Actinobacteria, a large group of Gram-positive bacteria, secrete a wide range of extracellular enzymes involved in the degradation of organic compounds and biopolymers including the ubiquitous aminopolysaccharides chitin and chitosan. While chitinolytic enzymes are distributed in all kingdoms of life, actinobacteria are recognized as particularly good decomposers of chitinous material and several members of this taxon carry impressive sets of genes dedicated to chitin and chitosan degradation. Degradation of these polymers in actinobacteria is dependent on endo- and exo-acting hydrolases as well as lytic polysaccharide monooxygenases. Actinobacterial chitinases and chitosanases belong to nine major families of glycosyl hydrolases that share no sequence similarity. In this paper, the distribution of chitinolytic actinobacteria within different ecosystems is examined and their chitinolytic machinery is described and compared to those of other chitinolytic organisms.Electronic supplementary materialThe online version of this article (10.1007/s00253-018-9149-4) contains supplementary material, which is available to authorized users.

The chitosanase from Streptomyces sp. N174 (CsnN174) catalyzes the hydrolysis of b-1,4-glycosidic links in chitosan, a water-soluble derivative of chitin composed of d-glucosamine (GlcN) with a variable but minor proportion of N-acetyl-d-glucosamine (GlcNAc) [1]. Research on the enzymatic hydrolysis of chitosan is driven by the fact that this polymer has numerous potential applications and that its properties often depend on its molecular mass [2]. CsnN174 belongs to family 46 of the glycoside hydrolases (GH46), endohydrolase-type enzymes acting via an inverting mechanism [3,4]. GH46 enzymes belong to the GH-I clan [5] together with lysozymes from family GH24 (the most studied being the lysozyme from T4 phage). Enzymes from these two families share the same catalytic mechanism and are folded similarly, with two globular The chitosanase from Streptomyces sp. N174 (CsnN174) is an inverting glycoside hydrolase belonging to family 46. Previous studies identified Asp40 as the general base residue. Mutation of Asp40 into glycine revealed an unexpectedly high residual activity. D40G mutation did not affect the stereochemical mechanism of catalysis or the mode of interaction with substrate. To explain the D40G residual activity, putative accessory catalytic residues were examined. Mutation of Glu36 was highly deleterious in a D40G background. Possibly, the D40G mutation reconfigured the catalytic center in a way that allowed Glu36 to be positioned favorably to perform catalysis. Thr45 was also found to be essential. Thr45 is thought to orientate the nucleophilic water molecule in a position to attack the glycosidic link. The finding that expression of heterologous CsnN174 in Escherichia coli protects cells against the antimicrobial effect of chitosan, allowed the selection of active chitosanase variants after saturation mutagenesis. Thr45 could be replaced only by serine, indicating the importance of the hydroxyl group. The newly identified accessory catalytic residues, Glu36 and Thr45 are located on a three-strand b sheet highly conserved in GH19, 22, 23, 24 and 46, all members of the 'lysozyme superfamily'. Structural comparisons reveal that each family has its catalytic residues located among a small number of critical positions in this b sheet. The position of Glu36 in CsnN174 is equivalent to general base residue in GH19 chitinases, whereas Thr45 is located similarly to the catalytic residue Asp52 of GH22 lysozyme. These examples reinforce the evolutionary link among these five GH families.Abbreviations (GlcN) n , b-D-glucosamine oligosaccharide with n monomer units; CsnN174, chitosanase from Streptomyces sp. N174; GH, glycoside hydrolase family; GlcN, D-glucosamine; GlcNAc, N-acetylglucosamine.

Chitosan, an N-deacetylated derivative of chitin, has attracted much attention as an antimicrobial agent against fungi, bacteria, and viruses. Chitosanases, the glycoside hydrolases responsible for chitosan depolymerisation, are intensively studied as tools for biotechnological transformation of chitosan. The chitosanase CsnA (SCO0677) from Streptomyces coelicolor A3(2) was purified and characterized. CsnA belongs to the GH46 family of glycoside hydrolases. However, it is secreted efficiently by the Tat translocation pathway despite its similarity to the well-studied chitosanase from Streptomyces sp. N174 (CsnN174), which is preferentially secreted through the Sec pathway. Melting point determination, however, revealed substantial differences between these chitosanases, both in the absence and in the presence of chitosan. We further assessed the role of CsnA as a potential protective enzyme against the antimicrobial effect of chitosan. A Streptomyces lividans TK24 strain in which the csnA gene was inactivated by gene disruption was more sensitive to chitosan than the wild-type strain or a chitosanase-overproducing strain. This is the first genetic evidence for the involvement of chitosanases in the protection of bacteria against the antimicrobial effect of chitosan.

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