The construction and characterization of two xylan‐degrading chimeric enzymes

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-alpha-L-arabinofuranoside or 2-nitrophenyl- beta-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 xyln-xylo 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 xyln-ara chimera released the same amounts of xylose from oat spelt xylan and approximately 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.

Multimeric Hemicellulases Facilitate Biomass Conversion

“…2A). A shift in pH optimum was observed previously for the xylanase-arabinofuranosidase and xylanase-xylosidase chimeras (5). We attribute this shift in pH optimum to either the change of pH in the microenvironment generated by the polymeric proteins or altered tertiary structure.…”

“…These enzymes commonly work in concert, and their synergistic effects in xylan degradation have been studied (4,7,17). We recently constructed two chimeric xylan-degrading enzymes, demonstrating that engineering bifunctional hemicellulases is a feasible strategy for reducing the number of proteins required for biomass conversion (5). In this study, we focused on producing a trifunctional hemicellulase and explored the feasibility of using a cellulose binding domain (CBD) as both a spacer and a functional module.…”

“…We and others have demonstrated that the creation of artificial, multifunctional, lignocellulosic hydrolases is a realistic and practical approach for the improvement of biomass conversion (5,8,9,12). These novel enzymes can be used alone, when a 1:1:1 stoichiometry is preferred, or in mixtures with small quantities of other enzymes to create an optimal enzyme cocktail when enzyme ratio FIG.…”

Contribution of a Xylan-Binding Module to the Degradation of a Complex Cellulosic Substrate by Designer Cellulosomes

“…In parallel with the designer cellulosomes approach, another interesting attempt to increase enzyme synergism has been reported recently in the form of multifunctional enzyme conjugates (15,16). These authors observed an increase in the degradation of natural substrates upon fusing two or three complementary xylan-degrading activities (xylanase, arabinofuranosidase, and xylosidase) into the same polypeptide chain.…”

Engineering Bifunctional Laccase-Xylanase Chimeras for Improved Catalytic Performance

“…A widely used approach involves the "end-to-end" fusion between the N and C termini of the parental enzymes; however, this technique may result in nonfunctional chimeras either due to misfolding or to restrictions resulting from steric hindrance between the domains (37). In some cases, these restrictions may be overcome by the insertion of a loop at the fusion site to increase inter-domain flexibility and maintain functionality of the parental enzymes (38,39).…”