Native Liquid Chromatography and Mass Spectrometry to Structurally and Functionally Characterize Endo-Xylanase Proteoforms.

Xylanases are of great value in various industries, including paper, food, and biorefinery. Due to their biotechnological production, these enzymes can contain a variety of post-translational modifications, which may have a profound effect on protein function. Understanding the structure-function relationship can guide the development of products with optimal performance. We have developed a workflow for the structural and functional characterization of an endo-1,4-ß-xylanase (ENDO-I) produced by Aspergillus niger with and without applying thermal stress. This workflow relies on orthogonal native separation techniques to resolve proteoforms. Mass spectrometry and activity assays of separated proteoforms permitted the establishment of structure-function relationships. The separation conditions were focus on balancing efficient separation and protein functionality. We employed size exclusion chromatography (SEC) to separate ENDO-I from other co-expressed proteins. Charge variants were investigated with ion exchange chromatography (IEX) and revealed the presence of low abundant glycated variants in the temperature-stressed material. To obtain better insights into the effect on glycation on function, we enriched for these species using boronate affinity chromatography (BAC). The activity measurements showed lower activity of glycated species compared to the non-modified enzyme. Altogether, this workflow allowed in-depth structural and functional characterization of ENDO-I proteoforms.

Substrate recognition by a bifunctional GH30-7 xylanase B from Talaromyces cellulolyticus.

Xylanase B, a member of subfamily 7 of the GH30 (glycoside hydrolase family 30) from Talaromyces cellulolyticus (TcXyn30B), is a bifunctional enzyme with glucuronoxylanase and xylobiohydrolase activities. In the present study, crystal structures of the native enzyme and the enzyme-product complex of TcXyn30B expressed in Pichia pastoris were determined at resolutions of 1.60 and 1.65 Å, respectively. The enzyme complexed with 22 -(4-O-methyl-α-d-glucuronyl)-xylobiose (U4m2 X) revealed that TcXyn30B strictly recognizes both the C-6 carboxyl group and the 4-O-methyl group of the 4-O-methyl-α-d-glucuronyl side chain by the conserved residues in GH30-7 endoxylanases. The crystal structure and site-directed mutagenesis indicated that Asn-93 on the ß2-α2-loop interacts with the non-reducing end of the xylose residue at subsite-2 and is likely to be involved in xylobiohydrolase activity. These findings provide structural insight into the mechanisms of substrate recognition of GH30-7 glucuronoxylanase and xylobiohydrolase.

Molecular organization and protein stability of the Clostridium thermocellum glucuronoxylan endo-ß-1,4-xylanase of family 30 glycoside hydrolase in solution.

Glucuronoxylan-ß-1,4-xylanohydrolase from Clostridium thermocellum (CtXynGH30) hydrolyzes ß-1,4-xylosidic linkages in 4-O-Methyl-D-glucuronoxylan. CtXynGH30 comprises an N-terminal catalytic domain, CtXyn30A, joined by a typical linker sequence to a family 6 carbohydrate-binding module, termed CtCBM6. ITC, mass spectrometric and enzyme activity analyses of CtXyn30A:CtCBM6 (1:1 M ratio), CtXyn30A and CtXynGH30 showed that the linker peptide plays a key role in connecting and orienting CtXyn30A and CtCBM6 modules resulting in the enhanced activity of CtXynGH30. To visualize the disposition of the two protein domains of CtXynGH30, SAXS analysis revealed that CtXynGH30 is monomeric and has a boot-shaped molecular envelope in solution with a Dmax of 18 nm and Rg of 3.6 nm. Kratky plot displayed the protein in a fully folded and flexible state. The ab initio derived dummy atom model of CtXynGH30 superposed well with the modelled structure.

Improved Thermal and Reusability Properties of Xylanase by Genipin Cross-Linking to Magnetic Chitosan Particles.

Enzymes are gradually increasingly preferred over chemical processes, but commercial enzyme applications remain limited due to their low stability and low product recovery, so the application of an immobilization technique is required for repeated use. The aims of this work were to produce stable enzyme complexes of cross-linked xylanase on magnetic chitosan, to describe some characteristics of these complexes, and to evaluate the thermal stability of the immobilized enzyme and its reusability. A xylanase was cross-linked to magnetite particles prepared by in situ co-precipitation of iron salts in a chitosan template. The effect of temperature, pH, kinetic parameters, and reusability on free and immobilized xylanase was evaluated. Magnetization, morphology, size, structural change, and thermal behavior of immobilized enzyme were described. 1.0 ± 0.1 µg of xylanase was immobilized per milligram of superparamagnetic chitosan nanoparticles via covalent bonds formed with genipin. Immobilized xylanase showed thermal, pH, and catalytic velocity improvement compared to the free enzyme and can be reused three times. Heterogeneous aggregates of 254 nm were obtained after enzyme immobilization. The immobilization protocol used in this work was successful in retaining enzyme thermal stability and could be important in using natural compounds such as Fe3O4@Chitosan@Xylanase in the harsh temperature condition of relevant industries.

The disruption of two salt bridges of the cold-active xylanase XynGR40 results in an increase in activity, but a decrease in thermostability.

Cold-active xylanases are of great interest due to their large potential for application in the food industry. In this study, salt bridges of the eight glycoside hydrolase (GH) family 10 cold-active xylanases reported to date were predicted and the salt bridges specific to the cold-active xylanase XynGR40 were identified. Seven mutants were constructed to disrupt salt bridges specific to XynGR40. The results suggested that five mutants lost their xylanase activity, while the other two mutants, D30N and D83N, displayed different properties when compared with the wild-type XynGR40. First, both mutations showed an obvious decrease in thermostability, with the T1/2 of D30N and D83N at 50 °C being about one half and one sixth of the wild-type, respectively. Second, both D30N and D83N had a higher specific activity than the wild-type, with activities about 13 and 163% higher, respectively. Third, both D30N and D83N had high kcat and Km values, which resulted in a higher catalytic efficiency of the mutant D83N, but a lower catalytic efficiency of the mutant D30N compared to the wild-type. Our results suggested that salt bridges play important roles in both the activity and thermostability of the cold-active xylanase XynGR40. The mutant D83N had a higher kcat and higher relative activity at low temperatures than the wild-type, and is a good candidate for application in the food industry.

Towards enzymatic breakdown of complex plant xylan structures: State of the art.

Significant progress over the past few years has been achieved in the enzymology of microbial degradation and saccharification of plant xylan, after cellulose being the most abundant natural renewable polysaccharide. Several new types of xylan depolymerizing and debranching enzymes have been described in microorganisms. Despite the increasing variety of known glycoside hydrolases and carbohydrate esterases, some xylan structures still appear quite recalcitrant. This review focuses on the mode of action of different types of depolymerizing endoxylanases and their cooperation with ß-xylosidase and accessory enzymes in breakdown of complex highly branched xylan structures. Emphasis is placed on the enzymatic hydrolysis of alkali-extracted deesterified polysaccharide as well as acetylated xylan isolated from plant cell walls under non-alkaline conditions. It is also shown how the combination of selected endoxylanases and debranching enzymes can determine the nature of prebiotic xylooligosaccharides or lead to complete hydrolysis of the polysaccharide. The article also highlights the possibility for discovery of novel xylanolytic enzymes, construction of multifunctional chimeric enzymes and xylanosomes in parallel with increasing knowledge on the fine structure of the polysaccharide.