Reconstitution of a Thermostable Xylan-Degrading Enzyme Mixture from the Bacterium Caldicellulosiruptor bescii

Abstract

Xylose, the major constituent of xylans, as well as the side chain sugars, such as arabinose, can be metabolized by engineered yeasts into ethanol. Therefore, xylan-degrading enzymes that efficiently hydrolyze xylans will add value to cellulases used in hydrolysis of plant cell wall polysaccharides for conversion to biofuels. Heterogeneous xylan is a complex substrate, and it requires multiple enzymes to release its constituent sugars. However, the components of xylan-degrading enzymes are often individually characterized, leading to a dearth of research that analyzes synergistic actions of the components of xylan-degrading enzymes. In the present report, six genes predicted to encode components of the xylan-degrading enzymes of the thermophilic bacterium Caldicellulosiruptor bescii were expressed in Escherichia coli, and the recombinant proteins were investigated as individual enzymes and also as a xylan-degrading enzyme cocktail. Most of the component enzymes of the xylan-degrading enzyme mixture had similar optimal pH (5.5 to ϳ6.5) and temperature (75 to ϳ90°C), and this facilitated their investigation as an enzyme cocktail for deconstruction of xylans. The core enzymes (two endoxylanases and a ␤-xylosidase) exhibited high turnover numbers during catalysis, with the two endoxylanases yielding estimated k cat values of ϳ8,000 and ϳ4,500 s ؊1 , respectively, on soluble wheat arabinoxylan. Addition of side chain-cleaving enzymes to the core enzymes increased depolymerization of a more complex model substrate, oat spelt xylan. The C. bescii xylan-degrading enzyme mixture effectively hydrolyzes xylan at 65 to 80°C and can serve as a basal mixture for deconstruction of xylans in bioenergy feedstock at high temperatures.

Two thermophilic fungal pectinases from Neosartorya fischeri P1: Gene cloning, expression, and biochemical characterization

“…3D and H), indicating that NfPG4 and NfPG5 are not very stable at these temperatures in absence of the substrate. The instability of thermophilic polygalacturonases at temperatures equal to or slightly lower to their optimums has been commonly found by other researchers [22,26], and this was also described for other thermophilic glycoside hydrolases such as xylanases [28]. However, when incubated at 55 • C, the two pectinases were rather stable, only slowly losing their activities to 85% (for NfPG4) and 75% (for NfPG5), respectively, after 1 h of incubation ( Fig.…”

The N-Terminal GH10 Domain of a Multimodular Protein from Caldicellulosiruptor bescii Is a Versatile Xylanase/β-Glucanase That Can Degrade Crystalline Cellulose

“…The E. coli Trans1 strain (Transgen, Beijing, China) was used for plasmid construction and propagation throughout this study. The genome sequence of C. bescii was uploaded and analyzed on the RAST (Rapid Annotation Using Subsystem Technology; http://rast.nmpdr.org) server (13) as described previously (3). A gene encoding a multimodular protein (GenBank accession number ACM60945) which contained a GH10 domain at its N terminus, two CBM3b modules in the middle, and a GH48 domain at the C terminus was identified to be cb1944, and the protein that it encoded was designated CbXyn10C/Cel48B.…”

“…The complete deconstruction of PCWPs into fermentable, simple mono-or oligosaccharides requires the concerted action of a complex array of glycoside hydrolases (GHs), including cellulases and hemicellulases (2,3). The genomes of Gram-positive bacteria of the genus Caldicellulosiruptor encode an arsenal of thermophilic plant cell wall polysaccharide-degrading enzymes (3)(4)(5), which are appealing candidates in the design of novel, robust enzyme cocktails for PCWP deconstruction.…”

Novel thermophilic hemicellulases for the conversion of lignocellulose for second generation biorefineries

“…These enzymes, named GH67-GA and GH67-GC, have, respectively, up to 57% and 77% amino acid sequence identity to GH67 enzymes characterized so far (Additional Table 2) [47,48]. Also for these enzymes, the analyses with the programs PRED-TAT (Additional Fig.…”