Enzyme engineering for valorization of agrowaste-derived levulinic acid to versatile 4-hydroxyvaleric acid

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Perspectives for biocatalytic lignin utilization: cleaving 4-O-5 and C??-C?? bonds in dimeric lignin model compounds catalyzed by a promiscuous activity of tyrosinase

Background: In the biorefinery utilizing lignocellulosic biomasses, lignin decomposition to value-added phenolic derivatives is a key issue, and recently biocatalytic delignification is emerging owing to its superior selectivity, low energy consumption, and unparalleled sustainability. However, besides heme-containing peroxidases and laccases, information about lignolytic biocatalysts is still limited till date. Results: Herein, we report a promiscuous activity of tyrosinase which is closely associated with delignification requiring high redox potentials (>1.4 V vs. normal hydrogen electrode [NHE]). The promiscuous activity of tyrosinase not only oxidizes veratryl alcohol, a commonly used nonphenolic substrate for assaying ligninolytic activity, to veratraldehyde but also cleaves the 4-O-5 and C??-C?? bonds in 4-phenoxyphenol and guaiacyl glycerol-??-guaiacyl ether (GGE) that are dimeric lignin model compounds. Cyclic voltammograms additionally verified that the promiscuous activity oxidizes lignin-related high redox potential substrates. Conclusion These results might be applicable for extending the versatility of tyrosinase toward biocatalytic delignification as well as suggesting a new perspective for sustainable lignin utilization. Furthermore, the results provide insight for exploring the previously unknown promiscuous activities of biocatalysts much more diverse than ever thought before, thereby innovatively expanding the applicable area of biocatalysis

Novel NAD-independent D-lactate dehydrogenases from Acetobacter aceti and Acidocella species MX-AZ02 as potential candidates for in vitro biocatalytic pyruvate production

Pyruvate is a significant platform chemical widely used in the agrochemical and pharmaceutical industries. We discovered FAD-containing lactate dehydrogenases (LDHs) from Acetobacter aceti (Aa-LDH) and Acidocella species MX-AZ02 (As-LDH), expressed them in Escherichia coil, optimized their FAD reconstitution, and characterized the recombinants as NAD-independent D-LDHs that are capable of the in vitro biocatalytic production of pyruvate from lactate. Instead of NAD, both Aa-LDH and As-LDH utilized various organic dyes as the electron acceptor. In addition, Aa-LDH and As-LDH exhibited substrate specificity for D-lactate only. Activity was optimized at pH 7.0 and 65 degrees C. The kinetic parameters of Aa-LDH and As-LDH were examined and both enzymes exhibited higher catalytic efficiency (k(cat)/K-m) for 2,6-dichlorophenolindophenol (DCIP), one of the electron acceptors, than D-lactate due to higher binding affinities. When using 10 mM D-lactate as the substrate with stepwise DCIP and D-LDH feeding, Aa-LDH and As-LDH produced 5.48 and 4.09 mM pyruvate, respectively, and the conversion was proportional to the DCIP concentration.clos

CO2 Reduction to Formate: An Electro-Enzymatic Approach Using a Formate Dehydrogenase from Rhodobacter capsulatus

CO2 utilization for producing value-added chemicals has recently emerged as a strategy to mitigate atmospheric CO2 levels. Given that (i) certain formate dehydrogenases are capable of interconverting CO2 and formate, and (ii) formate is versatile in various industries, we, herein, aimed to demonstrate FDH-driven formate production from CO2. Because of its O2 stability, we selected FDH from Rhodobacter capsulatus (RcFDH) and then constructed a mediated electro-enzymatic system. The mediated electro-enzymatic kinetic parameters (kred and kox) were calculated to optimize the reaction conditions favorable for CO2 reduction. Finally, a RcFDH-driven electro-enzymatic system successfully produced 6 mM of formate in 5 hours

Effect of manganese peroxidase on the decomposition of cellulosic components: Direct cellulolytic activity and synergistic effect with cellulase

Herein, it was unearthed that manganese peroxidase (MnP) from Phanerochaete chrysosporium, a lignin-degrading enzyme, is capable of not only directly decomposing cellulosic components but also boosting cellulase activity. MnP decomposes various cellulosic substrates (carboxymethyl cellulose, cellobiose [CMC], and Avicel (R)) and produces reducing sugars rather than oxidized sugars such as lactone and ketoaldolase. MnP with MnII in acetate buffer evolves the MnIII-acetate complex functioning as a strong oxidant, and the non-specificity of MnIII-acetate enables cellulose-decomposition. The catalytic mechanism was proposed by analyzing catalytic products derived from MnP-treated cellopentaose. Notably, MnP also boosts cellulase activity on CMC and Avicel (R), even considering the cellulolytic activity of MnP itself. To the best of the authors' knowledge, this is the first report demonstrating a previously unknown fungal MnP activity in cellulose-decomposition in addition to a known delignification activity. Consequently, the results provide a promising insight for further investigation of the versatility of lignin-degrading biocatalysts

Volatile Fatty Acids from Lipid-Extracted Yeast Provide Additional Feedstock for Microbial Lipid Production

Microbial lipid production from oleaginous yeasts is a promising process for the sustainable development of the microbial biodiesel industry. However, the feedstock cost poses an economic problem for the production of microbial biodiesel. After lipid extraction, yeast biomass can be used as an organic source for microbial biodiesel production. In this study, volatile fatty acids (VFAs), produced via anaerobic digestion of a lipid-extracted yeast (LEY) residue, were utilized as a carbon source for the yeast Cryptococcus curvatus. The response surface methodology was used to determine the initial pH and inoculum volume for the optimal VFA production. The experimental result for VFA concentration was 4.51 g/L at an initial pH of 9 and an inoculation 25%. The optimization results from the response surface methodology showed that the maximal VFA concentration was 4.58 g/L at an initial pH of 8.40 and an inoculation of 39.49%. This study indicates that VFAs from LEY can be used as a carbon source for microbial biodiesel production, with the potential to significantly reduce feedstock costs

A biosensor based on the self-entrapment of glucose oxidase within biomimetic silica nanoparticles induced by a fusion enzyme

We constructed a fusion protein (GOx-R5) consisting of R5 (a polypeptide component of silaffin) and glucose oxidase (GOx) that was expressed in Pichia pastoris. Silaffin proteins are responsible for the formation of a silica-based cell matrix of diatoms, and synthetic variants of the R5 protein can perform silicification in vitro [1]. GOx secreted by P. pastoris was self-immobilized (biosilicification) in a pH 5 citric buffer using 0.1 M tetramethoxysilane as a silica source. This self-entrapment property of GOx-R5 was used to immobilize GOx on a graphite rod electrode. An electric cell designed as a biosensor was prepared to monitor the glucose concentrations. The electric cell consisted of an Ag/AgCl reference electrode, a platinum counter electrode, and a working electrode modified with poly(neutral red) (PNR)/GOx/Nafion. Glucose oxidase was immobilized by fused protein on poly(neutral red) and covered by Nafion to protect diffusion to the solution. The morphology of the resulting composite PNR/GOx/Nafion material was analyzed by scanning electron microscopy (SEM). This amperometric transducer was characterized electrochemically using cyclic voltammetry and amperometry in the presence of glucose. An image produced by scanning electron microscopy supported the formation of a PNR/GOx complex and the current was increased to 1.58 mu A cm(-1) by adding 1 mM glucose at an applied potential of -0.5 V. The current was detected by way of PNR-reduced hydrogen peroxide, a product of the glucose oxidation by GOx. The detection limit was 0.67 mM (S/N = 3). The biosensor containing the graphite rod/PNR/GOx/Nafion detected glucose at various concentrations in mixed samples, which contained interfering molecules. In this study, we report the first expression of R5 fused to glucose oxidase in eukaryotic cells and demonstrate an application of self-entrapped GOx to a glucose biosensor.clos

Two-stage bioconversion of carbon monoxide to biopolymers via formate as an intermediate

Numerous industries discharge substantial amounts of carbon monoxide (CO) into the atmosphere as waste; utilizing CO-containing industrial waste gases to produce useful organic chemicals has recently attracted attention. Here, we constructed a two-stage biocatalytic CO-conversion system for producing poly-3-hydroxybutyrate (PHB), a promising degradable biopolymer. In the first stage, Acetobacterium woodii, an acetogenic bacterial strain containing CO dehydrogenase (CODH) and formate dehydrogenase (FDH), was used as a whole-cell biocatalyst to transform CO into formate independent of an external reducing agent, such as H2. The conversion yield and specificity were close to 100% when the strain???s energy metabolism was blocked to suppress acetate production. The resulting formate was fed to a second bioreactor, where it was converted to PHB by engineered Methylbacterium extorquens AM1. The two-stage bioconversion of CO to a valuable product via formate as an intermediate offers a novel and promising strategy for CO utilization

Comparison of Low-Temperature Alkali/Urea Pretreatments for Ethanol Production from Wheat Straw

NaOH/urea (NU) pretreatment at lower than 0 degrees C has been frequently applied for improving bio-conversion of lignocellulose, but the wastewater generated from the pretreatment process is hard to dispose. KOH/urea (KU) pretreatment for enhancing bioconversion of lignocellulose has recently attracted researchers' attention due to the recycling of wastewater for facilitating crops' growth. This study compared the effects of NU and KU pretreatments at cold conditions on the enzymatic hydrolysis and bioethanol yield from wheat straw (WS). By using response surface methodology, an optimal pretreatment with an equal ratio of alkali/urea (4% w/v) at -20 degrees C for 3 h was established. The enzymatic hydrolysis of KU-treated WS was 81.17%, which was similar to that of NU-treated WS (83.72%) under the same condition. It means that KU pretreatment has equal ability to NU pretreatment to improve enzymatic saccharification of lignocellulose. KU pretreatment has the promising potential to replace NU pretreatment for facilitating bioconversion of lignocellulose in cold conditions due to the clean way to recycle its wastewater as fertilizer for crop growth. Hence, KU pretreatment combined with enzymatic hydrolysis and fermentation could be a promising green way to cellulosic ethanol production with zero waste emission

Recent progress and challenges in biological degradation and biotechnological valorization of lignin as an emerging source of bioenergy: A state-of-the-art review

Due to concerns over climate change and the depletion of fossil fuels, recent studies have focused on biorefineries utilizing lignocellulosic biomass as a renewable feedstock for producing value-added fuels and chemicals. Among the components of lignocellulosic biomass, cellulosic/hemicellulosic components have mainly been utilized to obtain fermentable sugars in biorefineries, whereas lignin has commonly been treated as a byproduct thus far. Nonetheless, lignin is the most widely available macromolecule in nature; thus, recent biorefineries have paid special attention to lignin as an attractive source for renewable energy. Given that natural lignin is highly recalcitrant to breakdown, lignin degradation is the most significant issue for further valorization. Accordingly, biological approaches involving enzymes and microorganisms for lignin degradation are comprehensively reviewed in this study. Additionally, this review intensively addresses the recent progress in biotechnological lignin valorization including not only metabolic engineering for producing useful fuels and chemicals but also the use of lignin-based hybrid materials for biosensor and biomedical applications. Furthermore, future studies on challenging issues are suggested as an aid to developing feasible lignin-based biorefineries. The results discussed in this review might provide insights for zero-waste refineries utilizing all components of lignocellulosic biomass