Carbohydrate-binding properties of a separately folding protein module from β-1,3-glucanase Lic16A of Clostridium thermocellum

Abstract

The multi-modular non-cellulosomal endo-1,3(4)-b-glucanase Lic16A from Clostridium thermocellum contains a so-called X module (denoted as CBMX) near the N terminus of the catalytic module (191-426 aa). Melting of X-module-containing recombinant proteins revealed an independent folding of the module. CBMX was isolated and studied as a separate fragment. It was shown to bind to various insoluble polysaccharides, including xylan, pustulan, chitin, chitosan, yeast cell wall glucan, Avicel and bacterial crystalline cellulose. CBMX thus contains a hitherto unknown carbohydrate-binding module (CBM54). It did not bind soluble polysaccharides on which Lic16A is highly active. Ca 2+ ions had effects on the binding, e.g. stimulated complex formation with chitosan, which was observed only in the presence of Ca 2+ . The highest affinity to CBMX was shown for xylan (binding constant K53.1¾10 4 M "1 ), yeast cell wall glucan (K51.4¾10 5 M "1 ) and chitin (K53.3.10 5 M "1 in the presence of Ca 2+ ). Lic16A deletion derivatives lacking CBMX had lower affinity to lichenan and laminarin and a slight decrease in optimum temperature and thermostability. However, the specific activity was not significantly affected.

Novel Carbohydrate-Binding Module Identified in a Ruminal Metagenomic Endoglucanase

“…4, bottom) and not affected by the inclusion of laminarin and soluble starch (data not shown). The affinities of the XM in C5614-1 to insoluble substrates were similar to those of CBM37 from Ruminococcus albus (10) and CBM54 from Clostridium thermocellum (6), which could also bind various insoluble substrates. However, the binding of CBM54 and CBM37 to soluble substrates was either negative or not tested.…”

Biochemical and Domain Analyses of FSUAxe6B, a Modular Acetyl Xylan Esterase, Identify a Unique Carbohydrate Binding Module in Fibrobacter succinogenes S85

“…CBMs are protein folds that recognize specific polysaccharides and are often linked to a catalytic glycoside hydrolase domain through flexible loops (5). Many CBMs have been identified experimentally and classified into 54 families based on similarity of amino acid sequence, and some members from different families, such as families 2 (3), 4 (1), 6 (15, 23), 13 (6), 22 (8), 35 (4), 37 (45), and 54 (13) are known to bind to insoluble ␤-1,4-xylan. The F. succinogenes S85 FPm-1 is proposed as a novel CBM family because there is no characterized CBM that shares homology with its sequence.…”

Extremophiles and their Application to Biofuel Research

“…High-temperature optima, thermostability, solvent tolerance, and broad substrate specificity are characteristics of these enzymes. Bacterial variants have been described for members of the genera Clostridium and Rhodothermus (Hobel et al, 2005;Taylor et al, 2006;Dvortsov et al, 2009Dvortsov et al, ,2010.…”