a) showed a reduction in the signal of C6 and the appearance of additional peaks at ∼165–180 ppm suggesting the formation of a C6 oxidized chitosan derivative The new carbon resonance peaks appeared at 170.9 ppm and 174.9 ppm and are attributed to the ion carboxylate carbon (eCOO−) and to the aldehyde carbon (eCHO), respectively The signal detected at 165.9 ppm is typical of imine carbon (eC]Ne) due to the formation of Schiff’s base between the aldehyde and amine groups (Fig 3a) The reduction in C6 resonance and the signal corresponding to (eCHO) were also observed in Products 1, and (data not showed), however, the signal for (eCOO) was not evident probably due to less drastic reaction conditions The aldehyde-functionalization of chitosan by the TEMPO-laccase system as well as the formation of Schiff’s bases were also supported by 3.2.2 Degree of oxidation and aldehyde and carboxylate contents The oxidation degree (OD) and the carboxylate and aldehyde contents of modified chitosan products are shown in Table and support the NMR and FT-IR analysis The TEMPO-laccase oxidation introduced predominantly carbonyl groups and low amounts of carboxyl functions in chitosan High carbonyl-carboxyl ratios are typical for TEMPO-laccase oxidation and it is related to the forming of hemiacetal linkages between aldehydes and hydroxyl groups, which prevent further oxidation from carbonyl to carboxyl Similar results were reported by Jaušovec et al (2015), Mathew & Adlercreutz (2009) and Patel et al (2011), that used TEMPO-laccase to oxidize cellulose and potato starch The concentration of TEMPO and laccase used in the reaction significantly affected the degree of oxidation and the content of aldehydes and carboxyl groups Comparing Product with Product 2, a 10-fold higher TEMPO and laccase (same laccase-TEMPO ratio) increased 7fold the aldehyde content and the oxidation degree whereas, the carboxylate content doubled (Table 1) If the TEMPO concentration was increased 10-fold than the laccase-TEMPO ratio did not increase (comparing P1 with P3 and P4), the aldehyde content increases only 4fold and the carboxylate content remained statistically the same (Table 1) These results show the effect of fast TEMPO regeneration in TEMPO-laccase oxidation, mainly on the production of carboxylate, taken into account the range of concentrations tested Higher TEMPO concentration obviously demands more laccase for TEMPO regeneration Products and were obtained with the same TEMPO concentration as Product although with a lower laccase-TEMPO ratio, so a lower number of TEMPO regeneration sites was available As the regeneration of TEMPO was slower for Products and 4, less aldehyde 303 Carbohydrate Polymers 186 (2018) 299–309 S Botelho da Silva et al Fig 13 C NMR (a) and 1H NMR (b) spectra from untreated chitosan (DD = 82.8%) and from modified chitosan (Product 2) obtained by TEMPO-laccase selective oxidation of C6-carbonyl and C6-carboxyl groups along the polymer chain, and the random formation of aldehyde and carboxyl clusters, respectively Differences were observed between the type and substitution pattern of the enzyme-resistant oligomers obtained from the different Products, which is mainly related to the concentration of TEMPO and laccase used, as discussed above These are: has been produced and probably more hemiacetals were formed preventing the carboxylate groups formation Similar effects of TEMPO and laccase concentrations are also reported for cellulose by Jaušovec et al (2015) and Xu et al (2013) 3.2.3 Substitution pattern along the polymer chain To determine the distribution of the aldehyde and carboxyl groups along the polysaccharide chain, the modified chitosan products were subjected to enzymatic depolymerization and subsequently mass analysis of the degradation products For degradation of chitosan and the oxidized chitosan derivatives, a thermostable chitinase from Myceliophthora thermophila C1 (Chi1), able to cleave the glycosidic linkages GlcNAc-GlcNAc and GlcNAc-GlcN in chitin and chitosan, has been used The chitinase hydrolyzes the glycosidic linkages in a defined way, but the introduction of carbonyl and carboxyl groups on the polysaccharide chain sterically hinders the action of the enzyme (Krolicka et al., 2018) Thus, in this way enzyme-resistant oligosaccharides are released Identification of these enzyme-resistant oligosaccharides gives information about the substitution pattern of oxidized groups along the polymer chain MALDI-TOF-MS analysis of the chitosan and oxidized chitosan hydrolysates identified homo- and hetero-oligomers consisting of GlcNAc and GlcN units with low polymerization degree (DP), namely dimers (DP2) and trimers (DP3), as shown in Table The main unsubstituted chitosan oligosaccharides identified are (GlcN)2, (GlcNAc)2, (GlcNAc,GlcN), (GlcNAc)3 and ((GlcNAc)2,GlcN) On the other hand, the mass spectra of the digests of oxidized chitosan products contained in addition a diversity of homo- and hetero-oligomers consisting of GlcNAc and GlcN units with DP ranging from two to five, with aldehyde and carboxyl substitution ranging from one to four (Table 2) Surprisingly, all oxidized oligosaccharides contained either carbonyl or carboxyl groups; oligomers containing both carbonyl and carboxyl substitution were not identified The formation of larger oligosaccharides with one or multiple substitutions suggests a heterogeneous distribution • Low TEMPO, high enzyme (Product 1): low degree of oxidation, short oligomers (DP2, DP3), low carbonyl/carboxyl ratio • High TEMPO (10% wt vs chitosan) (Products 2, 3, 4): high degree of • oxidation, larger oligomers, high carbonyl/carboxyl ratio, random clusters of CHO and COO− substituents In addition, for Product low COO−, CHO clusters, but only one short COO− oxidized oligomer (GlcNAc, GlcN) was identified, indicating a homogeneous distribution of COOH along the chain The differences in the substitution pattern between Products and seems also to be related to the acid used to dissolve chitosan before the oxidation reaction, since the TEMPO and laccase concentration used in both reactions were the same (Table 1) According to Thevarajah, Bulanadi, Wagner, Gaborieau, and Castignolles (2016), the extent of chitosan dissolution in HCl solution is higher than in acetic acid solution, however, HCl can induce additional deacetylation and a higher depolymerization than acetic acid The protonation of NH2 is also higher with HCl than with acetic acid (Rinaudo, Pavlov, & Desbrières, 1999) These effects added to the formation of acetates in chitosan by dissolution in acetic acid are sufficient to induce different changes in the chain conformation depending on the used solvent Therefore, the regions susceptible to the attack of TEMPO during oxidation reaction would be different for Products and 4, as result of the different conformation of the chain in solution This would explain the difference in substitution pattern observed for the two products 304 Carbohydrate Polymers 186 (2018) 299–309 S Botelho da Silva et al Table MALDI-TOF-MS identification of chitosan fragments obtained after enzymatic hydrolysis with chitinase Chi1 from Myceliophthora thermophila C1 Ion composition Type of adducts Untreated Chitosan Product Product (GlcN)2 GlcNAc,GlcN (GlcNAc)2 (GlcNAc)2 (GlcNAc)2,GlcN (GlcNAc)3 GlcNAc,GlcN GlcNAc,GlcN (GlcNAc)2 (GlcNAc)2,GlcN GlcNAc, (GlcN)3 (GlcN)2 (GlcN)2 (GlcN)2 (GlcN)2 (GlcN)2 (GlcN)2 (GlcNAc)2,GlcN (GlcNAc)2,GlcN (GlcNAc)2,GlcN (GlcNAc)2,GlcN GlcNAc, (GlcN)2 (GlcNAc)3 (GlcNAc)3 (GlcN)4 GlcNAc, (GlcN)4 GlcNAc, (GlcN)4 [M+H]+ [M+Na]+ [M+H]+ [M+Na]+ [M+Na]+ [M+K]+ [M(COOH)+H]+ [M(COOH)2+K]+ [M(CHO)2+H]+ [M(COOH)+K]+ [M(CHO)4+H]+ [M(CHO)+H]+ [M(CHO)+Na]+ [M(CHO)2+K]+ [M(COOH)+K]+ [M(COOH)2+Na]+ [M(COOH)2+K]+ [M(CHO)+H]+ [M(CHO) + K]+ [M(COOH)+Na]+ [M(COOH)+K]+ [M(COOH)+Na]+ [M(CHO)3+Na]+ [M(COOH)3+Na]+ [M(CHO)4+K]+ [M(COOH)+Na]+ [M(CHO)4+Na]+ x x x x x Product Product x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x M (COOH), oxidized fragment: +14 Da mass shift, indicating a carboxyl group M (CHO), oxidized fragment: − Da mass shift, indicating an aldehyde group 3.3 Rheology of modified chitosan products in solution 3.4 Aqueous solubility and pH-responsive hydrogel Fig shows the rheological characterization of untreated and modified chitosan products with highest oxidation degree in 0.3 M CH3COOH/0.2 M CH3COONa aqueous solution (pH = 4.6) In acid pH, and at concentrations of less than 10 mg/mL, untreated and modified chitosans solutions showed a Newtonian behavior Modified products showed an expressive decrease in dynamic viscosity compared with the same concentration of untreated chitosan (Fig 4) This change is related to the modification of the chitosan structure by the introduction of hydrophilic carbonyl and carboxyl groups since the rheological parameters are strongly affected by the interaction between polymer and solvent Chitosans that produce low viscosity solutions are highly desirable for some biomedical applications such as blood thinning, cholesterol-lowering, and for application involving anti-oxidant, and antimicrobial properties (Lim, Lee, Israelachvili, Jho, & Hwang, 2015) Chitosan has typically poor solubility at physiological pH, it is soluble in aqueous acid diluted solutions and this limits several of its potential applications The TEMPO-laccase oxidation overcomes this problem since Products and show an increase in solubility in sodium phosphate buffer at pH 7.4 (Fig 5) As can be seen from the data in Table and Fig 5, the oxidation degree and the carboxylate content are important factors for improving the solubility of the chitosan derivatives Within the concentrations tested, an approximately 4% OD and 10 mmol/kg of carboxylate seem to be the minimum values to observe an increase in the solubility of the modified chitosan For Product 4, the oxidation of chitosan did not increase the solubility, in contrast, it produced a cross-linked structure capable of forming a hydrogel at pH 7.4 Product was produced using identical conditions as for Product 3, except for the use of 0.1 M HCl to dissolve chitosan instead of 0.1 M acetic acid before the oxidation reaction This caused a different substitution pattern of the aldehyde and carboxyl Fig Dynamic viscosity for solutions of untreated chitosan and oxidized products at different concentrations in 0.3 M CH3COOH/0.2 M CH3COONa (pH = 4.6) Untreated chitosan (square), Product (circle), Product (triangle) and Product (diamond) Fig Aqueous solubility of untreated chitosan and Products 1, and in 0.1 M sodium phosphate buffer (pH 7.4) Product formed a hydrogel under these conditions 305 Carbohydrate Polymers 186 (2018) 299–309 S Botelho da Silva et al groups added to chitosan by the TEMPO-laccase oxidation, as explained previously In Product 4, high ratio CHO/COO−, CHO clustered and low COO− favored the association of chains (minimized electrostatical repulsion) and the interaction between aldehyde and free amino groups via formation of imine bonds at pH 7.4 In Product 3, the association between chains through imine bonds was not possible since acetate groups were already bonded to amino groups Nunthanid et al (2004) used chitosan acetate as a drug delivery and performed delivery tests in pH 6.8 and in water They demonstrated the stability of the chitosan acetate in neutral pH, and in this condition, a sustained drug release was observed The designed reaction conditions used to obtain Product provide a completely different product with highly attractive characteristics for applications in the food and medical area At low pH, this chitosan derivative was fully soluble, as result of −NH2 protonation Increasing the pH from 4.5 to 6.5, the solution became gradually more viscous, and around pH 6.5–7.0 (over the pKa of chitosan) a hydrogel was obtained If more NaOH was added, the pH initially increased to and after that, the pH of the solution decreased spontaneously towards 7.0 and the solution got a little bit turbid At approximately pH 7.5, a product with a sponge-like appearance precipitated from the solution (Fig 6) Adding HCl, the pH decreased, and the “sponge” was maintained until ∼pH 4.0 Decreasing the pH even more with HCl, the “sponge” started to dissolve very slowly, and total dissolution was completed at a final ∼pH 3.7 The reversibility of the swelling/deswelling (sol-gel transition) of the polymer network was tested, showing the same pH-responsiveness for more cycles The structure of chitosan hydrogel is attributed to self-crosslinking amphiphilic chitosan network stabilized by dynamic imine bonds (Schiff base between aldehyde and amino groups) The responsiveness exhibited by chitosan derivative hydrogel is a consequence of a structure pH-dependent, which changes according to environmental stimuli, allowing formation and rupture of imine bonds and protonation and deprotonation of amino groups at different values of pH (Fig 6) The pH-responsiveness of this hydrogel was obtained due to higher amounts of aldehyde than carboxylate groups substitution on the primary OH- of C-6 of chitosan This high aldehyde/carboxylate substitution ratio allowed sufficient carboxylate groups to prevent precipitation of chitosan Fig SEM image of freeze-dried self-crosslinked hydrogel prepared with 3% TEMPOlaccase oxidized chitosan (Product 4) at pH at pH > 6, but low enough to allow self-crosslinking via imine bonds between aldehyde and amino groups Berger et al (2004) also reported a critical number of crosslinks per chain required to form a network and the influence of the type of the cross-linker Apart from that, and maybe more important, the carboxylate groups were not clustered as determined by MALDI-TOF MS analysis This finding has an important role in the network formation via imine bonds, since electrostatic repulsion due to carboxylate is not so strong and allow the attraction of the chains The self-crosslinked hydrogel obtained from oxidized chitosan (Product 4) is clear and transparent (Fig 6), and it can be formed with concentrations as low as 1.5% (w/v) It was possible to produce chitosan hydrogels (Product 4) with a swelling ratio (swollen weight/dry weight) as high as 30 (3000%) at pH 7.0 This swelling ratio is similar to the one reported by Singh, Narvi, Dutta, and Pandey (2006) for covalent hydrogels of chitosan prepared by crosslinking with formaldehyde as a crosslinking agent A representative SEM image of the freeze-dried gel that was produced with a concentration of 3% (w/v) at pH as described in Materials and Methods, is shown in Fig The Fig Self-crosslinked chitosan pH-responsive hydrogel (Product 4) 306 Carbohydrate Polymers 186 (2018) 299–309 S Botelho da Silva et al Fig TGA (a) and DTG (b) curves of untreated and oxidized chitosan products obtained under nitrogen atmosphere at a heating rate of 10 °C/ Untreated chitosan (blue solid), Product (purple long-dashed), Product (red short-dashed), Product (gray dash-dotted) and Product (green dash-double-dotted) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) pH the derivative is de-swelling, and the drug could be realized (Bhattarai et al., 2010; Du et al., 2015) Another potential application is anticancer hydrogels prepared in situ, delivering drugs directly to the tumor that have a slightly lower pH (6.5–6.8) than the normal physiological pH (Li, Hu et al., 2015; Li, Gu et al., 2015) oxidized chitosan gel exhibits a high surface area with a porous structure with random pore size distribution Similar porous structures are reported by Tan et al (2009) for crosslinked hydrogels obtained from the reaction between N-succinyl-chitosan and aldehyde hyaluronic acid Nevertheless, the authors did not report any information about pH responsiveness To the best of our knowledge, this is the first report of pH-responsive hydrogels formed by self-crosslinked chitosan Azevedo and Kumar (2012) modified the chitosan by introducing aldehyde groups into the molecule by nitrous oxide treatment and thereby, producing also hydrogels without the use of external cross-linkers although they did not report pH-responsiveness The use of external cross-linker represents a drawback since most of them can react irreversibly with −NH2 groups and prevent further stimuli-responsive effects (Bhattarai, Gunn, & Zhang, 2010) And, more important, some cross-linkers are toxic or not have recognized biocompatibility, therefore they cannot be used for application in biomedical and pharmaceutical areas (Bhattarai et al., 2010; Du, Liu, Yang, & Zhai, 2015) Among the potential applications of pH-responsive hydrogels, we highlight the platforms for gastrointestinal drug delivery (Du et al., 2015; Park, Kang, Lee, Kim, & Son, 2013; Woraphatphadung et al., 2016) and for injectable hydrogels prepared in situ (Li, Hu et al., 2015; Li, Gu et al., 2015) The pH-responsive chitosan hydrogel showed a solgel transition approximately around physiological pH (7.4) This finding indicates a potential application for this hydrogel to platforms for delivery drugs directed to the stomach (pH 1–3) for example, in this 3.5 Thermal stability To evaluate the effect of the TEMPO-laccase oxidation on the thermal stability of chitosan we performed thermogravimetric analysis (TGA-DTG) of untreated chitosan and its oxidized products TGA and DTG curves are shown in Fig Untreated chitosan, Products and show clearly two stages of weight loss, with approximately 65% of mass loss from 25 to 600 °C In the same interval, Products and lose more than 85% of their weight The first stage of weight loss of all samples occurs in the range between 25 and 100 °C, and it is mainly attributed to water loss The following stages are related to the thermal degradation of the polymer itself and include the deacetylation of chitosan, chain depolymerization, and disintegration of other intra and inter-molecular interactions (L Tian, Tan, Li, & You, 2015) The range where this thermal event occurs is characteristic for each product and shows that the derivatives are less thermally stable than their parent chitosan The maximum degradation temperature for untreated chitosan was observed at 300 °C, and it shifted to lower temperatures for the oxidized 307 Carbohydrate Polymers 186 (2018) 299–309 S Botelho da Silva et al products and tended to occur in broader ranges It is important to note that this decrease in the thermal stability is closely related to the oxidation degree (Table 1), meaning higher aldehyde and carboxylate groups by TEMPO-laccase oxidation leads to lower temperature of degradation Wei, Li, Tian, Xu, and Jin (2015) reported similar thermal degradation behavior for hypochlorite-oxidized starch nanocrystals treated by different concentrations of chlorine, in which higher oxidation also resulted in higher thermal degradation Although Products and showed the same oxidation degree (Table 1), the random distribution of oxidized groups and the consequent crosslinked structure in Product contributed for a lower temperature of degradation in the second stage, similar to effects reported by Neto et al (2005) for chitosan crosslinked by glutaraldehyde Nevertheless, in the range 300–600 °C, the thermal stability of Product was superior to Product and similar to untreated chitosan Another aspect that can be concluded from the thermal analysis is related to the changes in molecular weight induced by the TEMPOlaccase oxidation Mao et al (2004) and Tian et al (2015) showed that the thermal stability of chitosan significantly decreases with the reduction of the molecular weight Considering the small variation in degradation temperature observed for the oxidized products, it could be assumed that the depolymerization during the oxidation reaction was not intense and interactions in covalently and ionically crosslinked chitosan hydrogels for biomedical applications European Journal of Pharmaceutics and Biopharmaceutics, 57(1), 19–34 Bhattarai, N., Gunn, J., & Zhang, M (2010) Chitosan-based hydrogels for controlled, localized drug delivery Advanced Drug Delivery Reviews, 62(1), 83–99 Bordenave, N., Grelier, S., & Coma, V (2008) Advances on selective C-6 oxidation of chitosan by TEMPO Biomacromolecules, 9(9), 2377–2382 Bragd, P L., Besemer, A C., & van Bekkum, H (2000) Bromide-free TEMPO-mediated oxidation of primary alcohol groups in starch and methyl α-D-glucopyranoside Carbohydrate Research, 328(3), 355–363 Bragd, P L., van Bekkum, H., & Besemer, A C (2004) TEMPO-Mediated oxidation of polysaccharides: Survey of methods and applications Topics in Catalysis, 27(1–4), 49–66 Coseri, S., Biliuta, G., Simionescu, B C., Stana-Kleinschek, K., Ribitsch, V., & Harabagiu, V (2013) Oxidized cellulose—Survey of the most recent achievements Carbohydrate Polymers, 93(1), 207–215 Díaz-Rodríguez, A., Martínez-Montero, L., Lavandera, I., Gotor, V., & Gotor-Fernández, V (2014) Laccase/2,2,6,6-tetramethylpiperidinoxyl radical (TEMPO): An efficient catalytic system for selective oxidations of primary hydroxy and amino groups in aqueous and biphasic media Advanced Synthesis and Catalysis, 356(10), 2321–2329 DiFlavio, J.-L., Pelton, R., Leduc, M., Champ, S., Essig, M., & Frechen, T (2007) The role of mild TEMPO-NaBr-NaClO oxidation on the wet adhesion of regenerated cellulose membranes with polyvinylamine Cellulose, 14(3), 257–268 Du, H., Liu, M., Yang, X., & Zhai, G (2015) The design of pH-sensitive chitosan-based formulations for gastrointestinal delivery Drug Discovery Today, 20(8), 1004–1011 Filisetti-Cozzi, T M C C., & Carpita, N C (1991) Measurement of uronic acids without interference from neutral sugars Analytical Biochemistry, 197(1), 157–162 Hirai, A., Odani, H., & Nakajima, A (1991) Determination of degree of deacetylation of chitosan by 1H NMR spectroscopy Polymer Bulletin, 26(1), 87–94 Huang, J., Chen, W.-W., Hu, S., Gong, J.-Y., Lai, H.-W., Liu, P., Mao, J.-W (2013) Biochemical activities of 6-carboxy β-chitin derived from squid pens Carbohydrate Polymers, 91(1), 191–197 Jaušovec, D., Vogrinčič, R., & Kokol, V (2015) Introduction of aldehyde vs carboxylic groups to cellulose nanofibers using laccase/TEMPO mediated oxidation Carbohydrate Polymers, 116(13), 74–85 Kardas, I., Struszczyk, M H., Kucharska, M., van den Broek, L A., van Dam, J E., & Ciechańska, D (2013) Chitin and chitosan as functional biopolymers for industrial applications In P Navard (Ed.) The European polysaccharide network of excellence (EPNOE) (pp 329–373) Wien: Springer Kasaai, M R (2007) Calculation of Mark-Houwink-Sakurada (MHS) equation viscometric constants for chitosan in any solvent-temperature system using experimental reported viscometric constants data Carbohydrate Polymers, 68(3), 477–488 Kato, Y., Kaminaga, J., Matsuo, R., & Isogai, A (2004) TEMPO-mediated oxidation of chitin, regenerated chitin and N-acetylated chitosan Carbohydrate Polymers, 58(4), 421–426 Kierulff, J V (2000) Modification of polysaccharides by means of a phenol oxidizing enzyme Patent US6087135A Kong, M., Chen, X G., Xing, K., & Park, H J (2010) Antimicrobial properties of chitosan and mode of action: A state of the art review International Journal of Food Microbiology, 144(1), 51–63 Krolicka, M., Hinz, S W., Koetsier, M., Joosten, R., Eggink, G., van den Broek, L A., & Boeriu, C G (2018) Chitinase Chi1 from Myceliophthora thermophila C1, a thermostable enzyme for chitin and chitosan depolymerization Journal of Agriculture and Food Chemistry Accepted for publication Kumirska, J., Czerwicka, M., Kaczyński, Z., Bychowska, A., Brzozowski, K., Thöming, J., & Stepnowski, P (2010) Application of spectroscopic methods for structural analysis of chitin and chitosan Marine Drugs, 8(5), 1567–1636 Li, L., Gu, J., Zhang, J., Xie, Z., Lu, Y., Shen, L., Wang, Y (2015) Injectable and biodegradable pH-responsive hydrogels for localized and sustained treatment of human fibrosarcoma ACS Applied Materials & Interfaces, 7(15), 8033–8040 Li, J., Hu, W., Zhang, Y., Tan, H., Yan, X., Zhao, L., & Liang, H (2015) pH and glucose dually responsive injectable hydrogel prepared by in situ crosslinking of phenylboronic modified chitosan and oxidized dextran Journal of Polymer Science Part A: Polymer Chemistry, 53(10), 1235–1244 Lim, C., Lee, D W., Israelachvili, J N., Jho, Y., & Hwang, D S (2015) Contact time- and pH-dependent adhesion and cohesion of low molecular weight chitosan coated surfaces Carbohydrate Polymers, 117(0), 887–894 Luo, Y., & Wang, Q (2013) Recent advances of chitosan and its derivatives for novel applications in food science Journal of Food Procesing & Beverages, 1, 13 Mao, S., Shuai, X., Unger, F., Simon, M., Bi, D., & Kissel, T (2004) The depolymerization of chitosan: Effects on physicochemical and biological properties International Journal of Pharmaceutics, 281, 45–54 Mathew, S., & Adlercreutz, P (2009) Mediator facilitated, laccase catalysed oxidation of granular potato starch and the physico-chemical characterisation of the oxidized products Bioresource Technology, 100(14), 3576–3584 Mikhailov, G P., Tuchkov, S V., Lazarev, V V., & Kulish, E I (2014) Complexation of chitosan with acetic acid according to Fourier transform Raman spectroscopy data Russian Journal of Physical Chemistry A, 88(6), 936–941 Mourya, V K., Inamdar, N N., & Choudhari, Y M (2011) Chitooligosaccharides: Synthesis, characterization and applications Polymer Science Series A, 53(7), 583–612 Neto, C G T., Giacometti, J A., Job, A E., Ferreira, F C., Fonseca, J L C., & Pereira, M R (2005) Thermal analysis of chitosan based networks Carbohydrate Polymers, 62(2), 97–103 Niku-Paavola, M.-L., Karhunen, E., Salola, P., & Raunio, V (1988) Ligninolytic enzymes of the white-rot fungus Phlebia radiata Biochemical Journal, 254, 877–884 Conclusions In this work, the TEMPO-laccase catalytic redox system was successfully applied for the first time in C-6 oxidation of chitosan The derivative products were characterized and the effective formation of carboxyl and aldehyde groups on chitosan was demonstrated The oxidation degree and distribution of functional groups were affected by TEMPO and laccase concentration and by the acid solvent used to dissolve chitosan prior to TEMPO-oxidation The modification of chitosan structure by TEMPO-laccase oxidation provides an improvement in water solubility and a decrease in the viscosity of solutions of oxidized products in acid pH A slightly reduction on thermal stability was observed after TEMPO-laccase oxidation without showing evidence of intense depolymerization If chitosan was dissolved in hydrochloric acid prior to TEMPO-laccase oxidation than oxidized chitosan hydrogel was formed This hydrogel was clear and transparent and showed pH-responsiveness The reversibility exhibited by this chitosan hydrogel is a consequence of an amphiphilic structure pH-dependent stabilized by reversible covalent imine bonds This new material has a great potential for development of applications in medical and food area Acknowledgements The work of S Botelho da Silva was supported by the National Research Council of Brazil - CNPq [Process 249593/2013-0] We thank Sandra Hinz and Martijn Koetsier for their support by providing the crude chitinase and for useful discussions on chitinase purification The work of M Krolicka received funding from the Netherlands Organisation for Scientific Research (NWO) in the framework of the TASC Technology Area BIOMASS References Arends, I W C E., Li, Y.-X., & Sheldon, R A (2006) Stabilities and rates in the laccase/ TEMPO-catalyzed oxidation of alcohols Biocatalysis and Biotransformation, 24(6), 443–448 Azevedo, E P., & Kumar, V (2012) Rheological, water uptake and controlled release properties of a novel self-gelling aldehyde functionalized chitosan Carbohydrate Polymers, 90(2), 894–900 Azevedo, E P., Santhana Mariappan, S V., & Kumar, V (2012) Preparation and characterization of chitosans carrying aldehyde functions generated by nitrogen oxides Carbohydrate Polymers, 87(3), 1925–1932 Berger, J., Reist, M., Mayer, J M., Felt, O., Peppas, N A., & Gurny, R (2004) Structure 308 Carbohydrate Polymers 186 (2018) 299–309 S Botelho da Silva et al Sheldon, R A., & Arends, I W C E (2004) Organocatalytic oxidations mediated by nitroxyl radicals Advanced Synthesis & Catalysis, 346(9–10), 1051–1071 Singh, A., Narvi, S S., Dutta, P K., & Pandey, N D (2006) External stimuli response on a novel chitosan hydrogel crosslinked with formaldehyde Bullentin of Materials Science, 29(3), 233–238 Tan, H., Chu, C R., Payne, K A., & Marra, K G (2009) Injectable in situ forming biodegradable chitosan–hyaluronic acid based hydrogels for cartilage tissue engineering Biomaterials, 30(13), 2499–2506 Thevarajah, J J., Bulanadi, J C., Wagner, M., Gaborieau, M., & Castignolles, P (2016) Towards a less biased dissolution of chitosan Analytica Chimica Acta, 935, 258–268 Tian, M., Tan, H., Li, H., & You, C (2015) Molecular weight dependence of structure and properties of chitosan oligomers RSC Advances, 5(85), 69445–69452 Wei, B., Li, H., Tian, Y., Xu, X., & Jin, Z (2015) Thermal degradation behavior of hypochlorite-oxidized starch nanocrystals under different oxidized levels Carbohydrate Polymers, 124, 124–130 Wolf, B A (2007) Polyelectrolytes revisited: Reliable determination of intrinsic viscosities Macromolecular Rapid Communications, 28(2), 164–170 Woraphatphadung, T., Sajomsang, W., Gonil, P., Treetong, A., Akkaramongkolporn, P., Ngawhirunpat, T., & Opanasopit, P (2016) pH-Responsive polymeric micelles based on amphiphilic chitosan derivatives: Effect of hydrophobic cores on oral meloxicam delivery International Journal of Pharmaceutics, 497(1), 150–160 Xu, S., Song, Z., Qian, X., & Shen, J (2013) Introducing carboxyl and aldehyde groups to softwood-derived cellulosic fibers by laccase/TEMPO-catalyzed oxidation Cellulose, 20(5), 2371–2378 Nunthanid, J., Laungtana-anan, M., Sriamornsak, P., Limmatvapirat, S., Puttipipatkhachorn, S., Lim, L Y., & Khor, E (2004) Characterization of chitosan acetate as a binder for sustained release tablets Journal of Controlled Release, 99(1), 15–26 Park, B G., Kang, H S., Lee, W., Kim, J S., & Son, T I (2013) Reinforcement of pHresponsive γ-poly (glutamic acid)/chitosan hydrogel for orally administrable colontargeted drug delivery Journal of Applied Polymer Science, 127(1), 832–836 Patel, I., Ludwig, R., Haltrich, D., Rosenau, T., & Potthast, A (2011) Studies of the chemoenzymatic modification of cellulosic pulps by the laccase-TEMPO system Holzforschung, 65(4), 475 Pei, J., Yin, Y., Shen, Z., Bu, X., & Zhang, F (2016) Oxidation of primary hydroxyl groups in chitooligomer by a laccase–TEMPO system and physico-chemical characterisation of oxidation products Carbohydrate Polymers, 135, 234–238 Persin, Z., Stana-Kleinschek, K., Foster, T J., van Dam, J E G., Boeriu, C G., & Navard, P (2011) Challenges and opportunities in polysaccharides research and technology: The EPNOE views for the next decade in the areas of materials, food and health care Carbohydrate Polymers, 84(1), 22–32 Pierre, G., Salah, R., Gardarin, C., Traikia, M., Petit, E., Delort, A.-M., Michaud, P (2013) Enzymatic degradation and bioactivity evaluation of C-6 oxidized chitosan International Journal of Biological Macromolecules, 60(0), 383–392 Prashanth, K H., & Tharanathan, R (2007) Chitin/chitosan: Modifications and their unlimited application potential—An overview Trends in Food Science & Technology, 18(3), 117–131 Rinaudo, M., Pavlov, G., & Desbrières, J (1999) Influence of acetic acid concentration on the solubilization of chitosan Polymer, 40(25), 7029–7032 309 ... determined by Tukey’s test at 5% probability level Results and discussion 3.1 Oxidation of chitosan using the TEMPO-laccase catalytic redox system In order to perform the oxidation of chitosan, ... aldehyde-functionalization of chitosan by the TEMPO-laccase system as well as the formation of Schiff’s bases were also supported by 3.2.2 Degree of oxidation and aldehyde and carboxylate contents The oxidation degree... formed by self-crosslinked chitosan Azevedo and Kumar (2012) modified the chitosan by introducing aldehyde groups into the molecule by nitrous oxide treatment and thereby, producing also hydrogels