Introducing endo-xylanase activity into an exo-acting arabinofuranosidase that targets side chains.

The degradation of the plant cell wall by glycoside hydrolases is central to environmentally sustainable industries. The major polysaccharides of the plant cell wall are cellulose and xylan, a highly decorated ß-1,4-xylopyranose polymer. Glycoside hydrolases displaying multiple catalytic functions may simplify the enzymes required to degrade plant cell walls, increasing the industrial potential of these composite structures. Here we test the hypothesis that glycoside hydrolase family 43 (GH43) provides a suitable scaffold for introducing additional catalytic functions into enzymes that target complex structures in the plant cell wall. We report the crystal structure of Humicola insolens AXHd3 (HiAXHd3), a GH43 arabinofuranosidase that hydrolyses O3-linked arabinose of doubly substituted xylans, a feature of the polysaccharide that is recalcitrant to degradation. HiAXHd3 displays an N-terminal five-bladed ß-propeller domain and a C-terminal ß-sandwich domain. The interface between the domains comprises a xylan binding cleft that houses the active site pocket. Substrate specificity is conferred by a shallow arabinose binding pocket adjacent to the deep active site pocket, and through the orientation of the xylan backbone. Modification of the rim of the active site introduces endo-xylanase activity, whereas the resultant enzyme variant, Y166A, retains arabinofuranosidase activity. These data show that the active site of HiAXHd3 is tuned to hydrolyse arabinofuranosyl or xylosyl linkages, and it is the topology of the distal regions of the substrate binding surface that confers specificity. This report demonstrates that GH43 provides a platform for generating bespoke multifunctional enzymes that target industrially significant complex substrates, exemplified by the plant cell wall.

Characterization of substrate and product specificity of the purified recombinant glycogen branching enzyme of Rhodothermus obamensis.

BACKGROUND: Glycogen and starch branching enzymes catalyze the formation of α(1→6) linkages in storage polysaccharides by rearrangement of preexisting α-glucans. This reaction occurs through the cleavage of α(1→4) linkage and transfer in α(1→6) of the fragment in non-reducing position. These enzymes define major elements that control the structure of both glycogen and starch. METHODS: The kinetic parameters of the branching enzyme of Rhodothermus obamensis (RoBE) were established after in vitro incubation with different branched or unbranched α-glucans of controlled structure. RESULTS: A minimal chain length of ten glucosyl units was required for the donor substrate to be recognized by RoBE that essentially produces branches of DP 3-8. We show that RoBE preferentially creates new branches by intermolecular mechanism. Branched glucans define better substrates for the enzyme leading to the formation of hyper-branched particles of 30-70nm in diameter (dextrins). Interestingly, RoBE catalyzes an additional α-4-glucanotransferase activity not described so far for a member of the GH13 family. CONCLUSIONS: RoBE is able to transfer α(1→4)-linked-glucan in C4 position (instead of C6 position for the branching activity) of a glucan to create new α(1→4) linkages yielding to the elongation of linear chains subsequently used for further branching. This result is a novel case for the thin border that exists between enzymes of the GH13 family. GENERAL SIGNIFICANCE: This work reveals the original catalytic properties of the thermostable branching enzyme of R. obamensis. It defines new approach to produce highly branched α-glucan particles in vitro.

Characterization of hyperbranched glycopolymers produced in vitro using enzymes.

Asymmetrical flow field flow fractionation (AF4) has proven to be a very powerful and quantitative method for the determination of the macromolecular structure of high molar mass branched biopolymers, when coupled with multi-angle laser light scattering (MALLS). This work describes a detailed investigation of the macromolecular structure of native glycogens and hyperbranched α-glucans (HBPs), with average molar mass ranging from 2 × 10(6) to 4.3 × 10(7) g mol(-1), which are not well fractionated by means of classical size-exclusion chromatography. HBPs were enzymatically produced from sucrose by the tandem action of an amylosucrase and a branching enzyme mimicking in vitro the elongation and branching steps involved in glycogen biosynthesis. Size and molar mass distributions were studied by AF4, coupled with online quasi-elastic light scattering (QELS) and transmission electron microscopy. AF4-MALLS-QELS has shown a remarkable agreement between hydrodynamic radii obtained by online QELS and by AF4 theory in normal mode with constant cross flow. Molar mass, size, and dispersity were shown to significantly increase with initial sucrose concentration, and to decrease when the branching enzyme activity increases. Several populations with different size range were observed: the amount of small size molecules decreasing with increasing sucrose concentration. The spherical and dense global conformation thus highlighted was partly similar to native glycogens. A more detailed study of HBPs synthesized from low and high initial sucrose concentrations was performed using complementary enzymatic hydrolysis of external chains and chromatography. It emphasized a more homogeneous branching pattern than native glycogens with a denser core and shorter external chains.

In vitro synthesis of hyperbranched α-glucans using a biomimetic enzymatic toolbox.

Glycogen biosynthesis requires the coordinated action of elongating and branching enzymes, of which the synergetic action is still not clearly understood. We have designed an experimental plan to develop and fully exploit a biomimetic system reproducing in vitro the activities involved in the formation of α(1,4) and α(1,6) glycosidic linkages during glycogen biosynthesis. This method is based on the use of two bacterial transglucosidases, the amylosucrase from Neisseria polysaccharea and the branching enzyme from Rhodothermus obamensis . The α-glucans synthesized from sucrose, a low cost agroresource, by the tandem action of the two enzymes, have been characterized by using complementary enzymatic, chromatographic, and imaging techniques. In a single step, linear and branched α-glucans were obtained, whose proportions, morphology, molar mass, and branching degree depended on both the initial sucrose concentration and the ratio between elongating and branching enzymes. In particular, spherical hyperbranched α-glucans with a controlled mean diameter (ranging from 10 to 150 nm), branching degree (from 10 to 13%), and weight-average molar mass (3.7 × 10(6) to 4.4 × 10(7) g.mol(-1)) were synthesized. Despite their structure, which is similar to that of natural glycogens, the mechanisms involved in their in vitro synthesis appeared to be different from those involved in the biosynthesis of native hyperbranched α-glucans.

The structure and function of an arabinan-specific alpha-1,2-arabinofuranosidase identified from screening the activities of bacterial GH43 glycoside hydrolases.

Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo- and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific α-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave α-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific α-1,2-arabinofuranosidase, CjAbf43A, displays a five-bladed ß-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for α-1,2-l-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.

In situ tracking of enzymatic breakdown of starch granules by synchrotron UV fluorescence microscopy.

Synchrotron UV fluorescence microscopy was used for the first time to visualize the adsorption and diffusion of an enzyme while degrading a solid substrate. The degradation pathway of single starch granules by two amylases, optimized for biofuel production and industrial starch hydrolysis, was followed by tryptophan fluorescence (excitation at 280 nm, emission filter at 300-400 nm) and visible light imaging. Thus, both the adsorption of enzyme onto starch granules at 283 nm resolution and the resulting morphological changes were recorded at different stages of hydrolysis. It is the first time that amylases were localized on starch without staining or adding a fluorescent probe at such high resolution. This technique presents a very high potential for imaging proteins in complex systems. Its sensitivity was demonstrated by the detection of GBSS (the granular bound starch synthase) at high recording times, GBSS being present at very low levels in maize starch granules.

In depth study of a new highly efficient raw starch hydrolyzing α-amylase from Rhizomucor sp.

A new α-amylase from Rhizomucor sp. (RA) was studied in detail due to its very efficient hydrolysis of raw starch granules at low temperature (32 °C). RA contains a starch binding domain (SBD) connected to the core amylase catalytic domain by a O-glycosylated linker. The mode of degradation of native maize starch granules and, in particular, the changes in the starch structure during the hydrolysis, was monitored for hydrolysis of raw starch at concentrations varying between 0.1 and 31%. RA was compared to porcine pancreatic α-amylase (PPA), which has been widely studied either on resistant starch or as a model enzyme in solid starch hydrolysis studies. RA is particularly efficient on native maize starch and release glucose only. The hydrolysis rate reaches 75% for a 31% starch solution and is complete at 0.1% starch concentration. The final hydrolysis rate was dependent on both starch concentration and enzyme amount applied. RA is also very efficient in hydrolyzing the crystalline domains in the maize starch granule. The major A-type crystalline structure is more rapidly degraded than amorphous domains in the first stages of hydrolysis. This is in agreement with the observed preferential hydrolysis of amylopectin, the starch constituent that forms the backbone of the crystalline part of the granule. Amylose-lipid complexes present in most cereal starches are degraded in a second stage, yielding amylose fragments that then reassociate into B-type crystalline structures, forming the final resistant fraction.

Production of highly phosphorylated glycopolymers by expression of R1 in Escherichia coli.

The possible involvement of the starch bound R1 protein from potato (Solanum tuberosum L.) in the phosphorylation of starch was investigated by functional expression and characterisation of R1 in Escherichia coli. By expression of R1 in E. coli it is shown that it is possible to produce glycopolymers, e.g., glycogen, with an increased degree of phosphate substitution. The expression of R1 in E. coli resulted in a sixfold increase in glycogen bound phosphate and in an increased accumulation of glycogen leading to a glycogen excess (gex) phenotype. There was an overall shift in the unit-chain length of the isolated glycogen towards smaller degrees of polymerisation. The pleiotropic effects on the glycogen biosynthetic and amylolytic enzyme activities was investigated and showed an increase in ADPglucose pyrophosphorylase activity, as well as a decrease in exo-amylolytic activity. These results are discussed in relation to starch phosphorylation and a possible role of R1 in this respect.

Hydrolysis of concentrated raw starch: A new very efficient α-amylase from Anoxybacillus flavothermus.

A new α-amylase from Anoxybacillus flavothermus (AFA) was found to be effective in hydrolyzing raw starch in production of glucose syrup at temperatures below the starch gelatinization temperature. AFA is very efficient, leading to 77% hydrolysis of a 31% raw starch suspension. The final hydrolysis degree is reached in 2-3h at starch concentrations lower than 15% and 8-24h at higher concentrations. AFA is also very efficient in hydrolyzing the crystalline domains in the starch granule. The major A-type crystalline structure is more rapidly degraded than amorphous domains in agreement with the observed preferential hydrolysis of amylopectin. Amylose-lipid complexes are degraded in a second step, yielding amylose fragments which then re-associate into B-type crystalline structures forming the final α-amylase resistant fraction. The mode of action of AFA and the factors limiting complete hydrolysis are discussed in details.

Enzymatic xylose release from pretreated corn bran arabinoxylan: differential effects of deacetylation and deferuloylation on insoluble and soluble substrate fractions.

In the present work enzymatic hydrolysis of arabinoxylan from pretreated corn bran (190 degrees C, 10 min) was evaluated by measuring the release of xylose and arabinose after treatment with a designed minimal mixture of monocomponent enzymes consisting of alpha-L-arabinofuranosidases, an endoxylanase, and a beta-xylosidase. The pretreatment divided the corn bran material approximately 50:50 into soluble and insoluble fractions having A:X ratios of 0.66 and 0.40, respectively. Addition of acetyl xylan esterase to the monocomponent enzyme mixture almost doubled the xylose release from the insoluble substrate fraction and gave release of 1 mol of xylose/mol of acetic acid released, whereas addition of feruloyl esterase promoted release of only approximately 0.4 mol of xylose/mol of ferulic acid released. For the soluble substrate fraction up to 36% of the xylose could be released by the enzymatic treatment. Acetyl xylan esterase addition on top of the minimal monocomponent enzyme mixture resulted in liberation of up to 0.5 mol of xylose/mol of acetic acid released, whereas feruloyl esterase addition released 1 mol of xylose/mol of ferulic acid released from the soluble substrate. The results imply that on the insoluble material the acetyl xylan esterase was more important for the enzymatic degradation than feruloyl esterase, whereas on soluble arabinoxylan the feruloyl esterase seemed to be more important for the release of xylose.

A CBM20 low-affinity starch-binding domain from glucan, water dikinase.

The family 20 carbohydrate-binding module (CBM20) of the Arabidopsis starch phosphorylator glucan, water dikinase 3 (GWD3) was heterologously produced and its properties were compared to the CBM20 from a fungal glucoamylase (GA). The GWD3 CBM20 has 50-fold lower affinity for cyclodextrins than that from GA. Homology modelling identified possible structural elements responsible for this weak binding of the intracellular CBM20. Differential binding of fluorescein-labelled GWD3 and GA modules to starch granules in vitro was demonstrated by confocal laser scanning microscopy and yellow fluorescent protein-tagged GWD3 CBM20 expressed in tobacco confirmed binding to starch granules in planta.

The carbohydrate-binding module family 20--diversity, structure, and function.

Starch-active enzymes often possess starch-binding domains (SBDs) mediating attachment to starch granules and other high molecular weight substrates. SBDs are divided into nine carbohydrate-binding module (CBM) families, and CBM20 is the earliest-assigned and best characterized family. High diversity characterizes CBM20s, which occur in starch-active glycoside hydrolase families 13, 14, 15, and 77, and enzymes involved in starch or glycogen metabolism, exemplified by the starch-phosphorylating enzyme glucan, water dikinase 3 from Arabidopsis thaliana and the mammalian glycogen phosphatases, laforins. The clear evolutionary relatedness of CBM20s to CBM21s, CBM48s and CBM53s suggests a common clan hosting most of the known SBDs. This review surveys the diversity within the CBM20 family, and makes an evolutionary comparison with CBM21s, CBM48s and CBM53s, discussing intrafamily and interfamily relationships. Data on binding to and enzymatic activity towards soluble ligands and starch granules are summarized for wild-type, mutant and chimeric fusion proteins involving CBM20s. Noticeably, whereas CBM20s in amylolytic enzymes confer moderate binding affinities, with dissociation constants in the low micromolar range for the starch mimic beta-cyclodextrin, recent findings indicate that CBM20s in regulatory enzymes have weaker, low millimolar affinities, presumably facilitating dynamic regulation. Structures of CBM20s, including the first example of a full-length glucoamylase featuring both the catalytic domain and the SBD, are summarized, and distinct architectural and functional features of the two SBDs and roles of pivotal amino acids in binding are described. Finally, some applications of SBDs as affinity or immobilization tags and, recently, in biofuel and in planta bioengineering are presented.

Mode of action and properties of the beta-xylosidases from Talaromyces emersonii and Trichoderma reesei.

Enzymatic hydrolysis of arabinoxylan is an important prerequisite for the utilization of hemicellulose for ethanol fermentation or for making the low calorie sweetener xylitol by catalytic hydrogenation of the generated xylose. This study focus on cloning and characterization of two industrial relevant beta-xylosidases (1,4-beta-D-xylan xylohydrolase, EC 3.2.1.37) from Talaromyces emersonii (betaXTE) and Trichoderma reesei (betaXTR) and a comparison of these in relation to hemicellulose hydrolysis using an industrial relevant substrate. Both beta-xylosidases were expressed in A. oryzae and subsequently purified. During the enzymatic hydrolysis of xylobiose, the reaction product of both enzymes was found to be beta-D-xylose proving that the hydrolysis is proceeding via a retaining reaction mechanism. Based on sequence similarities and glycosyl hydrolases family membership, the active site residues of betaXTE and betaXTR are predicted to be Asp 242 and Glu 441, and Asp 264 and Glu 464, respectively. The involvement in catalysis of these carboxyls was examined by modification using the carbodiimide-nucleophile procedure resulting in a complete inactivation of both enzymes. The degree of xylose release from vinasse, an ethanol fermentation by-product, by betaXTE and betaXTR was 12.1% and 7.7%, respectively. Using the beta-xylosidases in combination with the multicomponent enzyme product Ultraflo L, resulted in 41.9% and 40.8% release of xylose, respectively indicating a strong synergistic effect between the exo-acting beta-xylosidases and the endo-1,4-beta-xylanases and alpha-L-arabinofuranosidase in Ultraflo L. There seems to be no measurable differences between the two beta-xylosidases when used in this specific application despite the differences in specific activity and kinetic properties.