Fast and reliable production, purification and characterization of heat-stable, bifunctional enzyme chimeras

Abstract

Der Abbau von komplexer Biomasse benötigt ein abgestimmtes Portfolio and Glykosidhydrolasen. Die LE-Klonierungsstrategie wurde dazu genutzt die beiden Cellulasen Cel5A (C) und Bgl1 (B) in zwei verschiedenen Reihenfolgen (CB und BC) zu verknüpfen und in rekombinanter Form in Escherichia coli zu produzieren. Die vollständige Reinigung der Enzymchimären und ihre Charakterisierung ergab, dass die katalytische Funktionalität beider Proteine durch die Reihenfolge der Fusion und damit verbunden durch die Konfirmation und Faltung beeinflusst wurde. Nichtsdestotrotz konnten verbesserte Eigenschaften bei dem Konstrukt BC beobachtet werden, welches einen besseren Umsatz von cellulolytische Substraten gewährleistete als eine Mischung beider einzelner Enzyme. Die Ergebnisse zeigen Vorteile von leicht zu produzierenden bifunktionellen Fusionsenzymen auf, um pflanzliche Biomasse abzubauen.

Degradation of complex plant biomass demands a fine-regulated portfolio of glycoside hydrolases. The LE (LguI/Eco81I)-cloning approach was used to produce two enzyme chimeras CB and BC composed of an endoglucanase Cel5A (C) from the extreme thermophilic bacterium Fervidobacterium gondwanense and an archaeal β-glucosidase Bgl1 (B) derived from a hydrothermal spring metagenome. Recombinant chimeras and parental enzymes were produced in Escherichia coli and purified using a two-step affinity chromatography approach. Enzymatic properties revealed that both chimeras closely resemble the parental enzymes and physical mixtures, but Cel5A displayed lower temperature tolerance at 100°C when fused to Bgl1 independent of the conformational order. Moreover, the determination of enzymatic performances resulted in the detection of additive effects in case of BC fusion chimera. Kinetic measurements in combination with HPLC-mediated product analyses and site-directed mutation constructs indicated that Cel5A was strongly impaired when fused at the N-terminus, while activity was reduced to a slighter extend as C-terminal fusion partner. In contrast to these results, catalytic activity of Bgl1 at the N-terminus was improved 1.2-fold, effectively counteracting the slightly reduced activity of Cel5A by converting cellobiose into glucose. In addition, cellobiose exhibited inhibitory effects on Cel5A, resulting in a higher yield of cellobiose and glucose by application of an enzyme mixture (53.1%) compared to cellobiose produced from endoglucanase alone (10.9%). However, the overall release of cellobiose and glucose was even increased by catalytic action of BC (59.2%). These results indicate possible advantages of easily produced bifunctional fusion enzymes for the improved conversion of complex polysaccharide plant materials.