A pH-dependent shift of redox cofactor specificity in a benzyl alcohol dehydrogenase of aromatoleum aromaticum EbN1.

We characterise a reversible bacterial zinc-containing benzyl alcohol dehydrogenase (BaDH) accepting either NAD+ or NADP+ as a redox cofactor. Remarkably, its redox cofactor specificity is pH-dependent with the phosphorylated cofactors favored at lower and the dephospho-forms at higher pH. BaDH also shows different steady-state kinetic behavior with the two cofactor forms. From a structural model, the pH-dependent shift may affect the charge of a histidine in the 2'-phosphate-binding pocket of the redox cofactor binding site. The enzyme is phylogenetically affiliated to a new subbranch of the Zn-containing alcohol dehydrogenases, which share this conserved residue. BaDH appears to have some specificity for its substrate, but also turns over many substituted benzyl alcohol and benzaldehyde variants, as well as compounds containing a conjugated C=C double bond with the aldehyde carbonyl group. However, compounds with an sp3-hybridised C next to the alcohol/aldehyde group are not or only weakly turned over. The enzyme appears to contain a Zn in its catalytic site and a mixture of Zn and Fe in its structural metal-binding site. Moreover, we demonstrate the use of BaDH in an enzyme cascade reaction with an acid-reducing tungsten enzyme to reduce benzoate to benzyl alcohol. KEY POINTS: •Zn-containing BaDH has activity with either NAD + or NADP+ at different pH optima. •BaDH converts a broad range of substrates. •BaDH is used in a cascade reaction for the reduction of benzoate to benzyl alcohol.

Construction of a Novel Vanillin-Induced Autoregulating Bidirectional Transport System in a Vanillin-Producing E. coli Cell Factory.

Vanillin is one of the world's most extensively used flavoring agents with high application value. However, the yield of vanillin biosynthesis remains limited due to the low efficiency of substrate uptake and the inhibitory effect on cell growth caused by vanillin. Here, we screened high-efficiency ferulic acid importer TodX and vanillin exporters PP_0178 and PP_0179 by overexpressing genes encoding candidate transporters in a vanillin-producing engineered Escherichia coli strain VA and further constructed an autoregulatory bidirectional transport system by coexpressing TodX and PP_0178/PP_0179 with a vanillin self-inducible promoter ADH7. Compared with strain VA, strain VA-TodX-PP_0179 can efficiently transport ferulic acid across the cell membrane and convert it to vanillin, which significantly increases the substrate utilization rate efficiency (14.86%) and vanillin titer (51.07%). This study demonstrated that the autoregulatory bidirectional transport system significantly enhances the substrate uptake efficiency while alleviating the vanillin toxicity issue, providing a promising viable route for vanillin biosynthesis.

Neodymium niobate nanospheres on functionalized carbon nanofibers: a nanoengineering approach for highly sensitive vanillin detection.

Vanillin (VAN), the primary aroma compound in vanilla, contributes significantly to sensory delight; however, its unrestrained presence poses notable health risks. In response to the demanding concern regarding food safety, researchers have directed their efforts towards the detection of VAN, seeking sustainable strategies for contamination prevention. A groundbreaking solution has emerged in the form of a novel sensing platform, whose core lies on a finely tuned electrode, crafted through the incorporation of nano-sized NdNbO4 spheres onto carbon nanofibers (CNFs). This incorporation serves to augment the capabilities of a glassy carbon electrode (GCE), transforming it into a highly sensitive detector primed for vanillin detection. The NdNbO4/f-CNF nanocomposite embodies a paradigm of synergistic collaboration, wherein the nonlinear cumulative effects of synergism and quantum confinement impart exceptional performance characteristics. Notably, the sensor achieves a low detection limit of 6.3 nmol L-1, indicative of its remarkable sensitivity of 2.3 µA µ(mol L-1)-1 cm-2 and precision of 1.519 and 4.72%. Moreover, the sensor boasts a wide linear range spanning from 0.001 to 63.101 µmol L-1. These attributes, coupled with its discerning selectivity and robust stability, underscore its efficacy as a versatile tool for vanillin detection. Indeed, its successful deployment in monitoring food samples underscores its applicability across diverse culinary contexts, further cementing its status as a pivotal asset in safeguarding food quality and consumer well-being.

Distal mutations enhance efficiency of free and immobilized NOV1 dioxygenase for vanillin synthesis.

Protein engineering is crucial to improve enzymes' efficiency and robustness for industrial biocatalysis. NOV1 is a bacterial dioxygenase that holds biotechnological potential by catalyzing the one-step oxidation of the lignin-derived isoeugenol into vanillin, a popular flavoring agent used in food, cleaning products, cosmetics and pharmaceuticals. This study aims to enhance NOV1 activity and operational stability through the identification of distal hotspots, located at more than 9 Šfrom the active site using Zymspot, a tool that predicts advantageous distant mutations, streamlining protein engineering. A total of 41 variants were constructed using site-directed mutagenesis and the six most active enzyme variants were then recombined. Two variants, with two and three mutations, showed nearly a 10-fold increase in activity and up to 40-fold higher operational stability than the wild-type. Furthermore, these variants show 90-100 % immobilization efficiency in metal affinity resins, compared to approximately 60 % for the wild-type. In bioconversions where 50 mM of isoeugenol was added stepwise over 24-h cycles, the 1D2 variant produced approximately 144 mM of vanillin after six reaction cycles, corresponding to around 22 mg, indicating a 35 % molar conversion yield. This output was around 2.5 times higher than that obtained using the wild-type. Our findings highlight the efficacy of distal protein engineering in enhancing enzyme functions like activity, stability, and metal binding selectivity, thereby fulfilling the criteria for industrial biocatalysts. This study provides a novel approach to enzyme optimization that could have significant implications for various biotechnological applications.

Production of vanillin via oxidation depolymerization of lignin over Fe- and Mn-modified TS-1 zeolites.

Converting lignin into specific aromatic chemicals for utilization through depolymerization of lignin is an effective way to achieve high-value applications. There are many depolymerization methods that can do this, but there are problems such as harsh reaction conditions, low depolymerization efficiency and uncontrollable target products that need to be solved. This study reports a novel system for the oxidative depolymerization of alkali lignin using Fe- and Mn- modified TS-1 as a catalyst to assist in the highly selective production of vanillin. We also proposed a possible reaction pathway for the oxidative depolymerization of alkali lignin to produce vanillin catalyzed by Fe-Mn/TS-1 catalyst. The catalytic effects of TS-1, Fe/TS-1, and Fe-Mn/TS-1 catalysts on the oxidative depolymerization of lignin to produce phenolic monomers and vanillin were investigated. The results show that the modified catalysts can effectively improve the efficiency of linkage bond breaking in lignin, especially the ß-O-4 bond, in which the inter-band transitions of Fe and Mn play an important role. The synergistic effect of the bimetallic-loaded catalyst (Fe-Mn/TS-1) could catalyze the oxidative depolymerization of lignin more efficiently than the monometallic-loaded catalyst (Fe/TS-1). This lignin oxidative depolymerization system produced 40.59 wt% bio-oil including 12.24 wt% phenolic monomers and 16.17 wt% re-lignin after the addition of Fe-Mn/TS-1 catalyst, owning the highest phenolic monomer yield. Surprisingly, this lignin oxidative depolymerization system exhibited high yield for vanillin (8.36 wt%) production. These results demonstrated that the Fe-Mn/TS-1 catalytic system has potential to produce vanillin from lignin under mild conditions.

Multienzymatic Cascade for Synthesis of Hydroxytyrosol via Two-Stage Biocatalysis.

Hydroxytyrosol, a naturally occurring compound with antioxidant and antiviral activity, is widely applied in the cosmetic, food, and nutraceutical industries. The development of a biocatalytic approach for producing hydroxytyrosol from simple and readily accessible substrates remains a challenge. Here, we designed and implemented an effective biocatalytic cascade to obtain hydroxytyrosol from 3,4-dihydroxybenzaldehyde and l-threonine via a four-step enzymatic cascade composed of seven enzymes. To prevent cross-reactions and protein expression burden caused by multiple enzymes expressed in a single cell, the designed enzymatic cascade was divided into two modules and catalyzed in a stepwise manner. The first module (FM) assisted the assembly of 3,4-dihydroxybenzaldehyde and l-threonine into (2S,3R)-2-amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropanoic acid, and the second module (SM) entailed converting (2S,3R)-2-amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropanoic acid into hydroxytyrosol. Each module was cloned into Escherichia coli BL21 (DE3) and engineered in parallel by fine-tuning enzyme expression, resulting in two engineered whole-cell catalyst modules, BL21(FM01) and BL21(SM13), capable of converting 30 mM 3,4-dihydroxybenzaldehyde to 28.7 mM hydroxytyrosol with a high space-time yield (0.88 g/L/h). To summarize, the current study proposes a simple and effective approach for biosynthesizing hydroxytyrosol from low-cost substrates and thus has great potential for industrial applications.

Biodegradation behavior of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) containing phenolic compounds in seawater in laboratory testing conditions.

Biodegradation in marine medium of PHBV films with or without 5 % wt. of phenolic compounds (catechin, ferulic acid, and vanillin) was assessed at laboratory scale. Respirometric analyses and film disintegration kinetics were used to monitor the process over a period of 162 days. Structural changes in the films were analyzed throughout the exposure period using FESEM, DSC, Thermogravimetric analyses, XRD, and FTIR spectra. Respirometric tests showed complete biodegradation of all materials during the exposure period (the biodegradation half-time ranged between 63 and 79 days) but at different rates, depending on the phenolic compound incorporated. Ferulic acid and vanillin accelerate the PHBV biodegradation, whereas catechin delayed the process. Disintegration kinetics confirmed these results and showed that degradation occurred from the surface to the interior of the films. This was controlled by the degradation rate of the polymer amorphous phase and the formation of a biomass coating on the film surface. This is the result of the compounds generated by polymer degradation in combination with excretions from microorganisms. This coating has the potential to affect the enzyme diffusion to the polymer substrate. Moreover, the cohesion forces of the amorphous phase (reflected in its glass transition temperature) affected its degradation rate, while the slower degrading crystalline fragments were released, thus contributing to the disintegration process on the film's surface. Ferulic acid, with its hydrolytic effect, enhanced degradation, as did vanillin for its plasticizing and weakening effect in the amorphous phase of polymer matrix. In contrast, catechin with cross-linking effect hindered the progress of the material degradation, considerably slowing down the process rate.

Heterojunction of branched benzopyrazine-based polymers coating on graphene for electrochemical sensing of vanillin.

Vanillin finds widespread applications in various industries, such as food, pharmaceuticals, and cosmetics. However, excessive intake of vanillin could pose risks to human health. This study detailed the successful creation of a heterojunction of branched benzopyrazine-based polymers coating on graphene (CMP-rGO) through the Sonogashira-Hagihara coupling reaction. Utilizing the CMP-rGO, a novel electrochemical sensor for vanillin detection was developed. Besides, the synthesized materials were validated using standard characterization techniques. Both cyclic voltammetry and differential pulse voltammetry techniques were employed to investigate vanillin's electrochemical characteristics on this sensor. The findings indicated a significant enhancement in vanillin's electrochemical signal responsiveness with the application of CMP-rGO. Under optimal conditions, the sensor demonstrated a linear response to vanillin concentrations ranging from 0.08 to 33 µM and achieved a detection limit as low as 0.014 µM. Also, the constructed electrochemical sensor exhibited excellent selectivity, stability, and reproducibility. It has been effectively employed to detect vanillin in real samples such as human serum, human urine, and vanillin tablets, with a recovery rate of 99.13-103.6 % and an RSD of 3.46-1.26 %. Overall, this innovative sensor offers a novel approach to the efficient and convenient detection of vanillin.

Engineering a coenzyme-independent dioxygenase for one-step production of vanillin from ferulic acid.

Vanillin is one of the world's most important flavor and fragrance compounds used in foods and cosmetics. In plants, vanillin is reportedly biosynthesized from ferulic acid via the hydratase/lyase-type enzyme VpVAN. However, in biotechnological and biocatalytic applications, the use of VpVAN limits the production of vanillin. Although microbial enzymes are helpful as substitutes for plant enzymes, synthesizing vanillin from ferulic acid in one step using microbial enzymes remains a challenge. Here, we developed a single enzyme that catalyzes vanillin production from ferulic acid in a coenzyme-independent manner via the rational design of a microbial dioxygenase in the carotenoid cleavage oxygenase family using computational simulations. This enzyme acquired catalytic activity toward ferulic acid by introducing mutations into the active center to increase its affinity for ferulic acid. We found that the single enzyme can catalyze not only the production of vanillin from ferulic acid but also the synthesis of other aldehydes from p-coumaric acid, sinapinic acid, and coniferyl alcohol. These results indicate that the approach used in this study can greatly expand the range of substrates available for the dioxygenase family of enzymes. The engineered enzyme enables efficient production of vanillin and other value-added aldehydes from renewable lignin-derived compounds. IMPORTANCE: The final step of vanillin biosynthesis in plants is reportedly catalyzed by the enzyme VpVAN. Prior to our study, VpVAN was the only reported enzyme that directly converts ferulic acid to vanillin. However, as many characteristics of VpVAN remain unknown, this enzyme is not yet suitable for biocatalytic applications. We show that an enzyme that converts ferulic acid to vanillin in one step could be constructed by modifying a microbial dioxygenase-type enzyme. The engineered enzyme is of biotechnological importance as a tool for the production of vanillin and related compounds via biocatalytic processes and metabolic engineering. The results of this study may also provide useful insights for understanding vanillin biosynthesis in plants.

Chemoenzymatic Synthesis of 2-Aryl Thiazolines from 4-Hydroxybenzaldehydes Using Vanillyl Alcohol Oxidases.

Nitrogen heterocycles are commonly found in bioactive natural products and drugs. However, the biocatalytic tools for nitrogen heterocycle synthesis are limited. Herein, we report the discovery of vanillyl alcohol oxidases (VAOs) as efficient biocatalysts for the one-pot synthesis of 2-aryl thiazolines from various 4-hydroxybenzaldehydes and aminothiols. The wild-type biocatalyst features a broad scope of 4-hydroxybenzaldehydes. Though the scope of aminothiols is limited, it could be improved via semi-rational protein engineering, generating a variant to produce previously inaccessible cysteine-derived bioactive 2-aryl thiazolines using the wild-type VAO. Benefiting from the derivatizable functional groups in the enzymatic products, we further chemically modified these products to expand the chemical space, offering a new chemoenzymatic strategy for the green and efficient synthesis of structurally diverse 2-aryl-thiazoline derivatives to prompt their use in drug discovery and catalysis.

Clinical Practice Patterns in Sickle Cell Disease Treatment: Disease-modifying and Potentially Curative Therapies.

Therapeutic options for sickle cell disease (SCD) have increased recently as well as the development of updated national guidelines. It is not known how these options are being offered or to what degree guidelines are incorporated into clinical practice. This study aimed to describe practice patterns for pediatric hematologists regarding the use of disease-modifying and potentially curative therapies for SCD. A 9-section, cross-sectional electronic survey was disseminated during a 3-month period via SurveyMonkey, to members of the American Society of Pediatric Hematology/Oncology Hemoglobinopathy Special Interest Group (ASPHO HSIG). A total of 88 physician members of the ASPHO HSIG were surveyed. Ninety percent of respondents (72/80) start hydroxyurea routinely in patients with HbSS and HbSß 0 thalassemia, regardless of disease severity. Laboratory monitoring was recommended every 3 months for stable dosing in 63.8% (51/80). New therapies were recommended for patients on hydroxyurea who were still experiencing SCD complications: L-glutamine 68.5% (37/54) or crizanlizumab 93.1% (54/58). Voxelotor was recommended for patients on hydroxyurea with low hemoglobin in 65.1% (43/66) of cases. Matched sibling transplant was considered for any disease severity by 55.1% (38/69). Gene therapy trials are offered on-site by 29% (20/69). Our study demonstrated the enhanced utilization of hydroxyurea while revealing the unexplored potential of other disease-modifying therapies in SCD. These findings underscore the importance of continued knowledge acquisition about the long-term efficacy of new medical therapies and addressing barriers to the use of proven therapies and guide the development of future studies of optimal SCD management.

Suppression of anthracnose disease by orsellinaldehyde isolated from the mushroom Coprinus comatus.

AIMS: Anthracnose caused by Colletotrichum species is one of the most devastating diseases of fruits and crops. We isolated and identified an antifungal compound from the mushroom Coprinus comatus and investigated its inhibitory potential against anthracnose disease-causing fungi with the goal of discovering natural products that can suppress anthracnose-caused plant disease. METHODS AND RESULTS: The culture filtrate of C. comatus was subjected to a bioassay-guided isolation of antifungal compounds. The active compound was identified as orsellinaldehyde (2,4-dihydroxy-6-methylbenzaldehyde) based on mass spectroscopy and nuclear magnetic resonance analyses. Orsellinaldehyde displayed broad-spectrum inhibitory activity against different plant pathogenic fungi. Among the tested Colletotrichum species, it exhibited the lowest IC50 values on conidial germination and germ tube elongation of Colletotrichum orbiculare. The compound also showed remarkable inhibitory activity against Colletotrichum gloeosporiodes. The staining of Colletotrichum conidia with fluorescein diacetate and propidium iodide demonstrated that the compound is fungicidal. The postharvest in-vivo detached fruit assay indicated that orsellinaldehyde suppressed anthracnose lesion symptoms on mango and cucumber fruits caused by C. gloeosporioides and C. orbiculare, respectively. CONCLUSIONS: Orsellinaldehyde was identified as a potent antifungal compound from the culture filtrate of C. comatus. The inhibitory and fungicidal activities of orsellinaldehyde against different Colletotrichum species indicate its potential as a fungicide for protecting various fruits against anthracnose disease-causing fungi.

Fabrication of chitosan-based fluorescent hydrogel membranes cross-linked with bisbenzaldehyde for efficient selective detection and adsorption of Fe2.

Chitosan, as a natural biomass material, is green, recyclable, sustainable and well biocompatible. The molecular chain is rich in active groups such as amino and hydroxyl groups, and its preparation of fluorescent probes has the advantages of biocompatibility and efficient detection performance. In this study, a bis(benzaldehyde) (BHD) fluorescent functional molecule was designed. Then a series of fluorescent chitosan-based hydrogel films (CSBHD) were prepared using chitosan as raw material and BHD as cross-linking agent. As a fluorescent probe for metal ions, CSBHD was able to efficiently detect Fe2+ with a linear correlation of fluorescence intensity in the range of 0-160 µM, and the limit of detection (LOD) was 0.55 µM. Moreover, it has excellent adsorption performance for Fe2+ ions, with a maximum adsorption capacity of 223.5 g/mg at 500 mg/L Fe2+ concentration. Finally, we characterised the structure and microscopic morphology of CSBHD films and found that CSBHD as a hydrogel film has a high cross-linking density, good water resistance, excellent thermal stability, strong resistance to swelling, and excellent stability in cycling tests. Hence, it has great potential for application in adsorption and detection of Fe2+ ions. It also provides a good strategy for the application of chitosan based fluorescent probe materials in environmental monitoring and heavy metal ion adsorption.

Evaluation of the effect of voxelotor and darbepoetin alfa on hemoglobin levels in patients with sickle cell disease.

BACKGROUND: To date, there is limited evidence on patients utilizing both voxelotor and darbepoetin alfa and its impact on hemoglobin levels. The objective is to evaluate the effect of voxelotor and darbepoetin alfa on hemoglobin levels in patients with SCD. RESEARCH DESIGN AND METHODS: This was a retrospective chart review study that assessed the primary independent variable as the utilization of either voxelotor alone, darbepoetin alfa alone, or the concurrent administration of voxelotor and darbepoetin alfa. Descriptive statistics were utilized to obtain the mean standard deviation for numerical variables and proportions for categorical variables. RESULTS: A total of 23 participants were included in this study. When comparing baseline to 2 months and 3 months, participants on voxelotor alone experienced a 3% decrease and a 6.6% increase in hemoglobin, darbepoetin alfa alone group a 4.3% decrease and a 0.6% increase in hemoglobin and voxelotor and darbepoetin group a 4.4% decrease and a 0.5% decrease in hemoglobin levels. Fifty percent of the participants in the voxelotor group and 6 (66.7%) participants in the voxelotor plus darbepoetin alfa group experienced adverse drug events. CONCLUSIONS: Voxelotor resulted in a clinically significant difference in the percent change of hemoglobin from baseline to 3 months.

Synergy of metallic Co and oxygen vacancy sites in Co/Ce-MOF catalysts for efficiently promoting lignin derived phenols and macromolecular lignin hydrodeoxygenation.

The enhanced utilization of biomass-derived chemicals for the generation of high value aromatics through an advanced catalytic strategy has captured considerable attention within the realm of eco-friendly manufacturing. This work presented four innovative three-dimensional rod-shaped mesoporous Ce-based MOF materials, which were coupled with a H-donor solvent to facilitate vanillin hydrodeoxygenation and macromolecular lignin. Under the optimized conditions (30 mg CoCe@C catalyst, 2 MPa N2 pressure, 15 mL isopropanol, 190 °C, and 5 h), the CoCe@C catalyst achieved nearly complete conversion of vanillin and demonstrated 87.8 % selectivity in the hydrogen-donor solvent. The characterization findings suggested that the synergy between metallic Co and oxygen vacancy sites enabled the effective activation of CHO group in vanillin, leading to formation of reactive product MMP. In addition, the optimal CoCe@C catalyst could also achieve macromolecular lignin hydrodeoxygenation to obtain high yield of lignin oil products with narrower molecular weight distribution. This study presented a viable approach for the concurrent utilization of lignin derivatives in the generation of high value fuels and chemicals.

A novel synthesized Vanillin-Based Deep Eutectic Agent (V-DEA) mitigates postharvest fungal decay and improve shelf life and quality of cherry tomatoes.

Fusarium oxysporum and Botrytis cinerea are the main pathogens that cause fruit decay and reduce the postharvest shelf life of cherry tomatoes. Boosting the potency of natural products requires implementing structural modification to combat postharvest pathogens. Herein, we developed a novel Vanillin-Deep Eutectic Agent (V-DEA) from natural compounds and evaluated its effectiveness against tomato fruit rot pathogens. The results demonstrated that V-DEA suppressed mycelium growth and spore germination of F. oxysporum and B. cinerea by enhancing cell membrane permeability, increasing lipid peroxidation, and inhibiting enzyme activities. Importantly, using 8-mM V-DEA successfully prevented postharvest decay in cherry tomatoes, while 4-mM significantly extended their shelf life by reducing weight loss and shriveling, and enhancing key fruit qualities such as total soluble solids, ascorbic acid, tartaric acid, and lycopene. In conclusion, V-DEA exhibits dual properties as a potent pathogen inhibitor and antioxidant activity, thus prolonging the shelf life of cherry tomatoes.

Effect of luminescent materials on the aquatic macrophyte Vallisneria natans and periphytic biofilm.

Luminescent materials can adjust the spectrum of light energy utilization by plants. However, current research on the effects of luminescent materials on aquatic plants and periphytic biofilms is limited. This study investigated the effects of the luminescent materials 4-(di-p-tolylamino) benzaldehyde-A (DTB-A) and 4-(di-p-tolylamino) benzaldehyde-M (DTB-M) on the submerged macrophyte Vallisneria natans (V. natans) and periphytic biofilm. Result demonstrated that low concentrations of DTB (0.1 µM) significantly promoted the growth and photosynthetic rate of V. natans. In terms of enzyme activity, exposure to a higher concentration of DTB (10 µM) increased the activities of peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT). A combination of DTB-A and DTB-M treatment significantly changed the V. natans morphology and physiological characteristics, reducing the thickness of the cell wall and subsequently, promoting protein accumulation in leaves. There was no difference in the removal of ammonia or phosphate by V. natans at the 0.1 µM concentration, and the removal of ammonia and phosphate by V. natans decreased significantly as the concentration of luminescent material increased. A total of 3563 OTUs were identified in the biofilm community. The microbial community was dominated by Pseudomonas and Fusobacteria. Furthermore, results showed that an obvious decrease in diversity in the DTB-A and DTB-M mixed treatment group. In addition, the migratory aggregation of DTB molecules in plants was observed by fluorescence imaging. Overall, these findings extend our understanding of the mechanism of effect of luminescent materials on submerged macrophytes and their periphytic microorganisms.

WOx boosted hollow Ni nanoreactors for the hydrodeoxygenation of lignin derivatives.

Reasonable design of non-noble metal catalysts with hollow open structure for hydrodeoxygenation (HDO) of lignin derivatives to value-added chemicals is of great significance but challenging. Herein, a novel MOF-derived multilayer hollow sphere coated nickel­tungsten bimetallic catalyst (Ni2-WOx@CN-700) was fabricated via by confined pyrolysis strategy using bimetallic MOFs as a self-sacrificial template, which exhibits robust activity for the typical model HDO of vanillin to 2-methoxy-4-methylphenol (Yield of 100 % at 140 °C for no less than 10 cycles). The characterizations revealed that WOx facilitated the dispersion of Ni nanoparticles and adjusted the acidic capacity of the catalyst through the formed Ni-WOx heterojunction. Density functional theory (DFT) calculations confirms that WOx species enhanced the electron-rich nature of the active sites, while the adsorption energies of H2 and vanillin on Ni-WOx decreased from -0.572 eV and - 0.622 eV on Ni to -3.969 eV and - 4.922 eV, respectively. These results further indicated that the high activity of Ni2-WOx@CN-700 was attributed to the Ni-WOx heterojunction. Based on the characterizations and the thermodynamic calculations, the reaction mechanism was proposed. In addition, the catalyst shows good substrate universality, which enables its good commercial application prospect.

Engineering a Carotenoid Cleavage Oxygenase for Coenzyme-Free Synthesis of Vanillin from Ferulic Acid.

One-pot biosynthesis of vanillin from ferulic acid without providing energy and cofactors adds significant value to lignin waste streams. However, naturally evolved carotenoid cleavage oxygenase (CCO) with extreme catalytic conditions greatly limited the above pathway for vanillin bioproduction. Herein, CCO from Thermothelomyces thermophilus (TtCCO) was rationally engineered for achieving high catalytic activity under neutral pH conditions and was further utilized for constructing a one-pot synthesis system of vanillin with Bacillus pumilus ferulic acid decarboxylase. TtCCO with the K192N-V310G-A311T-R404N-D407F-N556A mutation (TtCCOM3) was gradually obtained using substrate access channel engineering, catalytic pocket engineering, and pocket charge engineering. Molecular dynamics simulations revealed that reducing the site-blocking effect in the substrate access channel, enhancing affinity for substrates in the catalytic pocket, and eliminating the pocket's alkaline charge contributed to the high catalytic activity of TtCCOM3 under neutral pH conditions. Finally, the one-pot synthesis of vanillin in our study could achieve a maximum rate of up to 6.89 ± 0.3 mM h-1. Therefore, our study paves the way for a one-pot biosynthetic process of transforming renewable lignin-related aromatics into valuable chemicals.