Interaction Force of Chitin-Binding Domains onto Chitin Surface

Abstract

Interaction force of chitin-binding domains (ChBD1 and ChBD2) from a thermostable chitinase onto chitin surface was directly measured by atomic force microscopy (AFM) in a buffer solution. In the force curve measurement, multiple pull-off events were observed for the AFM tips functionalized with either ChBD1 or ChBD2, whereas the AFM tips terminated with nitrilotriacetic acid groups without ChBD showed no interaction peak, suggesting that the detected forces are derived from the binding functions of ChBDs onto the chitin surface. The force curve analyses indicate that the binding force of ChBD2 is stronger than that of ChBD1. This result suggests that ChBD1 and ChBD2 play different roles in adsorption onto chitin surface.

Binding ability of chitinase onto cellulose: an atomic force microscopy study

“…The results revealed that the binding force of ChBD2 onto cellulose was 84 ± 13 pN, which was almost identical to that of chitin at a similar loading rate (ca 2 nN s À1 ). 9 In our previous work, 9 we determined the binding force of ChBD1 and ChBD2 on the surface of chitin, and the results were compared with the tertiary structures of ChBDs, which were deduced from homology modeling. The binding force of ChBDs is proportional to the number of conserved, surface-exposed, aligned aromatic residues in the enzyme.…”

“…The frequency of binding events of ChBD2 onto cellulose (30%) was slightly lower than that of chitin (39%). 9 Currently, the origin of these differences and their effect on the binding of the enzyme to the polysaccharide surface remains unclear. To determine the mechanism of binding, the binding force of the enzyme on the polysaccharide surface must be further analyzed; thus, the energy landscape of enzymatic binding is under investigation.…”

“…Purification of recombinant ChBD2 from Escherichia coli cells and functionalization of the AFM cantilever tip were performed according to a previously reported method. 9 Briefly, a gold-coated AFM cantilever (OMCL-TR400PB-1, Olympus, Tokyo, Japan) was washed with ultravioletozone cleaner and was functionalized using HS(CH 2 ) 11 (OCH 2 CH 2 ) 6 OCH 2 COOH (PEG 6 -COOH; Prochimia, Gdansk, Poland). Subsequently, the carboxylic acid groups were connected with N-(5-amino-1-carboxypentyl) iminodiacetic acid (Dojindo, Kumamoto, Japan) through an amide bond.…”

“…[6][7][8] In our previous work, 9 we applied force spectroscopy to measure the interaction force between chitin and two different kinds of chitin-binding domains (ChBDs) of a multidomain chitinase. The chitinase is produced by Thermococcus kodakarensis KOD1, a hyperthermophilic archaeon, and is composed of five characteristic domains, including dual catalytic domains (CatD A and B) and triple ChBDs (ChBD1, ChBD2 and ChBD3).…”

“…Subsequently, the chain ends are transported by ChBD1 to exotype CatD A. 9 To further elucidate the substrate specificity and binding mechanism of ChBD, we investigated the effect of different substrates on the binding force of ChBD. Cellulose, which is structurally different from chitin due to the side chain group, was used as a substrate, as shown in Scheme 1.…”