A bifunctional gene was isolated from a rumen microbial metagenome with the sequence encoding two catalytic domains, a N-terminal 1,4-endo-�-mannanase and a C-terminal 1,3-1,4-�-glucanase.  The full-length gene and the individual domains were cloned into E. coli and expressed in active forms.  The bifunctional whole enzyme hydrolyzed glucomannan, galactomannan and �-glucan with Km and kcat values of 22.0, 28.4, 36.1 mg/ml, and 5.03, 2.27, 5.61 sec-1, respectively  The activity of the C-domain glucanase on �-glucan was decreased significantly with the addition of glucomannan to the substrate reaction.  Analogous result was not observed with the N-domain mannase when �-glucan was added to the glucomannan substrate reaction.  The bifunctional whole enzyme did not show synergistic advantage compared to the summation of the two individual domain enzymes on single or mixed substrates.

Gene cloning and characterization of a bifunctional endo-1,4-β-mannanase/1,3-1,4-β-glucanase enzyme and its individual domains

244th National Meeting (2012)

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