Modular organisation and functional analysis of dissected modular beta-mannanase CsMan26 from Caldicellulosiruptor Rt8B.4.
CsMan26 from Caldicellulosiruptor strain Rt8.B4 is a modular beta-mannanase consisting of two N-terminal family 27 carbohydrate-binding modules (CBMs), followed by a family 35 CBM and a family 26 glycoside hydrolase catalytic module (mannanase). A functional dissection of the full-length CsMan26 and a comprehensive characterisation of the truncated derivatives were undertaken to evaluate the role of the CBMs. Limited proteolysis was used to define biochemically the boundaries of the different structural modules in CsMan26. The full-length CsMan26 and three truncated derivatives were produced in Escherichia coli, purified and characterised. The systematic removal of the CBMs resulted in a decrease in the optimal temperature for activity and in the overall thermostability of the derivatives. Kinetic experiments indicated that the presence of the mannan-specific family 27 CBMs increased the affinity of the enzyme towards the soluble galactomannan substrate but this was accompanied by lower catalytic efficiency. The full-length CsMan26 and its truncated derivatives were unable to hydrolyse mannooligosaccharides with degree of polymerisation (DP) of three or less. The major difference in the hydrolysis pattern of larger mannooligosaccharides (DP >3) by the derivatives was determined by their abilities to further hydrolyse the intermediate sugar mannotetraose.
Sunna
Anwar
A
Department of Chemistry and Biomolecular Sciences, Faculty of Science, Macquarie University, Sydney, New South Wales 2109, Australia. asunna@els.mq.edu.au
D000595:Amino Acid Sequence; D001709:Biotechnology / Q000379:methods*; D003013:Clostridium / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D008351:Mannans / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D044902:beta-Mannosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D008351:Mannans; D009844:Oligosaccharides; D006026:Glycoside Hydrolases; D044902:beta-Mannosidase