Biomacromolecules 2010, 11, 3167–3171
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Heterogeneous Components of Chitosans Byung Y. Yang, Qiong Ding, and Rex Montgomery* Department of Biochemistry, College of Medicine, The University of Iowa, Iowa City, Iowa 52242, United States Received August 23, 2010; Revised Manuscript Received October 6, 2010
The main objectives of the research were to compare the components of partially N-deacetylated chitins prepared identically from native chitin and a chitin regenerated from a heavily deacetylated chitosan. Additionally, to determine if any of the water-soluble components would serve as substrates in a study of a Chitinase isolated from soy bean hull. The brief heating of suspended chitins in 20% (w/w) NaOH resulted in similar degrees of N-deacetylation, the native chitin giving DAc 0.84 and the regenerated chitin DAc 0.79-0.72, with DAc indicating the proportion of glucosamine residues that are acetylated. Evidence for the nature of the hydrolysis of acetamido groups was provided by analyses of the water-soluble and -insoluble Smith degradation products. The watersoluble fraction derived from the native chitin comprised very small amounts of erythrityl N-acetyl glucosaminoside (GlcNAc1E), erythrityl N,N′-diacetyl chitobioside (GlcNAc2E), and erythrityl N,N′,N′′-triacetyl chitotrioside (GlcNAc3E), each identified by MALDI-TOF mass spectrometry of the butanoyl derivative. The water-insoluble products, as analyzed by light scattering detection method of their butanoyl esters and corrected for their composition, had a molecular weight (Mw) of 25 kDa, corresponding to about 120 N-acetyl glucosaminyl repeating residues (DPw), contrasting to that of 140 kDa with DPw of 680 for the parent chitin. Much of the decrease in the molecular weight of the polymer occurs by the loss of sugar residues by alkaline peeling at reducing terminals. For the regenerated chitin (DAc 1.0), prepared by N-reacetylation of a commercial chitosan (DAc 0.15), the resulting Smith products comprised erythritol and a series of N-acetyl glucosaminyl erythritol homologues of up to at least 39 N-acetyl glucosaminyl repeating residues, reflecting greater heterogeneity in the hydrolysis of acetamido groups along the polymer chain than what was seen for the native chitin. Of the water-soluble Smith products, GlcNAc5-7E were good substrates for chitinase isolated from soybean hull.
Introduction Chitin is a naturally abundant linear polysaccharide of β-1,4 linked N-acetyl glucosaminyl repeating units. It occurs as a structural component of many organisms, such as fungi and crustaceans.1 Its copious amounts in nature and its biological activities have drawn enormous interest for research of chitin and its partially N-deacetylated derivatives, the chitosans, in biomedical and industrial applications.2,3 However, the insolubility of chitin in most solvents often hinders effective commercial and pharmaceutical uses of chitin without significant alteration of the polymer. The commercial production of the chitosans applies a heterogeneous N-deacetylation of chitin with NaOH at elevated temperatures, the conditions significantly affecting the final properties and composition of the resulting chitosan. These properties have been extensively examined by X-ray,4,5 NMR,6,7 nitrous acid deaamination,5,8,9 fractionation into acid soluble and insoluble parts,7 and thermal properties and swelling.5 The general conclusions from these various studies are that the degree of N-deacetylation increases with more drastic reaction conditions, the solubility in mild acid increases with the liberation of more free amino groups, the N-deacetylation is heterogeneous, and some crystallinity points to chitin-like parts of the chitosan. A further clarification of the definitive nature of the partially N-deacetylated chitin components was the main objective of the present studies. Consider the sequence of reactions on the solid particles of chitin. As the insoluble particle swells so the * To whom correspondence should be addressed. Tel.: +1 319 3357897. Fax: +1 319 335-9570. E-mail:
[email protected].
NaOH deacetylates the most exposed β-1,4-linked N-acetyl glucosaminyl residues and the reducing termini of the chitin chains degrade by a “peeling” process.10 These reactions continue as the NaOH diffuses into the particle of chitin, the rate depending to some extent on the nature of the chitin and its associated components like silica, calcium carbonate, and protein. For example, in the crab shell, the native chitin is intimately structured with several nonchitin components, the removal of which by alkali and acid may result in some slight N-deacetylation and change in the truly native state. One commercial chitin is a partially purified crab shell in which as little modification of the native state as possible has occurred. Extensive treatment of the crab shell to give “pure” chitin will change this native condition, even though the overall constitution of the chitin chains remain the same. Differences in the minimally deacetylated chitosans produced similarly from commercial native chitin and from a chitin produced by the N-reacetylation of a crab shell chitosan were demonstrated by applying the Smith degradation11 to each. The resulting chitooligosaccharides were significantly different. The insolubility of chitin presents difficulties for research on the properties of chitinase, where improved assays have turned to substrates in which chitin is modified to increase surface area for the enzyme to access more readily, such as the preparations of colloidal,12 amorphous, or superfine chitin.13 It is then a challenge to characterize all the released products from enzymic hydrolysis. Water-soluble nonreducing chito-oligosaccharides could be an ideal substitute for such assays, as isolated by the Smith degradation of N-deacetylated chitins. The Smith degradation products resulting from minimal N-deacetylation of
10.1021/bm100991s 2010 American Chemical Society Published on Web 10/19/2010
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Scheme 1. Fractionation of the Smith Degradation Products from Partially N-Deacetylated Chitins
regenerated chitins gave water-soluble chito-oligosaccharides that were examined as suitable chitinase substrates.
Experimental Section Materials and Reagents. Powdered chitin (C7170, practical grade), chitosan (C3646, >85% N-deacetylation), and N,N-dimethyl formamide (DMF) were purchased from Sigma-Aldrich (Milwaukee, WI, U.S.A.). All other chemicals were of analytical grade and reported in the previous work.14 Analytical and General Procedures. High pressure liquid chromatographic analysis (HPLC) for acetate and butyrate, high-pH anion exchange chromatography-pulsed amperometric detection analysis (HPAEC-PAD) for glucosamine, and light scattering detection method for chitin butyrate were presented in a previous report.14 The fractionation of the Smith degradation products from partially N-deacetylated chitins is presented in Scheme 1. Regenerated chitin was prepared by selective N-acetylation of commercial chitosan (C3646) in aqueous methanolic acetic acid with acetic anhydride.15 The regenerated chitin showed DAc 0.95-1.04. Partial Alkaline N-Deacetylation of Chitin. Partial N-deacetylation was performed with 20% NaOH, similar to the procedure by Kurita et al.4 Briefly, powdered chitin (C7170; 7.5 g) in 400 mL of 20% (w/w) NaOH was heated at 105 °C for 10 min (under constant flow of nitrogen or helium), and the insoluble reaction product, when chilled in an icebath, was filtered (glass-fiber filter) and washed with water, ethanol, and acetone before being air-dried (F1: 4.56 g, DAc 0.84). Regenerated chitin (5 g; DAc 0.95), prepared by selective Nacetylation, was treated as above. The dispersed product when chilled in an ice-water bath was recovered by centrifugation and washed with water before being freeze-dried (F2: 0.92 g, DAc 0.79). The combined solution of the supernatant and washings, when neutralized with HCl, resulted in the precipitation of another N-deacetylated product, which was recovered and dried (F3: 1.3 g, DAc 0.72), as for the F2. Smith Degradation Products of Partial N-Deacetylated Chitin. To a stirred suspension of partially N-deacetylated material (F1, DAc 0.84;
Yang et al. 0.5 to 3 g) from the native chitin in 0.1 M NaOAc (100 to 200 mL of buffer solution adjusted to pH 4.5) at 3 °C was added NaIO4 (to a final concentration of 0.25 M; 5-10 mol excess based on the available amino groups) and the stirring continued for 16 h. Excess NaIO4 was destroyed by the addition of ethylene glycol and the oxidized material was recovered by centrifugation, washed with water, and reduced with NaBH4 (0.5 g, 16 h, at room temperature (rt)). Excess NaBH4 was destroyed by the addition of glacial acetic acid. The oxidized-reduced product, recovered by centrifugation and washing with water, was suspended in 1 M TFA (100 mL) and stirred for 16 h at rt. The dispersed solid (F1-i) from the Smith degradation was recovered by centrifugation, washed, and freeze-dried. The combined solution of supernatant and washings was freeze-dried, and the resulting dried residue (F1-s) was mostly soluble in water. Chitosan (F2) from regenerated chitin was similarly oxidized and reduced. The borate was removed as methyl borate by rotary coevaporation with absolute MeOH before hydrolysis, followed by freezedrying. Resuspension of the freeze-dried product and centrifugation gave the water-insoluble material (F2-i), which was washed with water and freeze-dried. The water-soluble fraction (F2-s) of the Smith product was eluted on a BioGel P-2 gel column (2.5 × 86 cm, 400 mesh) to remove excess NaOAc, resulting from the reduction. The carbohydrate fractions were pooled before further analysis (Figure 1) on a BioGel P-4 column (66 × 5 cm,
