Isabelle Boucher

Purification and characterization of a chitosanase from Streptomyces N174

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.

Cloning and expression inStreptomyces lividans of a chitosanase-encoding gene from the actinomyceteKitasatosporia N174 isolated from soil

SummaryThe chitosanase gene from a new soil isolate, the actinomyceteKitasatosporia N174, was cloned inStreptomyces lividans TK24. The enzyme expressed from the cloned gene had a molecular weight of approximately 29,500; an isoelectric point of 7.5 and was indistinguishable from the purified N174 chitosanase.

Substrate binding to the inactive mutants of Streptomyces sp. N174 chitosanase: indirect evaluation from the thermal unfolding experiments

Oligosaccharide binding to chitosanase from Streptomyces sp. N174 was indirectly evaluated from thermal unfolding experiments of the protein. Thermal unfolding curves were obtained by fluorescence spectroscopy in the presence of d‐glucosamine oligosaccharides ((GlcN) n , n=3,4,5, and 6) using the inactive mutant chitosanase in which the catalytic residue, Glu22, is mutated to glutamine (E22Q), aspartic acid (E22D), or alanine (E22A). The midpoint temperature of the unfolding transition (T m) of E22Q was found to be 44.4°C at pH 7.0. However, the T m increased upon the addition of (GlcN) n by 1.3°C (n=3), 2.5°C (n=4), 5.2°C (n=5), or 7.6°C (n=6). No appreciable change in T m was observed when (GlcNAc)6 was added to E22Q. The effect of (GlcN) n on the thermal stability was examined using the other protein, RNase T1, but the oligosaccharide did not affect T m of the protein. Thus, we concluded that the stabilization effect of (GlcN) n on the chitosanase results from specific binding of the oligosaccharides to the substrate binding cleft. When E22D or E22A was used instead of E22Q, the increases in T m induced by (GlcN)6 binding were 2.7°C for E22D and 4.2°C for E22A. In E22D or E22A, interaction with (GlcN)6 seems to be partly disrupted by a conformational distortion in the catalytic cleft.

Thermal unfolding of chitosanase from Streptomyces sp. N174: role of tryptophan residues in the protein structure stabilization

Tryptophan residues in chitosanase from Streptomyces sp. N174 (Trp28, Trp101, and Trp227) were mutated to phenylalanine, and thermal unfolding experiments of the proteins were done in order to investigate the role of tryptophan residues in thermal stability. Four types of mutants (W28F, W101F, W227F and W28F/W101F) were produced in sufficient quantity in our expression system using Streptomyces lividans TK24. Each unfolding curve obtained by CD at 222 nm did not exhibit a two-state transition profile, but exhibited a biphasic profile: a first cooperative phase and a second phase that is less cooperative. The single tryptophan mutation decreased the midpoint temperature (Tm) of the first transition phase by about 7 degrees C, and the double mutation by about 11 degrees C. The second transition phase in each mutant chitosanase was more distinct and extended than that in the wild-type. On the other hand, each unfolding curve obtained by tryptophan fluorescence exhibited a typical two-state profile and agreed with the first phase of transition curves obtained by CD. Differential scanning calorimetry profiles of the proteins were consistent with the data obtained by CD. These data suggested that the mutation of individual tryptophan residues would partly collapse the side chain interactions, consequently decreasing Tm and enhancing the formation of a molten globule-like intermediate in the thermal unfolding process. The tryptophan side chains are most likely to play important roles in cooperative stabilization of the protein.