Cellulase-Xylanase Synergy in Designer Cellulosomes for Enhanced Degradation of a Complex Cellulosic Substrate

Abstract

Designer cellulosomes are precision-engineered multienzyme complexes in which the molecular architecture and enzyme content are exquisitely controlled. This system was used to examine enzyme cooperation for improved synergy among Thermobifida fusca glycoside hydrolases. Two T. fusca cellulases, Cel48A exoglucanase and Cel5A endoglucanase, and two T. fusca xylanases, endoxylanases Xyn10B and Xyn11A, were selected as enzymatic components of a mixed cellulase/xylanase-containing designer cellulosome. The resultant mixed multienzyme complex was fabricated on a single scaffoldin subunit bearing all four enzymes. Conversion of T. fusca enzymes to the cellulosomal mode followed by their subsequent incorporation into a tetravalent cellulosome led to assemblies with enhanced activity (~2.4-fold) on wheat straw as a complex cellulosic substrate. The enhanced synergy was caused by the proximity of the enzymes on the complex compared to the free-enzyme systems. The hydrolytic properties of the tetravalent designer cellulosome were compared with the combined action of two separate divalent cellulase- and xylanase-containing cellulosomes. Significantly, the tetravalent designer cellulosome system exhibited an ~2-fold enhancement in enzymatic activity compared to the activity of the mixture of two distinct divalent scaffoldin-borne enzymes. These results provide additional evidence that close proximity between cellulases and xylanases is key to the observed concerted degradation of the complex cellulosic substrate in which the integrated enzymes complement each other by promoting access to the relevant polysaccharide components of the substrate. The data demonstrate that cooperation among xylanases and cellulases can be augmented by their integration into a single designer cellulosome.

Optimization and Scale-Up of Enzymatic Hydrolysis of Wood Pulp for Cellulosic Sugar Production

“…However, even with the highest AF100L loading of 15 mg/g dry mass, the sugar production was only 65.8 g/L after 48 h, which was far lower than the sugar production using 15 mg/g dry mass of each one of CMax and AF100L, indicating that 15 mg/g dry mass of both enzymes was the limit for hydrolysis. The synergy between xylanase and cellulases may have enhanced the proximity and access of the substrate to the enzymes (Moraïs et al 2010). …”

Enhanced cellulose degradation by targeted integration of a cohesin-fused β-glucosidase into the Clostridium thermocellum cellulosome

“…The importance of substrate targeting to cellulosome efficiency has been discussed previously in the context of CBMmediated attachment of cellulosomal cellulases to the insoluble substrate (23)(24)(25). Nevertheless, our data suggest that in a cellfree system another type of targeting mechanism can play an important role, i.e., the targeting of the β-glucosidase to the cellobiose-susceptible cellulases.…”

“…Fierobe et al (24), thus, prepared a designer cellulosome composed of three cellulases arranged into an artificial CBM-bearing scaffoldin that enhanced the degradation of microcrystaline cellulose by 3.8 fold (compared to the noncomplexed enzymes). A similar design composed of two xylanases, two cellulases, and a xylan-binding module enhanced the degradation of a wheat straw substrate by 2.6 fold (25). Similar levels of enhanced ethanol production were achieved on phosphoric acid-swollen cellulose (PASC) by mini cellulosomes composed of two cellulases and β-glucosidase attached to yeast cells (35); however, to date, the efficiency of artificial cellulosome systems has been inferior to that of the native cellulosome possibly due to the larger variety of enzymes and preferred conformation of the native complex (24).…”

“…The reaction was incubated at 60°C for 3 h, sufficient to convert all cellobiose into glucose. Reducing sugars measurement (DNS) was performed as described previously (25).…”

“…To date, most of the designer cellulosome experiments try to mimic the enzymatic synergism observed for native cellulosome systems by fabricating complexes composed of an artificial chimeric cohesin-containing scaffoldin and a set of matching dockerin-containing cellulases (23)(24)(25).…”