Eudicot primary cell wall glucomannan is related in synthesis, structure, and function to xyloglucan.

Hemicellulose polysaccharides influence assembly and properties of the plant primary cell wall (PCW), perhaps by interacting with cellulose to affect the deposition and bundling of cellulose fibrils. However, the functional differences between plant cell wall hemicelluloses such as glucomannan, xylan, and xyloglucan (XyG) remain unclear. As the most abundant hemicellulose, XyG is considered important in eudicot PCWs, but plants devoid of XyG show relatively mild phenotypes. We report here that a patterned ß-galactoglucomannan (ß-GGM) is widespread in eudicot PCWs and shows remarkable similarities to XyG. The sugar linkages forming the backbone and side chains of ß-GGM are analogous to those that make up XyG, and moreover, these linkages are formed by glycosyltransferases from the same CAZy families. Solid-state nuclear magnetic resonance indicated that ß-GGM shows low mobility in the cell wall, consistent with interaction with cellulose. Although Arabidopsis ß-GGM synthesis mutants show no obvious growth defects, genetic crosses between ß-GGM and XyG mutants produce exacerbated phenotypes compared with XyG mutants. These findings demonstrate a related role of these two similar but distinct classes of hemicelluloses in PCWs. This work opens avenues to study the roles of ß-GGM and XyG in PCWs.

Evolution of glucuronoxylan side chain variability in vascular plants and the compensatory adaptations of cell wall-degrading hydrolases.

Polysaccharide structural complexity not only influences cell wall strength and extensibility but also hinders pathogenic and biotechnological attempts to saccharify the wall. In certain species and tissues, glucuronic acid side groups on xylan exhibit arabinopyranose or galactose decorations whose genetic and evolutionary basis is completely unknown, impeding efforts to understand their function and engineer wall digestibility. Genetics and polysaccharide profiling were used to identify the responsible loci in Arabidopsis and Eucalyptus from proposed candidates, while phylogenies uncovered a shared evolutionary origin. GH30-family endo-glucuronoxylanase activities were analysed by electrophoresis, and their differing specificities were rationalised by phylogeny and structural analysis. The newly identified xylan arabinopyranosyltransferases comprise an overlooked subfamily in the GT47-A family of Golgi glycosyltransferases, previously assumed to comprise mainly xyloglucan galactosyltransferases, highlighting an unanticipated adaptation of both donor and acceptor specificities. Further neofunctionalisation has produced a Myrtaceae-specific xylan galactosyltransferase. Simultaneously, GH30 endo-glucuronoxylanases have convergently adapted to overcome these decorations, suggesting a role for these structures in defence. The differential expression of glucuronoxylan-modifying genes across Eucalyptus tissues, however, hints at further functions. Our results demonstrate the rapid adaptability of biosynthetic and degradative carbohydrate-active enzyme activities, providing insight into plant-pathogen interactions and facilitating plant cell wall biotechnological utilisation.

Analysis of fungal high-mannose structures using CAZymes.

Glycoengineering ultimately allows control over glycosylation patterns to generate new glycoprotein variants with desired properties. A common challenge is glycan heterogeneity, which may affect protein function and limit the use of key techniques such as mass spectrometry. Moreover, heterologous protein expression can introduce nonnative glycan chains that may not fulfill the requirement for therapeutic proteins. One strategy to address these challenges is partial trimming or complete removal of glycan chains, which can be obtained through selective application of exoglycosidases. Here, we demonstrate an enzymatic O-deglycosylation toolbox of a GH92 α-1,2-mannosidase from Neobacillus novalis, a GH2 ß-galactofuranosidase from Amesia atrobrunnea and the jack bean α-mannosidase. The extent of enzymatic O-deglycosylation was mapped against a full glycosyl linkage analysis of the O-glycosylated linker of cellobiohydrolase I from Trichoderma reesei (TrCel7A). Furthermore, the influence of deglycosylation on TrCel7A functionality was evaluated by kinetic characterization of native and O-deglycosylated forms of TrCel7A. This study expands structural knowledge on fungal O-glycosylation and presents a ready-to-use enzymatic approach for controlled O-glycan engineering in glycoproteins expressed in filamentous fungi.

Two Subgroups within the GH43_36 α-l-Arabinofuranosidase Subfamily Hydrolyze Arabinosyl from Either Mono-or Disubstituted Xylosyl Units in Wheat Arabinoxylan.

Fungal arabinofuranosidases (ABFs) catalyze the hydrolysis of arabinosyl substituents (Ara) and are key in the interplay with other glycosyl hydrolases to saccharify arabinoxylans (AXs). Most characterized ABFs belong to GH51 and GH62 and are known to hydrolyze the linkage of α-(1→2)-Ara and α-(1→3)-Ara in monosubstituted xylosyl residues (Xyl) (ABF-m2,3). Nevertheless, in AX a substantial number of Xyls have two Aras (i.e., disubstituted), which are unaffected by ABFs from GH51 and GH62. To date, only two fungal enzymes have been identified (in GH43_36) that specifically release the α-(1→3)-Ara from disubstituted Xyls (ABF-d3). In our research, phylogenetic analysis of available GH43_36 sequences revealed two major clades (GH43_36a and GH43_36b) with an expected substrate specificity difference. The characterized fungal ABF-d3 enzymes aligned with GH43_36a, including the GH43_36 from Humicola insolens (HiABF43_36a). Hereto, the first fungal GH43_36b (from Talaromyces pinophilus) was cloned, purified, and characterized (TpABF43_36b). Surprisingly, TpABF43_36b was found to be active as ABF-m2,3, albeit with a relatively low rate compared to other ABFs tested, and showed minor xylanase activity. Novel specificities were also discovered for the HiABF43_36a, as it also released α-(1→2)-Ara from a disubstitution on the non-reducing end of an arabinoxylooligosaccharide (AXOS), and it was active to a lesser extent as an ABF-m2,3 towards AXOS when the Ara was on the second xylosyl from the non-reducing end. In essence, this work adds new insights into the biorefinery of agricultural residues.

A carbohydrate-binding family 48 module enables feruloyl esterase action on polymeric arabinoxylan.

Feruloyl esterases (EC 3.1.1.73), belonging to carbohydrate esterase family 1 (CE1), hydrolyze ester bonds between ferulic acid (FA) and arabinose moieties in arabinoxylans. Recently, some CE1 enzymes identified in metagenomics studies have been predicted to contain a family 48 carbohydrate-binding module (CBM48), a CBM family associated with starch binding. Two of these CE1s, wastewater treatment sludge (wts) Fae1A and wtsFae1B isolated from wastewater treatment surplus sludge, have a cognate CBM48 domain and are feruloyl esterases, and wtsFae1A binds arabinoxylan. Here, we show that wtsFae1B also binds to arabinoxylan and that neither binds starch. Surface plasmon resonance analysis revealed that wtsFae1B's Kd for xylohexaose is 14.8 µm and that it does not bind to starch mimics, ß-cyclodextrin, or maltohexaose. Interestingly, in the absence of CBM48 domains, the CE1 regions from wtsFae1A and wtsFae1B did not bind arabinoxylan and were also unable to catalyze FA release from arabinoxylan. Pretreatment with a ß-d-1,4-xylanase did enable CE1 domain-mediated FA release from arabinoxylan in the absence of CBM48, indicating that CBM48 is essential for the CE1 activity on the polysaccharide. Crystal structures of wtsFae1A (at 1.63 Å resolution) and wtsFae1B (1.98 Å) revealed that both are folded proteins comprising structurally-conserved hydrogen bonds that lock the CBM48 position relative to that of the CE1 domain. wtsFae1A docking indicated that both enzymes accommodate the arabinoxylan backbone in a cleft at the CE1-CBM48 domain interface. Binding at this cleft appears to enable CE1 activities on polymeric arabinoxylan, illustrating an unexpected and crucial role of CBM48 domains for accommodating arabinoxylan.

Glucuronoyl esterases: diversity, properties and biotechnological potential. A review.

Glucuronoyl esterases (GEs) belonging to the carbohydrate esterase family 15 (CE15) are involved in microbial degradation of lignocellulosic plant materials. GEs are capable of degrading complex polymers of lignin and hemicellulose cleaving ester bonds between glucuronic acid residues in xylan and lignin alcohols. GEs promote separation of lignin, hemicellulose and cellulose which is crucial for efficient utilization of biomass as an energy source and feedstock for further processing into products or chemicals. Genes encoding GEs are found in both fungi and bacteria, but, so far, bacterial GEs are essentially unexplored, and despite being discovered >10 years ago, only a limited number of GEs have been characterized. The first laboratory scale example of improved xylose and glucuronic acid release by the synergistic action of GE with cellulolytic enzymes was only reported recently (improved C5 sugar and glucuronic acid yields) and, until now, not much is known about their biotechnology potential. In this review, we discuss the diversity, structure and properties of microbial GEs and consider the status of their action on natural substrates and in biological systems in relation to their future industrial use.

ß-Mannanase-catalyzed synthesis of alkyl mannooligosides.

ß-Mannanases catalyze the conversion and modification of ß-mannans and may, in addition to hydrolysis, also be capable of transglycosylation which can result in enzymatic synthesis of novel glycoconjugates. Using alcohols as glycosyl acceptors (alcoholysis), ß-mannanases can potentially be used to synthesize alkyl glycosides, biodegradable surfactants, from renewable ß-mannans. In this paper, we investigate the synthesis of alkyl mannooligosides using glycoside hydrolase family 5 ß-mannanases from the fungi Trichoderma reesei (TrMan5A and TrMan5A-R171K) and Aspergillus nidulans (AnMan5C). To evaluate ß-mannanase alcoholysis capacity, a novel mass spectrometry-based method was developed that allows for relative comparison of the formation of alcoholysis products using different enzymes or reaction conditions. Differences in alcoholysis capacity and potential secondary hydrolysis of alkyl mannooligosides were observed when comparing alcoholysis catalyzed by the three ß-mannanases using methanol or 1-hexanol as acceptor. Among the three ß-mannanases studied, TrMan5A was the most efficient in producing hexyl mannooligosides with 1-hexanol as acceptor. Hexyl mannooligosides were synthesized using TrMan5A and purified using high-performance liquid chromatography. The data suggests a high selectivity of TrMan5A for 1-hexanol as acceptor over water. The synthesized hexyl mannooligosides were structurally characterized using nuclear magnetic resonance, with results in agreement with their predicted ß-conformation. The surfactant properties of the synthesized hexyl mannooligosides were evaluated using tensiometry, showing that they have similar micelle-forming properties as commercially available hexyl glucosides. The present paper demonstrates the possibility of using ß-mannanases for alkyl glycoside synthesis and increases the potential utilization of renewable ß-mannans.

Enzymatic degradation of lignin-carbohydrate complexes (LCCs): model studies using a fungal glucuronoyl esterase from Cerrena unicolor.

Lignin-carbohydrate complexes (LCCs) are believed to influence the recalcitrance of lignocellulosic plant material preventing optimal utilization of biomass in e.g. forestry, feed and biofuel applications. The recently emerged carbohydrate esterase (CE) 15 family of glucuronoyl esterases (GEs) has been proposed to degrade ester LCC bonds between glucuronic acids in xylans and lignin alcohols thereby potentially improving delignification of lignocellulosic biomass when applied in conjunction with other cellulases, hemicellulases and oxidoreductases. Herein, we report the synthesis of four new GE model substrates comprising α- and É£-arylalkyl esters representative of the lignin part of naturally occurring ester LCCs as well as the cloning and purification of a novel GE from Cerrena unicolor (CuGE). Together with a known GE from Schizophyllum commune (ScGE), CuGE was biochemically characterized by means of Michaelis-Menten kinetics with respect to substrate specificity using the synthesized compounds. For both enzymes, a strong preference for 4-O-methyl glucuronoyl esters rather than unsubstituted glucuronoyl esters was observed. Moreover, we found that α-arylalkyl esters of methyl α-D-glucuronic acid are more easily cleaved by GEs than their corresponding É£-arylalkyl esters. Furthermore, our results suggest a preference of CuGE for glucuronoyl esters of bulky alcohols supporting the suggested biological action of GEs on LCCs. The synthesis of relevant GE model substrates presented here may provide a valuable tool for the screening, selection and development of industrially relevant GEs for delignification of biomass.

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.

Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components.

The enzymatic degradation of recalcitrant plant biomass is one of the key industrial challenges of the 21st century. Accordingly, there is a continuing drive to discover new routes to promote polysaccharide degradation. Perhaps the most promising approach involves the application of "cellulase-enhancing factors," such as those from the glycoside hydrolase (CAZy) GH61 family. Here we show that GH61 enzymes are a unique family of copper-dependent oxidases. We demonstrate that copper is needed for GH61 maximal activity and that the formation of cellodextrin and oxidized cellodextrin products by GH61 is enhanced in the presence of small molecule redox-active cofactors such as ascorbate and gallate. By using electron paramagnetic resonance spectroscopy and single-crystal X-ray diffraction, the active site of GH61 is revealed to contain a type II copper and, uniquely, a methylated histidine in the copper's coordination sphere, thus providing an innovative paradigm in bioinorganic enzymatic catalysis.

Investigating the role of AA9 LPMOs in enzymatic hydrolysis of differentially steam-pretreated spruce.

BACKGROUND: To realize the full potential of softwood-based forest biorefineries, the bottlenecks of enzymatic saccharification of softwood need to be better understood. Here, we investigated the potential of lytic polysaccharide monooxygenases (LPMO9s) in softwood saccharification. Norway spruce was steam-pretreated at three different severities, leading to varying hemicellulose retention, lignin condensation, and cellulose ultrastructure. Hydrolyzability of the three substrates was assessed after pretreatment and after an additional knife-milling step, comparing the efficiency of cellulolytic Celluclast + Novozym 188 and LPMO-containing Cellic CTec2 cocktails. The role of Thermoascus aurantiacus TaLPMO9 in saccharification was assessed through time-course analysis of sugar release and accumulation of oxidized sugars, as well as wide-angle X-ray scattering analysis of cellulose ultrastructural changes. RESULTS: Glucose yield was 6% (w/w) with the mildest pretreatment (steam pretreatment at 210 °C without catalyst) and 66% (w/w) with the harshest (steam pretreatment at 210 °C with 3%(w/w) SO2) when using Celluclast + Novozym 188. Surprisingly, the yield was lower with all substrates when Cellic CTec2 was used. Therefore, the conditions for optimal LPMO activity were tested and it was found that enough O2 was present over the headspace and that the reducing power of the lignin of all three substrates was sufficient for the LPMOs in Cellic CTec2 to be active. Supplementation of Celluclast + Novozym 188 with TaLPMO9 increased the conversion of glucan by 1.6-fold and xylan by 1.5-fold, which was evident primarily in the later stages of saccharification (24-72 h). Improved glucan conversion could be explained by drastically reduced cellulose crystallinity of spruce substrates upon TaLPMO9 supplementation. CONCLUSION: Our study demonstrated that LPMO addition to hydrolytic enzymes improves the release of glucose and xylose from steam-pretreated softwood substrates. Furthermore, softwood lignin provides enough reducing power for LPMOs, irrespective of pretreatment severity. These results provided new insights into the potential role of LPMOs in saccharification of industrially relevant softwood substrates.

Fungal feruloyl esterases can catalyze release of diferulic acids from complex arabinoxylan.

Feruloyl esterases (FAEs, EC 3.1.1.73) catalyze the hydrolytic cleavage of ester bonds between feruloyl and arabinosyl moieties in arabinoxylans. Recently, we discovered that two bacterial FAEs could catalyze release of diferulic acids (diFAs) from highly substituted, cross-linked corn bran arabinoxylan. Here, we show that several fungal FAEs, notably AnFae1 (Aspergillus niger), AoFae1 (A. oryzae), and MgFae1 (Magnaporthe oryzae (also known as M. grisae)) also catalyze liberation of diFAs from complex arabinoxylan. By comparing the enzyme kinetics of diFA release to feruloyl esterase activity of the enzymes on methyl- and arabinosyl-ferulate substrates we demonstrate that the diFA release activity cannot be predicted from the activity of the enzymes on these synthetic substrates. A detailed structure-function analysis, based on AlphaFold2 modeled enzyme structures and docking with the relevant di-feruloyl ligands, reveal how distinct differences in the active site topology and surroundings may explain the diFA releasing action of the enzymes. Interestingly, the analysis also unveils that the carbohydrate binding module of the MgFae1 may play a key role in the diFA releasing ability of this enzyme. The findings contribute further understanding of the function of FAEs in the deconstruction of complex arabinoxylans and provide new opportunities for enzyme assisted upgrading of complex bran arabinoxylans.

Structural differences of cell walls in earlywood and latewood of Pinus sylvestris and their contribution to biomass recalcitrance.

Scots pine (Pinus sylvestris L.) is an evergreen coniferous tree with wide distribution and good growth performance in a range of habitats. Therefore, wood from P. sylvestris is produced in many managed forests and is frequently used in industry. Despite the importance of pine wood, we still do not fully understand its molecular structure what limits improvements in its processing. One of the basic features leading to variation in wood properties is the presence of earlywood and latewood which form annual growth rings. Here, we characterise biochemical traits that differentiate cell walls of earlywood and latewood in Scots pine. We discover that latewood is less recalcitrant to enzymatic digestion, with galactoglucomannan showing particularly pronounced difference in accessibility. Interestingly, characterisation of lignin reveals a higher proportion of coniferaldehydes in pine latewood and suggests the presence of a different linkage landscape in this wood type. With complementary analysis of wood polysaccharides this enabled us to propose the first detailed molecular model of earlywood and latewood and to conclude that the variation in lignin structure is likely the main determinant of differences in recalcitrance observed between the two wood types in pine. Our discoveries lay the foundation for improvements in industrial processes that use pine wood since we show clear pathways for increasing the efficiency of enzymatic processing of this renewable material. Our work will help guide future breeding of pine trees with desired timber properties and can help link molecular structure of softwood cell walls to function of the different types of xylem in conifers.

Structural and functional characterization of a multi-domain GH92 α-1,2-mannosidase from Neobacillus novalis.

Many secreted eukaryotic proteins are N-glycosylated with oligosaccharides composed of a high-mannose N-glycan core and, in the specific case of yeast cell-wall proteins, an extended α-1,6-mannan backbone carrying a number of α-1,2- and α-1,3-mannose substituents of varying lengths. α-Mannosidases from CAZy family GH92 release terminal mannose residues from these N-glycans, providing access for the α-endomannanases, which then degrade the α-mannan backbone. Most characterized GH92 α-mannosidases consist of a single catalytic domain, while a few have extra domains including putative carbohydrate-binding modules (CBMs). To date, neither the function nor the structure of a multi-domain GH92 α-mannosidase CBM has been characterized. Here, the biochemical investigation and crystal structure of the full-length five-domain GH92 α-1,2-mannosidase from Neobacillus novalis (NnGH92) with mannoimidazole bound in the active site and an additional mannoimidazole bound to the N-terminal CBM32 are reported. The structure of the catalytic domain is very similar to that reported for the GH92 α-mannosidase Bt3990 from Bacteroides thetaiotaomicron, with the substrate-binding site being highly conserved. The function of the CBM32s and other NnGH92 domains was investigated by their sequential deletion and suggested that whilst their binding to the catalytic domain was crucial for the overall structural integrity of the enzyme, they appear to have little impact on the binding affinity to the yeast α-mannan substrate. These new findings provide a better understanding of how to select and optimize other multi-domain bacterial GH92 α-mannosidases for the degradation of yeast α-mannan or mannose-rich glycans.

Enzymatic Cleavage of Diferuloyl Cross-Links in Corn Bran Arabinoxylan by Two Bacterial Feruloyl Esterases.

Corn bran is an abundant coprocessing stream of corn-starch processing, rich in highly substituted, diferuloyl-cross-linked glucurono-arabinoxylan. The diferuloyl cross-links make the glucurono-arabinoxylan recalcitrant to enzymatic conversion and constitute a hindrance for designing selective enzymatic upgrading of corn glucurono-arabinoxylan. Here, we show that two bacterial feruloyl esterases, wtsFae1A and wtsFae1B, each having a carbohydrate-binding module of family 48, are capable of cleaving the ester bonds of the cross-linkages and releasing 5-5', 8-5', 8-5' benzofuran, and 8-O-4' diferulate from soluble and insoluble corn bran glucurono-arabinoxylan. All four diferulic acids were released at similar efficiency, indicating nondiscriminatory enzymatic selectivity for the esterified dimer linkages, the only exception being that wtsFae1B had a surprisingly high propensity for releasing the dimers, especially 8-5' benzofuran diferulate, indicating a potential, unique catalytic selectivity. The data provide evidence of direct enzymatic release of diferulic acids from corn bran by newly discovered feruloyl esterases, i.e., a new enzyme activity. The findings yield new insight and create new opportunities for enzymatic opening of diferuloyl cross-linkages to pave the way for upgrading of recalcitrant arabinoxylans.

Advantages of isothermal titration calorimetry for xylanase kinetics in comparison to chemical-reducing-end assays.

In lignocellulosic raw materials for biomass conversion, hemicelluloses constitute a substantial fraction, with xylan being the primary part. Although many pretreatments reduce the amount or change the distribution of xylan, it is important to degrade residual xylan so as to improve the overall yield. Typically, xylanase reaction rates are measured in stopped assays by chemical quantification of the reducing ends. With isothermal titration calorimetry (ITC), the heat flow of the hydrolysis can be measured in continuous fashion, with the reaction rate being directly proportional to the heat flow. Reaction enthalpies for carbohydrate hydrolysis are typically below 5kJ/mol, which is the limiting factor for straight forward calorimetric quantification of enzymatic reaction rates using current ITC technology. To increase the apparent reaction enthalpy, we employed a subsequent oxidation of hydrolysis products by carbohydrate oxidase and catalase. Here we show that the coupled assay with carbohydrate oxidase and catalase can be used to measure enzyme kinetics of a GH10 xylanase from Aspergillus aculeatus on birch xylan and wheat arabinoxylan. Results are discussed in the light of a critical analysis of the sensitivity of four chemical-reducing-end quantification methods using well-characterized substrates.

Engineering the substrate binding site of the hyperthermostable archaeal endo-ß-1,4-galactanase from Ignisphaera aggregans.

BACKGROUND: Endo-ß-1,4-galactanases are glycoside hydrolases (GH) from the GH53 family belonging to the largest clan of GHs, clan GH-A. GHs are ubiquitous and involved in a myriad of biological functions as well as being widely used industrially. Endo-ß-1,4-galactanases, in particular hydrolyse galactan and arabinogalactan in pectin, a major component of the primary plant cell wall, with important functions in plant defence and application in the food and other industries. Here, we explore the family's biological diversity by characterizing the first archaeal and hyperthermophilic GH53 galactanase, and utilize it as a scaffold for engineering enzymes with different product lengths. RESULTS: A galactanase gene was identified in the genome of the anaerobic hyperthermophilic archaeon Ignisphaera aggregans, and the isolated catalytic domain expressed and characterized (IaGal). IaGal presents the typical (ßα)8 barrel structure of clan GH-A enzymes, with catalytic carboxylates at the end of the 4th and 7th barrel strands. Its activity optimum of at least 95 °C and melting point over 100 °C indicate extreme thermostability, a very advantageous property for industrial applications. If enzyme depletion is reduced, so is the need for re-addition, and thus costs. The main stabilizing features of IaGal compared to other structurally characterized members are π-π and cation-π interactions. The length of the substrate binding site-and thus produced oligosaccharide products-is intermediate compared to previously characterized galactanases. Variants inspired by the structural diversity in the GH53 family were rationally designed to shorten or extend the substrate binding groove, in order to modulate product length. Subsite-deleted variants produced shorter products than IaGal, as do the fungal galactanases inspiring the design. IaGal variants engineered with a longer binding site produced a less expected degradation pattern, though still different from that of wild-type IaGal. All variants remained extremely stable. CONCLUSIONS: We have characterized in detail the most thermophilic endo-ß-1,4-galactanase known to date and successfully engineered it to modify the degradation profile, while maintaining much of its desirable thermostability. This is an important achievement as oligosaccharide products length is an important property for industrial and natural GHs alike.

Physical constraints and functional plasticity of cellulases.

Enzyme reactions, both in Nature and technical applications, commonly occur at the interface of immiscible phases. Nevertheless, stringent descriptions of interfacial enzyme catalysis remain sparse, and this is partly due to a shortage of coherent experimental data to guide and assess such work. In this work, we produced and kinetically characterized 83 cellulases, which revealed a conspicuous linear free energy relationship (LFER) between the substrate binding strength and the activation barrier. The scaling occurred despite the investigated enzymes being structurally and mechanistically diverse. We suggest that the scaling reflects basic physical restrictions of the hydrolytic process and that evolutionary selection has condensed cellulase phenotypes near the line. One consequence of the LFER is that the activity of a cellulase can be estimated from its substrate binding strength, irrespectively of structural and mechanistic details, and this appears promising for in silico selection and design within this industrially important group of enzymes.

Characterization and kinetic analysis of a thermostable GH3 beta-glucosidase from Penicillium brasilianum.

A GH3 beta-glucosidase (BGL) from Penicillium brasilianum was purified to homogeneity after cultivation on a cellulose and xylan rich medium. The BGL was identified in a genomic library, and it was successfully expressed in Aspergillus oryzae. The BGL had excellent stability at elevated temperatures with no loss in activity after 24 h of incubation at 60 degrees C at pH 4-6, and the BGL was shown to have significantly higher stability at these conditions in comparison to Novozym 188 and to other fungal GH3 BGLs reported in the literature. The BGL had significant lower affinity for cellobiose compared with the artificial substrate para-nitrophenyl-beta-D-glucopyranoside (pNP-Glc) and further, pronounced substrate inhibition using pNP-Glc. Kinetic studies demonstrated the high importance of using cellobiose as substrate and glucose as inhibitor to describe the inhibition kinetics of BGL taking place during cellulose hydrolysis. A novel assay was developed to characterize this glucose inhibition on cellobiose hydrolysis. The assay uses labelled glucose-13C6 as inhibitor and subsequent mass spectrometry analysis to quantify the hydrolysis rates.

Comparative Characterization of Aspergillus Pectin Lyases by Discriminative Substrate Degradation Profiling.

Fungal genomes often contain several copies of genes that encode carbohydrate active enzymes having similar activity. The copies usually have slight sequence variability, and it has been suggested that the multigenecity represents distinct reaction optima versions of the enzyme. Whether the copies represent differences in substrate attack proficiencies of the enzyme have rarely been considered. The genomes of Aspergillus species encode several pectin lyases (EC 4.2.2.10), which all belong to polysaccharide lyase subfamily PL1_4 in the CAZy database. The enzymes differ in terms of sequence identity and phylogeny, and exhibit structural differences near the active site in their homology models. These enzymes catalyze pectin degradation via eliminative cleavage of the α-(1,4) glycosidic linkages in homogalacturonan with a preference for linkages between methyl-esterified galacturonate residues. This study examines four different pectin lyases (PelB, PelC, PelD, and PelF) encoded by the same Aspergillus sp. (namely A. luchuensis), and further compares two PelA pectin lyases from two related Aspergillus spp. (A. aculeatus and A. tubingensis). We report the phylogeny, enzyme kinetics, and enzymatic degradation profiles of the enzymes' action on apple pectin, citrus pectin, and sugar beet pectin. All the pectin lyases exerted highest reaction rate on apple pectin [degree of methoxylation (DM) 69%, degree of acetylation (DAc) 2%] and lowest reaction rate on sugar beet pectin (DM 56%, DAc 19%). Activity comparison at pH 5-5.5 produced the following ranking: PelB > PelA > PelD > PelF > PelC. The evolution of homogalacturonan-oligomer product profiles during reaction was analyzed by liquid chromatography with mass spectrometry (LC-MS) detection. This analyses revealed subtle differences in the product profiles indicating distinct substrate degradation preferences amongst the enzymes, notably with regard to acetyl substitutions. The LC-MS product profiling analysis thus disclosed that the multigenecity appears to provide the fungus with additional substrate degradation versatility. This product profiling furthermore represents a novel approach to functionally compare pectin-degrading enzymes, which can help explain structure-function relations and reaction properties of disparate copies of carbohydrate active enzymes. A better understanding of the product profiles generated by pectin modifying enzymes has significant implications for targeted pectin modification in food and biorefinery processes.