Alcohol Oxidase Research Articles

A unique AA5 alcohol oxidase fused with a catalytically inactive CE3 domain from the bacterium Burkholderia pseudomallei.

Copper radical oxidases (CROs) are redox enzymes able to oxidize alcohols or aldehydes, while only requiring a single copper atom as cofactor. Studied CROs are found in one of two subfamilies within the Auxiliary Activities family 5 (AA5) in the carbohydrate-active enzymes database. We here characterize an AA5 enzyme outside the subfamily classification from the opportunistic bacterial pathogen Burkholderia pseudomallei, which curiously was fused to a carbohydrate esterase family 3 domain. The enzyme was shown to be a promiscuous primary alcohol oxidase, with an activity profile similar to enzymes from subfamily 2. The esterase domain was inactive on all tested substrates, and structural predictions revealed this being an effect of crippling substitutions in the expected active site residues.

A conductometric enzymatic methanol sensor based on polystyrene - PAMAM dendritic polymer electrospun nanofibers

Methanol (MeOH) is a solvent and cleaning agent used in industry, but it is poisonous when ingested. The recommended release threshold for MeOH vapor is 200 ppm. We present a novel sensitive micro-conductometric MeOH biosensor created by grafting alcohol oxidase (AOX) onto electrospun polystyrene-poly(amidoamine) dendritic polymer blend nanofibers (PS-PAMAM-ESNFs) on interdigitated electrodes (IDEs). The analytical performance of the MeOH microsensor was evaluated using gaseous MeOH, ethanol, and acetone samples collected from the headspace above aqueous solution with known concentration. The sensor's response time (tRes) fluctuates from 13 s to 35 s from lower to higher concentrations. The conductometric sensor has a sensitivity of 150.53 μS.cm-1 (v/v) for MeOH and a detection limit of 100 ppm in the gas phase. The MeOH sensor is 7.3 times less sensitive to ethanol and 136.8 times less sensitive to acetone. The sensor was verified for detecting MeOH in commercial rubbing alcohol samples.

Eco-friendly detoxification of hazardous Congo red dye using novel fungal strain Trametes flavida WTFP2: Deduced enzymatic biomineralization process through combinatorial in-silico and in-vitro studies

Growing textile industry is a major global concern, owing to the presence of recalcitrant hazardous pollutants, like synthetic dyes in discharged effluents. To explore new bioresources for mycoremediation, a high laccase-producing novel white-rot fungus (WRF), Trametes flavida WTFP2, was employed. T. flavida is an underexplored member of Polyporales. Using bioinformatic tools, 8 different cis-acting RNA elements were identified in the 5.8 S ITS gene sequence, where CRISPR (CRISPR-DR15), sRNA (RUF1), and snoRNA (ceN111) are uniquely present. Molecular docking was adopted to predict the catalytic interaction of chosen toxic diazo colorant, Congo red (CR), with four dye-degrading enzymes (laccase, lignin peroxidase, azoreductase, and aryl alcohol oxidase). With 376.41 × 103 U/L laccase production, novel WRF exhibited dye-decolorization potential. WTFP2 effectively removed 99.48 ± 0.04% CR (100 mg/L) and demonstrated remarkable recyclability and persistence in consecutive remediation trials. Mycelial dye adsorption was not only substantial driver of colorant elimination; decolorization using active T. flavida was regulated by enzymatic catalysis, as outlined by in-vitro growth, induction of extracellular enzymes, and FESEM. Fifteen metabolites were identified using HRLCMS-QTOF, and novel CR degradation pathway was proposed. Furthermore, microbial and phyto-toxicity tests of metabolites suggested complete detoxification of toxic dye, making the process clean, green, and economically sustainable.

Expression and biochemical characterization of a Bacillus subtilis catalase in Pichia pastoris X-33

Catalase, which catalyzes the decomposition of H2O2 to H2O and O2, is widely used to reduce H2O2 in industrial applications, such as in food processing, textile dyeing and wastewater treatment. In this study, the catalase (KatA) from Bacillus subtilis was cloned and expressed in the yeast Pichia pastoris X-33. The effect of the promoter in the expression plasmid on the activity level of the secreted KatA protein was also studied. First, the gene encoding KatA was cloned and inserted into a plasmid containing an inducible alcohol oxidase 1 promoter (pAOX1) or a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP). The recombinant plasmids were validated by colony PCR and sequencing and then linearized and transformed into the yeast P. pastoris X-33 for expression. With the promoter pAOX1, the maximum yield of KatA in the culture medium reached 338.8 ± 9.6 U/mL in 2 days of shake flask cultivation, which was approximately 2.1-fold greater than the maximum yield obtained with the promoter pGAP. The expressed KatA was then purified from the culture medium by anion exchange chromatography, and its specific activity was determined to be 14826.58 U/mg. Finally, the purified KatA exhibited optimum activity at 25 °C and pH 11.0. Its Km for hydrogen peroxide was 10.9 ± 0.5 mM, and its kcat/Km was 5788.1 ± 25.6 s−1 mM−1. Through the work presented in this article, we have therefore demonstrated efficient expression and purification of KatA in P. pastoris, which might be advantageous for scaling up the production of KatA for use in a variety of biotechnological applications.

Confined Cascade Metabolic Reprogramming Nanoreactor for Targeted Alcohol Detoxification and Alcoholic Liver Injury Management

Alcoholic liver injury (ALI) is the leading cause of serious liver disease, whereas current treatments are mostly supportive and unable to metabolize alcohol directly. Here we report a metabolic reprogramming strategy for targeted alcohol detoxification and ALI management based on a confined cascade nanoreactor. The nanoreactor (named AA@mMOF) is designed by assembling natural enzymes of alcohol oxidase (AOx) and aldehyde dehydrogenase (ALDH) in the cavity of a mesoporous metal organic framework (mMOF) nanozyme with intrinsic catalase (CAT)-like activity. By conducting confined AOx/CAT/ALDH cascade reactions, AA@mMOF enables self-accelerated alcohol degradation (>0.5 mg·mL-1·h-1) with negligible aldehyde diffusion and accumulation, reprogramming alcohol metabolism and allowing high-efficiency detoxification. Administered to high-dose alcohol-intoxicated mice, AA@mMOF shows surprising liver targeting and accumulation performance and dramatically reduces blood alcohol concentration and rapidly reverses unconsciousness and acute liver injury to afford targeted alcoholism treatment. Moreover, AA@mMOF dramatically alleviates fat accumulation and oxidative stress in the liver of chronic alcoholism mice to block and reverse the progression of ALI. By conducting confined AOx/CAT/ALDH cascade reactions for high-efficiency alcohol metabolism reprogramming, AA@mMOF nanoreactor offers a powerful modality for targeted alcohol detoxification and ALI management. The proposed confined cascade metabolic reprogramming strategy provides a paradigm shift for the treatment of metabolic diseases.

Membrane-based approach for the removal of pigment impurities secreted by Pichia pastoris

During recombinant protein expression in Pichia pastoris under the alcohol oxidase promoter, the culture supernatant developed green and yellow pigments, complicating downstream processing. Currently, a chromatography-based purification approach is one of the most efficient strategies to eliminate these pigments, but this method is complicated and often the most expensive step during processing. In this study, we designed a sequential cross-flow filtration by the combination of a microfiltration membrane with a pore size of 0.2 micrometers, followed by an ultrafiltration membrane with a molecular weight cut-off of 10 and 2 kilodaltons (kDa), respectively, and finished with diafiltration of the 10 and 2 kDa retentate fractions. The results show that the microfiltration membrane eliminated host cell impurities and significantly reduced the green pigment, which was measured using the 1976 CIE LAB system, while the 10 and 2 kDa membranes allowed the yellow pigment to pass through. The diafiltration steps also significantly reduced the yellow pigment. The host cell proteins were removed by recirculating in the retentate fraction of the 10 kDa membrane, while the 4.1 kDa target peptide, the candidate cell-penetrating antimicrobial peptide, was recovered by recirculation in the retentate fraction of the 2 kDa membrane.

Structure-Based Redesign of a Methanol Oxidase into an "Aryl Alcohol Oxidase" for Enzymatic Synthesis of Aromatic Flavor Compounds.

Alcohol oxidases (AOxs) catalyze the aerobic oxidation of alcohols to the corresponding carbonyl products (aldehydes or ketones), producing only H2O2 as the byproduct. The majority of known AOxs, however, have a strong preference for small, primary alcohols, limiting their broad applicability, e.g., in the food industry. To broaden the product scope of AOxs, we performed structure-guided enzyme engineering of a methanol oxidase from Phanerochaete chrysosporium (PcAOx). The substrate preference was extended from methanol to a broad range of benzylic alcohols by modifying the substrate binding pocket. A mutant (PcAOx-EFMH) with four substitutions exhibited improved catalytic activity toward benzyl alcohols with increased conversion and kcat toward the benzyl alcohol from 11.3 to 88.9% and from 0.5 to 2.6 s-1, respectively. The molecular basis for the change of substrate selectivity was analyzed by molecular simulation.

In situ treatment of real textile effluent in constructed furrows using consortium of Canna indica and Saccharomyces cerevisiae and subsequent biochemical and toxicity evaluation

Emerging contaminants removals like dyes and heavy metals from the textile effluent have an immense challenge. The present study focuses on the biotransformation and detoxification of dyes and in situ textile effluent treatment by plants and microbes efficiently. A mixed consortium of perennial herbaceous plant Canna indica and fungi Saccharomyces cerevisiae showed decolorization of di-azo dye Congo red (CR, 100 mg/L) up to 97% within 72 h. Root tissues and Saccharomyces cerevisiae cells revealed induction of various dye-degrading oxidoreductase enzymes such as lignin peroxidase, laccase, veratryl alcohol oxidase and azo reductase during CR decolorization. Chlorophyll a, Chlorophyll b and carotenoid pigments were notably elevated in the leaves of a plant during the treatment. Phytotransformation of CR into its metabolic constituents was detected by using several analytical techniques, including FTIR, HPLC, and GC-MS and its non-toxic nature was confirmed by cyto-toxicological evaluation on Allium cepa and on freshwater bivalves. Mix consortium of plant Canna indica and fungi Saccharomyces cerevisiae efficiently treated textile wastewater (500 L) and reduced ADMI, COD, BOD, TSS and TDS (74, 68, 68, 78, and 66%) within 96 h. In situ textile wastewater treatment for in furrows constructed and planted with Canna indica, Saccharomyces cerevisiae and consortium-CS within 4 days reveals reduced ADMI, COD, BOD, TDS and TSS (74, 73, 75, 78, and 77%). Comprehensive observations recommend this is an intelligent tactic to exploit this consortium in the furrows for textile wastewater treatment.

Saponins modulates fatty alcohol oxidase to reduce myocardial remodeling and control heart failure by regulating cGMP-PKG signaling pathway

This study analyzed mechanism of saponins regulating fatty alcohol oxidase (FAO) to reduce myocardial remodeling and control heart failure. 30 Sprague-Dawley (SD) rats were randomly and equally assigned into control group, model group, and saponin group, followed by analysis of myocardial tissue pathology, cyclic guanosine monophosphate (cGMP), Recombinant Human Protein (PKG), Peroxisome proliferator-activated receptors (PPAR-α) expression levels, and cell apoptosis. Compared to control group, cGMP, PKG, PPAR-α, uncoupling protein 3 (UCP3), and cluster of differentiation 36 (CD36) mRNA levels in the model group were significantly decreased (P <0.001) and elevated in the saponin group (P <0.05). Oxidation rates of adenosine triphosphate (ATP), phosphocreatine/ATP, and palmitic acid in the model group were significantly decreased (P <0.001) and elevated in the saponin group (P <0.05). Apoptosis and level of Cleaved caspase-3 were significantly reduced in the model group (P <0.001) and increased in the saponin group (P <0.05). Levels of cGMP, PKG, PPAR-α, UCP3 and CD36 in the model group decreased (P <0.001) and increased in the saponin group (P <0.001), but lower than in the control group. Relative to the model group, Brain Natriuretic Peptide (BNP) level was significantly increased in the inhibitor group and decreased in the agonist group (P <0.001). Saponins activate cGMP-PKG signaling pathway, up-regulating cGMP and PKG, promoting PPAR-α expression, inhibiting myocardial cell necroptosis, thereby reducing inflammatory infiltration of myocardial cells, improving connective tissue hyperplasia, and reducing myocardial injury and myocardial remodeling, thus play an anti-heart failure role.

Improving Methanol Utilization by Reducing Alcohol Oxidase Activity and Adding Co-Substrate of Sodium Citrate in Pichia pastoris.

Methanol, which produced in large quantities from low-quality coal and the hydrogenation of CO2, is a potentially renewable one-carbon (C1) feedstock for biomanufacturing. The methylotrophic yeast Pichia pastoris is an ideal host for methanol biotransformation given its natural capacity as a methanol assimilation system. However, the utilization efficiency of methanol for biochemical production is limited by the toxicity of formaldehyde. Therefore, reducing the toxicity of formaldehyde to cells remains a challenge to the engineering design of a methanol metabolism. Based on genome-scale metabolic models (GSMM) calculations, we speculated that reducing alcohol oxidase (AOX) activity would re-construct the carbon metabolic flow and promote balance between the assimilation and dissimilation of formaldehyde metabolism processes, thereby increasing the biomass formation of P. pastoris. According to experimental verification, we proved that the accumulation of intracellular formaldehyde can be decreased by reducing AOX activity. The reduced formaldehyde formation upregulated methanol dissimilation and assimilation and the central carbon metabolism, which provided more energy for the cells to grow, ultimately leading to an increased conversion of methanol to biomass, as evidenced by phenotypic and transcriptome analysis. Significantly, the methanol conversion rate of AOX-attenuated strain PC110-AOX1-464 reached 0.364 g DCW/g, representing a 14% increase compared to the control strain PC110. In addition, we also proved that adding a co-substrate of sodium citrate could further improve the conversion of methanol to biomass in the AOX-attenuated strain. It was found that the methanol conversion rate of the PC110-AOX1-464 strain with the addition of 6 g/L sodium citrate reached 0.442 g DCW/g, representing 20% and 39% increases compared to AOX-attenuated strain PC110-AOX1-464 and control strain PC110 without sodium citrate addition, respectively. The study described here provides insight into the molecular mechanism of efficient methanol utilization by regulating AOX. Reducing AOX activity and adding sodium citrate as a co-substrate are potential engineering strategies to regulate the production of chemicals from methanol in P. pastoris.

Bacterial enzymes for azo dye degradation: an insight

Azo dye has a massive marketplace in various industries, biological laboratories and clinics. A significant drawback is the excessive flush-off of azo dyes and their intermediate products are that they pollute the environment. Several environmental bacteria degrade azo dye under certain specific ecological conditions. Decolourisation by different bacteria occurs under aerobic, anoxic and anaerobic conditions. Bacterial genera such as Pseudomonas, Bacillus and Rhodococcus are used for the rapid decolourisation of azo dyes. Bacterial enzymes play an essential role in the degradation of azo dyes by cleavage of azo bonds (-N=N-). It is degraded by both oxidative and reductive enzymes including azo reductase, laccase, lignin peroxidase, NADH-DCIP reductase, malachite green reductase, polyphenol oxidase (tyrosinase), veratrol alcohol oxidase. This review describes the role and mechanism of these enzymes in the degradation of azo dyes.

The Komagatella phaffii ACG1 gene, encoding β-1,6-N-acetylglucosaminyltransferase, is involved in the autophagy of cytosolic and peroxisomal proteins.

The methylotrophic yeast Komagataella phaffii is considered one of the most effective producers of recombinant proteins of industrial importance. Effective producers should be characterized by the maximal reduction of degradation of the cytosolic recombinant proteins. The mechanisms of degradation of cytosolic proteins in K. phaffii have not been elucidated; however, data suggest that they are partially degraded in the autophagic pathway. To identify factors that influence this process, a developed system for the selection of recombinant strains of K. phaffii with impaired autophagic degradation of the heterologous model cytosolic protein (yeast β-galactosidase) was used for insertional tagging of the genes involved in cytosolic proteins degradation. In one of the obtained strains, the insertion cassette disrupted the open reading frame of the gene encoding β-1,6-N-acetylglucosaminyltransferase. A recombinant strain with deletion of this gene was also obtained. The rate of degradation of the β-galactosidase enzyme was two times slower in the insertion mutant and 1.5 times slower in the deletion strain as compared to the parental strain with native β-1,6-N-acetylglucosaminyltransferase. The rate of degradation of native K. phaffii cytosolic and peroxisomal enzymes, formaldehyde dehydrogenase, formate dehydrogenase, and alcohol oxidase, respectively, showed similar trends to that of β-galactosidase-slower degradation in the deletion and insertional mutants as compared to the wild-type strain, but faster protein degradation relative to the strain completely defective in autophagy. We conclude that K. phaffii gene designated ACG1, encoding β-1,6-N-acetylglucosaminyltransferase, is involved in autophagy of the cytosolic and peroxisomal proteins.

Is heterogeneity in large-scale bioreactors a real problem in recombinant protein synthesis by Pichia pastoris?

Culture medium heterogeneity is inherent in industrial bioreactors. The loss of mixing efficiency in a large-scale bioreactor yields to the formation of concentration gradients. Consequently, cells face oscillatory culture conditions that may deeply affect their metabolism. Herein, cell response to transient perturbations, namely high methanol concentration combined with hypoxia, has been investigated usinga two stirred-tank reactor compartiments(STR-STR) scale-down system and a Pichia pastoris strain expressing the gene encoding enhanced green fluorescent protein (eGFP) under the control of the alcohol oxidase 1 (AOX1) promoter. Cell residence times under transient stressing conditions were calculated based on the typical hydraulic circulation times of bioreactors of tens and hundreds cubic metres. A significant increase in methanol and oxygen uptake rates was observed as the cell residence time was increased. Stressful culture conditions impaired biomass formation and triggered cell flocculation. More importantly, both expression levels of genes under the control of pAOX1 promoter and eGFP specific fluorescence were higher in those oscillatory culture conditions, suggesting that those a priori unfavourable culture conditions in fact benefit to recombinant protein productivity. Flocculent cells were also identified as the most productive as compared to ovoid cells. KEY POINTS: • Transient hypoxia and high methanol trigger high level of recombinant protein synthesis • In Pichia pastoris, pAOX1 induction is higher in flocculent cells • Medium heterogeneity leads to morphological diversification.

A Biological OR(XNOR) Logic Gate Couples Carbon Source and Transgene Expression Switching in a Komagataella phaffii (Pichia pastoris) Strain Co-producing Process-Enhancing Lipase and a Virus-like Particle (VLP) Vaccine.

We constructed a three-input biological logic gate: S OR (G XNOR M), where S is sorbitol, G is glycerol, and M is methanol, to optimize co-expression of two transgenes in Komagataella phaffii using batch-mode carbon source switching (CSS). K. phaffii was engineered to harbor transgenes encoding a Candida rugosa triacylglycerol lipase, which can enhance downstream processing by removing host cell lipids from homogenates, and the hepatitis B virus surface antigen (HBsAg), a protein that self-assembles into a virus-like particle (VLP) vaccine. Using the native alcohol oxidase 1 (PAOX1) and enolase 1 (PENO1) promoters to direct VLP vaccine and lipase expression, respectively, successfully provided an OR(XNOR) gate function with double-repression as the output. This logic gate functionality enabled use of CSS to ensure that approximately 80% of total VLP yield was accumulated before cells were burdened with lipase expression in 250 mL DasGip bioreactor cultivation.

Metasecretome and biochemical analysis of consortium PM-06 during the degradation of nixtamalized maize pericarp

Nixtamalized maize pericarp is an abundant by-product of the industrial processing of maize for tortilla production, and a potential source of prebiotics and biofuels. Consortium PM-06 is a bacterial community with the capacity to efficiently saccharify the nixtamalized pericarp; however, there is scarce information about the enzymatic mechanism involved. In this study, the soluble enzymes secreted by PM-06 in different stages of the degradation process were analyzed using biochemical approaches and label-free quantitative proteomics. Metasecretomic analysis revealed a marked abundance of glycosyl hydrolases in the initial stages of the process, mainly secreted by Bacillus and Paenibacillus. The main function of glycosyl hydrolases was the metabolism of soluble sugars released from maize pericarp. As degradation proceeded, substrate recalcitrance and oxidoreductase mass abundance increased. Actinobacteria, predominantly Microbacterium, secreted a great diversity of oxidoreductases with vanillyl alcohol oxidase as the most abundant enzyme. Lignin consumption in the last 48 h of culture and the lack of laccase and peroxidase sequences, suggest a role for Fenton chemistry. Different oxidoreductases secreted by PM-06 produce hydrogen peroxide, a co-substrate for various enzymes and a Fenton reactant. A strategy used by PM-06 to cope with substrate recalcitrance could be the use of hydroxyl radicals produced from H2O2 to attack polysaccharides and lignin. This study is the first report about a consortium secreting a great diversity of soluble oxidoreductases and provides information about possible synergistic interactions and functions of bacterial enzymes involved in lignocellulose degradation.

Rational engineering of AA5_2 copper radical oxidases to probe the molecular determinants governing their substrate selectivity.

Fungal copper radical oxidases (CROs) from the Auxiliary Activity family 5 (AA5) constitute a group of metalloenzymes that oxidize a wide panel of natural compounds, such as galactose-containing saccharides or primary alcohols, into product derivatives exhibiting promising biotechnological interests. Despite a well-conserved first copper-coordination sphere and overall fold, some members of the AA5_2 subfamily are incapable of oxidizing galactose and galactosides but conversely efficiently catalyse the oxidation of diverse aliphatic alcohols. The objective of this study was to understand which residues dictate the substrate preferences between alcohol oxidases and galactose oxidases within the AA5_2 subfamily. Based on structural differences and molecular modelling predictions between the alcohol oxidase from Colletotrichum graminicola (CgrAlcOx) and the archetypal galactose oxidase from Fusarium graminearum (FgrGalOx), a rational mutagenesis approach was developed to target regions or residues potentially driving the substrate specificity of these enzymes. A set of 21 single and multiple CgrAlcOx variants was produced and characterized leading to the identification of six residues (W39, F138, M173, F174, T246, L302), in the vicinity of the active site, crucial for substrate recognition. Two multiple CgrAlcOx variants, i.e. M4F (W39F, F138W, M173R and T246Q) and M6 (W39F, F138W, M173R, F174Y, T246Q and L302P), exhibited a similar affinity for carbohydrate substrates when compared to FgrGalOx. In conclusion, using a rational site-directed mutagenesis approach, we identified key residues involved in the substrate selectivity of AA5_2 enzymes towards galactose-containing saccharides.

Engineering of Hydroxymandelate Oxidase and Cascade Reactions for High-Yielding Conversion of Racemic Mandelic Acids to Phenylglyoxylic Acids and (R)- and (S)-Phenylglycines

An alcohol oxidase (AOx) for the (S)-enantioselective oxidation of 4-hydroxymandelic acid (4-HMA) to 4-hydroxyphenylglyoxylic acid (4-HPGA) with enhanced activity was developed by directed evolution of hydroxymandelate oxidase (HMO) through three rounds of iterative saturation mutagenesis. The engineered HMO mutant A80G-T159S-T162Q (HMOTM) catalyzed the oxidation of (S)-4-HMA to 4-HPGA with a 23-fold enhancement in catalytic efficiency (kcat/KM). Substrate docking simulation on HMOTM suggested that A80G reoriented FMN, while T159S and T162Q formed hydrogen bonds with the carboxylic group of the substrate, thus facilitating substrate binding and catalysis. (S)-Enantioselective HMOTM was used together with mandelic acid racemase (MR) and catalase (KatE) to achieve high-yielding oxidation of rac-4-HMA to 4-HPGA with either purified enzymes (up to 93% yield and 426 mM) or Escherichia coli (HMC) cells expressing the three enzymes (up to 93% yield and 140 mM). The HMOTM-MR-KatE cascades were applied for the oxidation of seven other substituted MAs, producing the corresponding phenylglyoxylic acids with 90–99% conversions using purified enzymes or whole cells. Efficient conversion of racemic α-hydroxy acids to (S)- or (R)-α-amino acids was achieved by combining HMOTM-MR-KatE with (S)-enantioselective transaminase (EcαTA) or (R)-enantioselective transaminase (DpgAT), together with glutamate dehydrogenase (GluDH). Coupling of E. coli (HMC) with E. coli (E-G) expressing EcαTA and GluDH for one-pot biotransformation of five rac-MAs produced the corresponding (S)-phenylglycines (PGs) in 91–99% ee with 73–98% conversions. Using E. coli (HMC) and E. coli (D-G) expressing DpgAT and GluDH enabled the production of five (R)-PGs with 93–99% ee and 73–98% conversions from the corresponding rac-MAs. The engineered AOx, AOx-MR-KatE cascades, and AOx-MR-Kat-GluDH-(S)- or (R)-TA cascades provide useful tools for highly active and enantioselective oxidation of racemic hydroxyacids and high-yielding conversion of racemic hydroxyacids to ketoacids and enantiopure (S)- or (R)-aminoacids, respectively, which are challenging and useful chemical reactions.

Bienzymatic Cascade Combining a Peroxygenase with an Oxidase for the Synthesis of Aromatic Aldehydes from Benzyl Alcohols

Aromatic aldehydes are important aromatic compounds for the flavour and fragrance industry. In this study, a parallel cascade combining aryl alcohol oxidase from Pleurotus eryngii (PeAAOx) and unspecific peroxygenase from the basidiomycete Agrocybe aegerita (AaeUPO) to convert aromatic primary alcohols into high-value aromatic aldehydes is proposed. Key influencing factors in the process of enzyme cascade catalysis, such as enzyme dosage, pH and temperature, were investigated. The universality of PeAAOx coupled with AaeUPO cascade catalysis for the synthesis of aromatic aldehyde flavour compounds from aromatic primary alcohols was evaluated. In a partially optimised system (comprising 30 μM PeAAOx, 2 μM AaeUPO at pH 7 and 40 °C) up to 84% conversion of 50 mM veratryl alcohol into veratryl aldehyde was achieved in a self-sufficient aerobic reaction. Promising turnover numbers of 2800 and 21,000 for PeAAOx and AaeUPO, respectively, point towards practical applicability.

A novel wearable biosensor for real-time alcohol detection

Alcohol misuse has become a major public health concern worldwide. The development of an accurate, non-invasive, and cost-effective method for real-time alcohol detection would have important applications for monitoring and preventing alcohol misuse. This paper proposes a novel wearable biosensor for real-time alcohol detection in sweat. The biosensor uses alcohol oxidase immobilized on a screen-printed electrode to amperometrically detect ethanol in sweat samples. An artificial sweat solution was used to optimize the sensor design and evaluate its analytical performance. The optimized biosensor demonstrated a linear response to ethanol in the concentration range of 0.1–1.0 g/dL, with a limit of detection of 0.02 g/dL. On-body trials of the wearable biosensor provided rapid measurements that strongly correlated with blood alcohol content. This approach could offer a convenient solution for continuous, non-invasive alcohol monitoring to help reduce alcohol misuse and promote healthy behaviors. Further research on long-term use, alternate electrode geometries, and fully integrated wearable designs is warranted.

Tandem metalloenzymes gate plant cell entry by pathogenic fungi

Global food security is endangered by fungal phytopathogens causing devastating crop production losses. Many of these pathogens use specialized appressoria cells to puncture plant cuticles. Here, we unveil a pair of alcohol oxidase-peroxidase enzymes to be essential for pathogenicity. Using Colletotrichum orbiculare, we show that the enzyme pair is cosecreted by the fungus early during plant penetration and that single and double mutants have impaired penetration ability. Molecular modeling, biochemical, and biophysical approaches revealed a fine-tuned interplay between these metalloenzymes, which oxidize plant cuticular long-chain alcohols into aldehydes. We show that the enzyme pair is involved in transcriptional regulation of genes necessary for host penetration. The identification of these infection-specific metalloenzymes opens new avenues on the role of wax-derived compounds and the design of oxidase-specific inhibitors for crop protection.