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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Bioproducts Research » Research » Publications at this Location » Publication #228726

Title: The Construction and Characterization of Two Xylan-degrading Chimeric Enzymes

item Lee, Charles
item KONG, QUE

Submitted to: Biotechnology and Bioengineering
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/26/2008
Publication Date: 2/15/2009
Citation: Fan, Z., Wagschal, K.C., Lee, C.C., Kong, Q., Shen, K.A., Maiti, I.B., Yuan, L. 2009. The Construction and Characterization of Two Xylan-degrading Chimeric Enzymes. Biotechnology and Bioengineering. 102:684-692

Interpretive Summary: Hemicellulose is a major component of the woody part of plants, and represents a significant renewable source of chemical energy. However, it would first be desirable to break the hemicellulose down into simpler components, that could be converted by other processes to a more useable form of the energy, e.g. bioethanol. Hemicellulose is structurally complex, and to enzymatically degrade it requires a suite of different enzymes. Three of the most important enzyme activities required for its degradation are xylanase, xylosidase, and arabinofuranosidase. We have taken the novel approach here of making two chimeric enzymes, one being a xylanase coupled to a xylosidase, and the other being a xylanase coupled to an arabinofuranosidase. This work allows the creation of bifunctional enzymes, which can lower enzyme production costs, and moreover may allow for synergetic action of the individual activities when they are thusly tethered. Our results demonstrate the feasibility and advantages of generating bifunctional enzymes for the improvement of enzymatic hemicellulose degradation.

Technical Abstract: Degradation of xylan requires several enzymes. Two chimeric enzymes, xyln-ara and xyln-xylo, were constructed by linking the catalytic portion of a xylanase (xyln) to either an arabinofuranosidase (ara) or a xylosidase (xylo) with a flexible peptide linker. The recombinant parental enzymes and chimeras were produced in E. coli at high levels and purified for characterization of their enzymatic and kinetic properties as well as activities on natural substrates. The chimeras closely resemble the parental enzymes or their mixtures with regard to protein properties. They share similar temperature profiles and have similar catalytic efficiencies as the parental enzymes when assayed using substrates 4-nitrophenyl-a-L- arabinofuranoside or 2-nitrophenyl-b-D-xylopyranoside. The chimeras also show unique enzymatic characteristics. In xylanase activity assays using Remazol Brilliant Blue-xylan, while the parental xylanase has a pH optimum of pH 8, the chimeras showed shifted pH optima as a consequence of significantly increased activity at pH 6 (the optimal pH for ara and xylo). Both chimeras exhibited additive effects of the parental enzymes when assayed at wide-ranges of pH and temperatures. The xylnxylo chimera had the same activities as the xyln/xylo mixture in hydrolyzing the natural substrates oat spelt xylan and wheat arabinoxylan. Compared to the xyln/ara mixture, the xylnara chimera released the same amounts of xylose from oat spelt xylan and ~30% more from wheat arabinoxylan at pH 6. Our results demonstrate the feasibility and advantages of generating bifunctional enzymes for the improvement of xylan bioconversion.