Molecular Catalysis Mechanism And Module Polymorphism Of Caldicellulosiruptor Kronotskyensis 2002 Endo-β-1,4-xylanase

The extreme thermophile Caldicellulosiruptor kronotskyensis 2002 can convert hemicelluloses to hydrogen and chemicals. It utilizes natural lignocellulosic biomass containing xylan. In the genome of this strain, there are many kinds of genes encoding xylanases composed of multi-functional domains, with catalytic domains and non-catalytic domains diversity. Two types of multi-modular xylanases belong to GH10 and GH11 glycoside hydrolase families, respectively. Meanwhile, carbohydrate binding modules （CBMs） exist in the structure of multi-modular xylanases. Xylanases are crucial for lignocellulosic biomass deconstruction, and generally xylanases containing non-catalytic domain CBMs can access recalcitrant polysaccharides easily. Understanding modular components and function of multi-modular xylanases can benefit enzyme engineering aiming at accommodating industrial production. In order to explore the mechanism of different GHs xylanases from C. kronotskyensis in hydrolysis of xylan and understand the roles of CBM for biochemical properties of xylanases, two types of different GHs xylanases and their truncations were cloned, expressed and characterized.1. The gene Calkro₀081 encoded a novel GH11 xylanase （Xyn11 A） with a catalytic domain （GH-CD） and a carbohydrate binding module （CBM6）. The native Xyn11A and two corresponded truncations Xyn11A-CD （catalytic domain of Xyn11 A） and Xyn11A-CBM （carbohydrate binding module of Xyn11 A） were cloned, heterologously expressed, and biochemically characterized. The recombinant Xyn11A showed the highest activity at 75℃, and active in a range of 40-95℃. Xynl 1A was stable for 6 h at 70℃, and more than 50% activity was retained after incubation for 6 h at 75℃. The optimum pH of Xyn11A was 6.0, and retained 100% activity after 15 h incubation in pH 5.5-7.5 at 70℃. As for Xyn11A-CD, the optimal pH value and temperature were 6.0 and 65℃, the residual activities retained 30% after 6 h at 60℃. The respective specific activity of Xyn11A, Xyn11 A-CD, and Xyn11A-CBM was 1752.0,986.8, and 0 IU/mg on beechwood xylan （BWX） at optimum conditions. The activity of Xyn11A is the highest reported activity at 75℃. Using BWX as substrate, the final products were xylose and xylobiose after hydrolysis with Xyn11A or Xyn11 A-CD. CBM6 plays a crucial part for thermostability and activity of Xyn11A. The characteristics of thermostability and high activity make Xyn11A a potential enzyme for industry application.2. The predicted extra （Xyn10A） and intracellular （Xyn10B） endoxylanase were comparatively characterized in this present study. Xyn10A and Xyn10B shared GH10 domain with similarity of 41%, while the former contains another two tandem N-terminus CBM22s. Xyn10A had a stronger hydrolysis capability than Xyn10B on both BWX and oat spelt xylan （OSX）, whereas Xynl OB showed preference for xylooligosaccharide （XOS） than Xyn10A. Truncation mutation experiments revealed the importance of CBMs for hydrolytic activity, substrate binding and thermostability of Xyn10A, while the number of CBMs was not directly related to binding and thermostability. Although CBM was considered to be crucial for substrate binding, Xyn10B and Xyn10A as well as truncations shared similar binding affinity to insoluble substrate OSX, and all of them displayed slight adsorption to Avicel and natural substrates. Point mutation analyses revealed the similar key residues Glu493, Glu601 and Trp658 of Xyn10A and Glu139, Glu247 and Trp305 of Xyn10B in active site. Both Xyn10B and Xyn10A exhibited hydrolytic activity on mechanical pretreated corncob, corn straw, and rice straw, and produced xylose containing oligosaccharides. Microstructure analysis revealed the emergence of holes on xylan-containing lignocellulosic biomass treated with Xyn10A or Xyn10B, and the cellulase accessibility might be enhanced thereafter. An enhanced hydrolytic performance for natural lignocellulosic biomass was observed when Xyn10A or Xyn10B pre-digested or synergized with commercial cellulase Cellic CTec2, which provided promising approaches to improve conversion or alleviate pretreatment intensity for biomass refinery. For the synergism with Cellic CTec2, the yield of glucose were increased 24.7/32.8% by Xyn10A/Xyn10B, and xylose increased 62.6/58.0%。...