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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
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Email: schalk@unf.edu
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Enzyme and Microbial Technology

  • Publisher: Elsevier
  • FAD Code: EZMT
  • CODEN: EMTED2
  • ISSN: 0141-0229
  • Abbreviation: Enzyme Microb. Technol.
  • DOI Prefix: 10.1016/j.enzmictec,10.1016/S0141-0229
  • Language: English
  • Comments: Fulltext from 1979 V1

Citations 24

"Laccase-based Biosensors For Monitoring Lignin"
Enzyme Microb. Technol. 2006 Volume 39, Issue 4 Pages 835-840
Sergey Shleev, Per Persson, Galina Shumakovich, Yulia Mazhugo, Alexander Yaropolov, Tautgirdas Ruzgas and Lo Gorton

Abstract: Four different ways to detect Kraft and soluble pine lignin using either soluble or immobilized Trametes hirsuta laccase have been investigated. In the first, the concentration of lignin was measured using laccase in homogeneous medium with a Clark electrode. In the second, laccase immobilized onto aminated porous glass beads was used in a reactor submerged into the lignin containing solution and the reaction was followed with a Clark electrode. The third and fourth techniques involved lignin detection using laccase modified spectrographic graphite electrodes, either used in flow injection mode or in batch mode, where lignin solutions were injected into an electrochemical cell. It was shown that the use of laccase modified graphite electrodes in conjunction with flow injection for amperometric lignin detection is an attractive perspective and our data form the basis for designing laccase-based biosensors to be applied for measuring lignin and its model compounds in waste water, e.g. from the pulp and paper industry. © 2006 Elsevier Inc. All rights reserved.

"Chemical Delivery Microsystem For Single-molecule Analysis Using Multilaminar Continuous Flow"
Enzyme Microb. Technol. 2006 Volume 39, Issue 3 Pages 519-525
Sang Wook Lee, Takatoki Yamamoto, Hiroyuki Noji and Teruo Fujii

Abstract: The fluidic operations, such as chemical delivery, hydrodynamic force control, solutions exchange, etc., to a desired location in a microfluidic channel are one of the most important technical aspects to progress single-molecule studies in microfluidic devices. This paper experimentally demonstrates multilaminar flow-based fluidic operations to supply and remove chemicals to an immobilized single-molecule on an inner surface of a microchannel to develop a future platform microfluidic device for single-molecule analysis. The microfluidic device, having multiple inlets and single outlet embedded with a micropump for each inlet, can generate continuous multiple laminar flow inside of the microchannel. The multiple laminar flow control capabilities were experimentally evaluated to deliver any single laminar flow to a desired location and to control flow speed to minimize hydrodynamic force to the target single-molecule. As a demonstration of single-molecule analysis using this format, ATP dependent rotation of F1-ATPase was investigated. As a result, the rotation of F1-ATPase was successfully investigated. This format showed many advantages and flexibility to realize the future platform of single-molecule studies.

"Use Of Laccase-modified Electrode For Amperometric Detection Of Plant Flavonoids"
Enzyme Microb. Technol. 2004 Volume 33, Issue 2-3 Pages 238-241
Anna Jarosz-Wilkolazka, Tautgirdas Ruzgasb and Lo Gorton

Abstract: In this study, laccase from Cerrena unicolor as a highly active enzyme was adsorptively immobilized on the surface of graphite electrode. The electrode inserted into flow-injection system served as a biosensor for detection of polyphenols, including flavonoids. We used catechin hydrate, epicatechin, epicatechin gallate, prodelphinidin, and caffeic acid as a target compounds in determination. The performed investigations demonstrate that laccase from white rot fungus C. unicolor can be applied for the construction of biosensors useful for determination of flavonoids and related compounds in flow-injection system. Electrodes modified with laccase yield responses for both simple compounds (caffeic acid) and compounds with three or more phenolic and non-phenolic rings, although with different sensitivity.

"Applications Of Laccases And Tyrosinases (phenoloxidases) Immobilized On Different Supports: A Review"
Enzyme Microb. Technol. 2002 Volume 31, Issue 7 Pages 907-931
Nelson Durán, Maria A. Rosa, Alessandro D'Annibale and Liliana Gianfred

Abstract: This review summarizes all the research efforts that have been spent to immobilize laccase and tyrosinase for various applications, including synthetic and analytical purposes, bioremediation, wastewater treatment, and must and wine stabilization. All immobilization procedures used in these areas are discussed. Considerations on the efficacy of immobilized copper oxidases and products, in addition to their kinetic parameters are also discussed. The available data indicate that the immobilization of laccase into cationic polymer cross-linked with epichlorohydrin appears to be a promising procedure for industrial applications. The development of laccase and tyrosinase-based biosensors to monitor a wide range of compounds appears to be at a mature stage of technology. (C) 2002 Elsevier Science Inc. All rights reserved.
Review

"Continuous Monitoring Of D-glucose And L-lactate By Flow Injection Analysis"
Enzyme Microb. Technol. 2002 Volume 30, Issue 1 Pages 129-133
Ahmed Haouz and Scott Stieg

Abstract: The D-glucose or the L-lactate oxidases were copolymerized by glutaraldehyde with bovine albumin and mounted on an oxygen electrode to form an enzyme electrode. This enzyme electrode was mounted on a Lachat QuikChem((R)) 8000 flow injection analysis (FIA) system. With this system the glucose and the L-lactate is simultaneously measured in the concentration range 0 to 40 g/liter. The enzymatic films can be used continuously for three months, at a rate of one measurement every two minutes. The stability of the enzyme electrode is greater than one year when stored in the dry state at 4°C. This FIA system can be used for monitoring simultaneously the concentration of D-glucose and L-lactate in the bioprocessing industries. (C) 2002 Elsevier Science Inc. All rights reserved.

"The Preparation And Characterization Of An Immobilized L-glutamic Decarboxylase And Its Application For Determination Of L-glutamic Acida"
Enzyme Microb. Technol. 2000 Volume 27, Issue 7 Pages 516-521
Daren Ling, Guoqi Wu, Chen Wang, Fan Wang and Guoqiang Song

Abstract: This paper is to study the preparation and characterization of an immobilized L-glutamic decarboxylase (GDC) and develop a sensitive method for the determination of L-glutamate using a new biosensor, which consists of an enzyme column reactor of GDC immobilized on a novel ion exchange resin (carboxymethyl-copolymer of allyl dextran and N,N-methylene-bisacrylamide CM-CADB) and ion analyzer coupled with a CO2 electrode. The conditions for the enzyme immobilization were optimized by the parameters: buffer composition and concentration, adsorption equilibration time, amount of enzyme, temperature, ionic strength and pH. The dynamic response of Na2HPO4-citric acid buffer system selected is much better than that of the others, 0.10 M HAc-0.10 hi NaAc and 0.10 M sodium citrate-0.10 M citric acid. The initial rate of the enzyme reaction v(0) in this buffer system is 1.76 mol . L-1 min-1, moreover, the rate of the enzyme reaction appears linear in the first 4 min. The optimum adsorption equilibrium time is around 6 h. The amount of enzyme adsorbed on CM-CADB resin affects the response to substrate L-glutamic acid, the widest range of linearity is obtained with over 30 mg (GDC)/g(resin). The GDC activity immobilized on CM-CADB reaches a maximum when the immobilization temperature was kept around 40°C. pH was kept at 4.4 when measuring the activity of the immobilized GDC. No variation of the activity of immobilized GDC is observed when the capacity is over 2.5 meq/g.(CM-CADB resin). The properties of the immobilized enzyme on CM-CADB were characterized. No significant improvement can be achieved when the substrate concentration exceeds 12.00 mmol/l, where the activity of immobilized GDC is equal to 1.58 mmol/l.min.g. The optimum pH is found to be 5.2, which changes 0.2 unit, comparing with that of the free GDC (5.0). The optimum temperature is found to be around 48°C, which is lower than that of free GDC (55°C). The critical temperature of the free GDC and the immobilized GDC is approximately 50°C and 45°C, respectively. The half-life of the activity is 127 days when the immobilized enzyme was stored in the cold (4°C). An immobilized GDC enzyme column reactor matched with a flow injection system-ion analyzer coupled with CO2 electrode-data collection system made up the original form of the apparatus of biosensor for determining of L-glutamic acid. The determination conditions are that the buffer solution is 0.10 M Na2HPO4-0.05 M citric acid at pH 4.4 and t = 37°C. The limit of detection is 1.0 x 10^-5 M. The linearity response is in the range of 5 x 10^-2 - 5 x 10^-5 M. The equation of linear regression of the calibration curve is y = 43.3x + 181.6 (y is the milli-volt of electrical potential response, x is the logarithm of the concentration of the substrate of L-glutamic acid). The correlation coefficient equals 0.99. The coefficient of variation equals 2.7%.

"Accelerated Prediction Of Recombinant Protein Production In Saccharomyces Cerevisiae By Using Rapid Monitoring Techniques"
Enzyme Microb. Technol. 2000 Volume 26, Issue 9-10 Pages 801-807
R. Browna, R. D. O'Kennedya, J. Helwighb, E. Maddena and M. Hoare

Abstract: The use of a stopped-flow analyzer. for the monitoring of the production of a secreted recombinant protein, a wild-type cutinase, from S. cerevisiae CEN.PK111-32D pUR7320 is described. Induction is through use of a galactose promoter, and the monitoring facility is used to record the formation of the cutinase and cell density with optical density measurements. A range of induction conditions was studied with a view to using the monitoring to predict the likely level of cutinase formation. Results achieved within 4 to 5 h of induction were of sufficient quality to allow the use of simple modelling relating cutinase formation and cell production to predict likely final specific activities of the product. The utility of such monitoring and prediction is discussed with regards to improved process confidence and definition during fermentation production. Copyright (C) 2000 Elsevier Science Inc. The use of a stopped-flow analyzer. for the monitoring of the production of a secreted recombinant protein, a wild-type cutinase, from S. cerevisiae CEN.PK111-32D pUR7320 is described. Induction is through use of a galactose promoter, and the monitoring facility is used to record the formation of the cutinase and cell density with optical density measurements. A range of induction conditions was studied with a view to using the monitoring to predict the likely level of cutinase formation. Results achieved within 4 to 5 h of induction were of sufficient quality to allow the use of simple modelling relating cutinase formation and cell production to predict likely final specific activities of the product. The utility of such monitoring and prediction is discussed with regards to improved process confidence and definition during fermentation production.

"Fed-batch Production Of A Soluble β-1,4-galactosyltransferase With Saccharomyces Cerevisiae"
Enzyme Microb. Technol. 1999 Volume 25, Issue 3-5 Pages 201-207
Jana Zigovaa, Matthias Mahlea, Holger Pascholda, Martine Malissard, Eric G. Berger and Dirk Weuster-Botz

Abstract: A feedback-controlled fed-batch process for the recombinant production of a soluble human β-1,4-galactosyltransferase (NdrGal-T) with Saccharomyces cerevisiae was developed and scaled up to the pilot scale. A 5000 U NdrGal-T fermentation run was performed on a 300 l scale. Indirect feedback control of the glucose feeding with RQ data at a set-point of RQ = 1.1 resulted in higher NdrGal-T activities (30 U/l) than direct feedback control of glucose with online flow injection analysis (9 U/l). This increase in final activity of NdrGal-T by a factor of 42 compared to published data makes the fed-batch production of glycosyltransferases with a Saccharomyces cerevisiae expression system competitive with cell-culture systems.
Kalman filter

"Monitoring Of The Glucose Concentration During Microbial Fermentation Using A Novel Mass-producible Biosensor Suitable For Online Use"
Enzyme Microb. Technol. 1997 Volume 20, Issue 8 Pages 590-596
Ibtisam E. Tothill, Jeffrey D. Newman, Stephen F. White and Anthony P. F. Turner

Abstract: A flow injection analysis system was combined with a mass producible, disposable biosensor and was used to monitor glucose concentrations during several microbial fermentations. The biosensor was manufactured using thick film, screen printing technology. Unlike previous devices of this type, the biosensor was designed to make multiple measurements over extended periods rather than to operate as a ''one-shot'' sensor. One yeast, one lactic acid, and three E. coli bioreactor cultures were tested using either defined or complex media. Results from the sensor were compared with a standard spectrophotometric test kit. In samples containing glucose concentrations within the range of the biosensor and the test kit, good correlations were obtained between the two methods. In addition to glucose, microbial growth and pH were recorded. (C) 1997 by Elsevier Science Inc. 17 References
Glucose Fermentation broth Sensor Amperometry Electrode Process monitoring Method comparison Interferences

"Online Monitoring Of Enzyme-catalyzed Biotransformations With Biosensors"
Enzyme Microb. Technol. 1997 Volume 20, Issue 6 Pages 432-436
Frank Lammers and Thomas Scheper

Abstract: A computer-controlled flow injection analysis system with an enzyme thermistor as detector is presented for online monitoring of enzyme-catalyzed syntheses performed in a simple batch reactor with immobilized biocatalyst. Reactions studied were (1) L-arginine to L-ornithine + urea, by arginase, (2) N-acetyl-L-methionine to L-methionine, by acylase [aminoacylase, EC 3.5.1.14], and (3) fructose from sucrose, by invertase + glucose isomerase [xylose isomerase from Streptomyces albus, EC 5.3.1.5]. All the enzymes were separately immobilized from phosphate-buffered solution at 4°C, pH 7, using VA-Epoxy-Biosynth from Riedel de Haen of Seelze in Germany. The method allowed continuous monitoring and optimization of enzymatic processes.
Sucrose Fructose l-arginine N-Acetyl-L-Methionine Sensor Thermistor Immobilized enzyme Process monitoring Optimization

"Enhancement Of The Response Of A Lactate Oxidase/peroxidase-based Fiberoptic Sensor By Compartmentalization Of The Enzyme Layer"
Enzyme Microb. Technol. 1994 Volume 16, Issue 11 Pages 979-984
Agn&eagrave;s Berger and Loïc J. Blum

Abstract: A bienzyme fiberoptic sensor for the chemiluminescent flow injection analysis of L-lactate was developed. Lactate oxidase and peroxidase were covalently immobilized on preactivated polyamide membrane either separately on different membranes or randomly co-immobilized on the same membrane. Hydrogen peroxide, generated by the lactate oxidase reaction in the presence of L-lactate, was the substrate of the second reaction, catalyzed by peroxidase, in the course of which light was emitted in the presence of luminol. Compartmentalization of the enzyme layer was obtained by stacking a peroxidase membrne on a lactate oxidase membrane at the sensing tip of the fiberoptic sensor. When using such a sensing tip, the response of the fiberoptic sensor for lactate was 22 times higher than the response obtained when using a membrane bearing the two enzymes randomly co-immobilized. With the compartmentalized sensor tip, the detection limit was 250 pmol lactate and the coefficient of variation was 1.7% for 10 replicates of 6.25 nmol lactate. The method was applied for lactate determination in reconstituted whey solutions, and the results were in good agreement with the analysis by an enzyme electrode.

"Optimization Of Enzyme Ratios In A Coimmobilized Enzyme Reactor For The Analysis Of D-xylose And D-xylulose In A Flow System"
Enzyme Microb. Technol. 1994 Volume 16, Issue 3 Pages 216-222
Elena Domínguez, György Marko-Varga, Bärbel Hahn-Hägerdal and Lo Gorton

Abstract: A coupled enzyme system for the detection of D-xylose and D-xylulose is presented. The system is based on three consecutive enzymatic steps. The enzymes xylose isomerase (XI), mutarotase (MT), and glucose dehydrogenase (GDH) are coimmobilized on controlled pore glass and packed in a bed reactor. The relative amount of enzymes, i.e., enzyme ratio, plays a critical role in driving the overall reaction, resulting in a system with linear response characteristics and an operational range of several orders of magnitude. Three different enzyme ratios are assayed to achieve maximum conversion efficiencies for xylose and xylulose. The highest enzyme unit ratio assayed, 13.4 of GDH to XI, gave the highest apparent pseudo-first-order rate constant showing the importance of the last enzymatic reaction in the coupled system to make the overall reaction thermodynamically favorable. A pH of 7.0 was found to be an optimum compromise for the multienzyme system. Sensitivity was dependent on NAD+ concentration. The study was carried out in a flow injection system. The optimized reactor has been applied for the catalytic detection of pentoses in flow injection analysis (FIA) and liquid chromatography (LC).
d-Xylose d-Xylulose Biological LC Kinetic Optimization Immobilized enzyme Controlled pore glass

"Detection Of Bacterial Contamination In Sterile UHT Milk Using An L-lactate Biosensor"
Enzyme Microb. Technol. 1993 Volume 15, Issue 6 Pages 508-512
Petr Skladal, Marco Mascini, Claudio Salvadori and Gianfranco Zannoni

Abstract: The fermentation of milk inoculated with bacteria was monitored amperometrically using an L-lactate biosensor and a flow injection system. This approach was compared with the measurement of pH. The effects of Enterococcus faecalis, Bacillus coagulans, Enterobacter sakazakii, Staphylococcus aureus, and Bacillus sphericus were investigated. The most promising results were obtained for Staphylococcus aureus, whose fermentation increases milk pH.
l-Lactate Milk Amperometry Sensor Process monitoring

"Online Chemiluminescence Assay Using FIA And Fiber Optics For Urinary And Blood Glucose"
Enzyme Microb. Technol. 1993 Volume 15, Issue 5 Pages 424-428
M. V. Cattaneo and J. H. T. Luong

Abstract: A chemiluminescence fiber optic system coupled to flow injection analysis (FIA) and ion exchange chromatography has been developed for determining glucose in blood and urine. Immobilized glucose oxidase acted on β-D-glucose to produce hydrogen peroxide, which was then reacted with luminol in the presence of ferricyanide to produce a light signal. Endogenous ascorbic acid and uric acid present in urine or blood samples were effectively retained by an upstream acetate anion exchanger. In addition, acetaminophen could also be adsorbed by this ion exchanger. The detection system exhibited a sensitivity of 1.315±0.044 RU µM-1 for glucose with a minimum detection level of 1 µM. When applied for the determination of urinary and blood glucose levels, the results obtained compared well with those of the reference hexokinase assay. Immobilized glucose oxidase was reused for over 500 analyzes without losing its original activity. A conservative estimate for the reuse of the acetate ion exchange column was about 100 analyzes.
Glucose Blood Urine Chemiluminescence Biotechnology HPIC Matrix removal Method comparison Optical fiber

"Chemiluminescent Flow Injection Analysis Of Glucose In Drinks With A Bienzyme Fiberoptic Biosensor"
Enzyme Microb. Technol. 1993 Volume 15, Issue 5 Pages 407-411
Loïc J. Blum

Abstract: A fiberoptic sensor for the chemiluminescent flow injection analysis of glucose in a one-step procedure was developed. Glucose oxidase and peroxidase were co-immobilized on a preactivated polyamide membrane. Hydrogen peroxide, generated by the glucose oxidase reaction in the presence of glucose, was detected by the chemiluminescence reaction of luminol catalyzed by peroxidase. The detection limit for hydrogen peroxide was 2.5 pmol at pH 8.5 and 0.25 pmol at pH 9.0. For 10 replicates of 0.1 nmol H2O2, the coefficient of variation was 2.5%. For glucose analysis, the detection limit was 0.25 nmol and the coefficient of variation was 3.8% for seven measurements of 2.5 nmol glucose. The method was applied for glucose analysis in soft drinks, and the results were in good agreement with the analysis by a standard spectrophotometric method. The analytical throughput was 40 samples per hour for hydrogen peroxide and 25 samples per hour for glucose.
Glucose

"Uses Of β-galactosidase Tag In Online Monitoring Production Of Fusion Proteins And Gene Expression In Escherichia Coli"
Enzyme Microb. Technol. 1993 Volume 15, Issue 1 Pages 66-71
A. Benito, F. Valero, J. Lafuente, M. Vidal, J. Cairo, C. Solà and A. Villaverde

Abstract: A simple method for monitoring and quantifying automatically the production by fermentation of β-galactosidase fusion proteins, making use of the remaining activity of the β-galactosidase part, is considered. A hybrid protein carrying the major antigenic domain of foot-and-mouth disease virus C1 joined at the N-terminus of β- galactosidase has been expressed in Escherichia coli. The yield of the chimeric protein has been monitored by flow injection analysis (FIA) during batch fermentations at laboratory scale, and a high correlation between values of product concentration from FIA and from immunological quantizations has been obtained. Because of the possibility of employing FIA in large-scale experiments, and the high sampling frequency, versatility, and reproducibility offered by this method, we propose FIA as a general, simple, quick, flexible, and reliable instrument for both monitoring the yield of recombinant proteins produced industrially, and performing basic research at laboratory scale.
Proteins Fermentation broth Process monitoring

"Novel Flow Injection Analysis Amperometric Biosensor System For The Determination Of Glutamine In Cell-culture Systems"
Enzyme Microb. Technol. 1993 Volume 15, Issue 1 Pages 26-32
K. B. Male, J. H. T. Luong*, R. Tom and S. Mercille

Abstract: A flow injection analysis (FIA) biosensor system has been developed for determining glutamine in insect cell and in murine hybridoma cell cultures. Glutamate oxidase and glutaminase, respectively, were covalently immobilized onto porous aminopropyl glass beads to form an immobilized enzyme column. The hydrogen peroxide produced by the enzyme reactions was detected by an amperometric electrode (platinum vs. silver/silver chloride) posied at +0.7 V. Among several anion exchange resins tested, endogenous glutamate was completely adsorbed onto acetate anion exchange resins. Such an ion exchanger also effectively adsorbed aspartic acid, uric acid, ascorbic acid, and acetaminophen. The FIA biosensor was linear up to 1 mM glutamine, with a lower detection limit of 10 µM, and possessed good reproducibility (relative error of ± 1.2%). Each analysis could be performed in 3.5 min including sampling and washing with a corresponding throughput of 17 h-1. The anion exchange column could be operated continuously for 12 h for 200 analyzes with diluted cell culture. The immobilized enzyme column was stable for at least 500 repeated analyzes without significant loss of activity. When the biosensor system was applied to glutamine measurement in insect cell and mammalian cell cultures, the results obtained compared well with those of HPLC.
Glutamine Fermentation broth Amperometry Sensor

"Colorimetric Determination Of Free And Total Cholesterol By Flow Injection Analysis With A Fiber Optic Detector"
Enzyme Microb. Technol. 1992 Volume 14, Issue 4 Pages 313-316
A. Krug, A. A. Suleiman, G. G. Guilbault and R. Kellner

Abstract: A flow injection method for the determination of total and free cholesterol is presented. Cholesterol esterase and cholesterol oxidase are immobilized on aminoalkyl glass beads. The beads are packed into a tubular glass reactor. The cholesterol esters traversing through the esterase reactor are cleaved to cholesterol and fatty acids. The oxidase reactor converts cholesterol to cholest-4-en-3-one and hydrogen peroxide is generated. The sample stream is merged with reagent streams consisting of a peroxidase solution and a solution of 2,2'-azinobis-(3- ethylbenzothiazoline-6-sulfonic acid)diammonium salt, and a hydrogen peroxide-dependent color reaction takes place in a short coiled reactor. The signal is monitored by means of fiber-optic instrumentation. Cholesterol concentration. can be related to the absorption of the oxidized dye form at a wavelength of 425 nm. The working range is 0.5-0.8 mmol/L, and the sample throughputs are 60 and 30/h for free and total cholesterol, respectively.
Cholesterol, free Cholesterol, total Sensor Spectrophotometry Immobilized enzyme Glass beads Optical fiber Indirect

"Development Of A Turbidimetric Immunoassay For Online Monitoring Of Proteins In Cultivation Processes"
Enzyme Microb. Technol. 1991 Volume 13, Issue 12 Pages 969-975
Ruth Freitag, Thomas Scheper and Karl Schügerl

Abstract: An online assay for a thermostable pullulanase and antithrombin III (AT III) is described. The assay is based on the formation of aggregates between the protein to be measured and antibodies raised against this protein. Assay automation was achieved by utilizing the flow injection analysis (FIA) principles. The apparatus, a stopped-flow, merging-zone manifold, is described in detail. Since the reaction used in an FIA system does not have to reach equilibrium, it was possible to reduce the time for an assay cycle to 2.5 min. A method for simulating cultivation conditions was developed for assay optimization. Using this method, a detection limit of 1 mg L-1 together with a standard deviation of 1.5 was found. A sandwich ELISA was used as reference assay in the case of AT III and an enzymatic activity assay in the case of pullulanase. Correlation coefficients of 0.988 (AT III) and 0.976 (pullulanase) were determined. The turbidimetric assay was successfully used for pullulanase monitoring during a 240-h cultivation of Clostridium thermosulfurogenes.
Proteins Fermentation broth Immunoassay Turbidimetry Merging zones Stopped-flow Optimization Process monitoring

"Biosensor System For Continuous-flow Determination Of Enzyme Activities. 2. Simultaneous Determination Of Plural Enzyme Activities"
Enzyme Microb. Technol. 1991 Volume 13, Issue 2 Pages 134-138
Hirokazu Okuma, Hitoshi Takahashi and Shuichi Sekimukai

Abstract: A biosensor system for continuous flow determination of plural enzyme activities was prepared from the combination of two pyruvate sensors, a prereactor and a flow cell. This system was applied to the simultaneous determination of lactic dehydrogenase (LDH) and glutamic-pyruvic transaminase (GPT) activities in the same sample. These enzyme activities can be determined by measuring pyruvate produced by the enzyme reactions as follows. The amount of pyruvic acid can also be determined from the amount of oxygen consumed upon oxidation of pyruvic acid by pyruvate oxidase. Therefore, both of the detectors for the determination of lactic dehydrogenase and glutamic-pyruvic transaminase activities were prepared from the combination of a pyruvate oxidase membrane and an oxygen electrode. Pyruvate oxidase was covalently immobilized on a membrane prepared from cellulose triacetate. A linear relation was obtained between the output current and LDH or GPT activities in the range of 50 to 3,600 IU L-1 or 6 to 1,000 IU l-1, respectively. Each assay of these enzyme activities was completed within 15 min. The results obtained had a precision of ~4%. The sensor was stable for more than 25 days at 5°C.
Enzyme, activity Sensor

"Biosensor System For Continuous-flow Determination Of Enzyme Activities. 1. Determination Of Glucose Oxidase And Lactate Dehydrogenase Activities"
Enzyme Microb. Technol. 1989 Volume 11, Issue 12 Pages 824-829
Hirokazu Okuma, Shuichi Sekimukai, Masakazu Hoshi, Kenzo Toyama and Etsuo Watanabe

Abstract: The system incorporates a flow cell divided into two compartments by a porous polycarbonate - polyester membrane (pore size 0.1 to 10 µm) and fitted with an O electrode. Buffer solution is pumped via the sample injection port through the cell on the electrode side of the membrane, and enzyme substrate is pumped through the other cell compartment. To assay glucose oxidase, the sample is injected into 0.05 M phosphate buffer of pH 7.8 at 30°C, and the substrate is 1 M glucose in the same buffer. For lactate dehydrogenase, a membrane containing immobilized pyruvate oxidase (prep. described) is fitted over the O electrode and covered by a dialysis membrane, and the substrate contains 8.9 mM L-lactic acid, 9.8 mM NAD+ and phosphate. The reaction is carried out at 35°C and pH 7.8. The decrease in current from its initial value to a minimum (taking 3 min) is measured in each instance. One assay of either enzyme takes ~15 min.
Enzyme, glucose oxidase Enzyme, lactate dehydrogenase Sensor Electrode Flowcell Buffer Injection technique Enzyme Immobilized enzyme Dialysis Membrane

"Online Measurement Of Extracellular Proteins In The Continuous Cellulase Production By Flow Injection Analysis"
Enzyme Microb. Technol. 1989 Volume 11, Issue 2 Pages 96-105
Wolfgang W. Stamm, Gunnar Pommerening, Christian Wandrey and Maria-Regina Kula

Abstract: The BCA protein assay was adapted to flow injection analysis to monitor the production of extracellular cellulases of Trichoderma species in batch and continuously operated fermentations. Response of the assay to protein (bovine serum albumin) was linear in the range of 0.1-2.0 g L-1; standard deviation of the calibration graph was 0.86%, accuracy and standard deviation were < +1.5% and 0.9% in the upper range, < +8% and < 2% in the lower range, respectively. Interferences from technical fermentation media were taken into consideration and can be calculated. The slow fermentation process was measured with a frequency of 0.5 samples h-1 over a period of 380 h. In each measuring cycle, recalibration was carried out. The necessary cell-free sample stream for the analytical system was obtained by installing the BIOPEMR, a steam-sterilizable sampling device which showed excellent performance even for long-term fermentations; membranes had to be changed after about 10 days only to keep the protein transmission constant (60-95%, depending on the special protein).
Proteins Albumin Cow Serum Enzyme Tecator

"Online Monitoring Of Enzymes In Downstream Processing By Flow Injection Analysis"
Enzyme Microb. Technol. 1985 Volume 7, Issue 12 Pages 607-612
Achim Recktenwald, Karl-Heinz Kroner and Maria-Regina Kula

Abstract: A Tecator flow injection analysis-5020 analyzer. was coupled to a spectrophotometer and fitted with a temperature control device (deviation <0.1°C) and a laboratory-built sampling device with a Millipore 0.45 µm membrane. The equipment was used in assays for formate dehydrogenase and leucine dehydrogenase in process control of enzyme-production processes. Both assays involved the formation of NADH and absorbance measurement at 340 nm; modifications necessary for the flow injection implementation are described. Calibration graphs for both enzymes were almost rectilinear up to ~11 iu mL-1.
Enzymes Enzyme, formate dehydrogenase Enzyme, leucine dehydrogenase Spectrophotometry Membrane Process control Tecator Millipore

"Rapid Online Protein Detection In Biotechnological Processes By Flow Injection Analysis"
Enzyme Microb. Technol. 1985 Volume 7, Issue 4 Pages 146-149
Achim Recktenwald, Karl-Heinz Kroner and Maria-Regina Kula

Abstract: Two rapid methods for on-line protein determination useful for control purposes in the automation of biotechnological processes such as fermentation and downstream processing are described. Both methods are derived from colorimetric laboratory biuret and Bradford protein assays adapted to a flow injection analyzer..
Protein Spectrophotometry Tecator