Search

Intracellular alcohol oxidase (AOX) was isolated from the basidiomycetous white rot fungus Cerrena unicolor FCL139. The enzyme was semi-purified (13-fold) using two-step chromatography with 30% activity recovery. The identity of the protein was confirmed by LC-MS/MS analysis, and its MW (72 kDa) and pI (6.18) were also determined. The kinetics parameters of the AOX reaction towards various substrates were analysed, which proved that, in addition to methanol (4.36 ± 0.27% of the oxidised substrate), AOX most potently oxidises aromatic alcohols, such as 4-hydroxybenzyl alcohol (14.0 ± 0.8%), benzyl alcohol (4.2 ± 0.3%), anisyl alcohol (7.6 ± 0.4%), and veratryl alcohol (5.0 ± 0.3%). Moreover, the influence of selected commercially available proteases on the biocatalytic properties of AOX from C. unicolor was studied. It was proved that the digested enzyme lost its catalytic potential properties except when incubated with pepsin, which significantly boosted its activity up to 123%. [ABSTRACT FROM AUTHOR]

Unspecific Peroxygenases (UPOs) have emerged as robust biocatalysts for selective oxygenation reactions, as they are easily produced at scale and require only hydrogen peroxide as the external oxidant. UPOs can catalyze the oxygenation of the primary benzylic carbons of toluenes to give alcohol, aldehyde and carboxylic acid products. They can also catalyze hydroxylation at the benzylic position of ethylbenzenes, and the subsequent oxidation of the secondary alcohols to ketones. In this study, we have investigated factors that affect the balance of products in UPO‐catalyzed benzylic oxygenations using a range of functionalised toluenes and ethyl benzenes, and a UPO from Agrocybe aegerita (rAaeUPO‐PaDa‐I‐H variant). The product distribution is dependent upon a mixture of steric and electronic effects and, in selected cases, controlling the reaction conditions permits products from each product series to be generated chemoselectively. In this way, electron poor toluenes were converted directly into carboxylic acids in isolated yields of 36–99 % on preparative scale. [ABSTRACT FROM AUTHOR]

Alcohol oxidase (AOX) oxidizes alcohols to produce carbonyl compounds and peroxides. Its promoter (PAOX1) is widely used in methylotrophic yeasts. A promising yeast expression system (Pichia sp. strain SO) was developed for bacterial lipase expression regulated by PAOX1 of Komagataella phaffii (previously known as Pichia pastoris). However, its unidentified AOX gene and the protein structure have deterred the search for the best inducer. This study was aimed to identify the yeast species and determine the best inducer for PAOX1 upregulation using in silico AOX protein analysis. Morphological (scanning and transmission electron microscopies) and carbon assimilation analyses confirmed isolate SO as Meyerozyma guilliermondii (previously known as Pichia guilliiermondii). Using Hidden-Markov model and degenerate PCR, the LCAO gene (2091 bp) was discovered in M. guilliermondii strain SO. The enzyme, MgFAO1 shared 14% similarity to K. phaffii AOX1 protein (KpAOX1). Molecular docking of MgFAO1 three-dimensional structure predicted using AlphaFold2 showed its preference toward long-chain 1-dodecanol as the substrate unlike KpAOX1 (short-chain methanol). While the alcohol-binding pocket in MgFAO1 was more hydrophobic compared to KpAOX1, 1-dodecanol could be a better inducer for protein expression in M. guilliermondii strain SO. Thus, in silico pipeline employed in this study can help identify homologous proteins in other expression hosts and their preferred substrates for promoter upregulation. However, the computational analyses were merely predictions and further wet-lab validation is required. Yet, this strategy allows cost-efficient screening of potential inducers for microbe-based protein production in the industries, reducing the production cost and offering cheaper options for consumers. [ABSTRACT FROM AUTHOR]

Bacterial and fungal copper radical oxidases (CROs) from Auxiliary Activity Family 5 (AA5) are implicated in morphogenesis and pathogenesis. The unique catalytic properties of CROs also make these enzymes attractive biocatalysts for the transformation of small molecules and biopolymers. Despite a recent increase in the number of characterized AA5 members, especially from subfamily 2 (AA5_2), the catalytic diversity of the family as a whole remains underexplored. In the present study, phylogenetic analysis guided the selection of six AA5_2 members from diverse fungi for recombinant expression in Komagataella pfaffii (syn. Pichia pastoris) and biochemical characterization in vitro. Five of the targets displayed predominant galactose 6-oxidase activity (EC 1.1.3.9), and one was a broad-specificity aryl alcohol oxidase (EC 1.1.3.7) with maximum activity on the platform chemical 5-hydroxymethyl furfural (EC 1.1.3.47). Sequence alignment comparing previously characterized AA5_2 members to those from this study indicated various amino acid substitutions at active site positions implicated in the modulation of specificity. [ABSTRACT FROM AUTHOR]

A high ethanol usage of alcohol oxidase (AOX) was required in industry. In this study, a "expand substrate pocket" strategy achieved a high activity AOX from Hansenula polymorpha (H. polymorpha) by Phe to Val residue (F/V) site-directed mutation to enlarge ethanol channel. Although H. Polymorpha AOX (HpAOX) possessed respectively 71.3% and 76.1% similarity with AOX (PpAOX) from Pichia pastoris (P. pastoris) in DNA and protein sequences, their active site structures including catalytic site and substrate channel were similar according to computer-aided analysis. After 3D structure analysis, Phe99 residue of their substrate channels was the most important residue to impact enzyme activity because of its large aromatic side chains. F99V mutation of HpAOX (HpAOXF99V) was designed and executed based on the enzyme catalytic mechanism and molecular computation in order to allow more larger size ethanol into active site. The highest enzyme activity of the fourth strains of HpAOXF99V mutant strain exhibited 12.06-folds increase than that of the host GS115 strain. Furthermore, kinetic studies indicated that the HpAOXF99V significantly promoted catalytic efficiency of ethanol than HpAOX, including Km, Vmax, kcat and kcat/Km. We also provided a new insight that the cofactor FAD irritated both active AOX octamer biosynthesis production and enzyme-catalysed ability due to help enzyme assembly and redox potential. [ABSTRACT FROM AUTHOR]

An amperometric bienzymatic biosensor was developed for the determination of aspartame in a flow injection analysis (FIA) system, consisting of two enzyme reactor columns packed with immobilized α-chymotrypsin (CHY) and alcohol oxidase (AOX) beads and a hydrogen peroxide electrode, connected in series. The CHY and AOX were separately immobilized on glutaraldehyde (GA)-activated beads through covalent bonding. The biosensor fabrication and operational conditions were optimized. The optimal fabrication conditions were: 2% GA with 120 min activation time; and 250 U/mL CHY and 100 U/mL AOX, with 180 min enzyme immobilization time. The optimal operational conditions were a flow rate of 0.5 mL/min and pH 8.0 at room temperature. The developed biosensor showed linearity over the aspartame concentration range 0.01–1.2 mM, with a detection limit of 0.005 mM. The developed biosensor was satisfactorily applied for detecting aspartame in beverage samples without any excessive pretreatments. [ABSTRACT FROM AUTHOR]

In this study, FeTOMPP (Tetrakis(4-methoxyphenyl) phenylporphyrinatoiron(III)) was synthesized and investigated for its peroxidase-like activity. The catalytic performance of FeTOMPP as a peroxidase mimic was explored by utilizing the colorless peroxidase substrate, 3,3',5,5'-tetramethylbenzidine (TMB), in the presence of hydrogen peroxide (H2O2). The oxidation of TMB by FeTOMPP resulted in the formation of its blue oxidized state. This enzymatic reaction was employed for the quantitative detection of H2O2, with a linear detection range spanning from 1.0 to 100 µM. The detection limit achieved using FeTOMPP as the catalyst was determined to be 2.4 µM. Furthermore, kinetic studies were conducted to investigate the interaction between FeTOMPP and H2O2. The results indicated that FeTOMPP exhibited favorable affinity towards H2O2, further highlighting its potential as a peroxidase mimic. In addition to its peroxidase-like activity, a simple and sensitive visual and colorimetric method was developed for the detection of ethanol by combining FeTOMPP with alcohol oxidase. The method relied on the use of TMB as the substrate. Ethanol-containing samples were subjected to the enzymatic reaction, resulting in the generation of a color change. The detection of ethanol was achieved with a minimum detection limit of 0.95 % v/v, and the linear range for quantification extended from 0 to 15 % v/v. This study demonstrates the potential application of FeTOMPP as a peroxidase mimic for the sensitive detection of H2O2 and the development of a colorimetric method for ethanol detection. The findings highlight the versatility and promising capabilities of FeTOMPP in various analytical and diagnostic applications. [ABSTRACT FROM AUTHOR]

Abstract Background As a cost-effective and eco-friendly approach, biocatalysis has great potential for the transformation of 5-hydroxymethylfurfural (HMF) into 2,5-furandicarboxylic acid (FDCA). However, the compatibility of each enzyme in the cascade reaction limits the transformation efficiency of HMF to FDCA. Results Coupled with an alcohol oxidase from Colletotrichum gloeosporioides (CglAlcOx), this study aims to study the potential of bacterial laccase from Bacillus pumilus (BpLac) in an enzymatic cascade for 2,5-furandicarboxylic acid (FDCA) biosynthesis from 5-hydroxymethylfurfural (HMF). BpLac showed 100% selectivity for HMF oxidation and generated 5-hydroxymethyl-2-furancarboxylic acid (HMFCA). CglAlcOx was capable of oxidizing HMFCA to 2-formyl-5-furancarboxylic acid (FFCA). Both BpLac and CglAlcOx could oxidize FFCA to FDCA. At the 5 mM scale, a complete transformation of HMF with a 97.5% yield of FDCA was achieved by coupling BpLac with CglAlcOx in the cascade reaction. The FDCA productivity in the reaction was 5.3 mg/L/h. Notably, BpLac could alleviate the inhibitory effect of FFCA on CglAlcOx activity and boost the transformation efficiency of HMF to FDCA. Moreover, the reaction was scaled up to 40 times the volume, and FDCA titer reached 2.6 mM with a yield of 58.77% at 168 h. Conclusions This work provides a candidate and novel insight for better design of an enzymatic cascade in FDCA production.

Background: As a cost-effective and eco-friendly approach, biocatalysis has great potential for the transformation of 5-hydroxymethylfurfural (HMF) into 2,5-furandicarboxylic acid (FDCA). However, the compatibility of each enzyme in the cascade reaction limits the transformation efficiency of HMF to FDCA. Results: Coupled with an alcohol oxidase from Colletotrichum gloeosporioides (CglAlcOx), this study aims to study the potential of bacterial laccase from Bacillus pumilus (BpLac) in an enzymatic cascade for 2,5-furandicarboxylic acid (FDCA) biosynthesis from 5-hydroxymethylfurfural (HMF). BpLac showed 100% selectivity for HMF oxidation and generated 5-hydroxymethyl-2-furancarboxylic acid (HMFCA). CglAlcOx was capable of oxidizing HMFCA to 2-formyl-5-furancarboxylic acid (FFCA). Both BpLac and CglAlcOx could oxidize FFCA to FDCA. At the 5 mM scale, a complete transformation of HMF with a 97.5% yield of FDCA was achieved by coupling BpLac with CglAlcOx in the cascade reaction. The FDCA productivity in the reaction was 5.3 mg/L/h. Notably, BpLac could alleviate the inhibitory effect of FFCA on CglAlcOx activity and boost the transformation efficiency of HMF to FDCA. Moreover, the reaction was scaled up to 40 times the volume, and FDCA titer reached 2.6 mM with a yield of 58.77% at 168 h. Conclusions: This work provides a candidate and novel insight for better design of an enzymatic cascade in FDCA production. [ABSTRACT FROM AUTHOR]

The analysis of alcohol represents an important parameter in food fermentation industries. The use of a biosensor would be an alternative to existing methods. An enzymatic sensor using alcohol oxidase (AOX) was used as the biorecognition element with an immobilized enzyme sensor in combination with an oxygen electrode, optimized in this study to determine the methanol content in cane brandy. The potentiometric signal obtained by oxygen depletion (oxygen consumed) during alcohol oxidation was carried out very quickly in 4.5 s using cassava biopolymer. AOX catalyzes the oxidation of alcohols using molecular oxygen as the electron acceptor, producing corresponding carbonyl compounds and hydrogen peroxide in the reaction medium. An optimal time sensor response to quantify the methanol content of 1.2 s was calculated from the slope of reaction rates.The analysis of methanol with the immobilized enzyme in cane brandy samples revealed very good agreement with the determination performed through standard HPLC methodology. The activity of alcohol oxidase used as a biorecognition element could be wholly retained in a non-dissolvable film (pore size

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Nanomaterials possessing artificial, enzyme-like catalytic activity (nanozymes, NZs) have a great potential for application in research, immunological assays, biosensors, in vivo imaging, and as therapeutic agents. Despite the obvious advances in construction and understanding of functional properties of NZs, there is still no clear evidence of whether they can complement the loss of corresponding enzymatic activity in vivo. Herein, we report the first, to the best to our knowledge, example of successful substitution of natural enzyme activity by catalase-like platinum (nPt) and platinum-gold (nPtAu) nanoparticles transferred to the cells of methylotrophic yeast Ogataea polymorpha. The nPt NZs were synthesized by the chemical reduction method and used as a seed to produce the nPt(core)Au(shell) particles. The produced nPt NZs were 68.1 and 91.3 nm in size, while the hydrids were of 531.2 and 615.1 nm. Both nPt and nPtAu demonstrated catalase activity in vitro. The catalase-deficient strain Ogataea polymorpha C-105 was shown to be able to grow on methanol and a mixture of glucose and methanol in the presence although not in the absence of NZs, this correlating with the decrease in intracellular hydrogen peroxide production. The results provide the first example of complementation of the natural enzyme function by synthetic NZs, the phenomenon which can further be used in a screening for new catalase-like nanozymes and as a fruitful tool to modify living cells by nanoparticles possessing catalytic activity and to use such modified cells as sensitive elements in cell-based biosensors. [ABSTRACT FROM AUTHOR]

Abstract In this study, we selected and engineered a flavin adenine dinucleotide (FAD)-dependent alcohol oxidase (AOX) to produce 1,4-cyclohexanedicarboxaldehyde (CHDA), an initial raw material for spiral compounds, from 1,4-cyclohexanedimethanol (CHDM). First, the structure of alcohol oxidase from Arthrobacter cholorphenolicus (AcCO) was analyzed, and the mechanism of AcCO-catalyzed primary alcohol oxidation was elucidated, demonstrating that the energy barrier of the hydride (H−) transfer (13.4 kcal·mol−1 and 20.4 kcal·mol−1) decreases the catalytic efficiency of the primary alcohol oxidation reaction. Therefore, we designed a protein engineering strategy to adjust the catalytically active conformation to shorten the distance of hydride (H−) transfer and further decreased the core energy barrier. Following this strategy, variant W4 (S101A/H351V/N378S/Q329N) was obtained with 112.5-fold increased catalytic efficiency to produce CHDA compared to that of the wild-type strain. The 3 L scale preparation of CHDA reached a titer up to 29.6 g·L−1 with a 42.2% yield by an Escherichia coli whole-cell catalyst, which demonstrates the potential of this system for industrial application. Graphical Abstract

Alcohol oxidases (AOXs) are ecologically important enzymes that facilitate a number of plant–fungal interactions. Within Ascomycota they are primarily associated with methylotrophy, as a peroxisomal AOX catalysing the conversion of methanol to formaldehyde in methylotrophic yeast. In this study we demonstrate that AOX orthologues are phylogenetically conserved proteins that are common in the genomes of nonmethylotrophic, plant‐associating fungi. Additionally, AOX orthologues are highly expressed during infection in a range of diverse pathosystems. To study the role of AOX in plant colonization, AOX knockout mutants were generated in the broad host range pathogen Sclerotinia sclerotiorum. Disease assays in soybean showed that these mutants had a significant virulence defect as evidenced by markedly reduced stem lesions and mortality rates. Chemical genomics suggested that SsAOX may function as an aromatic AOX, and growth assays demonstrated that ΔSsAOX is incapable of properly utilizing plant extract as a nutrient source. Profiling of known aromatic alcohols pointed towards the monolignol coniferyl alcohol (CA) as a possible substrate for SsAOX. As CA and other monolignols are ubiquitous among land plants, the presence of highly conserved AOX orthologues throughout Ascomycota implies that this is a broadly conserved protein used by ascomycete fungi during plant colonization. [ABSTRACT FROM AUTHOR]

The aim of the current research is to design alcohol oxidase-based amperometric biosensors (ABSs) using hybrid metallic nanoparticles as artificial peroxidases (PO) or PO-like nanozymes (NZs). A lot of metallic PO-like NZs were synthesized and tested with respect to their ability to substitute natural PO in solution and on amperometric electrode. The most effective PO mimetics were coupled with alcohol oxidase (AOX) on graphite electrodes (GEs) and characterized. Two types of modified GEs, namely, the AOX/nAuCePt/GE and the AOX/nFePtAu/GE show the highest sensitivities to ethanol (2600 A⋅M−1⋅m−2 and 1250 A⋅M−1⋅m−2, respectively), low limits of detection (1.5 µM and 2.2 µM), broad linear ranges (5 – 100 µM and 12 – 120 µM), as well as satisfactory storage stabilities. The most sensitive bioelectrode AOX/nAuCePt/GE was used as ABS for ethanol determination in real samples. The practical feasibility of the constructed ABS was demonstrated by determination of ethanol in beverages, human blood and saliva. [ABSTRACT FROM AUTHOR]

Extensive research efforts have been devoted to describing yeast alcohol oxidase (AO) and its promoter region, which is vastly applied in studies of heterologous gene expression. However, little is known about basidiomycetous AO and its physiological role in wood degradation. This review describes several alcohol oxidases from both white and brown rot fungi, highlighting their physicochemical and kinetic properties. Moreover, the review presents a detailed analysis of available AO-encoding gene promoter regions in basidiomycetous fungi with a discussion of the manipulations of culture conditions in relation to the modification of alcohol oxidase gene expression and changes in enzyme production. The analysis of reactions catalyzed by lignin-modifying enzymes (LME) and certain lignin auxiliary enzymes (LDA) elucidated the possible involvement of alcohol oxidase in the degradation of derivatives of this polymer. Combined data on lignin degradation pathways suggest that basidiomycetous AO is important in secondary reactions during lignin decomposition by wood degrading fungi. With numerous alcoholic substrates, the enzyme is probably engaged in a variety of catalytic reactions leading to the detoxification of compounds produced in lignin degradation processes and their utilization as a carbon source by fungal mycelium. [ABSTRACT FROM AUTHOR]

In this study, we selected and engineered a flavin adenine dinucleotide (FAD)-dependent alcohol oxidase (AOX) to produce 1,4-cyclohexanedicarboxaldehyde (CHDA), an initial raw material for spiral compounds, from 1,4-cyclohexanedimethanol (CHDM). First, the structure of alcohol oxidase from Arthrobacter cholorphenolicus (AcCO) was analyzed, and the mechanism of AcCO-catalyzed primary alcohol oxidation was elucidated, demonstrating that the energy barrier of the hydride (H−) transfer (13.4 kcal·mol−1 and 20.4 kcal·mol−1) decreases the catalytic efficiency of the primary alcohol oxidation reaction. Therefore, we designed a protein engineering strategy to adjust the catalytically active conformation to shorten the distance of hydride (H−) transfer and further decreased the core energy barrier. Following this strategy, variant W4 (S101A/H351V/N378S/Q329N) was obtained with 112.5-fold increased catalytic efficiency to produce CHDA compared to that of the wild-type strain. The 3 L scale preparation of CHDA reached a titer up to 29.6 g·L−1 with a 42.2% yield by an Escherichia coli whole-cell catalyst, which demonstrates the potential of this system for industrial application. [ABSTRACT FROM AUTHOR]

The commercial success of paper-based microfluidic biosensor devices with enzyme as recognition elements may be achieved greatly by improving the enzyme stability, averting the coffee-ring effect on the detection zone, and making it a stand-alone system. We have addressed these issues here by introducing silk-fibroin (SF) film for immobilizing enzymes and chromogenic reagent (ABTS), and wave-design microfluidic channels in the chromatographic paper for developing a hybrid microfluidic paper-based analytical device (μPAD), for methanol detection through peroxidase reaction. The activity of alcohol oxidase (AOx), used as a biorecognition element, could be wholly retained in a non-dissolvable SF film (pore size

There is significant contemporary interest in the application of enzymes to replace or augment chemical reagents toward the development of more environmentally sound and sustainable processes. In particular, copper radical oxidases (CRO) from Auxiliary Activity Family 5 Subfamily 2 (AA5_2) are attractive, organic cofactor-free catalysts for the chemoselective oxidation of alcohols to the corresponding aldehydes. These enzymes were first defined by the archetypal galactose-6-oxidase (GalOx, EC 1.1.3.13) from the fungus Fusarium graminearum. The recent discovery of specific alcohol oxidases (EC 1.1.3.7) and aryl alcohol oxidases (EC 1.1.3.47) within AA5_2 has indicated a potentially broad substrate scope among fungal CROs. However, only relatively few AA5_2 members have been characterized to date. Guided by sequence similarity network and phylogenetic analysis, twelve AA5_2 homologs have been recombinantly produced and biochemically characterized in the present study. As defined by their predominant activities, these comprise four galactose 6-oxidases, two raffinose oxidases, four broad-specificity primary alcohol oxidases, and two non-carbohydrate alcohol oxidases. Of particular relevance to applications in biomass valorization, detailed product analysis revealed that two CROs produce the bioplastics monomer furan-2,5-dicarboxylic acid (FDCA) directly from 5-hydroxymethylfurfural (HMF). Furthermore, several CROs could desymmetrize glycerol (a by-product of the biodiesel industry) to d- or l-glyceraldehyde. This study furthers our understanding of CROs by doubling the number of characterized AA5_2 members, which may find future applications as biocatalysts in diverse processes. [ABSTRACT FROM AUTHOR]

Copper radical alcohol oxidases (CRO-AlcOx), which have been recently discovered among fungal phytopathogens, are attractive for the production of fragrant fatty aldehydes. With the initial objective to investigate the secretion of CRO-AlcOx by natural fungal strains, we undertook time course analyses of the secretomes of three Colletotrichum species (C. graminicola, C. tabacum, and C. destructivum) using proteomics. The addition of a copper-manganese-ethanol mixture in the absence of any plantbiomass mimicking compounds to Colletotrichum cultures unexpectedly induced the secretion of up to 400 proteins, 29 to 52% of which were carbohydrate-active enzymes (CAZymes), including a wide diversity of copper-containing oxidoreductases from the auxiliary activities (AA) class (AA1, AA3, AA5, AA7, AA9, AA11, AA12, AA13, and AA16). Under these specific conditions, while a CRO-glyoxal oxidase from the AA5_1 subfamily was among the most abundantly secreted proteins, the targeted AA5_2 CRO-AlcOx were secreted at lower levels, suggesting heterologous expression as a more promising strategy for CRO-AlcOx production and utilization. C. tabacum and C. destructivum CROAlcOx were thus expressed in Pichia pastoris, and their preference toward both aromatic and aliphatic primary alcohols was assessed. The CRO-AlcOx from C. destructivum was further investigated in applied settings, revealing a full conversion of C6 and C8 alcohols into their corresponding fragrant aldehydes. [ABSTRACT FROM AUTHOR]

Ogataea minuta is a methylotrophic yeast that is closely related to Ogataea (Hansenula) polymorpha. Like other methylotrophic yeasts, O. minuta possesses strongly methanol-inducible genes, such as AOX1. We have focused on O. minuta as a host for the production of heterologous glycoproteins. However, it remained unknown how the AOX1 promoter is regulated in O. minuta. To elucidate regulation mechanisms of the AOX1 promoter, we adopted an assay system to quantitate AOX1 promoter activity using the PHO5 gene, which encodes an acid phosphatase, of Saccharomyces cerevisiae. The promoter activity assay revealed that glycerol, as well as glucose, cause strong catabolite repression of AOX1 expression in O. minuta. To investigate what factors are involved in transcription of the AOX1 promoter in O. minuta , we cloned three putative transcription factor genes, TRM1 , TRM2 , and MPP1 , as homologues of other methylotrophic yeast species. Deletion mutants of these genes all showed decreased induction of the AOX1 promoter when methanol was added as the sole carbon source, indicating that these genes are indeed involved in AOX1 promoter regulation in O. minuta. Double deletion and constitutive expression of these transcription factor genes indicated that TRM1 and MPP1 regulate the transcription of AOX1 in the same pathway, while TRM2 regulates it in another pathway. By reverse transcription-qPCR, we also found that these two pathways compensate for each other and have crosstalk mechanisms with each other. A possible model for regulation of the AOX1 promoter in O. minuta was shown. [ABSTRACT FROM AUTHOR]

This research was aimed at the fabrication of an improved enzyme-based amperometric biosensor for rapid quantification of ethanol. Alcohol oxidase (AOX) from Pichia pastoris was covalently immobilized on chemically treated polyvinylchloride (PVC) beaker and subsequently horseradish peroxidase (HRP), nafion (Nf), carboxylated multi-walled carbon nanotubes (c-MWCNTs), chitosan (CHIT) and gold nanoparticles (AuNPs) were immobilized onto Au electrode to fabricate a working electrode. The enzyme-coated PVC surface was analysed morphologically via scanning electron microscopy (SEM). At different stages of construction, the electrochemical properties of working electrode were deciphered by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The biosensor displayed optimal response in a short time span of 12 s at pH 7.5 and 35°C temperature. The working range exhibited by the proposed biosensor was 0.01–42 mM with a limit of detection (LOD) of 0.0001 µM and storage stability of 180 days at 4°C. When level of alcohol was evaluated in commercial samples via standard assay kit and existing biosensor, a good correlation (R2 = 0.98) was observed which authenticates its reliability. [ABSTRACT FROM AUTHOR]

The biocatalytic preparation of trans-hex-2-enal from trans-hex-2-enol using a novel aryl alcohol oxidase from Pleurotus eryngii (PeAAOx) is reported. As O2-dependent enzyme PeAAOx-dependent reactions are generally plagued by the poor solubility of O2 in aqueous media and mass transfer limitations resulting in poor reaction rates. These limitations were efficiently overcome by conducting the reaction in a flow-reactor setup reaching unpreceded catalytic activities for the enzyme in terms of turnover frequency (up to 38 s−1) and turnover numbers (more than 300000) pointing towards preparative usefulness of the proposed reaction scheme.

The current study investigated the efficiency of synergistic biological and Advanced Oxidation Process (AOPs) treatment (B-AOPs) using Aeromonas hydrophila SK16 and AOPs-H2O2 in the removal of Remazol Yellow RR dye. Singly, A. hydrophila and AOPs showed 90 and 63.07% decolourization of Remazol Yellow RR dye (100 mg L−1) at pH 6 and ambient temperature within 9 h respectively. However, the synergistic B-AOPs treatments showed maximum decolorization of Remazol Yellow RR dye within 4 h. Furthermore, the synergistic treatment significantly reduced BOD and COD of the textile wastewater by 84.88 and 82.76% respectively. Increased levels in laccase, tyrosinase, veratryl alcohol oxidase, lignin peroxidase and azo reductase activities further affirmed the role played by enzymes during degradation of the dye. UV–Visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), high-performance liquid chromatography (HPLC) and gas chromatography–mass spectroscopy (GC–MS) confirmed the biotransformation of dye. A metabolic pathway was proposed based on enzyme activities and metabolites obtained after GC–MS analysis. Therefore, this study affirmed the efficiency of combined biological and AOPs in the treatment of dyes and textile wastewaters in comparison with other methods. [ABSTRACT FROM AUTHOR]

Most of the alcohol analytical methods are robust and instrumentally expensive. An alternative of ethanol biosensor based on selected biofilm forming Acetobacter aceti bacteria producing alcohol oxidase was constructed on a screen-printed carbon electrode. The enzyme specifically oxidizes the ethanol and generate electrical current that then electrochemically detected and measured by cyclic voltammetry method. A scanning electron microscopic analysis indicated that the biofilm was formed firmly in the electrode. This constructed biosensor reached its optimum at biofilm formed by bacteria of 1.33 × 1010 cells/ml, temperature of 27°C, and pH 7. The enzyme kinetic had KM and Vmax AOX values of 3.5 mm and 125 μA respectively. The biosensor had detection and quantization limit of 0.003 and 0.009%, respectively, and a sensitivity of 57.29 μA (%)-1. A linearity and relative deviation value were revealed at 0.993 and 1.95% respectively. The biosensor was relatively specific and had no interferences with methanol, sodium chloride and citric acid as the common interferences of ethanol compounds. Furthermore, the biosensor had been stably for at least 55 days. Therefore, this constructed biosensor should be developed into a prototype for a practical effective analysis. [ABSTRACT FROM AUTHOR]

Komagataella phaffii yeast is one of the most important biocompounds producing microorganisms in modern biotechnology. Optimization of media recipes and cultivation strategies is key to successful synthesis of recombinant proteins. The complex effects of proline on gene expression in the yeast K. phaffii was analyzed on the transcriptome level in this work. Our analysis revealed drastic changes in gene expression when K. phaffii was grown in proline-containing media in comparison to ammonium sulphate-containing media. Around 18.9% of all protein-encoding genes were differentially expressed in the experimental conditions. Proline is catabolized by K. phaffii even in the presence of other nitrogen, carbon and energy sources. This results in the repression of genes involved in the utilization of other element sources, namely methanol. We also found that the repression of AOX1 gene promoter with proline can be partially reversed by the deletion of the KpPUT4.2 gene.

Glucose-Methanol-Choline (GMC) family enzymes are very important in catalyzing the oxidation of a wide range of structurally diverse substrates. Enzymes that constitute the GMC family, share a common tertiary fold but < 25% sequence identity. Cofactor FAD, FAD binding signature motif, and similar structural scaffold of the active site are common features of oxidoreductase enzymes of the GMC family. Protein functionality mainly depends on protein three-dimensional structures and dynamics. In this study, we used the normal mode analysis method to search the intrinsic dynamics of GMC family enzymes. We have explored the dynamical behavior of enzymes with unique substrate catabolism and active site characteristics from different classes of the GMC family. Analysis of individual enzymes and comparative ensemble analysis of enzymes from different classes has shown conserved dynamic motion at FAD binding sites. The present study revealed that GMC enzymes share a strong dynamic similarity (Bhattacharyya coefficient >90% and root mean squared inner product >52%) despite low sequence identity across the GMC family enzymes. The study predicts that local deformation energy between atoms of the enzyme may be responsible for the catalysis of different substrates. This study may help that intrinsic dynamics can be used to make meaningful classifications of proteins or enzymes from different organisms.Communicated by Ramaswamy H. Sarma.

Highly sensitive detection of ethanol concentrations in discrete brain regions of rats voluntarily accessing ethanol, with high temporal resolution, would represent a source of greatly desirable data in studies devoted to understanding the kinetics of the neurobiological basis of ethanol's ability to impact behavior. In the present study, we present a series of experiments aiming to validate and apply an original high-tech implantable device, consisting of the coupling, for the first time, of an amperometric biosensor for brain ethanol detection, with a sensor for detecting the microvibrations of the animal. This device allows the real-time comparison between the ethanol intake, its cerebral concentrations, and their effect on the motion when the animal is in the condition of voluntary drinking. To this end, we assessed in vitro the efficiency of three different biosensor designs loading diverse alcohol oxidase enzymes (AOx) obtained from three different AOx-donor strains: Hansenula polymorpha, Candida boidinii, and Pichia pastoris. In vitro data disclosed that the devices loading H. polymorpha and C. boidinii were similarly efficient (respectively, linear region slope [LRS]: 1.98 ± 0.07 and 1.38 ± 0.04 nA/mM) but significantly less than the P. pastoris-loaded one (LRS: 7.57 ± 0.12 nA/mM). The in vivo results indicate that this last biosensor design detected the rise of ethanol in the nucleus accumbens shell (AcbSh) after 15 minutes of voluntary 10% ethanol solution intake. At the same time, the microvibration sensor detected a significant increase in the rat's motion signal. Notably, both the biosensor and microvibration sensor described similar and parallel time-dependent U-shaped curves, thus providing a highly sensitive and time-locked high-resolution detection of the neurochemical and behavioral kinetics upon voluntary ethanol intake. The results overall indicate that such a dual telemetry unit represents a powerful device which, implanted in different brain areas, may boost further investigations on the neurobiological mechanisms that underlie ethanol-induced motor activity and reward. [ABSTRACT FROM AUTHOR]

A newly designed amperometric biosensor for the determination of ethanol through one-step electrochemical coating of (4,7-di(thiophen-2-yl)benzo[c][1,2,5]selenadiazole- co -1 H -pyrrole-3-carboxylic acid) (TBeSe- co -P3CA) on a graphite electrode is presented. It was aimed to propose a newly synthesized copolymer with enhanced biosensing properties as a novel sensor for the quantification of ethanol. The conjugated copolymer (TBeSe- co -P3CA) was prepared through electrochemical polymerization by potential cycling. After polymer modification, alcohol oxidase (AOx) was immobilized on a modified electrode surface for ethanol sensing. In the analytical investigation, the calibration plot is linear above large concentration range (0.085 to 1.7 mM), where sensitivity is around 16.44 μA/mMcm2 with a very low detection limit (LOD) of 0.052 mM based on the signal-to-noise ratio in short response time. Moreover, interfering effect of some possible compounds were examined and the capability of the biosensor in estimating ethanol content in commercial alcoholic beverages was also demonstrated. The results showed satisfactory accuracy of the developed sensor and confirm the proposed sensor has a potential for ethanol quantification compared to the currently used techniques. • A novel conducting copolymer for successful biosensor applications • A great surface architecture based on selenadiazole and pyrrole bearing polymers using one-step electrochemical polymerization • Sensitive, reliable and fast response ethanol biosensor [ABSTRACT FROM AUTHOR]

We cloned a set of genes encoding alcohol oxidase from Ochrobactrum sp. AIU 033 (OcAOD), which exhibits the appropriate substrate specificity for glyoxylic acid production from glycolic acid. The set of genes for OcAOD contained two open reading frames consisting of 555-bp (aodB) and 1572-bp (aodA) nucleotides, which encode the precursor for the β-subunit and α-subunit of OcAOD, respectively. We expressed the cloned genes as an active product in Escherichia coli BL21(DE3). The recombinant OcAOD oxidized glycolic acid and primary alcohols with C2–C8 but not glyoxylic acid (as is the case for native OcAOD), whereas the K m and V max values for glycolic acid and the pH stability were higher than those of native OcAOD. A consensus sequence for the twin-arginine translocation (Tat) pathway was identified in the N-terminal region of the precursor for the β-subunit, and the active form of OcAOD was localized in the periplasm of recombinant E. coli , which indicated that OcAOD would be transported from the cytoplasm to the periplasm by the hitchhiker mechanism through the Tat pathway. The OcAOD productivity of the recombinant E. coli was 24-fold higher than that of Ochrobactrum sp. AIU 033, and it was further enhanced by 1.2 times by the co-expression of additional tatABC from E. coli BL21(DE3). Our findings thus suggest a function of the β-subunit of OcAOD in membrane translocation, and that the recombinant OcAOD has characteristics that are suitable for the enzymatic synthesis of glyoxylic acid as well as native OcAOD. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Amperometric biosensors for ethanol and methylamine detection were designed. • Yeast alcohol oxidase and methylamine oxidase were used as biorecognition elements. • Nanoparticles of PtRu as peroxidase mimetic were chosen for biosensors construction. • Both biosensors exhibit high sensitivities and broad linear ranges to the substrates. • The constructed biosensors were tested on the real samples of food. Abstract Catalytically active nanomaterials have several advantages over their natural analogues when used as artificial enzymes (nanozymes), namely, higher stability and lower cost. Nanozymes with metallic nanocomposites are promising catalysts for biosensing applications. The aim of the current research is to construct oxidase-based bioelectrodes for food analysis using nanozymes as peroxidase mimetics. Bimetallic PtRu nanoparticles (nPtRu) coupled with alcohol oxidase (AO) and methylamine oxidase (AMO) were chosen to construct amperometric biosensors (ABSs) for primary alcohols and methylamine (MA). Both ABSs show high sensitivities (336 A·M−1·m−2 for the AO-ABS and 284 A·M−1·m−2 for the AMO-ABS), broad linear ranges (25–200 µM ethanol and 20–600 µM MA) and satisfactory storage stabilities. Practical feasibility of the constructed ABSs was demonstrated on food samples. High correlation between contents of MA and ethanol in foods determined by the ABSs and reference methods was observed. [ABSTRACT FROM AUTHOR]

Highlights • Alcohol oxidase of a fungus Pyricularia oryzae (PoAlcOX) contains a WSC domain. • Deletion of the WSC domain does not affect oxidation of alcohols by PoAlcOX. • PoAlcOX binds to xylans and fungal chitin/β-1,3-glucan. • Deletion of the WSC domain results in loss of the carbohydrate-binding property. Abstract The cell wall integrity and stress response component (WSC) domain was first described in the Wsc-family protein of the yeast Saccharomyces cerevisiae , and later found in diverse eukaryotic organisms. Due solely to their presence in the Wsc-family proteins working as a plasma membrane sensor for surface stress and in a fungal β-1,3-exoglucanse, WSC domains have been presumed to possess carbohydrate-binding property without any experimental evidence. Aiming at elucidation of function(s) of WSC domains, we characterized a WSC domain-containing alcohol oxidase from the rice blast pathogen Pyricularia oryzae (PoAlcOX). Recombinant PoAlcOX produced with Pichia pastoris showed alcohol oxidase activity toward a wide range of substrates including two aliphatic alcohols, a branched-chain alcohol, a diol, and a polyol. Deletion of the WSC domain virtually unaffected oxidation of these substrates by PoAlcOX, indicating that the domain makes no contribution to the catalytic activity. In analogy to some carbohydrate-binding modules, we inferred that the WSC domain plays a role in protein anchoring, and evaluated binding capability of PoAlcOX to a set of polysaccharide components of fungal and plant cell walls. This revealed that PoAlcOX binds to xylans and fungal chitin/β-1,3-glucan in the WSC domain-dependent manner, demonstrating for the first time the carbohydrate-binding property of the domain. Additionally, we provide evidence that PoAlcOX immobilized on birch wood xylan retains the catalytic activity. Overall, the data we collected suggest that the role of the WSC domain of PoAlcOX is not recognition of substrates but attaching the enzyme to plant and/or fungal cell wall. [ABSTRACT FROM AUTHOR]

Highlights • Novel on-line μ-stat approach is presented for regulating methanol feeding rate in fermenter. • Methanol feeding was controlled in fed-batch based on the on-line ammonia consumption rate. • MLP3 neural network was used to reconstruct the controller. • The designed controller was used for A1AT production process control in P. pastoris. • Control and maintaining μ at the optimal level led to increase in target protein production. Abstract Exact monitoring of the key process variables, such as specific growth rate (μ), is essential for effective process control, and maintenance of the optimal conditions for the production of recombinant proteins. In this study, a novel and confident on-line μ-stat approach for setting up methanol feeding strategy is described that is based on the consumption of ammonium hydroxide as feedback control. For this purpose a new and robust control with a time-based stable control structure was used, which had the ability to reconstruct the controller. Moreover, the MLP3 neural network was applied to adjust and optimize the performance of the robust control system. To evaluate the cited strategy, the overproduction of human recombinant alpha 1-antitrypsin (A1AT) under the control of alcohol oxidase 1 promoter (p AOX1) was investigated. On-line control of the μ at the optimal amount (0.03 1/h) led to 120 g/L dry cell weight and 324 mg/L of A1AT concentration. In comparison with traditional protocols of methanol feeding, the obtained product concentration demonstrated a significant improvement. It is suggested that this new presented method has a remarkable potential to be used for the overexpression of different recombinant proteins in the host Pichia pastoris as well as for process development in other hosts. [ABSTRACT FROM AUTHOR]

Abstract Alcohol oxidase (AOX), in the presence of oxygen, catalyzes the bioconversion of short-chained alcohols into their corresponding aldehydes and ketones. Alcohol oxidase can be used in the production of aldehydes that can serve as precursors of flavor compounds and fine chemicals. However due to AOX's poor stability, its practical industrial or analytical application is limited. Two kinetic inactivation regions suggest that one of AOX's isozymes is more resistant to thermal inactivation at atmospheric or high pressures. The resistant fraction was 4–34 times more stable than the labile fraction. High hydrostatic pressure (HHP) stabilized AOX against thermal inactivation. A 14-fold stabilization was observed at 49.4 °C for the labile fraction at 200 MPa relative to atmospheric pressure. The activation energy of inactivation at 40–160 MPa ranged from 95 to 184 kJ mol−1 for the resistant fraction and from 232 to 402 kJ mol−1 for the labile fraction. The activation volume of inactivation was independent of temperature and of approximately 20 cm3 mol−1. Highlights • High hydrostatic pressure (HHP) stabilized alcohol oxidase (AOX) from P. pastoris. • Kinetics of thermal inactivation suggest AOX isozymes have different thermal resistance. • HHP and the use of the heat resistant fraction of AOX increase potential industrial application. [ABSTRACT FROM AUTHOR]

Abstract A rapid and sensitive method has been devised in order to detect and estimate the synthesis of the enzyme alcohol oxidase (AOX) by fungi, by way of the use of two chemical complexes, namely, acetylacetone (3,5-diacetyl-1,4-dihydrolutidine) and acetylacetanilide (3,5-di- N -phenylacetyl-1,4-dihydrolutidine). This method involves the use of the AOX enzyme that could specifically oxidize methanol, giving rise to equimolar equivalents each of formaldehyde (HCHO) and hydrogen peroxide (H 2 O 2) as the end products. Further, the formaldehyde, thus produced was allowed to interact with the neutral solutions of acetylacetone and the ammonium salt, gradually developing a yellow color, owing to the synthesis and release of 3,5-diacetyl-1,4-dihydrolutidine (yellow product; λ = 420 nm; λ ex/em = 390/470 nm) and the product, so generated was quantified spectrophotometrically by measureing its absorbance at 412 nm. In another set up, the amount of formaldehyde produced as a sequel to the oxidation of methanol by the AOX enzyme was determined by allowing it to react with the acetylacetanilide reagent, after which the volume of the fluorescent product - 3,5-di- N -phenylacetyl-1,4-dihydrolutidine (colorless product; λ ex/em = 390/470 nm) that was generated was estimated by measuring its emission at 460 nm (excitation wavelength at 360 nm) in a spectrophotometer. Of the various substrates tested, a commercial source of the AOX enzyme appreciably oxidizes methanol, thereby generating formaldehyde, and further reacts with acetylacetone, to give rise to a bright yellow complex, displaying a maximum activity of 1402 U/mL. Determination of the AOX activity by the use of acetylacetone and acetylacetanilide could serve as a viable alternative to the conventional alcohol oxidase-peroxidase-2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (AOX-POD-ABTS) based method. In view of this, this method appears to be invaluable for application at the various food, pharmaceutical, fuel, biosensor, biorefinery, biopolymer, biomaterial, platform chemical, and biodiesel industries. Highlights • Alochol oxidase (AOX) oxidizes methanol and produces 1 μmol equivalent of HCHO and H 2 O 2 as products. • Methanol oxidizing enzymes detected using acetylacetone and acetylacetanilide. • Acetylacetone and ammonium salts react with HCHO and produces 3,5-Diacetyl-1,4-dihydroludine. • Also acetylacetanilide forms fluorescence product of 3,5-Di- N -phenylacetyl-1,4-dihydroludine. • The new AOX methods are replaces alcohol oxidase- peroxidase- ABTS (AOX-POD-ABTS) based method. [ABSTRACT FROM AUTHOR]

Alcohol oxidase catalyzes the oxidation of primary alcohols into the corresponding aldehydes, making it a potential biocatalyst in the chemical industry. However, the high production cost and poor operational stability of this enzyme are limitations for industrial application. Immobilization of enzyme onto solid supports is a useful strategy for improving enzyme stability. In this work, alcohol oxidase from the thermotolerant methylotrophic yeast Ogataea thermomethanolica (Oth AOX) was covalently immobilized onto barium ferrite (BaFe 12 O 19) magnetic microparticles. Among different conditions tested, the highest immobilization efficiency of 71.0 % and catalytic activity of 34.6 U/g was obtained. Immobilization of Oth AOX onto magnetic support was shown by Fourier-Transformed infrared microscopy, scanning electron microscopy and X-ray diffraction. The immobilized Oth AOX worked optimally at 55 °C and pH 8.0. Immobilization also improved thermostability, in which >65% of the initial immobilized enzyme activity was retained after 24 h pre-incubation at 45 °C. The immobilized enzyme showed a greater catalytic efficiency for oxidation of methanol and ethanol than free enzyme. The immobilized enzyme could be recovered by magnetization and recycled for at least three consecutive batches, after which 70% activity remained. The properties of the immobilized enzyme suggest its potential industrial application for synthesis of aldehyde. Graphical abstract Image 1 Highlights • Oth AOX was immobilized on BaFe 12 O 19 microparticles with >70% efficiency. • Immobilized Oth AOX was characterized by SEM, FT-IR, and XRD. • Immobilization led to improved thermal properties and kinetic parameters. • High ethanol conversion rate of 75.3% yield to acetaldehyde was obtained. • The enzyme was separated by magnetization and reused in consecutive batches. [ABSTRACT FROM AUTHOR]

Novel nanomaterials, including metallic nanoparticles obtained via green synthesis (gNPs), have a great potential for application in biotechnology, industry and medicine. The special role of gNPs is related to antibacterial agents, fluorescent markers and carriers for drug delivery. However, application of gNPs for construction of amperometric biosensors (ABSs) is not well documented. The aim of the current research was to study potential advantages of using gNPs in biosensorics. The extracellular metabolites of the yeast Ogataea polymorpha were used as reducing agents for obtaining gNPs from the corresponding inorganic ions. Several gNPs were synthesized, characterized and tested as enzyme carriers on the surface of graphite electrodes (GEs). The most effective were Pd-based gNPs (gPdNPs), and these were studied further and applied for construction of laccase- and alcohol oxidase (AO)-based ABSs. AO/GE, AO-gPdNPs/GE, laccase/GE and laccase-gPdNPs/GE were obtained, and their analytical characteristics were studied. Both gPdNPs-modified ABSs were found to have broader linear ranges and higher storage stabilities than control electrodes, although they are less sensitive toward corresponding substrates. We thus conclude that gPdNPs may be promising for construction of ABSs for enzymes with very high affinities to their substrates. [ABSTRACT FROM AUTHOR]

A new amperometric sensor has been fabricated for sensitive and rapid quantification of ethanol. The biosensor assembly was prepared by covalently immobilizing alcohol oxidase (AOX) from Pichia pastoris onto chemically modified surface of polyvinylchloride (PVC) beaker with glutaraldehyde as a coupling agent followed by immobilization of horseradish peroxidase (HRP), silver nanoparticles (AgNPs), chitosan (CHIT), carboxylated multi-walled carbon nanotubes (c-MWCNTs) and nafion (Nf) nanocomposite onto the surface of Au electrode (working electrode). Owing to properties such as chemical inertness, light weight, weather resistance, corrosion resistance, toughness and cost-effectiveness, PVC membrane has attracted a growing interest as a support for enzyme immobilization in the development of biosensors. The amperometric biosensor displayed optimum response within 8 s at pH 7.5 and 35°C temperature. A linear response to alcohol in the range of 0.01mM-50 mM and 0.0001 µM as a minimum limit of detection was displayed by the proposed biosensor with excellent storage stability (190 days) at 4°C. The sensitivity of the sensor was found to be 155 µA mM−1 cm−2. A good correlation (R2 = 0.99) was found between alcohol level in commercial samples as evaluated by standard ethanol assay kit and the current biosensor which validates its performance. [ABSTRACT FROM AUTHOR]

Abstract Hydrocarbon biosynthesis in insects involves the elongation of fatty acyl-CoAs to very-long chain fatty acyl-CoAs that are then reduced and converted to hydrocarbon, with the last step involving the oxidative decarbonylation of an aldehyde to hydrocarbon and carbon dioxide. Cytochromes P450 in the 4G family decarbonylate aldehydes to hydrocarbon. All insect acyl-CoA reductases studied to date reduce fatty acyl-CoAs to alcohols. The results of the work reported herein demonstrate that CYP4G55 and CYP4G56 from the mountain pine beetle, Dendroctonus ponderosae , expressed as fusion proteins with house fly cytochrome P450 reductase (CPR), convert both long chain aldehydes and long chain alcohols to hydrocarbons. CYP4G55 and CYP4G56 appear to prefer primary alcohols to aldehydes as substrates. These data strongly suggest that hydrocarbon biosynthesis in insects occurs by the two-step reduction of very long chain fatty acyl-CoAs to alcohols, which are then oxidized to aldehydes and then oxidatively decarbonylated to hydrocarbon by CYP4G enzymes. In addition, both CYP4G55 and CYP4G56 fusion proteins convert C10 alcohols and aldehydes to hydrocarbons, including the conversion of (Z)-7-decenal, a putative intermediate in the exo -brevicomin pheromone biosynthetic pathway, to (Z)-3-nonene. These data demonstrate that the highly conserved CYP4G enzymes accept a broad range of carbon chain lengths, including C10 and C18, and have evolved to function in cuticular hydrocarbon biosynthesis and pheromone production. Graphical abstract Image 1 Highlights • CYP4G55 and CYP4G56 synthesize hydrocarbons from primary alcohols. • CYP4G55 and CYP4G56 convert C10 aldehydes and C10 alcohols to hydrocarbon. • Both enzymes catalyze 7-decenal to 3-nonene, a step in exo -brevicomin biosynthesis. [ABSTRACT FROM AUTHOR]

Coniferyl alcohol is a valuable chemical. However, the current approaches to obtain coniferyl alcohol are either unefficient or expensive. Penicillium simplicissimum vanillyl alcohol oxidase (PsVAO) can be used to produce coniferyl alcohol. However, PsVAO intrinsically produces harmful byproduct H 2 O 2 . Utilizing catalase to decompose H 2 O 2 is a potential straightforward approach; however, catalase can also exhibit peroxidase activity to facilitate coniferyl alcohol over-oxidation. In this study, catalases exhibiting both high catalase activity and low peroxidase activity were found out, and introduced into the bioconversion systems. Our results showed that eliminating H 2 O 2 in situ released H 2 O 2 inhibition of PsVAO, improved coniferyl alcohol production and eliminated coniferyl alcohol over-oxidation. Finally, coniferyl alcohol titer, molar yield, and productivity reached 22.9 g/L, 78.7%, and 0.5 g/(L × h) respectively. An efficient coniferyl alcohol production method was developed by overcoming the intrinsic disadvantages of PsVAO. [ABSTRACT FROM AUTHOR]