Chitosanase was produced by the strain of Streptomyces lividans TK24 bearing the csn gene from Streptomyces sp. N174, and purified by S-Sepharose and Bio-Gel A column chromatography. Partially (25-35%) N-acetylated chitosan was digested by the purified chitosanase, and structures of the products were analysed by NMR spectroscopy. The chitosanase produced heterooligosaccharides consisting of D-GlcN and GlcNAc in addition to glucosamine oligosaccharides [(GlcN)n, n = 1, 2 and 3]. The reducing- and non-reducing-end residues of the heterooligosaccharide products were GlcNAc and GlcN respectively, indicating that the chitosanase can split the GlcNAc-GlcN linkage in addition to that of GlcN-GlcN. Time-dependent 1H-NMR spectra showing hydrolysis of (GlcN)6 by the chitosanase were obtained in order to determine the anomeric form of the reaction products. The chitosanase was found to produce only the alpha-form; therefore it is an inverting enzyme. Separation and quantification of (GlcN)n was achieved by HPLC, and the time course of the reaction catalysed by the chitosanase was studied using (GlcN)n (n = 4, 5 and 6) as the substrate. The chitosanase hydrolysed (GlcN)6 in an endo-splitting manner producing (GlcN)2, (GlcN)3 and (GlcN)4, and did not catalyse transglycosylation. Product distribution was (GlcN)3 >> (GlcN)2 > (GlcN)4. Cleavage to (GlcN)3 + (GlcN)3 predominated over that to (GlcN)2 + (GlcN)4. Time courses showed a decrease in rate of substrate degradation from (GlcN)6 to (GlcN)5 to (GlcN)4. It is most likely that the substrate-binding cleft of the chitosanase can accommodate at least six GlcN residues, and that the cleavage point is located at the midpoint of the binding cleft.

A highly efficient chitosanase producer, the actinomycete N174, identified by chemotaxonomic methods as belonging to the genus Streptomyces was isolated from soil. Chitosanase production by N174 was inducible by chitosan or D-glucosamine. In culture filtrates the chitosanase accounted for 50-60% of total extracellular proteins. The chitosanase was purified by polyacrylic acid precipitation, CM-Sepharose and gel permeation chromatography. The maximum velocity of chitosan degradation was obtained at 65 ° C when the pH was maintained at 5.5. The enzyme degraded chitosans with a range of acetylation degrees from 1 to 60% but not chitin or CM-cellulose. The enzyme showed an endo-splitting type of activity and the end-product of chitosan degradation contained a mixture of dimers and trimers of D-glucosamine.

The comparison of four sequences of prokaryotic chitosanases, belonging to the family 46 of glycosyl hydrolases, revealed a conserved N-terminal module of 50 residues, including five invariant carboxylic residues. To verify if some of these residues are important for catalytic activity in the chitosanase from Streptomyces sp. N174, these 5 residues were replaced by site-directed mutagenesis. Substitutions of Glu-22 or Asp-40 with sterically conservative (E22Q, D40N) or functionally conservative (E22D, D40E) residues reduced drastically specific activity and k cat , while K m was only slightly changed. The other residues examined, Asp-6, Glu-36, and Asp-37, retained significant activity after mutation. Circular dichroism studies of the mutant chitosanases confirmed that the observed effects are not due to changes in secondary structure. These results suggested that Glu-22 and Asp-40 are directly involved in the catalytic center of the chitosanase and the other residues are not essential for catalytic activity.

The antimicrobial properties of various chitosan-lactate polymers (ranging from 0.5 to 1.2 MDa in molecular weight) against two yeasts isolated from fermented vegetables and against three lactic acid bacteria from a mixed starter for sauerkraut on methylene blue agar (MBA) and in vegetable juice medium (VJM) were investigated. Chitosan-lactate reduced the growth of all microorganisms in solid (MBA) as well as in liquid (VJM) medium. In MBA, a concentration of 5 g/liter was needed to inhibit the growth of Saccharomyces bayanus, while 1 g/liter was sufficient to inhibit the growth of Saccharomyces unisporus. Lactic acid bacteria were also inhibited in this range of concentrations. The low-molecular-weight chitosan-lactate DP3 (0.5 kDa) was most efficient in solid medium (MBA), and inhibitory activities decreased with increasing hydrolysate lengths. In liquid medium (VJM), 0.5 g of chitosan-lactate per liter reduced the growth rates for both yeasts, but 10 g/liter was insufficient to prevent yeast growth. Intermediate-molecular-weight chitosan-lactate (5 kDa) was more efficient than chitosan of low molecular weight. Native chitosan (1.2 MDa) showed no inhibition in either medium. Microscopic examination of S. unisporus Y-42 after treatment with chitosan-lactate DP25 showed agglutination of a refractive substance on the entire cell wall, suggesting an interaction between chitosan and the cell wall. When chitosanase was added to the culture media containing chitosan-lactate, refractive substances could not be observed.

Strain N106, a newly isolated soil actinomycete classified in the genus Nocadioides on the basis of its chemotaxonomy, produced an extracellular chitosanase and was highly active in chitosan degradation. A gene library of Nocardioides sp. N106 was constructed in the shuttle vector pFD666 and recombinant plasmids carrying the chitosanase gene (csnN106) were identified using the 5'4erminal portion of the chitosanase gene from Streptomyces sp. N174 as a hybridization probe. One plasmid, pCSN106-2, was used to transform Streptomyces lividans TK24. The chitosanase produced by S. lividans(pCSN106-2) is a protein of 29.5 kDa, with a pl81, and hydrolyses chitosan by an endo-mechanism giving a mixture of dimers and trimers as endproducts. N-terminal sequencing revealed that the mature chitosanase is a mixture of two enzyme forms differing by one N-terminal amino acid. The csnN106 gene is 795% homologous to the csn gene from Streptomyces sp. N174. At the amino acid level, both chitosanases are homologous at 744% and hydrophobic cluster analysis revealed a strict conservation of structural features. This chitosanase is the third known member of family 46 of glycosyl hydro I ases .