Enzymatic Production and Enzymatic-Mass Spectrometric

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Enzymatic Production and Enzymatic-Mass Spectrometric Fingerprinting Analysis of Chitosan Polymers with Different Non-Random Patterns of Acetylation Jasper Wattjes, Anna Niehues, Laurent David, Thierry Delair, and Bruno M. Moerschbacher J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 23 Jan 2019 Downloaded from http://pubs.acs.org on January 24, 2019

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Journal of the American Chemical Society

Enzymatic Production and Enzymatic-Mass Spectrometric Fingerprinting Analysis of Chitosan Polymers with Different Non-Random Patterns of Acetylation Jasper Wattjes†‡, Anna Niehues†‡, Laurent David§, Thierry Delair§, and Bruno M. Moerschbacher†*. †

Institute for Biology and Biotechnology of Plants, University of Muenster, Schlossplatz 8, 48143 Münster, Germany

§

Laboratoire Ingénierie des Matériaux Polymères (IMP), CNRS UMR 5223, Univ. Lyon, Université Claude Bernard Lyon 1, 15 bd A. Latarjet, 69622 Villeurbanne France KEYWORDS: chitosan, chitin deacetylase, enzymatic fingerprinting.

ABSTRACT: Chitosans, a family of ß-(1,4)-linked, partially N-acetylated polyglucosamines, are considered to be among the most versatile and most promising functional biopolymers. Chemical analysis and bioactivity studies revealed that the functionalities of chitosans strongly depend on the polymers’ degree of polymerization and fraction of acetylation. More recently, the pattern of acetylation (PA) has been proposed as another important parameter to influence functionalities of chitosans. We therefore carried out studies on the acetylation pattern of chitosan polymers produced by three recombinant fungal chitin deacetylases (CDAs) originating from different species, namely Podospora anserina, Puccinia graminis f. sp. tritici, and Pestalotiopsis sp. We analyzed the chitosans by 1H-NMR, 13C-NMR, and SEC-MALS and established new methods for PA analysis based on enzymatic mass spectrometric fingerprinting and in silico simulations. Our studies strongly indicate that the different CDAs indeed produce chitosans with different PA. Finally, Zimm plot analysis revealed that enzymatically treated polymers differ with respect to their second virial coefficient and radius of gyration indicating an influence of PA on polymer-solvent interactions.

INTRODUCTION Sustainability is an important step towards a more ecological and considerate utilization of our planet’s resources. Biopolymers are unequivocal key players for this development and can be used to create novel materials and products that will help to advance our current economies to sustainable bioeconomies1–4. One of these promising biopolymers is chitin, a linear β-(1,4)-linked polysaccharide composed of N-acetylD-glucosamine (GlcNAc, A) residues. Chitin is widely distributed in nature and occurs i.a. in the exoskeletons of insects and crustaceans as well as in the cell walls of fungi. Partial de-N-acetylation converts it to chitosans, i.e. linear polymers composed of GlcNAc and D-glucosamine (GlcN, D) units, soluble in acidic aqueous media. They can be characterized by their average degree of polymerization (DP), fraction of acetylation (FA), and pattern of acetylation (PA). The structural diversity of these unique, partly polycationic and partly hydrophobic polymers has been implicated in their functional versatility that has led to many different applications reviewed in a number

of articles5,6. Among others, chitosans can be used for plant and harvest protection4, waste water purification7, as a food additive and food preservative8,9, for wound healing10 or drug and gene release control systems11,12. Commercially available chitosans are mainly produced by chemical deacetylation of shrimp and crab shell waste chitin under heterogeneous conditions resulting in heterogeneous products with varying DP and FA. While there is some controversy remaining, the PA of such chitosans appears to be close to random.13–16 As a biotechnological alternative, chitin and chitosan modifying enzymes (CCMEs) have been studied and were proposed as promising tools that not only allow production of novel and defined chitosan oligomers and polymers, possibly including some with non-random PA, but at the same time may help to understand structure function relationships17. Among these are chitinases18 and chitosanases19 that can be found in different glycoside hydrolase families and that are able to degrade polymeric chitosans to partially acetylated chito-oligosaccharides (paCOS).

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Similar to polymeric chitosan, paCOS were shown to possess numerous bioactivities and have already been implemented in different applications20–22. Another class of CCMEs are chitin deacetylases (CDAs, EC 3.5.1.41)23,24. CDAs are grouped into carbohydrate esterase family 4 (CE4), have been identified in bacteria, insects, and fungi, and catalyze the de-N-acetylation of polymeric or oligomeric chitosans. These enzymes are of special interest since they may potentially be used for the production of chitosans with defined, non-random PA.25 The mode of action of several fungal and bacterial CDAs on oligomeric substrates has already been investigated. Among the best studied are fungal CDAs from Aspergillus nidulans26,27, Colletotrichum lindemuthianum28,29 and Mucor rouxii30 as well as bacterial CDAs from Vibrio cholerae31,32 and Rhizobium spp.33 Recently, three fungal CDAs have been described in detail that originate from Podospora anserina (PaCDA)34, Puccinia graminis f. sp. tritici (PgtCDA)35, and Pestalotiopsis sp. (PesCDA)36. In these previous studies, recombinant enzymes were successfully produced in different expression systems and were used for the production of paCOS with different PA. We now wanted to investigate whether the different regio-selectivities of CDAs that were observed when acting on chitin oligomers can also influence the PA of the products when chitosan polymers are used as substrates. However, in contrast to the rather straightforward PA analysis of paCOS by the use of mass spectrometry and labeling techniques37,38, PA analysis of chitosan polymers is far more complex and only rarely done13– 15 . Weinhold et al.14 investigated different chitosans produced using chemical de- or re-N-acetylation by analyzing the diad frequencies of the C-5 carbon resonance area in 13C-NMR spectra based on former work of Vårum et al.15,39 and determined quantitatively whether the diad frequencies follow random (Bernoullian) statistics. In their study, all chitosans were described to have a random-dominated pattern. Possible alternatives and complements to NMR analysis are fingerprinting techniques. Such methods can be based e.g. on random40,41 or selective42,43 chemical degradation. Moreover, selective degradation of the polymer can be achieved using sequence-selective enzymes. The degradation products can then be analyzed to deduce information about a polymer’s PA44,45. Methods based on chemical degradation have been used to study substitution patterns of e.g. cellulose acetates41 or sulfates40 or the distribution of GlcN and GlcNAc in chitosans42,43. Enzymatic fingerprinting techniques have been used e.g. to investigate the block distribution in alginates46 or the structural composition of pectins47,48. We recently described the

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use of chitinosanase, a new chitosan degrading enzyme, for FA determination of polymeric chitosans based on enzymatic fingerprinting49,50. Due to its high substrate specificity, the enzyme produces unique degradation products and hence gives information about the FA and, as we show here, also the PA of the polymer. With the combined use of the already established 13C-NMR analysis and these new enzymatic fingerprinting approaches, we were able to investigate, characterize, and compare the PA of chitosan polymers produced by the three fungal enzymes PaCDA, PgtCDA, and PesCDA. MATERIALS AND METHODS Production of recombinant enzymes. PaCDA and PesCDA were produced as previously described34,36. Chitinosanase from Alternaria alternata was purified as previously described50. Chitinase ChiB51,52 from Serratia marcescens (SmChiB) was produced in E. coli Rosetta (DE3) pET22b:ChiBCStrepII37,53. Recombinant human chitotriosidase54–56 (HChT) and PgtCDA were produced in Schizosaccharomyces pombe using a modified pSLF172 vector57 encoding the thiamine dependent mmt1-promotor of fission yeast for controlled expression of the target genes. An N-terminal mel1 signal peptide from S. pombe was used for secretion and a C-terminal Histag for protein purification. Production of chitosans. Chemically N-acetylated chitosan samples of varying FA were produced from fully deacetylated chitosan (FA = 0) provided by Mahtani Chitosan Pvt. Ltd. (Veraval, Gujarat, India). N-Acetylation was carried out using acetic anhydride as previously described58. Enzymatically de-N-acetylated chitosan samples were produced by treating chemically N-acetylated chitosan (FA = 0.6) with PaCDA, PgtCDA, or PesCDA. To this end, the substrate chitosan was dissolved stoichiometrically overnight at 37°C in water/HCl to a final concentration of 2 mg/mL. To provide suitable conditions for enzyme activity, the solution was diluted 1:1 with 50 mM TEA buffer (pH 8.5). For de-Nacetylation, the desired amount of enzyme was added depending on batch size and CDA, as indicated. The solution was incubated at 37°C for 5 min up to 5 d to reach the intended FA. If necessary, additional enzyme was added. To stop the reaction, samples were boiled for 10 min at 98°C. Chitosan was precipitated by adding 25% (v/v) ammonia until a pH of 8-9. Finally, products were centrifuged and the pellets were washed several times with H2O to remove remaining buffer and enzymes. De-N-acetylated and washed


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chitosans were freeze-dried before being used for further analysis. Nuclear magnetic resonance (NMR) spectroscopy. NMR was used to investigate FA and PA of chitosan samples. FA was determined using proton NMR as described by Hirai et al.59. For a measurement, approximately 5 mg of chitosan were dissolved overnight in 1 mL acidic solution of D2O (1 mL 99.9% D2O and 2 µL DCl) and subsequently lyophilized. Samples were re-dissolved in D2O, and 1H-NMR spectra were recorded using an AV300 or DPX300 300 MHz spectrometer (Bruker, USA). For NMR-based PA analysis, 100 mg of purified chitosan were dissolved in 10 mL 0.07 M HCl and stirred overnight at room temperature. For partial depolymerization13, the solution was treated with 5 mg NaNO2, stirred for 4 h, and subsequently lyophilized. Samples were dissolved in 1 mL acidic solution of D2O (1 mL 99.9% D2O, 5 μl DCl) before 13C-NMR spectra were recorded on a 600 MHz DD2 instrument (Agilent, USA). Diad frequencies (FAA FAD FDA, FDD) were determined and analyzed as described by Weinhold and coworkers13,14. The diad frequencies were determined based on the C-5 resonances and possible deviation from random statistics was investigated by employing the formula = + , which was referred to as PA-value by Weinhold et al.14 Theoretical PΣ values for block copolymers. In order to facilitate interpretation of PΣ values derived from NMR measurements, theoretical diad frequencies of alternating and block-patterned chitosans were calculated as follows: frequency of diad A-A is given by = , with nA = mean A-block size and nD = mean D-block size, for series of different block sizes ranging from 1 to 100. Accordingly, the other diad (D-D, A-D, D-A) frequencies were calculated as = and = = . The fraction of acetylation was determined by

=

.

Theoretical diad frequency deviation from random statistics was calculated in the same way as for NMR derived diad frequencies. High Performance-Gel Permeation Chromatography (HP-GPC). HP-GPC was used to determine the weight-average molecular weight (Mw) of chitosan samples as well as for Zimm plot analysis. dn/dc Values used for calculations were deduced from a polynomial based on previous studies relating the dn/dc with the FA of samples with random PA60. All samples were prepared in 50 mM ammonium acetate buffer

(pH 4.5). For Mw determination, an Agilent HPLC system equipped with three TSKgel® columns (G6000PWXL-CP 13 µm, G5000PWXL-CP 10 µm, G300PWXL-CP 7µm, 7.8 mm diameter, 30 cm length) coupled online with a refractive index detector (Agilent Series 1200 RID®) and a multi-angle laser light scattering detector (PSS SLD 7000 MALLS®) was used. Depending on the Mw, 50-100 μl of sample (2 mg/mL) were injected to the system and the flow rate was kept constant at 0.6 mL/min. Data was evaluated using WinGPC software by PSS (GPC-MALS_1). Based on Mw and FA, the corresponding DP was calculated. For Zimm plot analysis, a DAWN® HELEOS® II MALS detector (WYATT Technologies) was used and Astra® software (Wyatt Technologies) was applied for data evaluation (GPC-MALS_2). Enzymatic hydrolysis for fingerprinting. Enzymatically de-N-acetylated chitosans and chemically N-acetylated chitosan FA = 0.3 were digested with chitinosanase, SmChiB, or HChT. For each reaction, 50 µg of each polymer were dissolved in 50 µL 200 mM ammonium acetate buffer, pH 4.2, and incubated at 37°C for 65 h with 3 µL (100 µg/µL) chitinosanase (10 replicates each), 1 µL (1 µg/µL) SmChiB (5 replicates each), and 2 µL (0.5 µg/µL) HChT (5 replicates each). All chitosans were enzymatically hydrolyzed using chitinosanase for subsequent analysis of the products by quantitative MS. Thirty µg of each polymer were dissolved in 30 µL 200 mM ammonium acetate buffer, pH 4.2, and incubated with 2 µL of chitinosanase (100 µg/µL) at 37°C for 65 h (5 replicates each). Sample preparation for MS analysis. All samples were freeze-dried and re-dissolved in H2O to a concentration of 1 µg/µL. For quantification of chitinosanase hydrolysis products, paCOS were [2H3]Nacetylated according to Cord-Landwehr et al.37. Since fully acetylated oligomers were not detected in chitinosanase hydrolysates (see Supplementary Figure S1) prior to [2H3]N-acetylation, chitin oligomers (DP 1-6, Megazyme International, Ireland) were added as internal standards for quantification. The final mixture contains 800 ng/µL [2H3]N-acetylated hydrolysate and 30 ng/µL of each standard chitin oligomer. Ultra-high performance liquid chromatography – electrospray ionization – mass spectrometry (UHPLC-ESI-MS). UHPLC-ESI-MS was used to semi-quantitatively and quantitatively determine the amounts of paCOS produced by chitinosanase treatments of chitosan polymers. All measurements were performed on a Dionex Ultimate 3000RS UHPLC system (Thermo Scientific, Milford, USA) equipped with an Acquity UPLC BEH Amide column (1.7 µm, 2.1 mm x 150 mm; Waters Corporation, Milford, MA, USA)



and a VanGuard precolumn (1.7 µm, 2.1 mm x 5 mm; Waters Corporation, Milford, MA, USA), and coupled to an amaZon speed ESI-MSn detector (Bruker Daltonik, Bremen, Germany). Two µL containing 2 µg of hydrolysate were injected into the system. Semiquantitative MS measurements of chitinosanase hydrolysis products and MS data analysis were performed using the same method and parameters as described by Niehues & Wattjes et al.49. Quantitative measurements were performed similarly as described by Cord-Landwehr et al.37. Two µL of each sample containing 1.6 µg of hydrolysate and 60 ng of each internal standard were injected into the system. Oligomers were separated over a 15 min gradient elution profile: 0-2.5 min isocratic 100% A (80:20 ACN:H2O with 10 mM NH4HCO2 and 0.1% (v/v) HCOOH); 2.512.5 min linear from 0% to 75% B (20:80 ACN:H2O with 10 mM NH4HCO2 and 0.1% (v/v) HCOOH); column re-equilibration: 12.5-13.5 min linear from 25% to 100% A; 13.5-15 min isocratic 100% A. The ESI-MSn detector’s target mass was set to 700 m/z. Quantitative MS data analysis was performed as described by CordLandwehr et al.37 and Niehues & Wattjes et al.49. Principal component analyses. Semi-quantitative MS data of enzymatic hydrolysis products (DP 110 paCOS) of three different hydrolases (SmChiB, HChT, chitinosanase) were subjected to principal component analyses (PCAs). The data was preprocessed as follows: only oligomers that occurred in at least 50% of all samples were used. Each fingerprint was normalized so that the sum of the oligomer amounts of each fingerprint equals one (L1 normalization). The variables (oligomer frequencies) were subsequently mean-centered. Due to the specific cleavage preference of chitinosanase50, each endpoint hydrolysis product represents one A-block followed by one D-block. Based on this, quantitative MS data of chitinosanase hydrolysis products were used to calculate the frequencies of A- and D-block sizes. PCA was performed on these block size fingerprints of different chitosan polymers. To this end, the data was preprocessed in the same manner as for the semiquantitative data. RESULTS To investigate the PA of chitosan polymers produced by PaCDA, PesCDA, and PgtCDA, we first prepared chitosan FA = 0.6 as a substrate for CDA treatment by partial N-acetylation of a fully de-N-acetylated chitosan polymer. Before and after enzymatic treatment, the polymers’ FA was determined by 1H-NMR. Previous studies showed that all enzymes are able to de-N-

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acetylate chitosan from FA = 0.6 to an endpoint of approximately 0.3. Importantly, all three enzymes were described to show activity on chemically produced chitosan with an FA of 0.334–36. This fact already gave a first indication that the enzymes produce PAs different from the random PA known for chemically produced chitosans. To provide sufficient material for 13 C-NMR measurements, the reactions were scaled up and yielded up to 500 mg of enzymatically de-N-acetylated chitosan polymers. To provide chemically produced controls, we produced three batches of chitosan FA = 0.3 (CS33), as well as one batch each with FA = 0.2 and 0.5, all from the same chitosan starting material. In all cases, FA and MW were determined by 1HNMR and HP-SEC-MALS, respectively (Supplementary Fehler! Verweisquelle konnte nicht gefunden werden.). First, the diad frequencies of enzymatically de-N-acetylated chitosans were determined using 13CNMR diad analysis as previously described13,15. To provide suitable measuring conditions and improve spectrum resolution, the Mw of CSPaCDA, CSPgtCDA, CSPesCDA, and CS33 was decreased by nitrous acid depolymerization13. The analysis of diad peaks in the C-5 resonance region revealed different intensities and ratios for the four possible diads, namely AA, AD, DA, and DD (Figure 1). Based on the formula previously used by Weinhold and coworkers, we determined whether the diad frequencies of CSPaCDA, CSPgtCDA, CSPesCDA, as well as CS33 deviate from random statistics (Figure 2A) and calculated corresponding PΣ values. With a value of 0.75, PgtCDA seems to shift the PA towards a more blockwise distribution, whereas the value determined for PaCDA-derived chitosan (1.31) indicates a tendency towards alternating PA. PesCDA-treated chitosan (1.15) turned out to be the most similar to the chemical control (1.06). To facilitate data interpretation, we calculated theoretical PΣ values for chitosans with alternating A and D blocks, varying block sizes, and plotted PΣ against FA. This allowed us to infer average A and D block sizes for distinct FA and PΣ combinations (Figure 2A). When looking at Figure 2A, the strong impact of FA on PA becomes obvious. A perfectly alternating pattern with a PΣ value of 2 can only be achieved for chitosans with FA = 0.5 with a blocksize of 1. Notably, an alternating polymer with a blocksize of 2 has a PΣ value of 1 and thus the same value as a polymer with random distribution of acetylated units. This shows that PΣ values alone do not allow unequivocal determination of PA. Due to this limitation of 13C-NMR, we decided to complement the analysis by enzymatic fingerprinting methods using different hydrolases.


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Figure 1. 13C-NMR analysis of CSPaCDA(FA = 0.29), CSPgtCDA (FA = 0.32), CSPesCDA (FA = 0.35), and chemically N-acetylated CS33. (A) Exemplary 13C-NMR spectrum (CS35PesCDA) with detailed view on the C-5 resonance region. The peak areas of the four diads (DA, DD, AA, and AD) are determined as integrals IDA, IDD, IAA, and IAD and were used to calculate the deviation from random statistics (PΣ). (B) C-5 resonance region comparison of the four different samples CSPaCDA, CSPgtCDA, CSPesCDA, and CS33. C-5 resonance peak areas used for diad analysis are listed in Supplementary Table S2.

Figure 2. Calculated values for the deviation from random statistics (P∑) of A-D diad frequencies depending on FA and block size in block-alternating chitosan polymers, and experimentally determined values for different chitosan polymers, as indicated (A). Enzymatic fingerprinting approach for PA analysis of chitosan samples. Dependency of chitinosanase fingerprints on the PA of the hydrolyzed polymer. paCOS derived from either random, alternating, or blockwise PA can be distinguished in terms of DP, FA, and PA. Chitinosanase cleavage occurs after each D-A sequence, as indicated (B).



Due to their cleavage specificities, hydrolytic enzymes produce different oligomeric fingerprints that inevitably depend on the PA of the polymeric substrate (Figure 2B). These differences are reflected in DP, FA, and PA of the paCOS produced. In this study, we tested three different chitosan hydrolyzing enzymes for their suitability for chitosan fingerprinting. Chemically N-acetylated and enzymatically de-Nacetylated polymers were hydrolyzed with chitinase B from Serratia marcescens (SmChiB), human chitotriosidase (HChT), and Alternaria alternata chitinosanase. SmChiB has an absolute preference for an

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acetylated unit at subsite (−1); additionally, it prefers GlcNAc at both subsites (−2) and (+2), but the preference is not absolute.37,61,62 Similarly, HChT has an absolute preference for GlcNAc at subsite (−1); at subsites (−2) and (+1), it has a strong and a weak preference for GlcNAc, respectively.56 Chitinosanase has an absolute preference for GlcN at subsite (-2) and for GlcNAc at subsite (-1), but no preference at subsites (+1) and (+2).50 Enzymatic hydrolysis products were analyzed by semi-quantitative HILIC-MS, and principal component analyses were carried out for a first data evaluation (Figure 3).

Figure 3. Principal component analyses of enzymatic fingerprints of chemically N-acetylated and enzymatically de-N-acetylated chitosans with FA ~ 0.3 using three different chitosan-hydrolyzing enzymes. (Top) Explained variance and cumulative explained variance of principle components. (Bottom) PCA score plots with PC 1 and PC 2 scores of fingerprint samples. PC loadings are shown in Supplementary Figure S3.

A detailed composition of the fingerprints is depicted in Supplementary Figure S1 in the supplementary material, along with examples of corresponding base peak chromatograms (Supplementary Figure S2). Figure 3 (top) shows that most of the data variance (~95%) can be explained by the first two components of each PCA. When HChT or chitinosanase is used for hydrolysis, PC1 and PC2 scores separate all chitosan samples in a similar manner. In case of the less specific SmChiB, chitosans de-N-acetylated with PaCDA or PesCDA cannot be separated. This shows that enzymes with high substrate specificities are better suited for enzymatic fingerprinting since their hy-

drolysis products capture potential differences between polymer samples better than less specific enzymes. A visualization of the loadings of the principal components from the three different PCAs (Supplementary Figure S3) supports these findings. Oligomers used for separation differ between all three PCAs and in case of chitinosanase treatment, a broader range of oligomers is used for fingerprint separation. This hydrolase treatment thus results in a better separation of samples. Chitinosanase and HChT separate the samples equally well. However, due to its absolute cleavage specificity50, chitinosanase allows direct conversion of paCOS frequencies to block size frequencies.


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Therefore, we decided to use this enzyme for a more detailed fingerprinting analysis. As additional controls, we included further chemically N-acetylated chitosans with FA values below and above 0.3. Chitinosanase hydrolysis products were analyzed by quantitative HILIC-MS. A detailed composition of the fingerprints is depicted in Supplementary Figure S4, along with examples of corresponding base peak chromatograms (Supplementary Figure S5). Based on the hydrolysis products, we calculated the block sizes represented by these oligomers (Supplementary Figure S6). The frequencies of the different A- and Dblocks were again used for PCA (Figure 4A). The chemically N-acetylated chitosans are clearly separated according to their FA by PC1 and PC2. Enzymatically de-N-acetylated chitosans do not cluster with the chemical samples of similar FA but instead were

separated from all chemical samples. This separation of the chemical samples with varying FA is mainly due to different frequencies in A-block sizes. Not surprisingly, A-blocks with size 1 are more frequent in chitosans with low FA while A-blocks with sizes 2, 3, and 4 are more frequent in samples with higher FA. Separation between chemical and enzymatic samples is mainly due to varying frequencies in D-block sizes. While D-blocks with a size of 1 are more frequent in chemical samples, D-blocks with the size of 2 are more frequent in the enzymatic samples. To provide additional controls, we simulated chitinosanase hydrolysis in silico on a series of chitosans with different FA and random PA. As done for the fingerprints measured in vitro, we calculated block size frequencies for these in silico samples.

Figure 4. (A) Principal component analysis of A- and D-block size fingerprints to compare different chitosan polymers based on quantitative HILIC-MS of chitinosanase hydrolysis products DP 2-6. Scores (colored dots) and loadings (black arrows with labels) for PC1 and PC2. (B) Average block sizes calculated from chitinosanase hydrolysis products DP 2-6 when different chitosan polymers were used as substrates, as indicated; theoretical average block sizes were determined from in silico data generated for chitosans of random PA and varying FA based on observable oligomers.

When looking at average block sizes in DP 2-6 chitinosanase hydrolysis products (Figure 4B), the differences between polymers produced by different CDAs become clearly visible. While average A-block sizes are larger in chitosan treated with PgtCDA (orange dots), they are smaller in PaCDA treated chitosan (yellow dots). This again proves that CDAs can influence the PA of chitosan polymers. To investigate whether the differences in the distribution of acetylated and de-N-acetylated units caused by PaCDA,

PesCDA, and PgtCDA have an influence on the physico-chemical properties of these polymers, we conducted GPC-MALS experiments under optimized conditions60 to investigate polymer conformation and polymer-solvent interactions. Figure 5 shows exemplary Zimm plots recorded for the enzymatically treated polymers and a chemical control, dissolved in ammonium acetate buffer (pH 4.3).



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Figure 5. Zimm plot analysis of enzymatically and chemically produced chitosans. Mw, Rg2, and A2 were determined for CSPaCDA, CSPesCDA, CSPgtCDA, and CS33 as a control with random PA.

Differently shaped Zimm plots were recorded for PaCDA, PesCDA, and PgtCDA samples indicating differences in polymer solvent interactions. Values for size (Rg) and second virial coefficient (A2) were calculated (Figure 5). While a very straight Zimm plot was observed for PaCDA-treated chitosan, giving an A2 value of almost zero, the PgtCDA-treated sample resulted in a Zimm plot with a negative initial slope and in consequence a negative A2 value (-2.58×10-2). The PesCDA-treated chitosan resulted in a Zimm plot with a slightly negative initial slope giving an A2 value of 6.864×10-3. Rg values revealed only minor differences between the different samples. However, the observed A2 values indicate differences in polymer solvent interactions between the three enzymatically treated polymers. The chemical control showed a similar A2 value as the PesCDA treated sample supporting our findings from NMR and fingerprinting

experiments that the two samples exhibit similar structural properties. DISCUSSION As shown in this study, it is possible to produce enzymatically de-N-acetylated chitosan polymers in the scale of 500 mg. By controlling and keeping constant FA and DP of the polymers, we were able to relate differences in the physico-chemical properties of the polymers to their PA. However, for the interpretation of the results, we need to consider that the enzymes have had a limited influence on the PA, given that the chemical N-acetylation of the starting material to a FA = 0.6 inevitably created an initial random PA in the substrates. This is probably the main reason why differences between the investigated samples can clearly be seen but are rather small. To study the PA produced by CDAs, fully acetylated chitin would be the most suitable substrate, since the obtained PA would fully result from enzymatic deacetylation. However, due to


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its high crystallinity, most CDAs show only marginal activity on this substrate, presumably acting only at the surface of the chitin nanofibrills, resulting in a heterogeneous deacetylation process63–65. Studies on CDAs with carbohydrate binding modules (CBMs) like PesCDA or PaCDA suggest that substrate accessibility can be facilitated to a certain extent 34,35. Still, a conversion of crystalline chitin to chitosan by a CDA has not been described so far. As stated before, the PA of chitosan polymers has only sparsely been studied. Several studies13–16 investigated chitosan samples derived from different chemical preparation methods including homo- and heterogeneous de-N-acetylation and homogeneous N-acetylation, but did not find evidences for a clear non-random PA in any of the samples. However, existing studies were almost66 exclusively carried out on chemically produced samples. In this study, we can show differences in the PA produced by three CDAs, namely PesCDA, PaCDA, and PgtCDA. As we discussed in this work, the evaluation of diad frequencies based on 13C-NMR has several drawbacks and misleading designations. For example, a chitosan with a di-block alternating acetylation pattern (AADDAADD etc.) cannot be distinguished from a chitosan with a random PA (P∑ = 1). 13C-NMR-based triad analysis could be used to distinguish these two patterns. However, the method does require very high resolution NMR spectra and even then, the accuracy was reported to be ±15% at best.15 We therefore propose to complement NMR-based methods with approaches that can be used to address not only the average A- and D-block size but also the distribution of block sizes. By the introduction of enzymatic fingerprinting techniques, we were able to establish such an alternative or complementation for 13C-NMR PA analysis. We have shown that due to its high substrate specificity, the recently described chitinosanase has advantages over the less specific SmChiB and HChT, although all enzymes are in principle suited for fingerprinting analysis and complement each other. For example, the increase in A-block size in PgtCDA treated chitosan determined by chitinosanase fingerprinting was confirmed by the increased amount of the A2 oligomer in SmChiB and HChT fingerprints (Supplementary Figure S1). Due to the preference of SmChiB and HChT to cleave between acetylated units, the presence of A2 in the hydrolysates even hints at larger acetylated blocks in the polymer. In contrast to 13C-NMR analysis that requires comparatively large sample amounts which are often not available for experimental chitosans, enzymatic fingerprinting is less time consuming and needs only small

amounts of sample. Moreover, in silico simulation allows the generation of large number of chitosan polymers and fingerprints that can be used as controls. Multivariate analysis turned out to be a highly effective complementation to mass spectrometry. Finally, the differences in solvent interaction that were seen for the polymers are a first hint that different PAs are indeed influencing intramolecular interactions of the chitosan chains. The presence and size of A- and D-blocks will impact the macromolecular conformation of the chains, yielding an apparent increase in intramolecular interactions with concomitantly reduced solvent interaction. While the enzymatic fingerprinting approach gives deeper insight into the distribution of block sizes than 13C-NMR alone, it will eventually be interesting to investigate the inter- vs. intramolecular67 patterns of acetylation. This will require a fractionation of the polymer sample according to its FA as well as PA, followed by characterization of the fractions. Recently, there have been some advances on this topic,68–70 but so far there is a lack of available polymer standards with defined FA, let alone PA. The use of CDAs to produce such standards will further advance this progress. ASSOCIATED CONTENT Supporting Information. Supporting_Information_S1.pdf containing the following tables and figures: Supplementary Table S1 – Chitosan sample overview Supplementary Table S2 – Intensities of 13C-NMR C-5 resonance peaks Supplementary Figure S1 – Enzymatic (SmChiB, HChT, chitinosanase) fingerprints determined by semi-quantitative HILIC-ESI-MS Supplementary Figure S2 – Base peak chromatograms (semiquantitative HILIC-ESI-MS1) of enzymatic hydrolysates (SmChiB, HChT, chitinosanase) Supplementary Figure S3 – Principal component analysis factor loadings Supplementary Figure S4 – Enzymatic (chitinosanase) fingerprints determined by quantitative HILIC-ESI-MS Supplementary Figure S5 – Base peak chromatograms (quantitative HILIC-ESI-MS1) of enzymatic hydrolysates (chitinosanase) Supplementary Figure S6 – D- and A-block size frequencies based on enzymatic (chitinosanase) fingerprints This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * Bruno M. Moerschbacher. E-Mail: [email protected]; Tel: +49 251 8324794; Fax: +49 251 8328371.

Author Contributions



The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡ J.W. and A.N. contributed equally.

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Funding Sources This work was supported by the European Comission under the FP7-KBBE programme [NANO3BIO, project ID 613931] [ERA-IB-2, project ID 291814].

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ACKNOWLEDGMENT We would like to thank Dr. Dominique Gillet (Mahtani Chitosan) for kindly providing chitosan, as well as Dr. Klaus Bergander (Institute for Organic Chemistry, University of Münster) for NMR measurements.

ABBREVIATIONS PaCDA, Podospora anserina chitin deacetylase; PesCDA, Pestalotiopsis sp. chitin deacetylase; PgtCDA, Puccinia graminis f. sp. tritici chitin deaectylase.

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