Degradation of Chitosans with a Family 46 Chitosanase from Streptomyces coelicolor A3(2) Ellinor B. Heggset, †,‡ Anette I. Dybvik, †,‡ Ingunn A. Hoell, § Anne Line Norberg, § Morten Sørlie, § Vincent G. H. Eijsink, § and Kjell M. Vårum* ,‡ Norwegian Biopolymer Laboratory (NOBIPOL), Department of Biotechnology, Norwegian University of Science and Technology, 7491 Trondheim, Norway, and Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, 1432 Ås, Norway Received June 17, 2010; Revised Manuscript Received July 21, 2010 We have studied the degradation of well-characterized soluble heteropolymeric chitosans by a novel family 46 chitosanase, ScCsn46A from Streptomyces coelicolor A3(2), to obtain insight into the enzyme’s mode of action and to determine its potential for production of different chitooligosaccharides. The degradation of both a fully deacetylated chitosan and a 32% acetylated chitosan showed a continuum of oligomeric products and a rapid disappearance of the polymeric fraction, which is diagnostic for a nonprocessive endomode of action. The kinetics of the degradation of the 32% acetylated chitosan demonstrated an initial rapid phase and a slower second phase, in addition to a third and even slower kinetic phase. The first phase reflects the cleavage of the glycosidic linkage between two deacetylated units (D-D), the primary products being fully deacetylated dimers, trimers, and tetramers, as well as longer oligomers with increasing degrees of acetylation. In the subsequent slower kinetic phases, oligomers with a higher degree of acetylated units (A) appear, including oligomers with A’s at the reducing or nonreducing end, which indicate that there are no absolute preferences for D in subsites -1 and +1. After maximum degradation of the chitosan, the dimers DA and DD were the dominant products. The degradation of chitosans with varying degrees of acetylation to a maximum degree of scission showed that ScCsn46A could degrade all chitosan substrates extensively, although to decreasing degrees of scission with increasing F A . The potential use of ScCsn46A to prepare fully deacetylated oligomers and more highly acetylated oligomers from chitosan substrates with varying degrees of acetylation is discussed. Introduction Chitin is an essential structural component in the exoskeleton of crustaceans and insects, and is also found in the cell walls of certain fungi and in algae. 1 This insoluble polymer is composed of (1-4)-linked units of 2-acetamido-2-deoxy--D- glucopyranose (GlcNAc; A-unit). Chitosans are a family of water-soluble linear binary heteropolysaccharides composed of (1f4)-linked A-units and 2-amino-2-deoxy--D-glucopyranose (GlcN, D-unit), which can be prepared from chitin with varying extents of deacetylation. Variation in the chemical composition and chain length of chitosans have been shown to affect their properties and functionalities. 1 From chitosans with a defined chemical composition, well-defined mixtures of chitooligosac- charides can be prepared, and we have recently reviewed some of their most promising applications. 2 Chitosans prepared by homogeneous de-N-acetylation of chitin, such as those used in the present study, have a random distribution of A- and D-units. 3-5 Chitinases and chitosanases are glycoside hydrolases that are capable of converting chitin and chitosans to low molecular weight products (chitooligosaccharides, CHOS) by hydrolyzing the glycosidic linkages between the sugar units. 2 Chitosanases (EC 3.2.1.132) are enzymes that can hydrolyze glycosidic linkages in chitosans and can be divided into subclasses, depending on their specificity toward A- or D-units bound to subsites -1 and +1. 6 Chitosanases are produced by various organisms such as fungi and bacteria and occur in six different families of the glycoside hydrolases (GHs), that is, families 5, 7, 8, 46, 75, and 80. 7 Families 5, 7, and 8 primarily contain nonchitosanases but include a few members for which chitosan- hydrolyzing activity has been detected. The other three families, GH46, GH75, and GH80, exclusively contain chitosanases. Chitinases occur in families GH18 and GH19. The genome of the Gram-positive bacterium Streptomyces coelicolor A3(2) 8 contains 13 chitinase genes (11 GH18 and 2 GH19 9 ), two genes putatively encoding GH46 chitosanases and one gene putatively encoding a chitosanase belonging to family GH75. Generally, glycoside hydrolases can either degrade polymeric substrates from the chain end (exo attack) or from a random point along the polymer chain (endo attack), and each of these mechanisms can occur in combination with a processive mode of action. The enzymes employ either a retaining “double- displacement” mechanism or an inverting “single displacement” mechanism. 10-12 There are several types of remote structural similarities between GH families. For example, family 46 chitosanases, family 19 chitinases, and lysozymes belonging to GH families 22-24 share a structural core consisting of two R-helixes and a three-stranded -sheet and are said to form “the lysozyme superfamily”. 13,14 We have previously performed in-depth studies of several chitinases, including the GH18 enzymes ChiA, ChiB, and ChiC from Serratia marcescens 15-20 and a family 19 chitinase, ChiG, from Streptomyces coelicolor A3(2), 21 using chitosans as substrates. These studies have provided protocols for the production of specific chitooligosaccharides as well * To whom correspondence should be addressed. E-mail: kjell.morten. vaarum@biotech.ntnu.no. † These authors contributed equally to this paper. ‡ Norwegian University of Science and Technology. § Norwegian University of Life Sciences. Biomacromolecules 2010, 11, 2487–2497 2487 10.1021/bm1006745  2010 American Chemical Society Published on Web 08/12/2010