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.     

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.     

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.     

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     

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     

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     

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.     

Thermal deactivation and inhibition of D‐Amino acid oxidase in permeabilized cells of the yeast Trigonopsis variabilis

The inhibition of D ‐amino acid oxidase contained in permeabilized cells of the yeast Trigonopsis variabilis by α‐keto acids (pyruvic acid, phenylpyruvic acid and 4‐methylthio‐2‐oxobutanoic acid), products of the transformation of the corresponding D ‐amino acids, was studied. In all cases, inhibition was of the mixed type and significant differences with respect to the inhibition shown by the enzyme from other sources such as pig kidney or the yeast Rhodotorula gracilis were observed. A study was also made of the thermal deactivation of the enzyme contained in permeabilized cells of T variabilis in the temperature range 30–50 °C in sodium phosphate and Tris hydroxylmethyl aminomethane + CaCl₂ buffers. A deactivation mechanism with two steps in series is proposed to account for the variation in activity with time. The results suggest that the enzyme shows greater stability in phosphate buffer, with half‐lives between 7.6 days at 30 °C and 8.6 h at 50 °C. Copyright © 2004 Society of Chemical Industry     

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.     

Construction of a choline biosensor through enzyme immobilization on a poly(2‐hydroxyethyl methacrylate)‐grafted Teflon film

An amperometric choline biosensor was constructed by immobilizing choline oxidase (ChO) on poly(2‐hydroxyethyl methacrylate) (PHEMA)‐grafted Teflon (polytetrafluoroethylene, PTFE) film. Grafting was achieved by γ irradiation. PHEMA‐grafted Teflon films were activated with epichlorohydrin or glutaraldehyde to achieve covalent immobilization of enzyme onto the film. To decrease the diffusional barrier caused by the enzyme‐immobilized film, the film was stretched directly on the electrode. The PHEMA‐grafted Teflon film, therefore, had to have appropriate mechanical properties. Glucose oxidase (GOD) was used in the determination of optimum immobilization conditions, then these were applied to ChO. With GOD, the effect of activation type and film position in electrode on enzyme activity was studied and the highest catalytic activity was obtained when the enzyme was immobilized using glutaraldehyde and the film was stretched over the electrode surface. Further studies revealed that the films activated with glutaraldehyde, immobilized in 2 mg/mL ChO concentration, and stretched directly on the electrode were suitable (specific activity, 0.427 ± 0.068 U mg^?1) for use in the choline biosensor. The linear working range of this biosensor was found to be 52–348 μM, with a 40 ± 5 μM minimum detection limit. The response of the sensor, however, decreased linearly upon repeated use. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2007     

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     

Effective biosynthesis of poly(3‐hydoxy‐ butyrate‐co‐4‐hydroxybutyrate) with high 4‐hydroxybutyrate fractions by Wautersia eutropha in the presence of α‐amino acids

BACKGROUND: Biopolymers produced by microbes are in demand as their biodegradable and biocompatible properties make them suitable for disposable products and for potential use as biomaterials for medical applications. The effective microbial production of copolyesters of 3‐hydroxybutyrate (3HB) and 4‐hydroxybutyrate(4HB) with high molar fractions of 4HB unit by a wild‐type Wautersia eutropha H16 was investigated in culture media containing 4‐hydroxybutyric acid (4HBA) and different carbon substrates in the presence of various α‐amino acids. RESULTS: The addition of carbon sources such as glucose, fructose and acetic acid to the culture medium containing 4HBA in the presence of α‐amino acids resulted in the production of random poly(3HB‐co‐4HB) with compositions of up to 77 mol% 4HB unit, but the yields of copolyesters with 60–77 mol% 4HB units were less than 15 wt% of dried cell weights. In contrast, when carbon sources such as propionic acid and butyric acid were used as the co‐substrates of 4HBA in the presence of α‐amino acids, poly(3HB‐co‐4HB) copolyesters with compositions of 72–86 mol% 4HB were produced at maximally 47.2 wt% of dried cell weight (11.3 g L^?1) and the molar conversion yield of 4HBA to 4HB fraction in copolyesters was as high as 31.4 mol%. Further, poly(3HB‐co‐4HB) copolyesters with compositions of 93–96 mol% 4HB were isolated at up to 35.2 wt% of dried cell weights by fractionation of the above copolymers with chloroform/n‐hexane. CONCLUSION: The productivity of copolyesters with over 80 mol% 4HB fractions was as high as 0.146 g L^?1 h^?1 (3.51 g L^?1 for 24 h) by flask batch cultivation. Copyright © 2007 Society of Chemical Industry     

Study on a carboxyl‐activated carrier and its properties for papain immobilization

BACKGROUND: Most enzymes, including protease, play a key role in biotechnology, but their use is quite limited due to poor recovery, limited reusability and instability. Immobilized enzymes offer advantages over free enzymes. This paper reports a simple method for the preparation of immobilized papain, an endolytic cysteine protease (EC: 3.4.22.2), on carboxyl‐activated silica nanoparticles. RESULTS: The carboxyl‐activated carriers produced reactive carboxyl groups which then react with the free amino groups of enzyme to give peptide bonds (? CO? NH? ). The results showed that the thermal and pH stabilities of the immobilized papain were higher than those of free enzyme. And the apparent K_m value of the immobilized papain was 1.26 times higher than that of free enzyme. Moreover, the immobilized papain retained more than 45% of the original activity after ten reuses continuously. CONCLUSION: The results indicated that papain was successfully immobilized on the surface of the activated carriers. The immobilized papain had not only higher activity recovery, but also better stability, reusability and environmental adaptability. Copyright © 2012 Society of Chemical Industry     

Four Amino Acid Residues Influence the Substrate Chain-Length and Regioselectivity of Siganus canaliculatus Δ4 and Δ5/6 Desaturases

Although ω3‐ and ω6‐ desaturases have been well studied in terms of substrate preference and regiospecificity, relatively little is known about the membrane‐bound, “front‐end” long chain fatty acid desaturases, such as ?4, Δ5 or Δ6 desaturases. The first vertebrate ?4 desaturase was recently identified in the marine teleost fish Siganus canaliculatus (S. canaliculatus), which also possesses a bifunctional Δ5/6 desaturase. These two long chain polyunsaturated fatty acid desaturases are very different in terms of regiospecificity and substrate chain‐length, but share an unusually high degree of amino acid identity (83 %). We took advantage of this similarity by constructing a series of chimeric enzymes, replacing regions of one enzyme with the corresponding sequence of the other. Heterologous expression of the chimeric series of enzymes in yeast indicated that the substitution of a four amino acid region was sufficient to convert a ?4 desaturase to an enzyme with ?6 desaturase activity, and convert a ?5/6 desaturase to an enzyme with a low level of ?4 desaturase activity. In addition, enzymes having both ?4 and ?6 desaturase activities were produced by single or double amino acid substitutions within this four‐amino acid region.     

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     

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.     

Identification and Characterization of Citrus Peel Uronic Acid Oxidase

Uronic acid-rich plant materials such as pectin are potential renewable feedstocks for the chemical industry. Uronic acid oxidase activity was first reported in extracts of citrus leaves, and was subsequently found to be widely distributed in plants, with the highest activity detected in extracts of the pectin-rich citrus peel. Herein we report the identification of the primary sequence of uronic acid oxidase from extracts of the peel of Citrus sinensis, by partial purification and protein mass spectrometry. Activity of the enzyme, a member of the berberine bridge family, was confirmed by recombinant expression in Pichia pastoris. Biochemical characterization of the recombinant enzyme is reported. Our findings facilitate further investigation of the biological function of uronic acid oxidation in the economically important orange fruit, and also provide a basis for the development of a catalyst for bioconversion of uronic acids.