The gene (manf-x10) encoding xylanase from an environmental genomic DNA library was cloned and expressed in Escherichia coli. The manf-x10 encoded a predicted protein of 467 amino acids residues with a molecular mass of 50.3 kD. Sequence analysis of manf-x10 gene revealed that the N-terminus had high homology to the catalytic domain of other bacterial xylanase enzymes. The optimal pH and temperature for xylanase activity were 7.0 and 40°C, respectively. In the presence of 1 mM solution of Co 2? , Fe 2? , Mg 2? and Zn 2? , the relative xylanase activity was enhanced; however, it had almost no activity in the presence of 10 mM solution of Cu 2? . The apparent K m and V max values obtained for the hydrolysis of rye arabinoxylan were 2.8 mg/ml and 49.5 lmol/min/mg, respectively. The C-terminus of the enzyme had high homology to a domain of unknown function found in several mannanase enzymes. Biochemical characterization of the C-terminus of the enzyme revealed a previously unrecognized carbohydrate binding module.

Hemicellulose biomass is a complex polymer with many different chemical constituents that can be utilized as industrial feedstocks. These molecules can be released from the polymer and transformed into value-added chemicals through multistep enzymatic pathways. Some bacteria produce cellulosomes which are assemblies composed of lignocellulolytic enzymes tethered to a large protein scaffold. Rosettasomes are artificial engineered ring scaffolds designed to mimic the bacterial cellulosome. Both cellulosomes and rosettasomes have been shown to facilitate much higher rates of biomass hydrolysis compared to the same enzymes free in solution. We investigated whether tethering enzymes involved in both biomass hydrolysis and oxidative transformation to glucaric acid onto a rosettasome scaffold would result in an analogous production enhancement in a combined hydrolysis and bioconversion metabolic pathway. Three different enzymes were used to hydrolyze birchwood hemicellulose and convert the substituents to glucaric acid, a top-12 DOE value added chemical feedstock derived from biomass. It was demonstrated that colocalizing the three different enzymes to the synthetic scaffold resulted in up to 40 % higher levels of product compared to uncomplexed enzymes.

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Lignocellulosic biomass represents a potentially large resource to supply the world's fuel and chemical feedstocks. Enzymatic bioconversion of this substrate offers a reliable strategy for accessing this material under mild reaction conditions. Owing to the complex nature of lignocellulose, many different enzymatic activities are required to function in concert to perform efficient transformation. In nature, large multienzyme complexes are known to effectively hydrolyze lignocellulose into constituent monomeric sugars. We created artificial complexes of enzymes, called rosettazymes, in order to hydrolyze glucuronoxylan, a common lignocellulose component, into its cognate sugar -xylose and then further convert the-xylose into -xylonic acid, a Department of Energy top-30 platform chemical. Four different types of enzymes (endoxylanase, α-glucuronidase, β-xylosidase, and xylose dehydrogenase) were incorporated into the artificial complexes. We demonstrated that tethering our enzymes in a complex resulted in significantly more activity (up to 71%) than the same amount of enzymes free in solution. We also determined that varying the enzyme composition affected the level of complex-related activity enhancement as well as overall yield.