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

Abstract

Conversion of components of the Thermobifida fusca free-enzyme system to the cellulosomal mode using the designer cellulosome approach can be employed to discover the properties and inherent advantages of the cellulosome system. In this article, we describe the conversion of the T. fusca xylanases Xyn11A and Xyn10B and their synergistic interaction in the free state or within designer cellulosome complexes in order to enhance specific degradation of hatched wheat straw as a model for a complex cellulosic substrate. Endoglucanase Cel5A from the same bacterium and its recombinant dockerin-containing chimera were also studied for their combined effect, together with the xylanases, on straw degradation. Synergism was demonstrated when Xyn11A was combined with Xyn10B and/or Cel5A, and ϳ1.5-fold activity enhancements were achieved by the designer cellulosome complexes compared to the free wild-type enzymes. These improvements in activity were due to both substrate-targeting and proximity effects among the enzymes contained in the designer cellulosome complexes. The intrinsic cellulose/xylan-binding module (XBM) of Xyn11A appeared to be essential for efficient substrate degradation. Indeed, only designer cellulosomes in which the XBM was maintained as a component of Xyn11A achieved marked enhancement in activity compared to the combination of wild-type enzymes. Moreover, integration of the XBM in designer cellulosomes via a dockerin module (separate from the Xyn11A catalytic module) failed to enhance activity, suggesting a role in orienting the parent xylanase toward its preferred polysaccharide component of the complex wheat straw substrate. The results provide novel mechanistic insight into the synergistic activity of designer cellulosome components on natural plant cell wall substrates.Thermobifida fusca is an aerobic thermophilic soil bacterium with strong cellulolytic activity (52). The T. fusca enzyme system is an extensively studied free cellulase system in which nearly all of the cellulolytic enzymes have been fully characterized, from the individual enzyme sequences to the threedimensional structures, as well as the biochemical activities of the native and recombinant proteins. The genome sequence has been published (36), and the number and types of carbohydrate-active enzymes produced by the organism are known. This actinomycete produces six different cellulases that have been well studied (29,31,32,50,52). T. fusca also has the ability to grow on xylan and produces several enzymes involved in xylan degradation, such as xylanases, ␤-xylosidase, ␣-L-arabinofuranosidase, and acetylesterases (1, 21).Previous research has suggested that the multienzyme cellulosome complex from Clostridium thermocellum is far more efficient than free cellulase systems that were tested in degrading polysaccharides (33). The cellulosome system is characterized by the strong bimodular interaction between the cohesin and dockerin modules that integrates the various enzymes into the complex (5,35,55). Scaffoldin subunits (nonenzymatic ...

Establishment of a Simple Lactobacillus plantarum Cell Consortium for Cellulase-Xylanase Synergistic Interactions

“…Wild-type enzymes Cel6A and Xyn11A were cloned from Thermobifida fusca genomic DNA as described previously (21,22). The enzyme constructs in pET28a were designed to contain a His tag for subsequent purification.…”

Toward combined delignification and saccharification of wheat straw by a laccase-containing designer cellulosome

“…This Lego-like complex has been used to test the effects of enzymatic compositions, the relative positioning of the enzymes within the complex, and their spatial proximity, which, together, generate the synergistic action of the cellulosomal components (33)(34)(35). The majority of the T. fusca biomass-degrading enzymes have been converted previously to the cellulosomal mode (19,20,(36)(37)(38)(39)(40)(41), including a pair of lytic polysaccharide monooxygenases (LPMOs) involved in the oxidative cleavage of crystalline cellulose fibers (22). Interestingly, in most cases, their inclusion into designer cellulosomes proved to be more efficient than the equivalent mixture of wild-type enzymes (20-22, 37, 40, 42).…”

Lignocellulases: a review of emerging and developing enzymes, systems, and practices

“…In parallel with the designer cellulosome approach, another interesting attempt to increase enzyme synergism, in the form of multifunctional enzyme conjugates, has been reported recently, and it is believed that this strategy may provide a component cost-reducing advantage over designer cellulosomes in future industrial applications (Moraïs et al 2010). However, the multifunctional enzyme strategy is limited to small numbers of enzymes and restricted to suboptimal equimolar ratios of enzymes.…”

“…This stems from the highly active-synergistic hydrolytic effect of the enzymes. To effectively evaluate the proposed benefits of cellulosomal enzymes over free enzymes, it is imperative to compare the optimized-state characteristics of each system on the same substrate (Moraïs et al 2010). Park et al (2014) reported 23-fold glucose production enhancement over that of free enzymes after their investigation of the effects of localization, surface accessibility, and functionality of synergetic enzymes on Scaf3-decorated bacteria outer membrane vesicles (OMVs) using phosphoric acid-swollen cellulose (PASC) as substrate.…”