Bioelectrocatalytic Carbon Ceramic Gas Electrode for Reduction of Dioxygen and Its Application in a Zinc–Dioxygen Cell

An enzyme‐modified carbon ceramic electrode was constructed and studied that is capable to reduce dioxygen supplied from the gas phase. The permeation of the electrode material and its hydrophobic silicate component was studied by scanning electrochemical microscopy. The mass‐transfer coefficient of dioxygen in methyltrimethoxysilane‐based silicate was estimated to be 6.44 × 10^–5 cm² s^–1. After modification of the electrode with bilirubin oxidase and immersion in deareated aqueous electrolyte, the dioxygen bioelectrocatalytic reduction is observed with onset potential at 0.45 V. The constructed electrode was successfully applied as cathode in a zinc–dioxygen cell.     

Enzymatic Conversion of Unnatural Amino Acids by Yeast D‐Amino Acid Oxidase

Unnatural amino acids, particularly synthetic α‐amino acids, are becoming crucial tools for modern drug discovery research. In particular, this application requires enantiomerically pure isomers. In this work we report on the resolution of racemic mixtures of the amino acids d,l ‐naphthylalanine and d,l ‐naphthylglycine by using a natural enzyme, D ‐amino acid oxidase from the yeast Rhodotorula gracilis. A significant improvement of the bioconversion is obtained using a single‐point mutant enzyme designed by a rational approach. With this D ‐amino acid oxidase variant the complete resolution of all the unnatural amino acids tested was obtained: in this case, the bioconversion requires a shorter time and a lower amount of biocatalyst compared to the wild‐type enzyme. The simultaneous production of the corresponding α‐keto acid, a possible precursor of the amino acid in the L ‐form, improves the significance of the procedure.     

Development of a new two‐enzyme biosensor based on poly(pyrrole‐co‐3,4‐ethylenedioxythiophene) for lactose determination in milk

A new lactose biosensor was developed by preparing a suitable copolymer of polypyrrole and poly(3,4‐ethylenedioxythiophene) synthesized using the electropolymerization method. Pyrrole and 3,4‐ethylenedioxythiophene monomers were deposited in the presence of sodium dodecylbenzene sulphonic acid on a platinum disc electrode, which was used as the working electrode. The sensor is based on the serial reactions of β‐galactosidase and galactose oxidase immobilized on a copolymer‐modified platinum disc electrode. Successful synthesis of the enzyme‐immobilized copolymer was confirmed by FT‐IR spectrometry, SEM, and electrochemical analysis. The response of the enzyme electrode to lactose was determined by cyclic voltammetry at + 0.40 V. The response time of the biosensor was found to be from 8 to 10 s, and the upper limit of the linear working portion was found to be at a lactose concentration of 2.30 mM with a detection limit of 1.4 × 10^?5 M. The apparent Michaelis–Menten constant was found to be 0.65 mM of lactose. The effects of interferents were also investigated. Lactose concentrations determined by the biosensor were in good agreement with those measured by the reference methods. Our results show that the developed biosensor has a significant potential to the determination of lactose concentration in milk. POLYM. ENG. SCI., 58:839–848, 2018. © 2017 Society of Plastics Engineers     

Expanding an Efficient,Electrically Driven and CNT‐Tagged P450 System into the Third Dimension: A Nanowired CNT‐Containing and Enzyme‐Stabilising 3 D Sol–Gel Electrode

Although electrochemically catalysed P450 reactions have been described, their efficiency and applicability remained limited. This is mostly due to low enzyme activity, laborious protein immobilisation and the small electrode surface. We established a novel protein immobilisation method for a determined orientation and electrical wiring of the enzyme without post‐expression modification. By genetic introduction of an anchor‐peptide our method is applicable for screening medium to large mutant libraries and detection by an electrode system. The system was expanded by using wired carbon nanotubes within a sol‐gel matrix to create a three dimensional electrode.     

Marine Molecular Machines: Heterocyclization in Cyanobactin Biosynthesis

Natural products that contain amino‐acid‐derived (Cys, Ser, Thr) heterocycles are ubiquitous in nature, yet key aspects of their biosynthesis remain undefined. Cyanobactins are heterocyclic ribosomal peptide natural products from cyanobacteria, including symbiotic bacteria living with marine ascidians. In contrast to other ribosomal peptide heterocyclases that have been studied, the cyanobactin heterocyclase is a single protein that does not require an oxidase enzyme. Using this simplifying condition, we provide new evidence to support the hypothesis that these enzymes are molecular machines that use ATP in a product binding or orientation cycle. Further, we show that both protease inhibitors and ATP analogues inhibit heterocyclization and define the order of biochemical steps in the cyanobactin biosynthetic pathway. The cyanobactin pathway enzymes, PatD and TruD, are thiazoline and oxazoline synthetases.     

Biodegradation of Leather Waste by Enzymatic Treatment

The treatment of shavings, trimmings and splits of leather waste from tanneries has a potential to generate value-added products. In this study enzymatic treatment of leather waste was performed. This method utilizes alkaline protease produced by Bacillus subtilis in our laboratory by submerged fermentation. Optimum conditions of pH, time duration, temperature and concentration of enzyme were determined for maximum degradation of leather waste. The amount of degradation was measured by the release of amino acid hydroxyproline. Amino acid composition in the hydrolysate obtained by the enzyme hydrolysis was determined. This relative simple biotreatment of leather waste may provide a practical and economical solution.     

Electrochemical immobilization of ascorbate oxidase in poly(3,4‐ethylenedioxythiophene)/multiwalled carbon nanotubes composite films

Immobilization of ascorbate oxidase (AO) in poly(3,4‐ethylenedioxythiophene) (PEDOT)/multiwalled carbon nanotubes (MWCNTs) composite films was achieved by one‐step electrochemical polymerization. The PEDOT/MWCNTs/AO modified electrode was fabricated by the entrapment of enzyme in conducting matrices during electrochemical polymerization. The PEDOT/MWCNTs modified electrodes were investigated by cyclic voltammetry and electrochemical impedance spectroscopy. The experimental results showed that the composite films exhibited better mechanical integrity, electrochemical activity, higher electronic and ionic conductivity, and larger redox capacitance compared with pure PEDOT films, which would be beneficial to the fabrication of PEDOT/MWCNTs/AO electrochemical biosensors. The scanning electron microscopy studies revealed that MWCNTs served as backbone for 3,4‐ethylenedioxythiophene (EDOT) electropolymerization. Furthermore, the resulting enzyme electrode could be used to determine L ‐ascorbic acid successfully, which demonstrated the good bioelectrochemical catalytic activity of the immobilized AO. The results indicated that the PEDOT/MWCNTs composite are a good candidate material for the immobilization of AO in the fabrication of enzyme‐based biosensor. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Properties of Soy Protein Produced by Countercurrent,Two-Stage,Enzyme-Assisted Aqueous Extraction

Enzyme-assisted aqueous extraction processing (EAEP) is an environmentally friendly technology where oil and protein can be simultaneously extracted from soybeans by using water and protease. Countercurrent, two-stage, EAEP was performed at a 1:6 solids-to-liquid ratio, 50 °C, pH 9.0, and 120 rpm for 1 h to extract oil and protein from soybeans. The skim fractions were produced by three methods: (1) by treating with 0.5 % protease (wt/g extruded flakes) in both extraction stages; (2) by treating with 0.5 % protease in the 2nd extraction stage only; and (3) by using the same two-stage extraction procedure without enzymes in either extraction stages. Countercurrent, two-stage, protein extraction of air-desolventized, hexane-defatted, soybean flakes was used as a control. Solubility profiles of the skim proteins were the typical U-shaped curves with the lowest solubility at the isoelectric point of soy protein (pH 4.5). The use of the enzyme slightly improved solubility of the recovered protein with hydrolyzed proteins having higher solubilities at acid pH. Emulsification and foaming properties were generally reduced by the use of enzyme during EAEP extractions. The skims produced with protease-extracted (hydrolyzed) proteins gave gels with lower hardness than did unhydrolyzed proteins when heated at 80 °C. The essential amino acid compositions and protein digestibilities were not adversely affected by either extrusion or extraction treatments.     

Kinetic and transport analysis of immobilized oxidoreductases that oxidize glycerol and its oxidation products

Glycerol has drawn increasing attention as a possible fuel, because it has many desirable qualities and is abundant due to the fact that it is a byproduct of biodiesel production. Previous research has shown that non-natural enzyme cascades can be used to create a bioanode that can stepwise oxidize glycerol to carbon dioxide. Two of these enzymes are pyrroloquinoline quinone (PQQ) dependant alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldDH) derived from Gluconobacter. The third enzyme, which is responsible for carbon bond cleavage, is oxalate oxidase (OxOx) derived from barley. Previous research has shown that all three enzymes have demonstrated the ability to undergo direct electron transfer to a carbon electrode which allows for a simple and efficient bioanode that completely oxidizes glycerol. In this study, each enzyme was individually immobilized within modified Nafion^® on a glassy carbon rotating disc electrode (GC-RDE) and voltammetric analysis was performed employing different rotation rates in a solution containing each enzyme's respective substrate. This substrate was glycerol for alcohol dehydrogenase, glyceraldehyde for aldehyde dehydrogenase, and mesoxalic acid for oxalate oxidase. From the voltammograms, Levich plots were produced and the solution diffusion coefficient (D_soln), the membrane diffusion coefficient (D_film), k_CAT, K_M, and V_MAX were determined.     

Electrochemical poly(1,3-phenylenediamine) synthesis as enzyme immobilization media

Electrochemical polymerization of the 1,3-phenylenediamine in the presence of glucose oxidase with KCl aqueous electrolyte at a potential of 0.800 V versus Ag–AgCl produces adherent poly(1,3-phenylenediamine) containing enzyme (glucose oxidase) film on a platinum electrode. Polymeric sensor prepared in this one-step procedure can be used to determine hydrogen peroxide formed as the result of the enzymatic reaction between glucose and glucose oxidase in the presence of O₂. The amperometric responses of the resultant enzyme electrode to glucose were rapid, reaching steady-state values within 4–5 s, and there was a linear relationship between glucose concentration and obtained current up to 6 mM. Polymeric sensor was stable for more 3 months. The glucose selectivity of enzyme electrode was determined in the presence of some interfering substances, such as lactose, sucrose, urea, uric acid, paracetamol, and ascorbic acid. Also, the effects of buffer concentration, storage conditions, and temperature on the steady-state amperometric responses were studied. Moreover, the Arrhenius activation energy for the enzymatic reaction was calculated. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 68:145–152, 1998     

Poly(phenylenediamine) film for the construction of glucose biosensors based on platinized glassy carbon electrode

Three phenylenediamine isomers (including ortho-, meta- and para-derivatives) were electrochemically polymerized to give polyphenylenediamine (PPD) films on platinized glassy carbon electrodes. Amperometric glucose sensors were developed by immobilizing glucose oxidase (GOx) into these polymer matrices during polymerization. Effects of the polymerization potential, polymerization charge, monomer concentration, GOx concentration and Pt deposition charge on the performance of the enzyme electrode to glucose were investigated. These resulting GC/Pt/PPD-GOx electrodes showed rapid electrochemical responses to hydrogen peroxide and glucose, and very good anti-interference ability to ascorbic acid. Correlation between the electroanalytical behaviors of the enzyme electrodes and the polymer structures was examined.     

Properties of the enzyme electrode fabricated with a film of polythiophene derivative and its application to a glucose fuel cell

A polymer electrode in the form of a thin film was prepared by electrochemical copolymerization of 3‐methylthiophene and thiophene‐3‐acetic acid. Glucose oxidase (GOx) was immobilized by covalent binding to the carboxyl groups on the electrode, and the GOx‐immobilized electrode (GOx‐electrode) was used as an anode in a glucose fuel cell. It was demonstrated by cyclic voltametry that in the presence of p‐benzoquinone (BQ), which was adopted as an electron mediator, the GOx‐electrode generated a significant glucose‐oxidation current depending on the concentrations of both glucose and BQ. A large surface area of the GOx‐electrode was considered to afford effective environment for the enzyme reaction and electron transfer. The fuel cell using the GOx‐electrode as an anode gave a power output of 42 μW/cm²‐anode at 30°C, when its anodic compartment contained 100 mM glucose and 10 mM BQ. The performance of the cell was influenced by the concentrations of glucose and BQ in the anodic compartment. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007     

Single-Molecule Studies of HIV-1 Protease Catalysis Enabled by Chemical Protein Synthesis

Single-molecule fluorescence studies of the proteolytic activity of the enzyme HIV-1 protease were performed using FRET-pair dye labeled peptide substrates and substrate-derived inhibitors prepared by solid phase peptide synthesis. Chemical protein synthesis was used to prepare homodimeric HIV-1 protease in soluble form and to prepare a covalent dimer 203 amino acid residue HIV-1 protease containing a biotin tether at the mid-point of the synthetic protein molecule. The biotin-tagged HIV-1 protease was immobilized on a neutravidin-coated glass slide. Total internal reflection excitation multiwavelength fluorescence spectroscopy was used to monitor substrate binding and cleavage by the synthetic enzyme molecules. Single-molecule traces for the dye-labeled peptide substrate showed distinct binding and cleavage events; the corresponding dye-labeled peptide inhibitor showed only the binding event. These results constitute strong proof-of-principle for the utility of chemical peptide and protein synthesis for single-molecule studies of enzyme catalysis.     

Specific Modulation of Protein Activity by Using a Bioorthogonal Reaction

Unnatural amino acids with bioorthogonal reactive groups have the potential to provide a rapid and specific mechanism for covalently inhibiting a protein of interest. Here, we use mutagenesis to insert an unnatural amino acid containing an azide group (Z) into the target protein at positions such that a “click” reaction with an alkyne modulator (X) will alter the function of the protein. This bioorthogonally reactive pair can engender specificity of X for the Z‐containing protein, even if the target is otherwise identical to another protein, allowing for rapid target validation in living cells. We demonstrate our method using inhibition of the Escherichia coli enzyme aminoacyl transferase by both active‐site occlusion and allosteric mechanisms. We have termed this a “clickable magic bullet” strategy, and it should be generally applicable to studying the effects of protein inhibition, within the limits of unnatural amino acid mutagenesis.     

Effect of enzymatic pretreatment on acid fermentation of food waste

Although food waste is a valuable carbon source for biological nutrient removal systems with low organic wastewater because of high C/N and C/P ratios, it must be pretreated to promote the hydrolysis of particulates, which is considered as a rate‐limiting step. This study investigated the effects of enzymatic pretreatment on hydrolytic solubilization of food waste with commercial enzyme. Both acidification efficiency and volatile fatty acid (VFA) production potential of enzymatically pretreated food waste were examined under controlled laboratory conditions. Experimental results indicated that protease exhibited the highest VSS reduction rate among three types of enzymes: carbohydrase, protease and lipase. A mixed enzyme treatment showed better reduction efficiency than a single enzyme treatment, and the highest volatile suspended solids (VSS) reduction was observed at an enzyme mixture ratio of 1:2:1 with carbohydrase:protease:lipase, respectively. It has been noted that pretreatment resulted in both maximum VFA production and the highest VFA content of soluble chemical oxygen demand at an enzyme mixture dosage of 0.1% (v/v). VFA production at this dosage revealed a 3.3 times higher rate than that of no‐enzyme added fermenter. The dominant VFAs were n‐butyrate followed by acetate. Copyright © 2006 Society of Chemical Industry     

Purification and characterization of an organic solvent and detergent‐tolerant novel protease produced by Bacillus sp. RKY3

BACKGROUND: Purification and characterization of a novel protease produced by Bacillus sp. RKY3, has been investigated, with special emphasis on the stability of the enzyme in the presence of different oxidizing and reducing agents as well as organic solvents. The enzyme was purified in two steps through concentration of the crude enzyme by ammonium sulfate precipitation, followed by anion exchange chromatography. RESULTS: The purified protease had a molecular mass of approximately 38 kDa, which was highly active over a broad range of pH between 7.0 and 9.0 and was also stable over a wide pH range from 5.0 to 11.0. Although the optimum temperature for enzyme activity was found to be 60 °C, it was rapidly deactivated at temperatures above 60 °C. It also showed good stability at 50 °C, with a 70 min half‐life. Ca²⁺ ions did not greatly enhance the activity or the stability of the enzyme. PMSF (1 mmol L^?1) completely inhibited the protease activity, and thus the purified protease was considered to be serine protease. The purified protease was stable with oxidants (H₂O₂, 2%), reducing agents (sodium dodecyl sulfate, 2%), and organic solvents (25%) such as benzene, hexane, and toluene. CONCLUSION: The purified enzyme, protease, seems to possess potential applications in protease‐based detergent and bleaching industries. The enzymatic activity against a wide variety of substrates suggests that the purified enzyme should be investigated for a range of commercial applications, especially for soy protein and gelatin hydrolysis in the food processing industry. Copyright © 2008 Society of Chemical Industry     

EDTA modified glassy carbon electrode: Preparation and characterization

EDTA-phenoxyamide modified glassy carbon electrode (EDTA-GC) was prepared at a glassy carbon electrode by surface synthesis. In the first step, nitrophenyl was grafted to the glassy carbon (GC) surface via the electrochemical reduction of its tetraflouroborate diazonium salt. In the second step, nitrophenyl-modified electrode (NP-GC) was subjected to the cathodic potential scan to reduce the nitro to amine group. p-Aminophenyl modified glassy carbon electrode (AP-GC) was dipped into a EDTA solution containing 1-ethyl-3(3-(dimethlyamino)propyl)-carbodiimide (EDC) as an activating agent. Thus formed ((2-anilino-2-oxoethyl){2-[bis(carboxymethyl)amino]-ethyl}amino)acetic acid modified GC electrode was denoted as EDTA-GC and characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), ellipsometry and X-ray photoelectron spectroscopy (XPS). Complexation of the EDTA-GC surface with Pb²⁺ ions was investigated if this electrode could be used as a metal sensor.     

High-resolution crystal structure of M-protease: phylogeny aided analysis of the high-alkaline adaptation mechanism

M-protease is a subtilisin-family serine protease produced by an alkaliphilic Bacillus sp. strain. Optimal enzymatic activity of the protein occurs at pH 12.3. The crystal structure of M-protease (space group P2(1)2(1)2(1), a = 62.3, b = 75.5, c = 47.2 A) has been refined to a crystallographic R-factor of 17.2% at 1.5 A resolution. The alkaline adaptation mechanism of the enzyme was analyzed. Molecular phylogeny construction was used to determine the amino acid substitutions that occurred during the high-alkaline adaptation process. This analysis revealed a decrease in the number of negatively charged amino acids (aspartic acid and glutamic acid) and lysine residues and an increase in arginine and neutral hydrophilic amino acids (histidine, asparagine and glutamine) residues during the course of adaptation. These substitutions increased the isoelectric point of M- protease. Some of the acquired arginine residues form hydrogen bonds or ion pairs to combine both N- and C-terminal regions of M-protease. The substituted residues are localized to a hemisphere of the globular protein molecule where positional shifts of peptide segments, relative to those of the less alkaliphilic subtilisin Carlsberg, are observed. The biased distribution and interactions caused by the substituted residues seem to be responsible for stabilization of the conformation in a high-alkaline condition.      

Direct electrochemistry of dinuclear CuA fragment from cytochrome c oxidase of Thermus thermophilus at surfactant modified glassy carbon electrode

Cytochrome c oxidase is ubiquitous enzyme involved in the terminal step of respiratory electron transfer process. The unique binuclear copper center containing bis-dithiolato bridges form a valance delocalized [Cu^1.5+-Cu^1.5+] state of the metal center located at the subunit II of cytochrome c oxidase. This metal center acts as the electron entry site of the enzyme and accepts electrons from cytochrome c. Direct electrochemistry of this binuclear copper center containing the water soluble protein obtained by genetically truncating the membrane bound part of the subunit II from Thermus thermophilus was achieved by favorable orientation of the protein on glassy carbon electrode surface promoting efficient electron transfer in the presence of various surfactants. Very reproducible, Nernstian responses are obtained with Cu_A. The redox potential and the electrochemical response were enhanced prominently in case of cationic surfactant CTAB indicating that the nature of the surfactant has a significant effect on the microenvironment of the protein-electrode interface. The results have been used to understand the mechanism of electron transfer from cytochrome c to the copper center during the enzymatic reaction.     

Photoisomerization of electroactive polyimide/multiwalled carbon nanotube composites on the effect of electrochemical sensing for ascorbic acid

We present the first investigation of photoisomerization of the azo‐based electroactive polyimide (PI)/amino‐functionalized multiwalled carbon nanotube (MWCNT) composite electrode on the effect of electrochemical sensing for ascorbic acid (AA). First, MWCNTs were grafted with 4‐aminobenzoic acid in a medium of polyphosphoric acid/phosphorous pentoxide to obtain MWCNTs functionalized with 4‐aminobenzoyl groups (AF‐MWCNTs). Subsequently, photoactive and electroactive PI/AF‐MWCNT composites (PEPACCs) were prepared by introducing pendant conjugated oligoaniline (amino‐capped aniline trimer) in the main chain and azobenzene chromophores in the side chain, in the presence of AF‐MWCNTs. Photoactive and electroactive PI (PEPI) and PEPACCs were characterized by ¹H NMR spectra, UV?visible absorption spectra, cyclic voltammetry (CV) and transmission electron microscopy. The CV study shows that the PEPACCs have higher electroactivity than PEPI. The redox and reversible photoisomerization (i.e. cis ? trans) behavior of PEPACCs was analyzed by in situ monitoring through systematic studies of CV and UV?visible spectroscopy. The light of the UV lamp was 365 nm. It should be noted that the sensor constructed from a trans‐PEPACC‐modified carbon‐paste electrode (CPE) demonstrated a higher electrocatalytic activity by 2.75‐fold and 1.12‐fold towards the oxidation of AA compared with those constructed using a PEPI‐ and cis‐PEPACC‐modified CPE, respectively. The detection limit of the trans‐PEPACC‐modified electrode was 1.73‐fold and 1.70‐fold lower than that of PEPI‐ and cis‐PEPACC‐modified CPE. Moreover, the differential pulse voltammetry data showed that the trans‐PEPACC‐modified electrode had high electrochemical sensing ability for the determination of AA, dopamine and uric acid. © 2014 Society of Chemical Industry