Expression, Characterization, and Engineering Arabinofuranosidase-xylanase for Degrading Hemicelluloses for Biofuel Production
Date Issued
2009
Date
2009
Author(s)
Lee, Pei-Yun
Abstract
Due to a composite structure of polysaccharides, containing celluloses and hemicelluloses, depolymerization of the plant cell wall requires diversely sequential enzyme actions. Xylan, the major component of hemicelluloses, consists of β-1,4-linked xylopyranose units usually including different substituent groups, such as α-1,2- and/or α-1,3- arabinofuranosyl and acetyl, 4-O-methyl-D-glucuronosyl residues. An important enzyme in heteroxylan digestion is α-L-arabinofuranosidase (α-L-AFase), which releases L-arabinofuranosyl residues from side chains. n previous study, the crystal structure of an α-L-AFase (EC 3.2.1.55), Araf51A from Clostridium thermocellum, called “CtAraf51A” has been solved [1]. The co-crystal structures of the complexes reveal seven critical residues responsible for catalysis and substrate binding, which are Glu27, Asn72, Asn172, Trp178, Tyr244, Glu292 (nucleophile), and Gln352. The X-ray crystal structure of CtAraf51A shows Trp178 and Tyr244 are located at the head of the active center, which may be responsible for substrate specificity of CtAraf51A. In order to confirm this hypothesis, the single mutant, W178A and Y244A, and W178A/Y244A double mutant were constructed in this study. W178A mutant and Y244A mutant showed much lower α-L- AFase activity than the wild-type CtAraf51A (W178A with a 40-fold and Y244A with 70-fold lower kcat, respectively). Furthermore, double mutant W178A/Y244A had abolished activity of α-L-AFase. Interestingly, wild-type CtAraf51A could accommodate xylopyranosidic substrates, but all of the mutants could not hydrolyze the pyranosidic synthetic substrates. egradation of xylan also needs a key component, xylanase which cleaves internal glycosidic bonds by random hydrolysis of xylan backbone, resulting in xylo-oligosaccharides and xylobiose. In order to degrade xylan more efficiently, a hybrid enzyme with CtAraf51A in the N-terminal and truncated form of Xyn10Z (tXyn10Z) from C. thermocellum (Araf-tXyn) in the C-terminal, containing xylanase, β-xylosidase, and α-L-AFase activities was further constructed. The individual and hybrid enzymes shared similar pH and temperature profiles when assays were performed with 4-nitrophenyl-α-L-arabinofuranoside (4NPA) and beechwood xylan. This hybrid enzyme had the optimum for α-L-AFase activity at pH 6.5 and 65 oC and the highest xylanase activity at pH 6.5 and 65 oC similar to two individual enzymes, CtAraf51A and tXyn10Z. Moreover, the Km and kcat values of the individual CtAraf51A were determined to be 294±43
Subjects
arabinofuranosidase
xylanase
bifunctional enzyme
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