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.
Background Over the last few years, valorization of lignocellulosic biomass has been expanded beyond the production of second-generation biofuels to the synthesis of numerous platform chemicals to be used instead of their fossil-based counterparts. One such well-researched example is 5-hydroxymethylfurfural (HMF), which is preferably produced by the dehydration of fructose. Fructose is obtained by the isomerization of glucose, which in turn is derived by the hydrolysis of cellulose. However, to avoid harsh reaction conditions with high environmental impact, an isomerization step towards fructose is necessary, as fructose can be directly dehydrated to HMF under mild conditions. This work presents an optimized process to produce fructose from beechwood biomass hydrolysate and subsequently convert it to HMF by employing homogeneous catalysis. Results The optimal saccharification conditions were identified at 10% wt. solids loading and 15 mg enzyme/gsolids, as determined from preliminary trials on pure cellulose (Avicel® PH-101). Furthermore, since high rate glucose isomerization to fructose requires the addition of sodium tetraborate, the optimum borate to glucose molar ratio was determined to 0.28 and was used in all experiments. Among 20 beechwood solid pulps obtained from different organosolv pretreatment conditions tested, the highest fructose production was obtained with acetone (160 °C, 120 min), reaching 56.8 g/100 g pretreated biomass. A scale-up hydrolysis in high solids (25% wt.) was then conducted. The hydrolysate was subjected to isomerization eventually leading to a high-fructose solution (104.5 g/L). Dehydration of fructose to HMF was tested with 5 different catalysts (HCl, H3PO4, formic acid, maleic acid and H-mordenite). Formic acid was found to be the best one displaying 79.9% sugars conversion with an HMF yield and selectivity of 44.6% and 55.8%, respectively. Conclusions Overall, this work shows the feasibility of coupling bio- and chemo-catalytic processes to produce HMF from lignocellulose in an environmentally friendly manner. Further work for the deployment of biocatalysts for the oxidation of HMF to its derivatives could pave the way for the emergence of an integrated process to effectively produce biobased monomers from lignocellulose.
Polyphenol oxidases (PPOs) are a group of Cu-containing enzymes exhibiting two activities: catechol oxidase and tyrosinase. Their precise mechanism of action and the structural elements that determine the distinction between the two activities are yet to be fully understood. In nature, PPOs catalyze the oxidation of several phenols to o-quinones, considerably affecting the color and nutritional properties of numerous agricultural products. On the other hand, PPOs have been widely employed as biocatalysts in food, pharmaceutical, and cosmetic industries. TtPPO is a PPO from the thermophilic fungus Thermothelomyces thermophila (TtPPO), capable of degrading of chlorophenols (CPs), contagious by-products of various pesticides. The present work aims to clarify the structural determinants of TtPPO function, by performing protein-ligand docking experiments via YASARA software. The docking results are compared with biochemical data, and the role of specific amino acids in TtPPO function is investigated. The identification of the amino acids involved in binding of the different substrates to the active site of the enzyme would allow the structure-based design of a more efficient biocatalyst for wastewater treatment.
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