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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.

Ethanol production at high-gravity promise to achieve concentrations over the threshold for an economical distillation process and concurrently reduce water consumption. However, a persisting limitation is the poor mass transfer conditions resulting in low ethanol yields and concentrations. Hereby, the combination of an acetone/water oxidation pretreatment process (AWO) with a liquefaction/saccharification step, using a free-fall mixer, before simultaneous saccharification and fermentation (SSF) can realize ethanol concentrations of up to ca. 74 g/L at a solids content of 20 wt%. The free-fall mixer achieved a biomass slurry viscosity reduction by 87% after only 2 h of enzymatic saccharification, indicating the efficiency of the mixing system. Furthermore, the direct enzymatic treatment of AWO pretreated corn stover (CS) by a GH11 recombinant xylanase, led to the production of xylooligosaccharides (XOS) with prebiotic potential and the removal of insoluble fibers of hemicellulose improved the glucose release of AWOCS by 22%.

Polyphenol oxidases (PPOs) are an industrially relevant family of enzymes, being involved in the post-harvest browning of fruits and vegetables, as well as in human melanogenesis. Their involvement lies in their ability to oxidize phenolic or polyphenolic compounds, that subsequently form pigments. PPO family includes tyrosinases and catechol oxidases, which in spite of their high structural similarity, exhibit different catalytic activities. Long-standing research efforts have not yet managed to decipher the structural determinants responsible for this differentiation, as every new theory is disproved by a more recent study. In the present work, we combined biochemical along with structural data, in order to rationalize the function of a previously characterized PPO from Thermothelomyces thermophila (TtPPO). The crystal structure of a TtPPO variant, determined at 1.55 Å resolution, represents the second known structure of an ascomycete PPO. Kinetic data of structure-guided mutants prove the implication of “gate” residue L306, residue HB1+1 (G292) and HB2+1 (Y296) in TtPPO function against various substrates. Our findings demonstrate the role of L306 in the accommodation of bulky substrates and that residue HB1+1 is unlikely to determine monophenolase activity as suggested from previous studies.IMPORTANCEPPOs are enzymes of biotechnological interest. They have been extensively studied both biochemically and structurally, with a special focus on the plant-derived counterparts. Even so, explicit description of the molecular determinants of their substrate specificity is still pending. Especially for ascomycete PPOs, only one crystal structure has been determined so far, thus limiting our knowledge on this tree branch of the family. In the present study, we report the second crystal structure of an ascomycete PPO. Combined with site-directed mutagenesis and biochemical studies, we depict the amino acids in the vicinity of the active site that affect enzyme activity, and perform a detailed analysis on a variety of substrates. Our findings improve current understanding of structure-function relations of microbial PPOs, which is a prerequisite for the engineering of biocatalysts of desired properties.

Polyphenol oxidases (PPOs) are an industrially relevant family of enzymes, being involved in the postharvest browning of fruits and vegetables, as well as in human melanogenesis. Their involvement lies in their ability to oxidize phenolic or polyphenolic compounds, which subsequently form pigments. The PPO family includes tyrosinases and catechol oxidases, which, in spite of their high structural similarity, exhibit different catalytic activities. Long-standing research efforts have not yet managed to decipher the structural determinants responsible for this differentiation, as every new theory is disproved by a more recent study. In the present work, we combined biochemical along with structural data in order to better understand the function of a previously characterized PPO from Thermothelomyces thermophila (TtPPO). The crystal structure of a TtPPO variant, determined at 1.55 A resolution, represents the second known structure of an ascomycete PPO. Kinetic data for structure-guided mutants prove the implication of “gate” residue L306, residue HB1+1 (G292), and HB2+1 (Y296) in TtPPO function against various substrates. Our findings demonstrate the role of L306 in the accommodation of bulky substrates and show that residue HB1+1 is unlikely to determine monophenolase activity, as was suggested from previous studies. IMPORTANCE PPOs are enzymes of biotechnological interest. They have been extensively studied both biochemically and structurally, with a special focus on the plant-derived counterparts. Even so, explicit description of the molecular determinants of their substrate specificity is still pending. For ascomycete PPOs, only one crystal structure has been determined so far, thus limiting our knowledge on this tree branch of the family. In the present study, we report the second crystal structure of an ascomycete PPO. Combined with site-directed mutagenesis and biochemical studies, we depict the amino acids in the vicinity of the active site that affect enzyme activity and perform a detailed analysis on a variety of substrates. Our findings improve current understanding of structure-function relations of microbial PPOs, which is a prerequisite for the engineering of biocatalysts of desired properties.

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.

The constant depletion of fossil fuels along with the increasing need for novel materials, necessitate the development of alternative routes for polymer synthesis. Lignocellulosic biomass, the most abundant carbon source on the planet, can serve as a renewable starting material for the design of environmentally-friendly processes for the synthesis of polyesters, polyamides and other polymers with significant value. The present review provides an overview of the main processes that have been reported throughout the literature for the production of bio-based monomers from lignocellulose, focusing on physicochemical procedures and biocatalysis. An extensive description of all different stages for the production of furans is presented, starting from physicochemical pretreatment of biomass and biocatalytic decomposition to monomeric sugars, coupled with isomerization by enzymes prior to chemical dehydration by acid Lewis catalysts. A summary of all biotransformations of furans carried out by enzymes is also described, focusing on galactose, glyoxal and aryl-alcohol oxidases, monooxygenases and transaminases for the production of oxidized derivatives and amines. The increased interest in these products in polymer chemistry can lead to a redirection of biomass valorization from second generation biofuels to chemical synthesis, by creating novel pathways to produce bio-based polymers.