Degradation of Chitosans with a Family 46 Chitosanase from Streptomyces coelicolor A3(2)

Abstract

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.

Structural and biochemical insights into the degradation mechanism of chitosan by chitosanase OU01

“…The catalytic mechanism and three-dimensional protein structures have been well studied, especially for chitosanases from the GH46 family [10,[14][15][16][17]. Previous studies demonstrated that the chitosanases from GH46 and GH75 acted in a non-processive mode, and its hydrolysis process contained an initial rapid phase and followed by a second slower phase [18,19].…”

Chemical Organization of the Cell Wall Polysaccharide Core of Malassezia restricta

“…Cellulase, amyloglucosidase, and amylase from Aspergillus niger were purchased from Sigma (stock solution at 1 mg/ml). Five recombinant enzymes were used: mutanase, an ␣-(1,3)-glucanase from Trichoderma harzianum, produced in Escherichia coli, a kind gift from C. Fuglsang (Novozymes A/S, Bagwaerd, Denmark) (18); laminarinase A, an endo-␤-(1,3)-glucanase from Thermotoga neapolitana expressed in E. coli (19); an endo-␤-(1,6)-glucanase (member of GH-5 family) from Schizosaccharomyces pombe expressed in Pichia pastoris (20,21); a chitinase from Serratia marcescens produced in E. coli (22), and a chitosanase (Csn46A) from Streptomyces coelicolor expressed in E. coli (23). Specific activities and optimum pH values are indicated in supplemental Table 1.…”

Uncoupling chitosanase production from chitosan

“…K17 12 and in an extensive investigation of the mechanism of action of the GH46 chitosanase ScCsn46A from Streptomyces coelicolor A3(2). 13 While very useful for basic research purposes, E. coli recombinant strains are less often proposed for large scale production of chitosanases, as the cost of downstream processing (enzyme recovery and purification) becomes a bottleneck. In a recent study, 14 the gene encoding a GH8 chitosanase from a newly isolated Bacillus strain was expressed in E. coli under the control of the promoter of T7 bacteriophage.…”