Background The main representatives of hemicellulose are xylans, usually decorated β-1,4-linked d -xylose polymers, which are hydrolyzed by xylanases. The efficient utilization and complete hydrolysis of xylans necessitate the understanding of the mode of action of xylan degrading enzymes. The glycoside hydrolase family 30 (GH30) xylanases comprise a less studied group of such enzymes, and differences regarding the substrate recognition have been reported between fungal and bacterial GH30 xylanases. Besides their role in the utilization of lignocellulosic biomass for bioenergy, such enzymes could be used for the tailored production of prebiotic xylooligosaccharides (XOS) due to their substrate specificity. Results The expression of a putative GH30_7 xylanase from the fungus Thermothelomyces thermophila (synonyms Myceliophthora thermophila , Sporotrichum thermophile ) in Pichia pastoris resulted in the production and isolation of a novel xylanase with unique catalytic properties. The novel enzyme designated Tt Xyn30A, exhibited an endo- mode of action similar to that of bacterial GH30 xylanases that require 4- O -methyl- d -glucuronic acid (MeGlcA) decorations, in contrast to most characterized fungal ones. However, Tt Xyn30A also exhibited an exo-acting catalytic behavior by releasing the disaccharide xylobiose from the non-reducing end of XOS. The hydrolysis products from beechwood glucuronoxylan were MeGlcA substituted XOS, and xylobiose. The major uronic XOS (UXOS) were the aldotriuronic and aldotetrauronic acid after longer incubation indicating the ability of Tt Xyn30A to cleave linear parts of xylan and UXOS as well. Conclusions Hereby, we reported the heterologous production and biochemical characterization of a novel fungal GH30 xylanase exhibiting endo- and exo-xylanase activity. To date, considering its novel catalytic properties, Tt Xyn30A shows differences with most characterized fungal and bacterial GH30 xylanases. The discovered xylobiohydrolase mode of action offers new insights into fungal enzymatic systems that are employed for the utilization of lignocellulosic biomass. The recombinant xylanase could be used for the production of X2 and UXOS from glucuronoxylan, which in turn would be utilized as prebiotics carrying manifold health benefits. Electronic supplementary material The online version of this article (10.1186/s13068-019-1455-2) contains supplementary material, which is available to authorized users.

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Glucuronoyl esterases (GEs) are recently discovered enzymes that are suggested to cleave the ester bond between lignin alcohols and xylan-bound 4-O-methyl-D-glucuronic acid. Although their potential use for enhanced enzymatic biomass degradation and synthesis of valuable chemicals renders them attractive research targets for biotechnological applications, the difficulty to purify natural fractions of lignin-carbohydrate complexes hampers the characterization of fungal GEs. In this work, we report the synthesis of three aryl alkyl or alkenyl D-glucuronate esters using lipase B from Candida antarctica (CALB) and their use to determine the kinetic parameters of two GEs, StGE2 from the thermophilic fungus Myceliophthora thermophila (syn. Sporotrichum thermophile) and PaGE1 from the coprophilous fungus Podospora anserina. PaGE1 was functionally expressed in the methylotrophic yeast Pichia pastoris under the transcriptional control of the alcohol oxidase (AOX1) promoter and purified to its homogeneity (63 kDa). The three D-glucuronate esters contain an aromatic UV-absorbing phenol group that facilitates the quantification of their enzymatic hydrolysis by HPLC. Both enzymes were able to hydrolyze the synthetic esters with a pronounced preference towards the cinnamyl-D-glucuronate ester. The experimental results were corroborated by computational docking of the synthesized substrate analogues. We show that the nature of the alcohol portion of the hydrolyzed ester influences the catalytic efficiency of the two GEs.

The endomannanase gene em26a from the thermophilic fungus Myceliophthora thermophila, belonging to the glycoside hydrolase family 26, was functionally expressed in the methylotrophic yeast Pichia pastoris. The putative endomannanase, dubbed MtMan26A, was purified to homogeneity (60 kDa) and subsequently characterized. The optimum pH and temperature for the enzymatic activity of MtMan26A were 6.0 and 60 °C, respectively. MtMan26A showed high specific activity against konjac glucomannan and carob galactomannan, while it also exhibited high thermal stability with a half-life of 14.4 h at 60 °C. Thermostability is of great importance, especially in industrial processes where harsh conditions are employed. With the aim of better understanding its structure-function relationships, a homology model of MtMan26A was constructed, based on the crystallographic structure of a close homologue. Finally, the addition of MtMan26A as a supplement to the commercial enzyme mixture Celluclast® 1.5 L and Novozyme® 188 resulted in enhanced enzymatic hydrolysis of pretreated beechwood sawdust, improving the release of total reducing sugars and glucose by 13 and 12 %, respectively.

BackgroundLignocellulosic biomass is an abundant and inexpensive resource for biofuel production. Alongside its biotechnological conversion, pretreatment is essential to enable efficient enzymatic hydrolysis by making cellulose susceptible to cellulases. Wet oxidation of biomass, such as acetone/water oxidation, that employs hot acetone, water, and oxygen, has been found to be an attractive pretreatment method for removing lignin while producing less degradation products. The remaining enriched cellulose fraction has the potential to be utilized under high gravity enzymatic saccharification and fermentation processes for the cost-competing production of bioethanol.ResultsBeech wood residual biomass was pretreated following an acetone/water oxidation process aiming at the production of high concentration of cellulosic ethanol. The effect of pressure, reaction time, temperature, and acetone-to-water ratio on the final composition of the pretreated samples was studied for the efficient utilization of the lignocellulosic feedstock. The optimal conditions were acetone/water ratio 1:1, 40 atm initial pressure of 40 vol% O2 gas, and 64 atm at reaction temperature of 175 °C for 2 h incubation. The pretreated beech wood underwent an optimization step studying the effect of enzyme loading and solids content on the enzymatic liquefaction/saccharification prior to fermentation. In a custom designed free-fall mixer at 50 °C for either 6 or 12 h of prehydrolysis using an enzyme loading of 9 mg/g dry matter at 20 wt% initial solids content, high ethanol concentration of 75.9 g/L was obtained.ConclusionThe optimization of the pretreatment process allowed the efficient utilization of beech wood residual biomass for the production of high concentrations of cellulosic ethanol, while obtaining lignin that can be upgraded towards high-added-value chemicals. The threshold of 4 wt% ethanol concentration that is required for the sustainable bioethanol production was surpassed almost twofold, underpinning the efficient conversion of biomass to ethanol and bio-based chemicals on behalf of the biorefinery concept.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0737-9) contains supplementary material, which is available to authorized users.

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