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Streptomyces olivaceoviridis secretes a so far unique protein of 18.7 kDa (CHB1) which lacks catalytic activity. It interacts highly specifically with α-chitin, but not with β-chitin, chitosan, or cellulose. Each of the five codons for tryptophan (Trp) in the chb1 gene was replaced by those for leucine (Leu) or tyrosine (Tyr). Eight corresponding mutant proteins and the wild-type protein were purified to homogeneity and their binding capacity to α-chitin was determined. The relative affinities to anti-CHB1 antibodies, the kinetics of binding, the dissociation constants, circular dichroism, and fluorescence emission spectra for three mutant types were compared to the characteristics of CHB1. The presented data lead to the following conclusions. (a) CHB1 presents a highly flexible protein lacking α-helices. (b) Replacement of each of the buried Trp residues (Trp134 and Trp184) leads to conformational alterations and, in due course, to a considerably reduced binding affinity of the protein. (c) The exchange of the exposed Trp 57 by either Leu or Tyr results in relatively slight topological changes, but entails a loss of binding capacity of about 90%. (d) The dissociation constant was highest for the mutant protein [L57]CHB1 (2.17 µM), followed by [LI34]CHB1 (0.91 µM) and [LI84]CHB1 (0.26 µM), and lowest for the progenitor CHB1 (0.11 µM), indicating its strong affinity to the unsoiuble substrate. (e) The data suggest that the exposed Trp57 contributes directly and significantly to the interaction of CHB1 with α-chitin. [ABSTRACT FROM AUTHOR]