Abstract:

Cellulase can convert cellulose into fermentable sugars. In order to enrich the sequences and structure resources of cellulase and reveal the relationship between cellulase structure and function, two novel multi-domain endoglucanases belonging to glycosyl hydrolases（GH）family 5 TlCel5 and ReCel5 were cloned and expressed. Their enzymatic properties were determined and the mutation of structure domain was studied. Sequence and structure analysis showed that Tlcel5 and Recel5 encoded 655-residue and 632-residue polypeptides with theoretical molecular weights of 68.3 kD and 65.9 kD respectively. They wee both multi-domain cellulases, comprising a carbohydrate-binding module grouped into family 1（CBM1）and a catalytic module of family 5 glycoside hydrolase（CD）at N-terminal, a family X2 carbohydrate binding module（CBMX2）and an unknown domain（UM）at the C-terminal, which made them significantly different from most single-domain or dual-domain cellulases reported previously. With the purpose of understanding the effect of additional domains on enzyme function, the truncated mutant without the N-terminal CBM1 domain（TM1）and the truncated mutant without the C-terminal unknown domain（TM2）were constructed based on wild-type ReCel5. The assays of enzymatic properties demonstrated that the optimal pH and temperature of TlCel5 and ReCel5 were pH 3.0, 50℃ and pH 4.0, 70℃, respectively. They remained fine stability at 50℃ and 70℃, and showed certain hydrolytic ability to a variety of cellulose and hemicellulose substrates. Their hydrolysis specific viabilities to sodium carboxymethyl cellulose, barley β-glucan, and lichenan reduced by 23% to 68% although the enzymatic properties of TM1 and TM2 did not change significantly compared with the wild-type proteins. These results suggested that there was a close relationship between the additional domains and the hydrolysis ability of multi-domain enzymes.

Key words: multi-domain endoglucanase, cloning expression, enzymatic property, structure and function