Abstract
Degradation of complex plant biomass demands a fine-regulated portfolio of glycoside hydrolases. The LE (LguI/Eco81I)-cloning approach was used to produce two enzyme chimeras CB and BC composed of an endoglucanase Cel5A (C) from the extreme thermophilic bacterium Fervidobacterium gondwanense and an archaeal β-glucosidase Bgl1 (B) derived from a hydrothermal spring metagenome. Recombinant chimeras and parental enzymes were produced in Escherichia coli and purified using a two-step affinity chromatography approach. Enzymatic properties revealed that both chimeras closely resemble the parental enzymes and physical mixtures, but Cel5A displayed lower temperature tolerance at 100°C when fused to Bgl1 independent of the conformational order. Moreover, the determination of enzymatic performances resulted in the detection of additive effects in case of BC fusion chimera. Kinetic measurements in combination with HPLC-mediated product analyses and site-directed mutation constructs indicated that Cel5A was strongly impaired when fused at the N-terminus, while activity was reduced to a slighter extend as C-terminal fusion partner. In contrast to these results, catalytic activity of Bgl1 at the N-terminus was improved 1.2-fold, effectively counteracting the slightly reduced activity of Cel5A by converting cellobiose into glucose. In addition, cellobiose exhibited inhibitory effects on Cel5A, resulting in a higher yield of cellobiose and glucose by application of an enzyme mixture (53.1%) compared to cellobiose produced from endoglucanase alone (10.9%). However, the overall release of cellobiose and glucose was even increased by catalytic action of BC (59.2%). These results indicate possible advantages of easily produced bifunctional fusion enzymes for the improved conversion of complex polysaccharide plant materials.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-015-0122-7) contains supplementary material, which is available to authorized users.
Highlights
As the major component of the plant cell wall, cellulose is the most abundant renewable biomass resource on earth
A consortium of cellulases and hemicellulases is mandatory for the efficient degradation of complex lignocellulosic plant materials (Bornscheuer et al 2014; Khandeparker and Numan 2008; Rizk et al 2012)
There are two main strategies to circumvent the problem of high molecular weights: (1) enzymes can be truncated or even reduced to their catalytic site, or (2) proteins are chosen that do not contain domains in addition to the catalytic region
Summary
As the major component of the plant cell wall, cellulose is the most abundant renewable biomass resource on earth. In addition to efficient pretreatment methods to separate lignin, hemicellulose and cellulose, the efficient degradation of the latter polysaccharides into fermentable monosaccharide sugars, by the synergistic action of enzymes, is a bottleneck in lignocellulose. Most cellulases that exhibit enzymatic properties for industrial applications were isolated and Neddersen and Elleuche. Conventional isolation and application techniques reached their limits in recent years, resulting in the development of versatile molecular biology techniques to engineer tailored biocatalysts (Bornscheuer et al 2012). These candidates are being designed to overcome main drawbacks including limits in enzymatic specificity, efficiency and thermal instability
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