Characterization of Chitin and Its Hydrolysis to GlcNAc and GlcN

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Biomacromolecules 2008, 9, 1870–1875

Characterization of Chitin and Its Hydrolysis to GlcNAc and GlcN Aslak Einbu and Kjell M. Vårum* Norwegian Biopolymer Laboratory (NOBIPOL), Department of Biotechnology, Norwegian University of Science and Technology, 7491 Trondheim, Norway Received February 1, 2008; Revised Manuscript Received April 14, 2008

Proton NMR spectra of chitin dissolved in concentrated and deuterated hydrochloric acid (DCl) were found to be a simple and powerful method for identifying chitin from samples of biological origin. During the first hour after dissolving chitin in concentrated DCl (25 °C), insignificant de-N-acetylation occurred, meaning that the fraction of acetylated units (FA) of chitin could be determined. FA of demineralized shrimp shell samples treated with 1 M NaOH at 95 °C for 1-24 h were determined and were found to decrease linearly with time from 0.96 to 0.91 during the treatment with NaOH. Extrapolation to zero time suggested that chitin from shrimp shells has a FA of 0.96, that is, contains a small but significant fraction of de-N-acetylated units. Proton NMR spectra of chitin (FA ) 0.96) dissolved in concentrated DCl were obtained as a function of time until the samples were almost quantitatively hydrolyzed to the monomer glucosamine (GlcN). The initial phase of the reaction involves mainly depolymerization of the chitin chains, resulting in that almost 90% (molar fraction) of the chitin is converted to the monomer N-acetyl-glucosamine (GlcNAc).Thus, effective conversion of chitin to GlcNAc in concentrated acid is reported for the first time. GlcNAc is then further de-N-acetylated to GlcN. A new theoretical model was developed to simulate the experimental data of the kinetics of hydrolysis of chitin in concentrated acid. The model uses three different rate constants; two for the hydrolysis of the glycosidic linkages following an N-acetylated or a de-N-acetylated sugar unit and one for the de-N-acetylation reaction. The three rate constants were estimated by fitting model data to experimental results. The rate of hydrolysis of a glycosidic linkage following an N-acetylated unit was found to be 54 times higher as compared to the rate of de-N-acetylation and 115 times higher than the rate of hydrolysis of a glycosidic linkage following a de-N-acetylated unit. Two chitin samples with different FA values (0.96 and 0.70) were incubated in concentrated DCl until the samples were converted to the maximum yield of GlcNAc and the oligomer composition analyzed, showing that the maximum yield of GlcNAc was much higher when prepared from the chitin with the highest FA value.

Introduction Chitin is one of the most abundant biopolymers in nature and the most important nonplant structural biopolymer. It is widely distributed in nature as a main component of the exoskeleton of animals with an outer skeleton such as crustaceans and insects. It is also found in some microorganisms, for example, in the cell walls of fungi, yeast, and green algae.1 The main use of chitin is as a raw material to produce chitinderived products, such as chitosans, oligosaccharides, and glucosamine. Chemical characterization of chitin with respect to its degree of acetylation (FA) is complicated by the insolubility of chitin in aqueous solvents. A number of methods have been reported for determining FA of chitin, including hydrolytic and pyrolytic techniques,2,3 IR spectroscopy,4 elemental analysis,5 and UV6 and solid state NMR spectroscopy.7,8 We have recently reported a new method to characterize the chain length of chitin.9 Chitin can be solubilized in concentrated acids, although the solubilization is accompanied by depolymerization and to a more limited extent also de-N-acetylation.10 The monomer glucosamine (GlcN) is commercially prepared by acid hydrolysis of chitin using, for example, concentrated hydrochloric or sulfuric acid, and is an important commercial product as the number one dietary supplement in the U.S.A. The most used * To whom correspondence should [email protected].

be

addressed.

E-mail:

method for production is acid hydrolysis followed by purification of the hydrolysate and recrystallization of the product.11 The present procedure for large-scale production of the monomer N-acetylglucosamine (GlcNAc) is by chemical reacetylation of glucosamine using acetic anhydride.12 Other methods for production of GlcNAc, such as enzymatic degradation of chitin, have also been reported,13 and work is in progress to develop a new industrial scale microbial fermentation process using strains of Escherichia coli modified by metabolic engineering.14 Falk and co-workers15 studied the hydrolysis of chitin in concentrated hydrochloric acid (10.2 M) more than 30 years ago and found that the reaction occurred in three distinct steps, that is, the depolymerization of the polysaccharide to smaller polymeric units, the production of N-acetyl-glucosamine from the latter, and the conversion of N-acetyl-glucosamine into glucosamine and acetic acid. The acid-catalyzed hydrolysis of chitin involves two main acid-catalyzed reactions, that is, the hydrolysis of the glycosidic linkage (depolymerization) and the N-acetyl linkage (de-Nacetylation). In a recent publication,17 we found that the acid concentration, but not the temperature, could be used to control the rate of depolymerization (kglyc) relative to the rate of deN-acetylation (kacetyl) when the dimer GlcNAc2 and the monomer GlcNAc were used as model substances to study kglyc and kacetyl, respectively. It was found that the ratio of the rate

10.1021/bm8001123 CCC: $40.75  2008 American Chemical Society Published on Web 06/10/2008

Chitin and Its Hydrolysis to GlcNAc and GlcN

Figure 1. Schematic illustration of the acid catalysed cleavage of the different linkages present in chitin (shown as a polymer of A- and D-units).

constants kglyc/kacetyl increases strongly with increasing acid concentration. The rate of acid-catalyzed hydrolysis of the glycosidic linkage following an N-acetylated unit has been found to be 2-3 orders of magnitude faster as compared to the glycosidic linkage following a de-N-acetylated unit,10,19 and can be explained by two main factors. The first is a catalytic role of the N-acetyl group at carbon 2, which stabilizes the oxocarbonium ion transition state resulting in an enhanced rate of cleavage of the glycosidic linkage following the N-acetylated residue,22 and the second is the presence of a positively charged amino group at carbon 2 in a glucosamine residue that would shield the glycosidic oxygen from protonation and, thereby, result in a decreased rate of cleavage of the glycosidic linkage following a glucosamine residue.23 In this work we report on the characterization of chitin with respect to FA showing that chitin from shrimp shells contains a small but significant fraction of de-N-acetylated units. Furthermore, we developed a theoretical model for the kinetics of the hydrolysis of chitin in concentrated acid, where we find that the monomer GlcNAc constitutes more than 90% (molar fraction) of the reaction mixture during the initial phase of hydrolysis of chitin to glucosamine.

Materials and Methods Extraction of Chitin from Shrimp Shells. Chitin was extracted from shrimp shell (for NMR analysis) essentially as described by Percot et al.16 and Rødde et al.,21 as follows: Demineralization was performed by treatment (2×) with cold 0.25 M HCl (300 mL to 50.0 g thawed shrimp shells that was not dried) for 5 and 35 min, followed by washing with distilled water (300 mL). After demineralization, deproteinization was performed by extraction (3×) with 100 mL of 1 M NaOH at 95 °C (for 2, 2, and 1 h). Then the suspension was cooled to room temperature, filtered, and washed with water until neutrality was achieved. The pellet was finally washed with ethanol (96%) and dried at 80 °C. For the experiment with increasing extraction time (see Figure 3), demineralized shrimp shells were extracted with 1 M NaOH at 95 °C for 1-24 h without change of NaOH, and the suspension was cooled to room temperature, filtered, and washed with water until neutrality was achieved. The pellet was finally washed with ethanol (96%) and dried at 80 °C.

Biomacromolecules, Vol. 9, No. 7, 2008

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Figure 2. 1H NMR spectrum (400 MHz) of chitin in concentrated DCl at 25 °C.

Figure 3. FA values of chitin samples isolated from demineralized shrimp shells treated with 1 M NaOH at 95 °C for 1-24 h. FA determined from 1H NMR spectra of chitin dissolved in concentrated DCl at 25 °C.

Proton NMR on Chitin Dissolved in Concentrated DCl. Finely ground chitin was first wetted with dilute DCl and then dissolved in concentrated DCl acid (37 wt % from Sigma) to a concentration of 20 mg/mL. Solution-state proton NMR spectra were obtained with Bruker Avance DPX spectrometers. As a result of the high ionic strength of the solvent, matching and tuning of the probe was a nontrivial task for the sample in concentrated acid. Because of the effect of radiation dampening, the radio frequency pulses used had to be significantly longer than the pulses used with water as a solvent. To ensure quantitative uptake parameters, the relaxation times (T1) of the different protons were determined, using the inversion-recovery method. T1 relaxation times varied between 0.1 and 0.4 s, except for the acetate protons, which were found to be significantly longer (2.9 s), as expected. Chemical shifts were measured with 1% sodium 3-(trimethylsilyl)propionate-d4 (TSP) from Merck as an internal standard (at 0.00 ppm). Chitin may be dissolved in DCl and stored at -20 °C, as proton NMR spectra of samples stored for several weeks at -20 °C showed no significant difference when compared with spectra of the same samples analyzed directly after dissolving in DCl. [Aint] and [Dint] can be determined from the resonances at 4.91 and 3.44 ppm, respectively, and [Ared] and [Dred] from the R- and β-reducing end resonances. The monomer GlcNAc exists in equilibrium with the glucofuranosyl oxazolinium ion in concentrated HCl,17 and the resonance from the ion H-1* in Figure 4 are included in the molar fraction of acetylated reducing end units. The number average chain length (DPn) was determined from the integrated area of all H-1 protons divided by the integrated area of the reducing end protons (as assigned in Table 1 and Figure 4). These

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illustration of the acid-catalyzed cleavage of the different linkages in chitin with their respective rate constants used in our model. In this model two different rate constants for the hydrolysis of internal glycosidic linkages are taken into account, depending on whether the preceding unit is an A-unit (kglycA) or a D-unit (kglycD). These two rate constants can be determined as the resonances of H-1 of new reducing ends (N-acetylated or deN-acetylated) appear well separated in the proton NMR spectrum (Figure 4). Assuming that kacetyl is the same for all N-acetylated sugar units, that is, independent of DP, four differential equations can be derived to express the change in the molar concentration of the different sugar units as a function of time. An N-acetylated internal sugar unit can either be de-Nacetylated or it can become a new reducing end by hydrolysis of the following glycosidic linkage. Equation 1 expresses the change in the molar concentration of internal N-acetylated sugar units, [Aint] as a function of time, [Aint], and the rate constants kglycA and kacetyl

d[Aint] ) -(kglycA + kacetyl) · [Aint](t) dt

(1)

A de-N-acetylated internal sugar unit can either be formed by de-N-acetylation of an internal N-acetylated sugar unit, or it can become a new reducing end by the hydrolysis of the following glycosidic linkage. Equation 2 expresses the change in the molar concentration of internal de-N-acetylated sugar units, [Dint], as a function of time, [Dint], [Aint], and the rate constants kglycD and kacetyl:

Figure 4. 500 MHz 1H NMR spectra of chitin dissolved in concentrated DCl at 40 °C (relative to resonance of TSP at 0.00 ppm). The resonance from H-1 of the glucofuranosyl oxazolinium ion is denoted with an asterisk.

d[Dint] ) (kacetyl · [Aint]) - (kglycD · [Dint](t)) dt

(2)

An N-acetylated reducing end sugar unit can be formed by hydrolysis of the glycosidic linkage following an internal N-acetylated unit or it can be de-N-acetylated (to a new reducing end D-unit). Equation 3 expresses the change in the molar concentration of reducing end N-acetylated sugar units, [Ared], as a function of time, [Aint], [Ared], and rate constants kglycA and kacetyl

integrated areas also include the resonance of H-1 of the glucofuranosyl oxazolinium ion17 (denoted with an asterisk in Figure 4). Size-Exclusion Chromatography (SEC) of Reaction Products. The oligomers from the neutralized reaction mixtures of two chitin samples (FA of 0.70 and 0.96) that were degraded to the maximum yield of GlcNAc (as determined from the proton NMR spectra) were separated on three Superdex 30 columns connected in series, as previously described18 obtained from Amersham Pharmacia Biotech, overall dimensions 2.6 × 180 cm. The columns were eluted with 0.15 M ammonium acetate, pH 4.5, at a flow rate of 0.8 mL/min. The effluent was monitored with an online refractive index (RI) detector (Shimadzu RID 6A, Shimadzu Schweiz GmbH, Reinach, Switzerland) coupled to a data logger.

d[Ared] ) (kglycA · [Aint]) - (kacetyl · [Ared](t)) dt

(3)

De-N-acetylated reducing end sugar units can either be formed by de-N-acetylation of a reducing end acetylated unit or by the hydrolysis of a glycosidic linkage following a de-N-acetylated sugar unit. Equation 4 expresses the change in the molar concentration of reducing end de-N-acetylated sugar units, [Dred], as a function of time, [Dint], [Ared], and the rate constants kglycD and kacetyl

Theory A new mathematical model was developed to evaluate the hydrolysis of chitin in concentrated acid. In this model the different sugar units present in the sample are divided into four groups: N-acetylated (A-unit) or de-N-acetylated (D-unit), which could each be located at the reducing end (Ared or Dred) or as an internal unit (Aint or Dint). Figure 1 shows a schematic

d[Dred] ) (kglycD · [Dint]) + (kacetyl · [Ared]) dt

(4)

By solving these differential equations, we obtain four equations (eqs 5–8) expressing the theoretical molar concentra-

Table 1. Chemical Shifts (Relative to TSP at 0.00 ppm) of Proton Resonances for Chitina proton H-1 (reducing end)

H-2 (reducing end)

residue

H-1

R

β

H-2

R

β

H-2/6

acetyl-H

GlcNAc (A) GlcN (D)

4.91 5.07

5.43 5.65

5.05 5.21

3.57

3.32

3.5-4.4 3.5-4.4

2.62

3.44

a

In concentrated and deuterated hydrochloric acid at 25 °C.



tions of the sugar units ([Aint], [Ared], [Dint], and [Dred]) during hydrolysis of chitin as a function of time and the rate constants kacetyl, kglycA, and kglycD.

[Aint](t) ) [Aint]0 · e-t(kglycA+kacetyl) [Dint](t) ) e-t·kglycD

(

(5)

)

kacetyl[Aint]0 · (1 - e-t(kglycA+kacetyl-kglycD)) [Dint]0 + (6) kglycA + kacetyl - kglycD

[Ared](t) ) e-t·kacetyl(1 + [Aint]0 - [Aint]0 · e-t·kglycA)

(7)

[Dred](t) ) (kglycD - kglycA - kacetyl) · (1 + [Aint]0 + [Dint]0) - kacetyl · [Aint]0 · e-t(kglycA+kacetyl) kglycD - kglycA - kacetyl (1 + [Aint]0 · (kglycD - kglycA - kacetyl) · e-t·kacetyl + kglycD - kglycA - kacetyl (kacetyl([Aint]0 + [Dint]0) - [Dint]0(kglycD - kglycA)) · e-t·kglycD kglycD - kglycA - kacetyl

(8)

From eqs 58, the theoretical molar fractions of each sugar unit can be determined as a function of time and the rate constants.

Results and Discussion 1

H NMR Spectrum of Chitin in Concentrated DCl. Figure 2 shows a 400 MHz 1H NMR spectrum of R-chitin isolated from shrimp shells and dissolved in concentrated DCl at 25 °C. The resonances were assigned by comparison with spectra of chitin oligomers in the same solvent.17 The assignment of the resonances and their chemical shifts are given in Table 1. One advantage of using concentrated DCl as solvent is that the resonance of the solvent (HDO) does not interfere with any of the carbohydrate protons, as HDO resonates at 9.2 ppm (probably a weighted average of the signals from water and acid protons due to the fast exchange between the two). The spectrum shows the characteristic resonances in the anomeric region of the acetylated R-and the β-anomer at 5.43 and 5.05 ppm, respectively. H-1 of internal de-N-acetylated units resonate at 5.07, overlapping with the β-anomeric proton, while H-1 of internal acetylated units resonate at 4.91 ppm. H-2 of internal de-N-acetylated units resonate at 3.44 ppm. Acetyl-protons are found at 2.62 ppm, while the remaining ring protons appear between 3.6 and 4.4 ppm. The R- and β-anomer reducing end resonances from de-N-acetylated units, which would be expected to appear at 5.65 ppm and 5.21 ppm, are completely absent in the spectrum, which can be explained by the much higher rate of hydrolysis of the glycosidic linkage compared to the N-acetyl linkage.17,19 Upon de-N-acetylation of the sample, the resonances from the protons of acetic acid appears at 2.24 ppm, meaning that any de-N-acetylation of the chitin after its dissolution in concentrated DCl can easily be observed and quantified (Figure 4). Determination of the Chemical Composition of Chitin Dissolved in Concentrated DCl by 1H NMR. The extent of de-N-acetylation of the chitin after 24 h at 25 °C occurring in concentrated DCl was determined to be only 2%. Thus, the fraction of acetylated units (FA) of chitin samples can be determined from the NMR-spectra, as the very limited de-Nacetylation that occurs can be quantified from the resonance of acetic acid at 2.24 ppm. From the assignment of the resonances in Table 1, FA of chitin can be determined. The integral representing all H-1 protons is IRH1A + IβH1A+H1D + IH1A. By subtracting the integral from H-2 from deacetylated units (IH2D),

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we obtain the integral representing only acetylated units. Thus, FA can be determined from eq 9

FA )

(IRH1A + IβH1A+H1D + IH1A) - IH2D (IRH1A + IβH1A+H1D + IH1A)

(9)

To evaluate this new method for characterization of FA of the chitin, we analyzed samples of chitin extracted at different conditions. When extracting chitin from crustacean shell, the deproteinization step, involving typically 1 M NaOH at elevated temperatures, is expected to be more important with respect to de-N-acetylation of the chitin upon isolation.16,20 We investigated the time of the alkali treatments of shrimp shells (demineralized by treatment with cold 0.25 M HCl for 35 min) by varying the time of treatment with 1 M NaOH at 95 °C. The FA values of the isolated chitin samples were analyzed by 1 H NMR spectroscopy in concentrated DCl, and the results are shown in Figure 3. The uncertainty of the determined value of FA by this method depends on the precision of the integration of the NMR spectra (Figure 2). From our spectra, integration of the different resonances could be done with less than 1% uncertainty, giving a final uncertainty of the determined FA of about (1.5%. This illustrates that our method is relatively sensitive and can be used to detect a small decrease in the FA of chitin, such as treatment with 1 M NaOH at 95 °C. It should be noted that the samples treated with alkali for less than 5 h were only partially soluble in concentrated DCl, probably due to insufficient deproteinization, as methyl proton resonances from proteins were seen in the spectra between 1.0 and 1.5 ppm. However, extrapolation to 0 h (Figure 3) gives a FA value of 0.96, suggesting that the chitin in shrimp shells do contain a small but significant fraction of deacetylated units. Previous studies of the optimization of chitin extraction from shrimp shell16 reported that the deproteinization time and temperature did not influence the FA of the chitin when using 1 M NaOH at 70 °C and a reaction time of 24 h. However, to determine FA of the extracted chitins, solid state 13C cross-polarization magic-angle spinning (CP-MAS) NMR was used, with a lower resolution as compared to chitin samples dissolved in concentrated DCl. Thus, a limited de-Nacetylation (1-5%) would probably not be observed from solid NMR spectra. Acid Hydrolysis of Chitin in Concentrated DCl Monitored by 1H NMR. The hydrolysis of chitin down to the end product glucosamine in concentrated DCl was monitored by proton NMR. Figure 4 shows the1H NMR spectra of chitin in concentrated DCl as a function of time (at 40 °C). The resonances were identified (Table 1) by comparison with the previous assignment on chitin oligomers in concentrated DCl.17 From the increase in both the reducing end resonances and the acetic acid resonance in Figure 4, it can be seen that the chitin chain is both depolymerised and de-N-acetylated. The formation of the monomer GlcNAc can be seen from the H-1 resonances of the glucofuranosyl oxazolinium ion (denoted with asterisk), which exists in equilibrium with the monomer N-acetylglucosamine in concentrated DCl.17 From the proton NMR spectrum obtained 30 min after dissolution, the number average chain length (DPn) was found to be 24 units. Due to the much faster rate of hydrolysis of the glycosidic linkages following an acetylated unit compared to the hydrolysis of the N-acetyl linkage in concentrated acid,19 the initial phase of the reaction (less than 5 h) mainly results in depolymerization of chitin to chitin oligomers and to the acetylated monomer, N-acetylglucosamine, which is subsequently de-N-acetylated to the monomer glucosamine. This is

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Figure 5. Molar fractions of the different sugar units as a function of time in a sample of chitin dissolved in concentrated DCl at 40 °C (from Figure 4). Theoretical values for molar fractions are indicated by continuous lines (using kglycA ) 1.5 × 10-4 s-1 and kglycD ) 1.3 × 10-6 s-1 and kacetyl ) 2.8 × 10-6 s-1).

clearly seen from the relative intensities of the resonances from reducing end signals. After approximately 450 h, the chitin sample is fully depolymerized and de-N-acetylated to glucosamine. Experimental Results Compared with the New Theoretical Model for Acid Hydrolysis of Chitin. By integration of the different resonances from H-1 in the proton NMR spectra in Figure 4, we can obtain experimental values of the molar fraction of each of the four sugar units in a sample. Figure 5 shows the theoretical and experimental molar fractions of [Aint], [Ared], [Dint] and [Dred] obtained from 1H NMR spectra of chitin dissolved in concentrated DCl at 40 °C as a function of time. The continuous lines represent the optimal fit of the theoretical molar fractions from the model (molar fractions obtained from eqs 58 assuming an initial DPn of 24 and using kglycA ) 1.5 × 10-4 s-1, Figure 5 shows an excellent fit between the theoretical and experimental values when kglycA is about 54 times higher than kacetyl and 115 times higher than kglycD. This is in accordance with our previously published results using chitin oligomers as model compounds to study the acid hydrolysis of chitin.9,17 From kglycA of the chitin dimer (GlcNAc2) and kacetyl of GlcNAc in concentrated DCl,17 we found a ratio kglycA/kacetyl of approximately 130. This value is 2.5 times higher than the ratio of 54 (see Figure 4). However, in a recent publication on the hydrolysis of chitin/chitosan tetramers in concentrated hydrochloric acid9 we found that the rate of hydrolysis of the glycosidic linkage next to a nonreducing end is 2-2.5 times faster than the hydrolysis of an internal linkage, which can explain the differences in the ratio kglyc/kacetyl. To more clearly see the details of the initial stages of the reactions, we expanded the first 10 h of the chitin hydrolysis, which is shown in Figure 6. The results clearly show the rapid depolymerization in the early stages of the reaction, showing the number-average chain length (DPn) and the molar fractions of N-acetylated reducing ends ([Ared]) and de-N-acetylated reducing ends ([Dred]) as a function of time. The results also show that DPn decreases from 24 down to 1.0 during the first 6 h, and the molar fraction of Ared reaches a maximum of more than 0.9 after about 5 h and then decreases almost linearly. This implies that the molar fraction of Ared is almost exclusively composed of the monomer GlcNAc (in equilibrium with the glucosfuranosyl oxazolinium ion) around the maximum value of [Ared] (Figure 6).

Einbu and Vårum

Figure 6. Experimental data of the number-average chain length (DPn) and molar fractions of N-acetylated reducing ends ([Ared]) and de-N-acetylated reducing ends ([Dred]) as a function of time.

Falk and co-workers15 concluded that the maximum concentration of N-acetylglucosamine was obtained when 50% of the chitin was hydrolyzed (using 10.2 M HCl) and that good yields of N-acetylglucosamine could not be obtained by controlled acid hydrolysis of chitin. The difference in acid concentration (10.2 M as compared to 12 M herein) could possibly explain the deviation with our results, as we have recently found that the ratio kglyc/kacetyl increases strongly with increasing acid concentration.17 The lower acid concentration used by Falk et al. would result in reduced kglycA, while kacetyl does not change. The result would be an increase in the relative amount of internal de-N-acetylated units that is much more stable against cleavage (kglycA . kglycD), leading to a lower yield of N-acetylglucosamine. Interpolation of the kglycA/kacetyl ratios determined17 to 10.2 M acid concentration would result in a ∼50% decrease in the ratio of kglyc/kacetyl. Using our new theoretical model with relevant rate constants (kglycA and kglycD) while keeping kacetyl constant, resulted in a predicted reduced yield of N-acetylglucosamine down to 70%. Although this is higher than the yields obtained by Falk and co-workers,15 it clearly shows the large effect of the acid concentration on the yield of N-acetylglucosamine. Another contribution to the discrepancies between the results of Falk et al. and the present result could be a lower FA of the chitin used by Falk and co-workers. Also, most acidcatalyzed reactions proceed more readily in deuterated water (D2O) as compared to H2O.24 As deuterium oxide has a smaller autoprotolysis constant as compared to water, there is reason to believe that it is less basic than water,24 and because the concentration of the conjugate acid of the substrate will then be higher in D2O, the rate of the reaction should also be higher in D2O than in H2O. It has been found that the increase in the reaction rate due to the use of D2O as compared to H2O as solvent are mostly in the order of 1-3 times.24 After 2 h, the DPn levels out at 1.1 before decreasing to 1.0 after 6 h (see Figure 6). This means that the reaction mixture mainly consists of monomer and dimer during the time interval between 2 and 6 h. We therefore degraded two chitin samples of varying FA to the maximum yield of the acetylated monomer (GlcNAc, A), and the samples were characterized with respect to the size distribution of oligomers (Figure 7). From the chromatograms it can be seen that the sample obtained from the chitin with the highest FA (0.96) contains a much lower amount of the higher DP oligomers (e.g., DA and DDA) as compared to the sample obtained from the chitin with the lower



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N-acetylglucosamine can be obtained by controlled acid hydrolysis of chitin. Acknowledgment. This work was supported by the Norwegian Biopolymer Laboratory (NOBIPOL) and the Norwegian Research Council, Grant 143184/140 (Calanus). We thank Hans Grasdalen for valuable discussions and Finn Aachmann for technical assistance in the NMR-laboratory.

References and Notes

Figure 7. Chromatograms showing the size distribution of oligomers after degradation of two different chitin samples (FA ) 0.70 and 0.96) in concentrated HCl to the maximum yield of GlcNAc (A). Bold line: Obtained from chitin with FA ) 0.96. Thin line: Obtained from chitin with FA ) 0.70. DA: Dimer with D at the nonreducing end and A at the reducing end. DDA: Trimer with D at the nonreducing end, D at the middle unit, and A at the reducing end.

FA (0.70). Note that the chromatographic system can separate the dimer AA from the dimer DA.18 It was not possible to quantify the de-N-acetylated monomer, glucosamine, as it was eluting together with the large salt peak (resulting from large amounts of NaCl from neutralization of the samples of concentrated HCl) in our chromatographic system.10 This demonstrates that FA of the chitin is important to the yield of the monomer GlcNAc, as an increasing content of D units in the chitin would result in the formation of increased amounts of the dimer DA and higher DP oligomers with an acetylated reducing end. An experiment where kacetyl was compared with the monomer GlcNAc and the dimer DA as substrates (in concentrated hydrochloric acid) showed that the values of kacetyl were the same (data not shown). Thus, the assumption that kacetyl is relatively independent of the chitin chain length is not critical, as the de-N-acetylation reaction will almost exclusively occur at the reducing end when kglyc is much larger than kacetyl.

Conclusions Concentrated and deuterated hydrochloric acid is a suitable solvent in order to characterize the chemical composition of the chitin by proton NMR spectroscopy and the fraction of acetylated units (FA) in the chitin samples can be determined accurately. The rate of hydrolysis of a glycosidic linkage following an N-acetylated unit was found to be 54 times higher than the rate of de-N-acetylation and 115 times higher than the rate of hydrolysis of a glycosidic linkage following a de-N-acetylated unit. The monomer N-acetylglucosamine was found to constitute more than 90% (molar fraction) of the reaction mixture at the initial time of the reaction, meaning that good yields of

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BM8001123

Characterization of Chitin and Its Hydrolysis to GlcNAc and GlcN

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