High Substrate Concentrations Research Articles

Photodecarboxylative Ring Annulation of α‐ and β‐Functionalized Phthaloyl‐GABA Derivatives: Bioactive Pyrroloisoindolinones with High Quantum Efficiency

AbstractThe triplet‐sensitized (by the solvent acetone) as well as the direct (λex=300–320 nm) photochemical decarboxylation of N‐phthaloylated γ‐aminobutyric acid (GABA) derivatives are versatile and high‐yielding routes to benzopyrrolizidines via intramolecular electron transfer initiated decarboxylation followed by radical coupling. The ß‐mono‐ and ß,ß'‐disubstituted N‐phthaloyl GABA derivatives 7 a–7 g, respectively, were applied as substrates. Decarboxylative photocyclization yielded hydroxy benzopyrrolizidines 8 a–8 g in high chemical yields and with moderate diastereoselectivities from the ß‐monosubstituted substrates. The analogous α‐substituted GABA derivatives 11 a–11 c were also applied as potential substrates for memory of chirality effects. The reaction quantum yields of the photodecarboxylation reactions for the parent GABA derivative 13 and for the new substrates 7 h and 11 a were determined by the quantum yield determination system (QYDS) and showed a remarkable concentration dependency indicating aggregation at higher substrate concentrations. Inhibition studies on the atherogenic human serine hydrolase cholesterol esterase showed derivatives 8 a and 8 d to exhibit a hyperbolic mode of inhibition with moderate IC50 values of about 60–80 μM.

Optimizing Continuous Flow Biocatalysis with 3D‐Printing and Inline IR Monitoring

Enzymatic biocatalysis typically generates less waste, uses less water, and minimizes energy consumption compared to traditional chemical methods. Efficient, cell‐free biosynthesis relies on the reuse of its valuable biocatalysts. Immobilization of enzymes on solid supports, such as enzyme carrier resins (ECRs), offers a reliable and widely deployed approach to maximize enzyme turnover in cell‐free biosynthesis. We focus on two major bottlenecks associated with optimizing cell‐free biocatalysis. First, we apply our lab’s 3D‐printed labware to screen ECRs in 96‐well mini‐reactors to optimize enzyme immobilization conditions. Second, we introduce inline infrared spectroscopy to monitor bioreactor output and maximize enzyme productivity. Urease provides a model system for examining immobilization conditions and continuous assessment of biocatalyst performance. As required for the high substrate concentrations to improve process efficiency and minimize waste, urease was studied in unusually high concentrations of its substrate—molar concentrations of urea. The optimized reactor processed 3.24 L of 4.00 M urea at an average volumetric productivity of 13 g·L‐1·h‐1 over 18 h and achieved an estimated productivity number of >17.4 kg urea processed per g of immobilized urease Type‐IX. This workflow can be generalized to most biocatalytic processes and could accelerate adoption of cell‐free biosynthesis for greater chemical sustainability.

Facile Asymmetric Syntheses of Non-Natural Amino Acid (S)-Cyclopropylglycine by the Developed NADH-Driven Biocatalytic System

A self-sufficient bifunctional enzyme integrating reductive amination and coenzyme regeneration activities was developed and successfully employed to synthesize (S)-cyclopropylglycine with an improved reaction rate 2.1-fold over the native enzymes and a short bioconversion period of 6 h at a high substrate concentration of 120 g·L−1 and space–time yield of (S)-cyclopropylglycine up to 377.3 g·L−1·d−1, higher than that of any previously reported data. Additionally, (S)-cyclopropylglycine could be continuously synthesized for 90 h with the enzymes packed in a dialysis tube, providing 634.6 g of (S)-cyclopropylglycine with >99.5% ee and over 95% conversion yield up to 12 changes. These results confirmed that the newly developed NADH-driven biocatalytic system could be utilized as a self-sufficient biocatalyst for industrial application in the synthesis of (S)-cyclopropylglycine, which provides a chiral center and cyclopropyl fragment for the frequent synthesis of preclinical/clinical drug molecules.

Optimizing light-driven ene-reductase reactions with g-C3N4 and electron mediators

In the realm of green chemistry and sustainable industrial processes, the integration of enzymes with photocatalytic materials offers a promising avenue for achieving efficient, light-driven catalysis while minimizing environmental impact. This study introduces an enzyme-photo-coupled catalytic system (EPCS) using graphitic carbon nitride (g-C3N4) as a photocatalyst and methyl viologen (MV2+) as an electron mediator to drive the reduction of 2-cyclohexen-1-one by the Old Yellow Enzyme (OYE), reducing the reliance on expensive and scarce bioelectronic carriers and demonstrating a sustainable alternative for biocatalytic applications. Furthermore, we explored the incorporation of a Pickering emulsion system to enhance mass transfer, especially in reactions involving highly concentrated substrates. This adaptation not only improves the yield of the EPCS at higher substrate concentrations but also offers a scalable and environmentally benign alternative to traditional photocatalytic systems.

Pullulanase pretreatment of highly concentrated maltodextrin solution improves maltose yield during β-amylase-catalyzed saccharification

Increasing the substrate concentration can effectively reduce energy consumption and result in more economic benefits in the industrial production of maltose, but this process remarkably increases the viscosity, which has a negative effect on saccharification. To improve saccharification efficiency, pullulanase is usually employed. In the conventional process of maltose production, pullulanase is added at the same time with β-amylase or later, but this process seems inefficient when the substrate concentration is high. Herein, a novel method was introduced to enhance the maltose yield under high substrate concentration. The results indicated that the pullulanase pretreatment of highly concentrated maltodextrin solution for 2 h greatly affects the final conversion rate of β-amylase-catalyzed saccharification. The maltose yield reached 80.95 %, which is 11.8 % above the control value. Further examination confirmed that pullulanase pretreatment decreased the number of branch points of maltodextrin and resulted in a high content of oligosaccharides. These linear chains were suitable for β-amylase-catalyzed saccharification to produce maltose. This research offers a new effective and green strategy for starch sugar production.

A protective shell on the surface of Chlorella cells promotes long-term stable production of hydrogen

The Chlorella cells exhibit excellent application potential in the field of environmental governance and bioenergy development. By selecting a bionic coating on the cell surface, it is possible to significantly enhance the cells' viability and stability within polluted environments. In this study, we employed catechol to induce the native Chlorella cells and Tannic acid (TA)-Fe3+@laccase coated cells to produce hydrogen. This protective coating effectively shielded the cells from external stressors, enhancing their tolerance in alkaline environments and higher substrate concentrations, ensuring long-term stable hydrogen production, achieving a 1.7-fold increase compared to the native cell hydrogen production in 7 days (Optical density, OD750 = 2.5). Meanwhile, the degradation rate of catechol and the accumulation of biomass were also improved, and the accumulation of biomass increased by 8%. This strategy is expected to provide new solutions and possibilities for utilizing environmental pollutants to produce clean energy.

Probing the Mechanisms Underlying the Transport of the Vinca Alkaloids by P-glycoprotein

The efficacy of many cancer drugs is hindered by P-glycoprotein (Pgp), a cellular pump that removes drugs from cells. To improve chemotherapy, drugs capable of evading Pgp must be developed. Despite similarities in structure, vinca alkaloids (VAs) show disparate Pgp-mediated efflux ratios. ATPase activity and binding affinity studies show at least two binding sites for the VAs: high- and low-affinity sites that stimulate and inhibit the ATPase activity rate, respectively. The affinity for ATP from the ATPase kinetics curve for vinblastine (VBL) at the high-affinity site was 2- and 9-fold higher than vinorelbine (VRL) and vincristine (VCR), respectively. Conversely, VBL had the highest Km (ATP) for the low-affinity site. The dissociation constants (KDs) determined by protein fluorescence quenching were in the order VBL < VRL< VCR. The order of the KDs was reversed at higher substrate concentrations. Acrylamide quenching of protein fluorescence indicate that the VAs, either at 10 µM or 150 µM, predominantly maintain Pgp in an open-outward conformation. When 3.2 mM AMPPNP was present, 10 µM of either VBL, VRL, or VCR cause Pgp to shift to an open-outward conformation, while 150 µM of the VAs shifted the conformation of Pgp to an intermediate orientation, between opened inward and open-outward. However, the conformational shift induced by saturating AMPPNP and VCR condition was less than either VBL or VRL in the presence of AMPPNP. At 150 µM, atomic force microscopy (AFM) revealed that the VAs shift Pgp population to a predominantly open-inward conformation. Additionally, STDD NMR studies revealed comparable groups in VBL, VRL, and VCR are in contact with the protein during binding. Our results, when coupled with VAs-microtubule structure-activity relationship studies, could lay the foundation for developing next-generation VAs that are effective as anti-tumor agents. A model that illustrates the intricate process of Pgp-mediated transport of the VAs is presented.

Characterization of Thermostable Cellulase Enzyme Isolated from a Hot Spring Bacterium: Bacillus sp.

Cellulase is a complex of enzymes which consists of β-1,4-endoglucanase, cellobiohydrolase, and β-glucosidase. Cellulase contributes a significant share to the world enzyme market and is used in number of industries viz; paper and pulp, food and beverage, bioethanol, detergent and textile. The harsh industrial conditions such as high temperature, extreme pH levels and high substrate concentration etc. which is used in such industries adversely effect on structure and activity of enzymes. Therefore, huge amount of chemical catalysts which cause chemical wastes are used in industrial settings instead of enzymes. Since the chemical wastes adversely affect the ecosystem, isolation and characterization of thermostable cellulase enzyme-producing bacteria and exploring the industrial perspective of thermostable enzymes is a better approach towards green chemistry. The present study focused on characterization of cellulase enzyme produced by Bacillus sp., which shows optimum activity at 60 C under neutral conditions, isolated from Gomarankadavala hot spring in Sri Lanka. The effect of different Carbon sources: glucose and lactose, Nitrogen sources: peptone, tryptone, yeast extract and urea on enzyme production and different Carboxymethylcellulose (CMC) concentrations; 0.5%, 1.5% and 2%, on enzyme activity were measured using the DNS method. Under the optimum conditions, cellulase enzyme activity on different substrates: corn cob, rice bran and raw leaves were measured. The highest enzyme production was recorded in the culture medium which added tryptone as the nitrogen source and adding carbon sources to the culture medium showed an increase of cellulase enzyme production. The optimum CMC concentration for the enzyme activity was recorded as 1% and from all the optimized parameters, the substrate; raw leaves showed the highest enzyme activity of 11.305 U/ml. Further, a considerable amount of enzyme activity was recorded on corn cobs and rice bran as well. Thus, the thermostable cellulase enzyme produced by bacterium identified as Bacillus cereus isolated from Gomarankadavala hot spring could be successfully used in industrial settings such as kitchen/industrial waste management, bioethanol production, paper and pulp industry and textile industry which use high temperatures and cellulosic substrates as raw materials. &#x0D; Keywords: Thermostable enzymes, Cellulase, Industrial applications, Hot springs

Loss of function and reduced levels of sphingolipid desaturase DEGS1 variants are both relevant in disease mechanism

The last step of ex novo ceramide biosynthesis consists of the conversion of dihydroceramide into ceramide catalyzed by sphingolipid Δ4-desaturase DEGS1. DEGS1 variants were found to be responsible for heterogeneous clinical pictures belonging to the family of hypomyelinating leukodystrophies. To investigate the mechanisms making such variants pathogenic, we designed a procedure for the efficient detection of desaturase activity in vitro using LC-MS/MS and prepared a suitable cell model knocking out DEGS1 in HEK-293T cells through CRISPR-Cas9 genome editing (KO-DES-HEK). Transfecting KO-DES-HEK cells with DEGS1 variants, we found that their transcripts were all overexpressed as much as the WT transcripts, while the levels of cognate protein were 40%–80% lower. In vitro desaturase activity was lost by many variants except L175Q and N255S, which maintain a catalytic efficiency close to 12% of the WT enzyme. Metabolic labeling of KO-DES-HEK with deuterated palmitate followed by LC-MS/MS analysis of the formed sphingolipids revealed that the ceramide/dihydroceramide and sphingomyelin/dihydrosphingomyelin ratios were low and could be reverted by the overexpression of WT DEGS1 as well as of L175Q and N255S variants, but not by the overexpression of all other variants. Similar analyses performed on fibroblasts from a patient heterozygous for the N255S variant showed very low variant DEGS1 levels and a low ratio between the same unsaturated and saturated sphingolipids formed upon metabolic labeling, notwithstanding the residual activity measured at high substrate and homogenate protein concentrations. We conclude that loss of function and reduced protein levels are both relevant in disease pathogenesis.

Racemization of the substrate and product by serine palmitoyltransferase from Sphingobacterium multivorum yields two enantiomers of the product from d-serine

Serine palmitoyltransferase (SPT) catalyzes the pyridoxal-5'-phosphate (PLP)-dependent decarboxylative condensation of l-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine (KDS). Although SPT was shown to synthesize corresponding products from amino acids other than l-serine, it is still arguable whether SPT catalyzes the reaction with d-serine, which is a question of biological importance. Using high substrate and enzyme concentrations, KDS was detected after the incubation of SPT from Sphingobacterium multivorum with d-serine and palmitoyl-CoA. Furthermore, the KDS comprised equal amounts of 2S and 2R isomers. 1H-NMR study showed a slow hydrogen-deuterium exchange at Cα of serine mediated by SPT. We further confirmed that SPT catalyzed the racemization of serine. The rate of the KDS formation from d-serine was comparable to those for the α-hydrogen exchange and the racemization reaction. The structure of the d-serine-soaked crystal (1.65Å resolution) showed a distinct electron density of the PLP-l-serine aldimine, interpreted as the racemized product trapped in the active site. The structure of the α-methyl-d-serine-soaked crystal (1.70Å resolution) showed the PLP-α-methyl-d-serine aldimine, mimicking the d-serine-SPT complex prior to racemization. Based on these enzymological and structural analyses, the synthesis of KDS from d-serine was explained as the result of the slow racemization to l-serine, followed by the reaction with palmitoyl-CoA, and SPT would not catalyze the direct condensation between d-serine and palmitoyl-CoA. It was also shown that the S.multivorum SPT catalyzed the racemization of the product KDS, which would explain the presence of (2R)-KDS in the reaction products.

Rapid Degradation of Hazardous Amides by Immobilized Engineered Pseudomonas putida KT2440 Based on a Novel Gene Expression Vector.

The amides 4-trifluoromethylnicotinamide, acrylamide, and benzamide are widely used in agriculture and industry, posing hazards to the environment and animals. Immobilized bacteria are preferred in wastewater treatment, but degradation of these amides by immobilized engineered bacteria has not been explored. Here, engineered Pseudomonas putida KT2440 pLSJ15-amiA was constructed by introducing a new amidase gene expression vector into environmentally safe P. putida KT2440. P. putida KT2440 pLSJ15-amiA had high amidase activity, even at 80 °C. P. putida KT2440 pLSJ15-amiA immobilized with calcium alginate exhibited a greater environmental tolerance than free cells. The amides were rapidly degraded by the immobilized cells, but the activity was inhibited by high concentrations of substrates. The substrate inhibition model revealed that the optimum initial concentrations of 4-trifluoromethylnicotinamide, acrylamide, and benzamide for degradation by immobilized cells were 197.65, 350.76, and 249.40 μmol/L, respectively. This study develops a novel and excellent immobilized biocatalyst for remediation of wastewater containing hazardous amides.

Enhancing the Enantioselectivity and Catalytic Efficiency of Esterase from Bacillus subtilis for Kinetic Resolution of l-Menthol through Semirational Design.

Enzymatic kinetic resolution is a promising way to produce l-menthol. However, the properties of the reported biocatalysts are still unsatisfactory and far from being ready for industrial application. Herein, a para-nitrobenzylesterase (pnbA) gene from Bacillus subtilis was cloned and expressed to produce l-menthol from d,l-menthyl acetate. The highest enantiomeric excess (ee) value of the product generated by pnbA was only approximately 80%, with a high conversion rate (47.8%) of d,l-menthyl acetate with the help of a cosolvent, indicating high catalytic activity but low enantioselectivity (E = 19.95). To enhance the enantioselectivity and catalytic efficiency of pnbA to d,l-menthyl acetate in an organic solvent-free system, site-directed mutagenesis was performed based on the results of molecular docking. The F314E/F315T mutant showed the best catalytic properties (E = 36.25) for d,l-menthyl acetate, with 92.11% ee and 30.58% conversion of d,l-menthyl acetate. To further improve the properties of pnbA, additional mutants were constructed based on the structure-guided triple-code saturation mutagenesis strategy. Finally, four mutants were screened for the best enantioselectivity (ee > 99%, E > 300) and catalytic efficiency at a high substrate concentration (200 g/L) without a cosolvent. This work provides several generally applicable biocatalysts for the industrial production of l-menthol.

Hepatic glucuronidation of tetrabromobisphenol A and tetrachlorobisphenol A: interspecies differences in humans and laboratory animals and responsible UDP-glucuronosyltransferase isoforms in humans.

Tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA), bisphenol A (BPA) analogs, are endocrine-disrupting chemicals predominantly metabolized into glucuronides by UDP-glucuronosyltransferase (UGT) enzymes in humans and rats. In the present study, TBBPA and TCBPA glucuronidation by the liver microsomes of humans and laboratory animals (monkeys, dogs, minipigs, rats, mice, and hamsters) and recombinant human hepatic UGTs (10 isoforms) were examined. TBBPA glucuronidation by the liver microsomes followed the Michaelis-Menten model kinetics in humans, rats, and hamsters and the biphasic model in monkeys, dogs, minipigs, and mice. The CLint values based on the Eadie-Hofstee plots were mice (147) > monkeys (122) > minipigs (108) > humans (100) and rats (98) > dogs (81) > hamsters (47). TCBPA glucuronidation kinetics by the liver microsomes followed the biphasic model in all species except for minipigs, which followed the Michaelis-Menten model. The CLint values were monkeys (172) > rats (151) > mice (134) > minipigs (104), dogs (102), and humans (100) > hamsters (88). Among recombinant human UGTs examined, UGT1A1 and UGT1A9 showed higher TBBPA and TCBPA glucuronidation abilities. The kinetics of TBBPA and TCBPA glucuronidation followed the substrate inhibition model in UGT1A1 and the Michaelis-Menten model in UGT1A9. The CLint values were UGT1A1 (100) > UGT1A9 (42) for TBBPA glucuronidation and UGT1A1 (100) > UGT1A9 (53) for TCBPA glucuronidation, and the activities at high substrate concentration ranges were higher in UGT1A9 than in UGT1A1 for both TBBPA and TCBPA. These results suggest that the glucuronidation abilities toward TBBPA and TCBPA in the liver differ extensively across species, and that UGT1A1 and UGT1A9 expressed in the liver mainly contribute to the metabolism and detoxification of TBBPA and TCBPA in humans.

A pushed biosynthesis of 2,6-dihydroxybenzoic acid by the recombinant 2,3-dihydroxybenzoic acid decarboxylase immobilized on novel amino-modified lignin-containing cellulose nanocrystal aerogel

Production of 2,6-dihydroxybenzoic acid (2,6-DHBA) via enzymatic carboxylation of resorcinol by decarboxylases is of great promising but shows depressed equilibrium conversion. In this study, 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus oryzae (2,3-DHBD_Ao) pushing the conversion towards carboxylation for efficient 2,6-DHBA biosynthesis was achieved. Meanwhile, a novel amino-modified and lignin-doped cellulose nanocrystal aerogel (A-LCNCA) with high specific surface area and prominent CO2 capture was prepared for 2,3-DHBD_Ao immobilization. 2,3-DHBD_Ao@A-LCNC contributed a further enhanced conversion of carboxylation with the maximal conversion of 76.2 %, which was correlated to both the activity of 2,3-DHBD_Ao and the high CO2 loading capacity of A-LCNCA. Moreover, 2,3-DHBD_Ao@A-LCNC exhibited superior performances in a wider range of temperature and higher concentrations of substrate, with a prolonged storage period longer than 30 days. After seven cycles reuse, 2,3-DHBD_Ao@A-LCNCA could retain 85.3 % of its original activity. These results suggest a considerable potential of 2,3-DHBD_Ao@A-LCNCA in the selective biosynthesis of 2,6-DHBA.

Understanding atrazine elimination via treatment of the enzyme-based Fenton reaction: Kinetics, mechanism, reaction pathway, and metabolites toxicity

The degradation kinetics and mechanism of atrazine (ATZ) via an enzyme-based Fenton reaction were investigated at various substrate concentrations and pH values. Toxicological assessment was conducted on ATZ and its degradation products, and the associated reaction pathway was examined. The in situ production of hydrogen peroxide (H2O2) was monitored within the range of 3–15 mM, depending on the increase in glucose concentration, while decreasing the pH to 3.2–5.1 (initial pH of 5.8) or 6.5–7.4 (initial pH of 7.7). The degradation efficiency of ATZ was approximately 2–3 times higher at an initial pH of 5.8 with lower glucose concentrations than at an initial pH of 7.7 with higher substrate concentrations during the enzyme-based Fenton reaction. The apparent pseudo-first-order rate constant for H2O2 decomposition under various conditions in the presence of ferric citrate was 1.9–6.3 × 10−5 s−1. The •OH concentration ([•OH]ss) during the enzyme-based Fenton reaction was 0.5–4.1 × 10−14 M, and the second-order rate constant for ATZ degradation was 1.5–3.3 × 109 M−1 s−1. ATZ intrinsically hinders the growth and development of Arabidopsis thaliana, and its inhibitory effect is marginal, depending on the reaction time of the enzyme-based Fenton process. The ATZ transformation during this process occurs through dealkylation, hydroxylation, and dechlorination via •OH-mediated reactions. The degradation kinetics, mechanism, and toxicological assessment in the present study could contribute to the development and application of enzyme-based Fenton reactions for in situ pollutant abatement. Moreover, the enzyme-based Fenton reaction could be an environmentally benign and applicable approach for eliminating persistent organic matter, such as herbicides, using diverse H2O2-producing microbes and ubiquitous ferric iron with organic complexes.

Structural and mechanistic insights into ALS patient derived mutations in D-amino acid oxidase

The D-amino acid oxidase protein modulates neurotransmission by controlling the levels of D-serine, a co-agonist of N-methyl-D-aspartate receptors. Mutations in the DAO gene have been associated with ALS, with some studies reporting pathogenic mechanisms of the R199W mutation. We have characterized two novel mutations R38H and Q201R found in ALS patients and report certain novel findings related to the R199W mutation. We report the first instance of crystal structure analysis of a patient-derived mutant of DAO, R38H, solved at 2.10 Å. The structure revealed significant perturbations and altered binding with the cofactor (FAD) and the inhibitor benzoate, supported by biochemical assays. Q201R-DAO also exhibited significantly lower ligand binding efficiency. Furthermore, kinetic analysis across all variants revealed reduced oxidase activity and substrate binding. Notably, R38H-DAO exhibited near-WT activity only at high substrate concentrations, while R199W-DAO and Q201R-DAO displayed drastic activity reduction. Additionally, structural perturbations were inferred for R199W-DAO and Q201R-DAO, evident by the higher oligomeric state in the holoenzyme form. We also observed thermal instability in case of R199W-DAO mutant. We hypothesize that the mutant enzymes may be rendered non-functional in a cellular context, potentially leading to NMDAR-associated excitotoxicity. The study provides novel insights into structural and functional aspects of DAO mutations in ALS.

Optimization of methane production from co-digestion of citrus agro-industrial waste in a two-stage system: Investigation of the effects of organic matter concentration and alkalinization

The essential oils in citrus waste poses challenges for the anaerobic digestion application to energy recovery from these wastes due to the potential to inhibit the bioprocess. The two-stage system is a practical alternative to overcome these limitations of anaerobic co-digestion of citrus peel waste and wastewater. This study proposes to overcome drawbacks associated with the use of these substrates in anaerobic digestion by separating the phases, performing parameters optimization of the methanogenic stage. To obtain optimized conditions for the methanogenic stage, the Rotational Central Composite Design (RCCD) and Response Surface Methodology (RSM) were applied, evaluating the effects of diluting the Fermentation Reactor Content (FRC) of the first stage (35.7%–100%) and alkalinization (up to 0.60 g sodium bicarbonate.gCOD−1), in methane production and organic matter removal. Values from 2100.4 to 6320.6 mLCH4.L−1 were obtained in the experimental design assays, with no inhibition identified by the high substrate concentration, nor by the limonene content (1.5–5.0 mg.L−1). Lower dilution of the fermentation reactor content (90%–100%) resulted in higher accumulated methane production, while the variation in the alkalinization did not imply significant effects on this response. The optimized condition for maximum production potential was obtained with 99.3% of fermentation reactor content and 0.58 gNaCHO3.gCOD−1, reaching a production of 6549.7 mLCH4.L−1, with organic matter removal (rCOD) of 87.1%, and 58.6% of reduction in solid waste content. For this condition, acetogenic metabolism predominance was observed, in the conversion of butyric acid and propionic acid into acetic acid. In the analysis of the microbial community of the optimized condition assay, a high relative abundance of hydrolytic and acidogenic bacteria (AUTHM297, Lachnospiraceae, and Hungateiclostridiaceae) was observed, as well as methanogenic archaea related to acetoclastic, hydrogenotrophic, and methylotrophic methanogenesis (Methanoregula, Methanolinea, Methanomassiliicoccus, Candidatus Methanomethylicus, and Methanofolis).

Statistical optimization of the yield of the oxidation of 5-hydroxymethylfurfural to 2,5-furandicarboxylic acid catalyzed by laccase from Trametes versicolor

Lignocellulosic residues are an interesting alternative to produce furans as 5-hydroxymethylfurfural (HMF), which can be oxidized to produce 2,5-furandicarboxylic acid (FDCA). According to the latter, this research aims to optimize the oxidation yield of HMF to FDCA catalyzed by a Trametes versicolor laccase using TEMPO as a mediator. For yield optimization, a Box–Behnken experimental design was developed with three factors and three levels. The model achieved 90 % fit (R2) with a maximum yield of 100 % FDCA, using 30 mM HMF, 55 mM 2,2,6,6-Tetramethylpiperidine 1-oxyl (TEMPO), and 6 mg laccase. The oxidation kinetics (at 30 and 100 mM HMF) evidenced the presence of the intermediate compound 5-formyl-2-furancarboxylic acid (FFCA), being higher when working at a higher concentration of substrate (100 mM HMF). Optimizing the operational conditions resulted in a notable improvement, boosting the yield of FDCA by approximately 10 % compared to the initial experimentation. Finally, the values of the variables predicted by the program were validated experimentally, reaching 100 % performance.

A Recombinant Alginate Lyase Algt1 with Potential in Preparing Alginate Oligosaccharides at High-Concentration Substrate.

Alginate lyase has been demonstrated as an efficient tool in the preparation of functional oligosaccharides (AOS) from alginate. The high viscosity resulting from the high concentration of alginate poses a limiting factor affecting enzymatic hydrolysis, particularly in the preparation of the fragments with low degrees of polymerization (DP). Herein, a PL7 family alginate lyase Algt from Microbulbifer thermotolerans DSM 19189 was developed and expressed in Pichia pastoris. The recombinant alginate lyase Algt1 was constructed by adopting the structural domain truncation strategy, and the enzymatic activity towards the alginate was improved from 53.9 U/mg to 212.86 U/mg compared to Algt. Algt1 was stable when incubated at 40 °C for 90 min, remaining with approximately 80.9% of initial activity. The analyses of thin-layer chromatography (TLC), fast protein liquid chromatography (FPLC), and electrospray ionization mass spectrometry (ESI-MS) demonstrated that the DP of the minimum identifiable substrate of Algt1 was five, and the main hydrolysis products were AOS with DP 1-4. Additionally, 1-L the enzymatic hydrolysis system demonstrated that Algt1 exhibited an effective degradation at alginate concentrations of up to 20%, with the resulting products of monosaccharides (14.02%), disaccharides (21.10%), trisaccharides (37.08%), and tetrasaccharides (27.80%). These superior properties of Algt1 make it possible to efficiently generate functional AOS with low DP in industrial processing.

Genomic potential and physiological characteristics of C1 metabolism in novel acetogenic bacteria.

Acetogenic bacteria can utilize C1 compounds, such as carbon monoxide (CO), formate, and methanol, via the Wood-Ljungdahl pathway (WLP) to produce biofuels and biochemicals. Two novel acetogenic bacteria of the family Eubacteriaceae ES2 and ES3 were isolated from Eulsukdo, a delta island in South Korea. We conducted whole genome sequencing of the ES strains and comparative genome analysis on the core clusters of WLP with Acetobacterium woodii DSM1030T and Eubacterium limosum ATCC8486T. The methyl-branch cluster included a formate transporter and duplicates or triplicates copies of the fhs gene, which encodes formyl-tetrahydrofolate synthetase. The formate dehydrogenase cluster did not include the hydrogenase gene, which might be replaced by a functional complex with a separate electron bifurcating hydrogenase (HytABCDE). Additionally, duplicated copies of the acsB gene, encoding acetyl-CoA synthase, are located within or close to the carbonyl-branch cluster. The serum bottle culture showed that ES strains can utilize a diverse range of C1 compounds, including CO, formate, and methanol, as well as CO2. Notably, ES2 exhibited remarkable resistance to high concentrations of C1 substrates, such as 100% CO (200 kPa), 700 mM formate, and 500 mM methanol. Moreover, ES2 demonstrated remarkable growth rates under 50% CO (0.45 h-1) and 200 mM formate (0.34 h-1). These growth rates are comparable to or surpassing those previously reported in other acetogenic bacteria. Our study introduces novel acetogenic ES strains and describes their genetic and physiological characteristics, which can be utilized in C1-based biomanufacturing.