Abstract
Glycoside hydrolase family 9 (GH9) endoglucanases are important enzymes for cellulose degradation. However, their activity on cellulose is diverse. Here, we cloned and expressed one GH9 enzyme (CalkGH9T) from Clostridium alkalicellulosi in Escherichia coli. CalkGH9T has a modular structure, containing one GH9 catalytic module, two family 3 carbohydrate binding modules, and one type I dockerin domain. CalkGH9T exhibited maximal activity at pH 7.0–8.0 and 55 °C and was resistant to urea and NaCl. It efficiently hydrolyzed carboxymethyl cellulose (CMC) but poorly degraded regenerated amorphous cellulose (RAC). Despite strongly binding to Avicel, CalkGH9T lacked the ability to hydrolyze this substrate. The hydrolysis of CMC by CalkGH9T produced a series of cello-oligomers, with cellotetraose being preferentially released. Similar proportions of soluble and insoluble reducing ends generated by hydrolysis of RAC indicated non-processive activity. Our study extends our knowledge of the molecular mechanism of cellulose hydrolysis by GH9 family endoglucanases with industrial relevance.
Highlights
IntroductionCellulose is the most abundant polysaccharide in plant cell walls
The genome of C. alkalicellulosi was sequenced, and analysis showed that it contained one gene, CloalDRAFT_2759, which encodes a protein belonging to glycoside hydrolase family 9
Our results indicate that dockerin-borne CalkGH9T is a cellulosomal enzymatic subunit of the C. alkalicellulosi cellulosome, and it possibly shows cross-reactivity with C. thermocellum cellulosome based on sequence similarity [21]
Summary
Cellulose is the most abundant polysaccharide in plant cell walls It is composed of glucose units linked by β-glucosidic bonds. Each glucose residue is rotated 180◦ relative to its adjacent residues, yielding cellobiose as the structural repeating unit of cellulose [1] This structural configuration makes the glucan chain linear. This chain linearity allows the glucan chains to contact other chains, forming intra- and inter-hydrogen bonding within and between the chains. This close association between cellulose chains results in a tightly packed, crystalline polymeric structure. This rigid and strong cellulose polymer provides structural integrity to plants and recalcitrance to microbial attack [2]
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