Improvement of Cellulomonas fimi endoglucanase CenA by multienzymatic display on a decameric structural scaffold

Abstract

The development of multifunctional particles using polymeric scaffolds is an emergingtechnology for many nanobiotechnological applications. Here we present a system forthe production of multifunctional complexes, based on the high affinity non-covalentinteraction of cohesin and dockerin modules complementary fused to decamericBrucella abortus lumazine synthase (BLS) subunits, and selected target proteins,respectively. The cohesin-BLS scaffold was solubly expressed in high yield inEscherichia coli, and revealed a high thermostability. The production of multienzymaticparticles using this system was evaluated using the catalytic domain of Cellulomonasfimi endoglucanase CenA recombinantly fused to a dockerin module. Coupling of theenzyme to the scaffold was highly efficient and occurred with the expectedstoichiometry. The decavalent enzymatic complexes obtained, showed highercellulolytic activity and association to the substrate compared to equivalent amounts ofthe free enzyme. This phenomenon was dependent on the multiplicity and proximity ofthe enzymes coupled to the scaffold, and was attributed to an avidity effect in thepolyvalent enzyme interaction with the substrate. Our results highlight the usefulness ofthe scaffold presented in this work for the development of multifunctional particles, andthe improvement of lignocellulose degradation among other applications.