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Effects of N-acetylation degree on N-acetylated chitosan hydrolysis with commercially available and modified pectinases

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Abstract

Three types of N-acetylated chitosans (NACs) with different degrees of acetylation (DA) were prepared and used as a substrate for enzymatic hydrolysis with a commercially available pectinase and a modified one. Pectinase modification was conducted using polyalkyleneoxide–maleic anhydride copolymer (PEO–MA copolymer). The effects of DA on enzymatic reaction with native and modified pectinases were investigated experimentally. Initial hydrolysis rate and Michaelis–Menten kinetic parameters were measured by analysis of reducing sugars. DA of NAC strongly affected the hydrolytic characteristics of native and modified pectinases. N-acetylation of chitosan increased the initial hydrolysis rate and the enzyme-substrate affinity with respect to both pectinases: NACs with DA over 0.3 showed high initial hydrolysis rate and strong affinity between enzyme and substrate. Especially, when NAC with DA over 0.3 was treated with modified pectinase, the affinity became much stronger than the native pectinase.

Introduction

Chitosan is deacetylated derivative of chitin, which is an abundant natural polysaccharide in an exoskeleton in creatures, such as crustaceans, insect and fungi, etc. Chitinous materials are obtained from the marine product industry as solid-waste.

Chitosan is a linear heteropolysaccharide composed of β-1,4-linked d-glucosamine (GlcN) and N-acetyl-d-glucosamine (GlcNAc) with various compositions of these two monomers. Commercially available chitosan is generally produced by heterogeneous N-deacetylation of chitin and the chitosan has low GlcNAc content from 0 to 0.3 and block type distribution of the GlcNAc residue. Chitosan is soluble only in aqueous acidic solution and insoluble in aqueous neutral and alkaline solution [1].

Recently, chitosanous materials have received considerable amount of attention because it has various biological activities of interest. According to the relationship between biological activities and degree of polymerization, depolymerized chitosan also has comparable biological activities to chitosan polymer. In addition, chitosan depolymerization enhances its water-solubility and reduces the solution viscosity. Therefore, depolymerization of chitosan could facilitate the application of chitosanous materials in a variety of fields.

Enzymatic hydrolysis has been considered as a promising method for preparing depolymerized chitosan. The enzymatic hydrolysis can be generally performed under gentle conditions compared to the chemical hydrolysis. However, enzymatic hydrolysis of chitosan has not been used in industrial scale because of the cost of the specific enzymes, chitosanase and chitinase.

Recently, several hydrolytic enzymes, such as lysozyme, cellulase, papain, lipase and pectinase were found to catalyze the cleavage of glycosidic linkage in chitosan polymer [2], [3], [4]. These enzymes are widely used in industry and are inexpensive. Moreover, we reported that modified pectinase with polyethyleneoxide–maleic anhydride copolymer (PEO–MA) had high thermo-stability and strong affinity between enzyme and chitosan [5]. Therefore, characterization of these enzymes is required for the practical application.

In this study, N-acetylated chitosans (NACs) with different degree of acetylation (DA) were used as the substrate of the enzymatic hydrolysis and the effects of DA on enzymatic hydrolysis of NAC were investigated using native and modified pectinases.

Section snippets

Materials

Chitosan 10B with a low DA (Funakoshi Co., Japan) was used for preparation of NACs with various DA. Commercially available pectinase (MERCK, Germany) was purchased and used for enzymatic hydrolysis of NACs without further purification. Modifier of pectinase, PEO–MA, was kindly supplied from Nippon Oil and Fat Co., Japan. Other chemicals were of reagent grade.

Preparation of NACs

NACs with various DA were prepared under homogeneous condition as reported by Kubota and Eguchi [6] with a slight modification. Chitosan

DA of NACs

DA and distribution of N-acetyl groups significantly affect the solution properties of chitosan [6], [9]. These facts might also affect the enzymatic hydrolysis of NACs. So, N-acetylation of chitosan was carried out under homogeneous condition in the present work. Therefore, N-acetyl groups are considered to be distributed randomly in chitosan polymer chain. NACs with different DA were prepared by varying the amount of acetic anhydride. DA of NACs was determined by 1H-NMR method, as listed in

Conclusions

NACs hydrolyses with native and modified pectinases were carried out with respect to NACs with different DA. From the results, DA of NACs strongly affected the hydrolytic characteristics of both enzymes. In addition, compared to the native pectinase, the modified pectinase showed somewhat different hydrolytic behavior when NACs with higher DA were used as the substrate.

When the native pectinase was used, initial hydrolysis rate gradually increased with increase in DA up to 0.3 while it did not

Acknowledgements

This work was partially supported by The Cosmetology Research Foundation, Japan.

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