Direct electron transfer and electrochemical study of hemoglobin immobilized in ZnO hollow spheres

ZnO hollow spheres were firstly prepared. A new type of amperometric hydrogen peroxide biosensor was fabricated by entrapping Hemoglobin (Hb) through the ZnO hollow spheres (ZHS) nanoparticles. The composition morphology and size were studied by transmission electron microscopy. The surface topography of the prepared films was imaged by atomic force microscope (AFM). Several techniques, including UV–vis absorption spectroscopy, cyclic voltammetry, chronoamperometry were employed to characterize the performance of the biosensor. The results indicated that the ZHS nanoparticles had enhanced the performance of the hydrogen peroxide sensors. The electrochemical parameters of Hb in the ZHS were calculated by the results of the electron-transfer coefficient (α) and the apparent heterogeneous electron-transfer rate constant K _s as 0.5 and 3.1 s⁻¹, respectively. The resulting biosensors showed a wide linear range from 2.1 × 10⁻⁶ to 5.18 × 10⁻³ M, with a low detection limit of 7.0 × 10⁻⁷ M (S/N = 3) under optimized experimental conditions. The results demonstrated that the ZHS matrix may improve the protein loading with the retention of bioactivity and greatly promote the direct electron transfer, which can be attributed to its unique morphology, high specific surface area, and biocompatibility. The biosensor obtained from this study possesses high sensitivity, good reproducibility, and long-term stability.     

Amperometric biosensor for hydrogen peroxide based on horseradish peroxidase onto gold nanowires and TiO2 nanoparticles

An electrochemical biosensor for determination of hydrogen peroxide (H₂O₂) was fabricated, based on the electrostatic immobilization of horseradish peroxidase (HRP) with one-dimensional gold nanowires (Au NWs) and TiO₂ nanoparticles (nano-TiO₂) on a gold electrode. The nano-TiO₂ can give a biocompatible microenvironment and compact film, and the Au NWs can provide fast electron transferring rate and greatly add the amount of HRP molecules immobilized on the electrode surface. Au NWs were characterized by ultraviolet–visible spectra and transmission electron microscope. The electrode modification process was probed by cyclic voltammetry and electrochemical impedance spectroscopy. Chronoamperometry was used to study the electrochemical performance of the resulting biosensor. Under optimal conditions, the linear range for the determination of H₂O₂ was from 2.3 × 10⁻⁶ to 2.4 × 10⁻³ M with a detection limit of 7.0 × 10⁻⁷ M (S/N = 3). Moreover, the proposed biosensor showed superior stability and high sensitivity.     

Immobilizing Pt nanoparticles and chitosan hybrid film on polyaniline naofibers membrane for an amperometric hydrogen peroxide biosensor

A convenient and effective way for fabricating amperometric hydrogen peroxide (H₂O₂) biosensor was designed in this paper. First, the polyaniline (PANI) nanofibers membrane with good conductance and high surface area was electropolymerized on a gold electrode surface. Then, Pt nanoparticle (PtNP) was electrochemically deposited on the PANI nanofibers membrane. Finally, the hybrid film of gold nanoparticle, chitosan, and horseradish peroxidase (HRP) was cast onto the modified electrode to form a stable biofunctional film, which was also employed as a protective layer to PtNP. The proposed biosensor exhibited a rapid response to H₂O₂ with the linear range from 7.0 × 10⁻⁶ to 1.4 × 10⁻² M and a detection limit of 2.8 × 10⁻⁶ M (S/N = 3). The sensitivity of 558 μA mM⁻¹ cm⁻² was obtained. The Michaelis–Menten constant, K_\textM^\textapp K_{\text{M}}^{\text{app}} value was 1.90 mM suggesting a high affinity. Moreover, it displayed a good reproducibility and long-term stability.     

Electrochemical properties of heme-protein in lauric acid films and its application as a biosensor

In this research, the enhancement of electron-transfer activity of hemoglobin (Hb) in lauric acid film was investigated for the first time. This type of composite film was made on a glassy carbon electrode by a casting method. Cyclic voltammetric result of the modified electrode displays a well-defined redox peak, which was attributed to the direct electrochemical response of Hb. Our results illustrate that Hb exchange electrons directly with electrode and exhibits the characteristics of peroxidase. When we use this modified electrode as a biosensor, it gives excellent performance in the electrocatalytic reduction of hydrogen peroxide (H₂O₂). The parameters such as pH and applied potential of the biosensor influencing in H₂O₂ detection were optimized carefully. Through the optimal conditions, the proposed biosensor shows the linear range for H₂O₂ determination was from 1×10⁻⁵ to 1.25×10⁻⁴ mol L⁻¹ with a detection limit of 1×10⁻⁷ mol L⁻¹. The biosensor retained more than 90% of the initial response after 14 d.     

Hydrogen peroxide sensor based on Prussian blue electrodeposited on (3-mercaptopropyl)-trimethoxysilane polymer-modified gold electrode

A hydrogen peroxide (H₂O₂) sensor was developed by electrodepositing Prussian blue (PB) on a gold electrode modified with (3-mercaptopropyl)-trimethoxysilane (MPS) polymer. The characterization of the self-assembled electrode was investigated by cyclic voltammetry and electrochemical impedance spectroscopy. The results of electrochemical experiments showed that such constructed sensor had a favorable catalytic ability to reduce H₂O₂. The MPS film on the modified gold electrode greatly enhanced the pH-adaptive range of PB. Large surface-to-volume ratio property of double-layer 2d-network MPS-modified PB electrode enabled stable and highly sensitive performance of the non-enzymatic H₂O₂ sensor. The linear range of H₂O₂ determined is from 2.0 × 10⁻⁶ to 2.0 × 10⁻⁴ mol L⁻¹ with a correlation coefficient of 0.9991 and a detection limit for H₂O₂ of 1.8 × 10⁻⁶ mol L⁻¹. The influences of the potentially interfering substances on the determination of H₂O₂ were investigated. This modified electrode exhibits a good selectivity and high sensitivity with satisfactory results.     

A mediator-free amperometric hydrogen peroxide biosensor based on HRP immobilized on a nano-Au/poly 2,6-pyridinediamine-coated electrode

A mediator-free amperometric hydrogen peroxide biosensor was prepared by immobilizing horseradish peroxidase (HRP) enzyme on colloidal Au modified platinum (Pt) wire electrode, which was modified by poly 2,6-pyridinediamine (pPA). The modified process was characterized by electrochemical impedance spectroscopy (EIS), and the electrochemical characteristics of the biosensor were studied by cyclic voltammetry, linear sweep voltammetry and chronoamperometry. The biosensor displayed an excellent electrocatalytical response to reduction of H₂O₂ without the aid of an electron mediator, the linear range was 4.2 × 10⁻⁷–1.5 × 10⁻³ mol/L (r = 0.9977), with a detection limit of 1.4 × 10⁻⁷ mol/L. Moreover, the performance and factors influencing the resulted biosensor were studied in detail. The studied biosensor exhibited permselectivity, good stability and good fabrication reproducibility.     

Effect of nitrite on growth and microcystins production of <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> PCC7806

As a common pollutant, nitrite concentrations can approach 15 mg NO₂⁻-N L⁻¹ in some aquatic systems. Microcystis aeruginosa blooms are common and widespread in eutrophic freshwater bodies. In this study, M. aeruginosa was exposed to nitrite concentrations ranging from 0 to 15 mg NO₂⁻-N L⁻¹, and the responses of M. aeruginosa were investigated. The specific growth rates, maximum cell densities, light-saturated photosynthetic rates (P_m ^chla ), dark respiration rates (R_d ^chla ), and apparent photosynthetic efficiencies (α^chla ) showed a significant decline with nitrite concentrations increasing. Electrical conductivity and malondialdehyde contents investigation revealed cell membrane damage and apparent leakage of intracellular contents under high nitrite level conditions due to oxidative stress enhancement. Intracellular microcystin (MC)-LR content reached the highest value at 10 mg NO₂⁻-N L⁻¹; however, extracellular MC-LR contents showed a continuous increase until 15 mg NO₂⁻-N L⁻¹ owing to the increasing leakage of intracellular contents. These results elucidated that the high-level nitrite inhibited M. aeruginosa growth by rising oxidative stress, damaging cell membrane, and reducing photosynthesis. However, the moderate increase in nitrite concentrations promoted toxin production and release of toxin.     

Isoniazid and rifampicin inhibit allosterically heme binding to albumin and peroxynitrite isomerization by heme–albumin

Human serum heme–albumin (HSA-heme) displays globin-like properties. Here, the allosteric inhibition of ferric heme [heme-Fe(III)] binding to human serum albumin (HSA) and of ferric HSA–heme [HSA-heme-Fe(III)]-mediated peroxynitrite isomerization by isoniazid and rifampicin is reported. Moreover, the allosteric inhibition of isoniazid and rifampicin binding to HSA by heme-Fe(III) has been investigated. Data were obtained at pH 7.2 and 20.0 °C. The affinity of isoniazid and rifampicin for HSA [K ₀ = (3.9 ± 0.4) × 10⁻⁴ and (1.3 ± 0.1) × 10⁻⁵ M, respectively] decreases by about 1 order of magnitude upon heme-Fe(III) binding to HSA [K _h = (4.3 ± 0.4) × 10⁻³ and (1.2 ± 0.1) × 10⁻⁴ M, respectively]. As expected, the heme-Fe(III) affinity for HSA [H ₀ = (1.9 ± 0.2) × 10⁻⁸ M] decreases by about 1 order of magnitude in the presence of saturating amounts of isoniazid and rifampicin [H _d = (2.1 ± 0.2) × 10⁻⁷ M]. In the absence and presence of CO₂, the values of the second-order rate constant (l _on) for peroxynitrite isomerization by HSA-heme-Fe(III) are 4.1 × 10⁵ and 4.3 × 10⁵ M⁻¹ s⁻¹, respectively. Moreover, isoniazid and rifampicin inhibit dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) in the absence and presence of CO₂. Accordingly, isoniazid and rifampicin impair in a dose-dependent fashion the HSA-heme-Fe(III)-based protection of free l-tyrosine against peroxynitrite-mediated nitration. This behavior has been ascribed to the pivotal role of Tyr150, a residue that either provides a polar environment in Sudlow’s site I (i.e., the binding pocket of isoniazid and rifampicin) or protrudes into the heme-Fe(III) cleft, depending on ligand binding to Sudlow’s site I or to the FA1 pocket, respectively. These results highlight the role of drugs in modulating heme-Fe(III) binding to HSA and HSA-heme-Fe(III) reactivity.     

Properties of native and hydrophobic laccases immobilized in the liquid-crystalline cubic phase on electrodes

Both native Trametes hirsuta laccase and the same laccase modified with palmytic chains to turn it more hydrophobic were prepared and studied with cyclic voltammetry and Raman spectroscopy. Native laccase immobilized in the monoolein cubic phase was characterized with resonance Raman spectroscopy, which demonstrated that the structure at the “blue” copper site of the protein remained intact. The diamond-type monoolein cubic phase prevents denaturation of enzymes on the electrode surface and provides contact of the enzyme with the electrode either directly or through the mediation by electroactive probes. Direct electron transfer for both laccases incorporated into a lyotropic liquid crystal was obtained under anaerobic conditions, whereas bioelectrocatalytic activity was shown only for the native enzyme. The differences in electrochemical behavior of native and hydrophobic laccase as well as possible mechanisms of direct and mediated electron transfers are discussed. The Michaelis constant for 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS²⁻), K _M^app, and the maximal current, I _max, for the native enzyme immobilized onto the electrode were estimated to be 0.24 mM, and 5.3 μA, respectively. The maximal current density and the efficiency of the catalysis, I _max/K _M^app, were found to be 73 μA cm⁻² and 208.2 μA cm⁻² mM⁻¹, respectively, and indicated a high efficiency of oxygen electroreduction by the enzyme in the presence of ABTS²⁻ in the cubic-phase environment. Rate constants were calculated to be 7.5 × 10⁴ and 3.6 × 10⁴ M⁻¹ s⁻¹ for native and hydrophobic laccase, respectively.     

An altered camelid-like single domain anti-idiotypic antibody fragment of HM-1 killer toxin: acts as an effective antifungal agent

Phage-display and competitive panning elution leads to the identification of minimum-sized antigen binders together with conventional antibodies from a mouse cDNA library constructed from HM-1 killer toxin neutralizing monoclonal antibody (nmAb-KT). Antigen-specific altered camelid-like single-domain heavy chain antibody (scFv K2) and a conventional antibody (scFv K1) have been isolated against the idiotypic antigen nmAb-KT. The objectives of the study were to examine (1) their properties as compared to conventional antibodies and also (2) their antifungal activity against different pathogenic and non-pathogenic fungal species. The alternative small antigen-binder, i.e., the single-domain heavy chain antibody, was originated from a conventional mouse scFv phage library through somatic hyper-mutation while selection against antigen. This single-domain antibody fragment was well expressed in bacteria and specifically bound with the idiotypic antigen nmAb-KT and had a high stability and solubility. Experimental data showed that the binding affinity for this single-domain antibody was 272-fold higher (K _d = 1.07 × 10⁻¹⁰ M) and antifungal activity was three- to fivefold more efficient (IC₅₀ = 0.46 × 10⁻⁶ to 1.17 × 10⁻⁶ M) than that for the conventional antibody (K _d = 2.91 × 10⁻⁸ M and IC₅₀ = 2.14 × 10⁻⁶ to 3.78 × 10⁻⁶ M). The derived single-domain antibody might be an ideal scaffold for anti-idiotypic antibody therapy and the development of smaller peptides or peptide mimetic drugs due to their less complex antigen-binding site. We expect that such single-domain synthetic antibodies will find their way into a number of biotechnological or medical applications.     

The effects of copper on the photosynthetic response of <Emphasis Type="Italic">Phaeocystis cordata</Emphasis>

We investigated the effects of limiting (1.96 × 10⁻⁹ mol l⁻¹ total Cu, corresponding to pCu 14.8; where pCu = −log [Cu²⁺]) and toxic Cu concentrations up to 8.0 × 10⁻⁵ mol l⁻¹ total Cu (equivalent to pCu 9.5) on growth rates and photosynthetic activity of exponentially grown Phaeocystis cordata, using batch and semi-continuous cultures. With pulse amplitude modulated (PAM) fluorometry, we determined the photochemical response of P. cordata to the various Cu levels, and showed contrasting results for the batch and semi-continuous cultures. Although maximum photosystem II (PSII) quantum yield (Φ_M) was optimal and constant in the semi-continuous P. cordata, the batch cultures showed a significant decrease in Φ_M with culture age (0–72 h). The EC50 for the batch cultures was higher (2.0 × 10⁻¹⁰ mol l⁻¹, pCu9.7), than that for the semi-continuous cultures (6.3 × 10⁻¹¹ mol l⁻¹, pCu10.2). The semi-continuous cultures exhibited a systematic and linear decrease in Φ_M as Cu levels increased (for [Cu²⁺] < 1.0 × 10⁻¹² mol l⁻¹, pCu12.0), however, no effect of high Cu was observed on their operational PSII quantum yield (Φ′_M). Similarly, semi-continuous cultures exhibited a significant decrease in Φ_M, but not in Φ′_M, because of low-Cu levels. Thus, Cu toxicity and Cu limitation damage the PSII reaction centers, but not the processes downstream of PSII. Quenching mechanisms (NPQ and Q _n) were lower under high Cu relative to the controls, suggesting that toxic Cu impairs photo-protective mechanisms. PAM fluorometry is a sensitive tool for detecting minor physiological variations. However, culturing techniques (batch vs. semi-continuous) and sampling time might account for literature discrepancies on the effects of Cu on PSII. Semi-continuous culturing might be the most adequate technique to investigate Cu effects on PSII photochemistry.     

Bioelectrical activity in the heart of the lugworm Arenicola marina

Standard microelectrode technique was used to study electrical activity of the isolated heart of the polychaete annelid, Arenicola marina. Typical pacemaker activity with slow diastolic depolarization was observed in all recordings. The average maximum diastolic potential (−58.4 ± 3.2 mV), the average amplitude of the action potential (28.7 ± 4.7 mV) and the average total duration of the action potential (2,434 ± 430 ms) were determined. There has been no gradient of automaticity observed in our studies, which suggests that all regions of the Arenicola heart could possess pacemaker functions. Acetylcholine (ACh) produced a concentration dependent (5 × 10⁻⁸–5 × 10⁻⁵ M) increase of the beating rate via increase in the rate of the diastolic depolarization. ACh (5 × 10⁻⁵ M) increased beating rate by 2.5-fold compared to the control rate. A stronger action of ACh resulted in depolarization, block of action potential generation and contracture of the heart. The non-hydrolysable ACh analog carbacholine (10⁻⁸–10⁻⁶ M) produced similar effects. All effects of ACh and carbacholine were abolished by 5 × 10⁻⁶ M atropine. d-Tubocurarine (5 × 10⁻⁵ M) did not significantly alter effects of ACh or carbacholine. Epinephrine (10⁻⁸–10⁻⁶ M) caused the slowing of pacemaker activity and marked decrease of action potential duration. 10⁻⁶ M epinephrine produced complete cardiac arrest. The effects of epinephrine were not significantly altered by the β-blocker propranolol (5 × 10⁻⁶ M). The β-agonist isoproterenol (10⁻⁷–10⁻⁵ M) and the α-agonist xylometazoline (10⁻⁶–10⁻⁵ M) did not produce significant effects. Thus, cholinergic effects in the Arenicola heart are likely to be mediated via muscarinic receptors, while the nature of adrenergic effects needs further investigation.     

A novel bifunctional endo-/exo-type cellulase from an anaerobic ruminal bacterium

An anaerobic microorganism termed AN-C16-KBRB was isolated from the bovine rumen and demonstrated cellulolytic activity on a NB agar plate containing azo-carboxymethyl cellulose. The 16S rRNA gene of the strain was 98% similar to that of Clostridiaceae bacterium SK082 (AB298754) as the highest homology. A novel celEdx16 gene encoding a bifunctional endo-/exocellulase (CelEdx16) was cloned by the shotgun method from AN-C16-KBRB, and the enzyme was characterized. The celEdx16 gene had an open reading frame of 1,104-base pairs, which encoded 367 amino acids to yield a protein of molecular mass 40.4 kDa. The amino acid sequence was 53% identical to that of an endoglucanase from Clostridium thermocellum. CelEdx16 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The specific endocellulase and exocellulase activities of CelEdx16 were 15.9 and 3.6 × 10⁻² U mg⁻¹, respectively. The Michaelis–Menten constant (K _m values) and the maximal reaction velocities (V _max values) of CelEdx16 were 47.1 μM and 9.6 × 10⁻³ μmole min⁻¹ when endocellulase activity was measured and 106.3 μM and 2.1 × 10⁻⁵ μmole min⁻¹ when exocellulase activity was assessed. CelEdx16 was optimally active at pH 5.0 and 40°C.     

Characterisation of acidic sites of Pseudomonas biomass capable of binding protons and cadmium and removal of cadmium via biosorption

Summary The ability of Pseudomonas aeruginosa to accumulate Cd(II) ions from wastewater industries was experimentally investigated and mathematically modelled. From the potentiometric titration and non-ideal competitive analysis (NICA) model, it was found that the biomass contains three acidic sites. The values of proton binding (pK _i =1.66±3.26×10⁻³, 1.92±1.63×10⁻⁴ and 2.16±3.79×10⁻⁴) and binding constant of cadmium metal ions (pK _M1=1.99±2.45×10⁻³ and pK _M2=1.67±4.08×10⁻³) on the whole surface of biomass showed that protonated functional groups and biosorption of Cd(II) ions could be attributed to a monodentate binding to one acidic site, mainly the carboxylic group. From the isothermal sorption experimental data and Langmuir model, it was also found that the value of Langmuir equilibrium (pK _f) constant is 2.04±2.1×10⁻⁵ suggesting that the carboxyl group is the main active binding site. In addition, results showed that the maximum cadmium capacity (q _max) and affinity of biomass towards cadmium metal ions (b) at pH 5.1 and 20 min were 96.5±0.06 mg/g and 3.40×10⁻³± 2.10×10⁻³, respectively. Finally, interfering metal ions such as Pb(II), Cu(II), Cr(III), Zn(II), Fe(II), Mn(II), Ca(II) and Mg(II) inhibited Cd(II) uptake. Comparing the biosorption of Cd(II) by various Pseudomonas isolates from contaminated environment samples (soil and sewage treatment plant) showed that maximum capacities and equilibrium times were different, indicating that there was a discrepancy in the chemical composition between biomasses of different strains.     

Copper (II) complex of 1,10-phenanthroline and <Emphasis Type="SmallCaps">l</Emphasis>-tyrosine with DNA oxidative cleavage activity in the gallic acid

Copper (II) complex of formulation [Cu–Phen–Tyr](H₂O)](ClO₄) (Phen = 1,10-phenanthroline, l-Tyr = l-tyrosine), has been prepared, and their induced DNA oxidative cleavage activity studied. The complex binds to DNA by intercalation, as deduced from the absorption and fluorescence spectral data. Scatchard plots constructed from the absorption titration data gave binding constant 2.44 × 10⁴ M⁻¹ of base pairs. Extensive hypochromism, broadening, and red shifts in the absorption spectra were observed. Upon binding to DNA, the fluorescence from the DNA–ethidium bromide system was efficiently quenched by the copper (II) complex. Stern–Volmer quenching constant 0.61 × 10³ M⁻¹ obtained from the linear quenching plots. [Cu–Phen–Tyr] complex efficiently cleave the supercoiled DNA to its nicked circular form with gallic acid as biological reductant at appropriate complex concentration. The gallic acid as reductant could observably improve copper (II) complex to DNA damage. The pseudo-Michaelis–Menten kinetic parameters (k _cat, K _M) were calculated to be 1.32 h⁻¹ and 5.46 × 10⁻⁵ M for [Cu–Phen–Tyr] complex. Mechanistic studies reveal the involvement of superoxide anions and hydroxyl radical (HO^·) as the reactive species under an aerobic medium.     

Evaluation and optimization of factors affecting the performance of a flow-through system based on immobilized acetylcholineesterase as a biosensor

A flow-through system based on acetylcholineesterase (AChE) was studied. The system was prepared by mixing AChE and a multiwall carbon nanotube (MWCNT). Two important parameters, the ratios of AChE:MWCNT (X₁) and AChE-MWCNT:sol-gel (X₂) were optimized using response surface methodology. The results revealed that an enzyme immobilized within the MWCNT-sol-gel was more effective compared to one conducted with sol-gel. The optimum feed flow rate was 0.4 mL/min and ATChI concentration was found to be 1 mM. The optimum ratios of X₁ and X₂ for immobilization on ceramic packing were 1.07 and 0.43, respectively. The sensitivity of this flow-through system was 1.82 × 10⁻⁵/μM and long-term stability analyzed after 120 days was 74% of initial absorbance. With respect to an incubation time of 14 min, the detection limit for paraoxon was 7.3 × 10⁻¹² mol.     

A hydrogen peroxide biosensor based on the direct electrochemistry of hemoglobin modified with quantum dots

Direct electron transfer of hemoglobin modified with quantum dots (QDs) (CdS) has been performed at a normal graphite electrode. The response current is linearly dependent on the scan rate, indicating the direct electrochemistry of hemoglobin in that case is a surface-controlled electrode process. UV–vis spectra suggest that the conformation of hemoglobin modified with CdS is little different from that of hemoglobin alone, and the conformation changes reversibly in the pH range 3.0–10.0. The hemoglobin in a QD film can retain its bioactivity and the modified electrode can work as a hydrogen peroxide biosensor because of its peroxidase-like activity. This biosensor shows an excellent response to the reduction of H₂O₂ without the aid of an electron mediator. The catalytic current shows a linear dependence on the concentration of H₂O₂ in the range 5 × 10⁻⁷–3 × 10⁻⁴ M with a detection limit of 6 × 10⁻⁸ M. The response shows Michaelis–Menten behavior at higher H₂O₂ concentrations and the apparent Michaelis–Menten constant is estimated to be 112 μM.     

Mycelial cultivation of <Emphasis Type="Italic">Phellinus linteus</Emphasis> using cheese-processing waste and optimization of bioconversion conditions

A medicinal mushroom, Phellinus linteus, was successfully cultivated using a cheese-processing waste, whey, and the optimal bioconversion conditions for the maximum mycelial growth rate was also estimated through solid-state cultivation experiments. Response surface analysis with a face-centered design (center point replication = 5) was applied to statistically approximate the simultaneous effects of the three variables, i.e., substrate concentration (10–30 g lactose l⁻¹), temperature (20–30°C), and pH (4–6), on the mycelial growth rate of P. linteus. The following is a partial cubic model where η is the mycelial growth rate (K _r ) and x _k is the corresponding variable term (k = substrate concentration, temperature, and pH in order): η = −23.8 + 8.67 × 10⁻² x ₁ + 1.48x ₂ + 1.77x ₃ + 8.00 × 10⁻⁴ x ₁ x ₂ + 7.25 × 10⁻² x ₁ x ₃ + 5.13 × 10⁻² x ₂ x ₃ −1.28 × 10⁻² x ₁² –3.18 × 10⁻² x ₂². −2.64 × 10⁻¹ x ₃² −3.28 × 10⁻³ x ₁ x ₂ x ₃ + 4.68 × 10⁻⁴ x ₁² x ₂. The produced response surface model proved to be significant (r ² > 0.99, P-value <0.0001, coefficient of variation <5%) to describe the explored space. Temperature was found to be the most significant factor of dominant effects on the mycelial growth rate, and other variables such as temperature², pH, pH², and (substrate concentration² × temperature) also showed significant effects on the model output. The maximum mycelial growth rate was predicted to be 2.80 mm d⁻¹ at 29.7 g lactose l⁻¹, 26.2°C, and pH 5. Our results proved a good potential of whey to serve as an alternative growth medium for cultivating P. linteus mycelia. This may provide another potential for managing this nutrient-rich waste in a cost-effective way.     

Stimulation and oxidative catalytic inactivation of thermolysin by copper•Cys-Gly-His-Lys

[Cu²⁺•Cys-Gly-His-Lys] stimulates thermolysin (TLN) activity at low concentration (below 10 μM) and inhibits the enzyme at higher concentration, with binding affinities of 2.0 and 4.9 μM, respectively. The metal-free Cys-Gly-His-Lys peptide also stimulates TLN activity, with an apparent binding affinity of 2.2 μM. Coordination of copper through deprotonated imine nitrogens, the histidyl nitrogen, and the free N-terminal amino group is consistent with the characteristic absorption spectrum of a Cu²⁺–amino-terminal copper and nickel binding motif (λ _max ∼ 525 nm). The lack of thiol coordination is suggested by both the absence of a thiol to Cu²⁺ charge transfer band and electrochemical studies, since the electrode potential (vs. Ag/AgCl) 0.84 V (ΔE = 92 mV) for the Cu^3+/2+ redox couple obtained for [Cu²⁺•Cys-Gly-His-Lys] was found to be in close agreement with that of a related complex [Cu²⁺•Lys-Gly-His-Lys]⁺ (0.84 V, ΔE = 114 mV). The N-terminal cysteine appears to be available as a zinc-anchoring residue and plays a critical functional role since the [Cu²⁺•Lys-Gly-His-Lys]⁺ homologue exhibits neither stimulation nor inhibition of TLN. Under oxidizing conditions (ascorbate/O₂) the catalyst is shown to mediate the complete irreversible inactivation of TLN at concentrations where enzyme activity would otherwise be stimulated. The observed rate constant for inactivation of TLN activity was determined as k _obs = 7.7 × 10⁻² min⁻¹, yielding a second-order rate constant of (7.7 ± 0.9) × 10⁴ M⁻¹ min⁻¹. Copper peptide mediated generation of reactive oxygen species that subsequently modify active-site residues is the most likely pathway for inactivation of TLN rather than cleavage of the peptide backbone.     

Interfacial Tension of the Lipid Membrane Formed from Phosphatidylcholine–Decanoic Acid and Phosphatidylcholine–Decylamine Systems

Interfacial tension has been determined for phosphatidylcholine (PC)–decanoic acid (DA) and PC–decylamine (DE) membranes. PC (lecithin), DA and DE were used in the experiments; the interfacial tension values of the pure components are 1.62 × 10⁻³, −2.38 × 10⁻² and −3.88 × 10⁻² N/m (hypothetical values for DA and DE), respectively. The 1:1 complexes were formed during formation of PC–DA and PC–DE membranes. The following parameters describing the complexes were determined: the surface concentrations of the lipid membranes formed from these complexes, A₃ ^{- 1} A_{3}^{ - 1} ; the interfacial tensions of such membranes, γ ₃; and the stability constants of these complexes, K.