Immobilization of AMP-deaminase in acrylamide-glycidylmethacrylate copolymer

The enzyme AMP-deaminase was entrapped in an acrylamide-glycidylmethacrylate copolymer. No leakage of AMP-deaminase was observed from the copolymer. The optimum pH of native AMP-deaminase was 5.6. The pH was displaced to 5.0 with immobilization in the copolymer. The immobilized AMP-deaminase column maintained more than 95% of initial activity after 10 days of operation.     

Ammonium ion sensor based on immobilized nitrifying bacteria and a cation-exchange membrane

The ammonium ion sensor is based on nitrifying bacteria isolated from activated sludge. The sensor comprises a cation-exchange membrane, an alkaline solution layer (pH 10), a gas-permeable membrane, an immobilized microbial membrane, and an oxygen electrode. This novel combination provides accurate amperometric determinations of ammonium ions in aqueous solutions within 7 min in the range 10^-4– 4.5 × 10^-2 M. Volatile amines or other ions do not interfere. The relative error is within 4% and the sensor can be used continually for more than 10 days.     

Alcohol-FET sensor based on a complex cell membrane enzyme system

An alcohol -FET sensor was developed by use of a complex enzyme system in a cell membrane and an ion-sensitive field effect transistor (ISFET). The cell membrane of Gluconobacter suboxydans IFO 12528, which converts ethanol to acetic acid, was immobilized on the gate of an ISFET with calcium alginate gel coated with nitrocellulose. This ISFET (1), a reference ISFET without the cell membrane (ISFET 2) and an Ag/AgCl reference electrode were placed in 5 mM Trismalate buffer (pH 5.5, 25°C), and the differential output between ISFETS 1 and 2 was measured. The output of the sensor was stabilized by adding pyrroloquinoline quinone. The response time was ca. 10 min., and there was a linear relationship between the differential output voltage and the ethanol concentration up to 20 mg l^?1. The output of the sensor was stable for 40 h below 30°C. The sensor responded to ethanol, propan- 1-ol and butan- 1-ol, but not to methanol, propan-2-ol and butan-2-ol. The sensor was used to determine blood ethanol.     

Integrated-circuit bio-calorimetric sensor for glucose

Two integrated-circuit temperature-sensitive devices, each composed of three Darlington-connected npn transistors and a CMOS constant-current circuit, are used for calorimetric determination of glucose concentration. One device is modified with a cellulose triacetate/1,8-diamino-4-aminomethyloctane membrane and used for glutaraldehyde immobilization of glucose oxidase. The difference in steady-state output voltage of the enzyme-modified sensor compared to the unmodified sensor after addition of glucose is related to the temperature change induced by the enzyme-catalyzed reaction. Glucose is determined in the range 5–100 mM after signal analysis by fast-Fourier transform. The enthalpy change approximates to that ofa spherical silicon conduction model.     

Amperometric determination of acetic acid with immobilized Trichosporon brassicae

A microbial sensor consisting of immobilized Trichosporon brassicae, a gas-permeable Teflon membrane and an oxygen electrode is suitable for the continuous determination of acetic acid in fermentation broths. When an acetic acid solution is pumped through the flow system, the current decreases to a steady state with a response time of 8 min; shorter pumping times give peaks which can also be measured. The relationship between the current decrease and the acetic acid concentration is linear up to 54 mg l^-1, with a relative standard deviation of about 6% at the higher concentrations. Selectivity is satisfactory. Results obtained with this sensor and by gas chromatography for a glutamic acid fermentation broth were in good agreement (regression coefficient 1.04). The sensor was stable for more than 3 weeks and 1500 assays.     

Alcohol sensor based on membrane-bound alcohol dehydrogenase

Ethanol is determined by a sensor system using purified, immobilized mernbrane-bound alcohol dehydrogenase frorn Gluconobacter suboxydans, attached to a platinum disk electrode (3 mm diameter), and covered with a dialysis membrane. Hexacyanoferrate (III) is used as the redox acceptor. To correct for the influence of interfering substances, this alcohol sensor is compensated by a control electrode which has no immobilized enzyme. The potential of these platinum electrodes was set at + 350 mV vs. Ag/AgCl. Linearity was observed in the range 0.1–5 mM ethanol, the response time was less than 5 min, the maximum sensitivity was obtained at 45°C and the optimum pH was in the range 4.5–5.5. The sensitivity decreased to 80% of the initial value after 1 month at 30°C. When the alcohol sensor system was applied to the determination of ethanol in alcoholic beverages, a good correlation was obtained between the results and those obtained by gas chromatography.     

Synthesis and characterization of α,α'-bis(aminomethyl)oligothiophenes and their related compounds

We have synthesized and characterized a series of novel compounds of α,α'-bis(arninomethyl)oligothiophenes and their related compounds whose degree of polymerization spans two (dimer) to five (pentamer). The compounds presented in our studies are α,α'-bis[(2,2,5,5-tetramethyl-1-aza-2,5-disilacyclopenthyl)methyl]oligothiophene, α,α'-bis(aminomethyl)oligothiophene dihydrochloride and α,α'-bis(aminomethyl)oligothiophene. These compounds exhibit desired chemical activity while maintaining controllable electronic properties. The synthetic processes of the oligothiophenes are as follows: 2-aminomethylthiophene is first ‘protected’ and the thiophene rings are coupled using standard Grignard methods. The protected groups are removed at the final stage of the reaction. The compounds show unique properties; for example α,α'-bis(aminomethyl)oligothiophene dihydrochloride is soluble in water. The results of electronic spectra and titration are also presented.     

Facile isolation ofEndo-pectate lyase fromErwinia carotovora based on electrostatic interaction

Endo-pectate lyase (PATE) from Erwinia carotovora was selectively cosedimented with extracellularly produced lipopolysaccharide-lipid complex (LPSLC) through dialysis of the cell free culture broth. The selective isolation of PATE was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The cosedimentation of the PATE with LPSLC was initiated by decreasing conductivity of the solution and terminated at approx 1 m siemens (mScm-1). As much as 62% of PATE activity in the culture broth was removed by precipitation. PATE was isolated from the precipitate by gel chromatography. The cosedimentation of PATE with LPSLC was remarkably affected by pH or ionic strength. The addition of polycationic peptide polymyxin B sulfate or a metal chloride affected the interaction. The cosedimentation was diminished by acetylation of the free amino groups of PATE. From these results, it was confirmed that the cosedimentation was induced by electrostatic interaction.     

Amperometric determination of choline and acetylcholine with enzymes immobilized in a photocross-linkable polymer

Choline and acetylcholine sensors were prepared by using choline oxidase and acetylcholinesterase, entrapped in photocross- linkable poly(vinyl alcohol) bearing styrylpyridinium (PVA-SbQ). The measurements were based on the detection of hydrogen peroxide liberated by an enzyme reaction (choline oxidase) or two sequential enzyme reactions (acetylcholine esterase and choline oxidase). The determination range for choline was 2.5-2-150 αmol 1^-1 and for acetylcholine 20-2-750 αmol 1^-1. The response times were 2-2-4 min. The immobilized enzyme membranes stored in a dry state were very stable and no loss of activity was observed after storage for 60 days.     

Application of microbiological sensors in fermentation processes

A review is presented of microbiological sensors which are composed of micro-organisms immobilized in a membrane and coupled to a sensing element. Conventional microbiological sensors such as those for biochemical oxygen demand (BOD), ethanol and acetic acid are discussed briefly. Novel sensors are then described. The sensor for carbon dioxide is based on a chemoautotrophic bacterium, that for alcohol on cell membranes of the acetic acid bacteria, Gluoconabacter suboxydans. Sensors for BOD carbon dioxide are based on thermophilic bacteria. Finally, a microbial field effect transistor sensor (FET) for alcohol sensor is described. For all the sensors, the ranges of linear response and their long-term stabilities are reported.