Design and response characteristics of an enzyme electrode for measurement of penicillin in fermentation broth

A principle for the construction of an autoclavable enzyme electrode is presented. Some characteristics of a penicillin electrode constructed according to this principle are given. The response time is ˜1 min. The response to increasing concentrations of penicillin is linear in buffered samples but logarithmic in unbuffered samples. The reason for the linearity is discussed. A local pH decrease in the enzyme, which is a is a consequence of the enzymatic reaction, reduces the buffering capacity within the electrode and thus increases the sensitivity. It is suggested that this increased sensitivity eliminates the logarithmic response predicted by the Nernst equation for a pH-based enzyme electrode.     

Microbial sensor for penicillins using a recombinant strain of Escherichia coli.

E. coli harboring the multicopy plasmid pBR327, which codes for beta-lactamase synthesis, was immobilized on acetylcellulose membranes. These were placed in the vicinity of a flat pH electrode, thus constituting a penicillin biosensor based on the detection of changes in pH using a reference pH electrode. A response time of 8 min was obtained with at least 2 mg of cells cm-2 of membrane. A linear detection range between 5 and 30 mM of penicillin was achieved. Buffering capacity decreased the sensitivity, but to a lower extent as when compared with probes using purified enzyme. The sensor was completely stable for at least 13 days and proved to be useful for penicillin estimation in complex media such as fermentation broths and milk.     

Optimization of liquid-state fermentation conditions for the glyphosate degradation enzyme production of strain Aspergillus oryzae by ultraviolet mutagenesis

This study aimed to obtain strains with high glyphosate-degrading ability and improve the ability of glyphosate degradation enzyme by the optimization of fermentation conditions. Spore from Aspergillus oryzae A-F02 was subjected to ultraviolet mutagenesis. Single-factor experiment and response surface methodology were used to optimize glyphosate degradation enzyme production from mutant strain by liquid-state fermentation. Four mutant strains were obtained and named as FUJX 001, FUJX 002, FUJX 003, and FUJX 004, in which FUJX 001 gave the highest total enzyme activity. Starch concentration at 0.56%, GP concentration at 1,370?mg/l, initial pH at 6.8, and temperature at 30°C were the optimum conditions for the improved glyphosate degradation endoenzyme production of A. oryzae FUJX 001. Under these conditions, the experimental endoenzyme activity was 784.15?U/100?ml fermentation liquor. The result (784.15?U/100?ml fermentation liquor) was approximately 14-fold higher than that of the original strain. The result highlights the potential of glyphosate degradation enzyme to degrade glyphosate.     

Extended-gate FET-based enzyme sensor with ferrocenyl-alkanethiol modified gold sensing electrode

We developed a field-effect transistor (FET)-based enzyme sensor that detects an enzyme-catalyzed redox-reaction event as an interfacial potential change on an 11-ferrocenyl-1-undecanethiol (11-FUT) modified gold electrode. While the sensitivity of ion-sensitive FET (ISFET)-based enzyme sensors that detect an enzyme-catalyzed reaction as a local pH change are strongly affected by the buffer conditions such as pH and buffer capacity, the sensitivity of the proposed FET-based enzyme sensor is not affected by them in principle. The FET-based enzyme sensor consists of a detection part, which is an extended-gate FET sensor with an 11-FUT immobilized gold electrode, and an enzyme reaction part. The FET sensor detected the redox reaction of hexacyanoferrate ions, which are standard redox reagents of an enzymatic assay in blood tests, as a change in the interfacial potential of the 11-FUT modified gold electrode in accordance with the Nernstian response at a slope of 59 mV/decade at 25 degrees C. Also, the FET sensor had a dynamic range of more than five orders and showed no sensitivity to pH. A FET-based enzyme sensor for measuring cholesterol level was constructed by adding an enzyme reaction part, which contained cholesterol dehydrogenase and hexacyanoferrate (II)/(III) ions, on the 11-FUT modified gold electrode. Since the sensitivity of the FET sensor based on potentiometric detection was independent of the sample volume, the sample volume was easily reduced to 2.5 microL while maintaining the sensitivity. The FET-based enzyme sensor successfully detected a serum cholesterol level from 33 to 233 mg/dL at the Nernstian slope of 57 mV/decade.     

Oxygen-stabilized enzyme electrode for d-glucose analysis in fermentation broths

A new principle for the construction of oxygen-dependent enzyme electrodes is presented. The enzyme electrode is based on a galvanic oxygen electrode which is furnished with an electrolysis anode covered by immobilized enzyme and placed close to the oxygen-sensing surface. An ordinary oxygen electrode is used as a reference electrode. The enzymatic consumption of oxygen in the enzyme electrode generates a potential difference between the electrodes which is utilized to control electrolytic generation of oxygen from water in such a way that zero differential potential is maintained. Thus, the enzyme electrode operates under ambient oxygen tension and does not suffer from oxygen limitation. The electrolytic current in this system gives a measure of the concentration of substrate surrounding the enzyme electrode. The electrode has been applied for continuous d-glucose analysis in situ during batch cultures of Candida utilis.     

新的分离纯化青霉素酰化酶方法的研究

按0．6％(w／v)的比例将皂土加到青霉索酰化酶发酵上清液中,可将酶100％吸附,而吸附的蛋白质仅占发酵上清液中的10％左右。吸附时的pH和无机盐对酶的吸附影响不大。使用不同pH和种类的缓冲液洗涤皂土－酶复合物,不能将酶洗脱,但可洗脱15％左右吸附的杂蛋白。使用含10％以上的PEG和NaCl的磷酸缓冲液可将酶全部洗脱．酶纯化25倍,浓缩6倍左右。此法特点是简便,酶活力收率高,可在常温下操作,也可直接从未除菌体的发酵液中提取酶,具有工业应用价值。     

Biosensor for direct determination of organophosphate nerve agents. 1. Potentiometric enzyme electrode

A potentiometric enzyme electrode for the direct measurement of organophosphate (OP) nerve agents was developed. The basic element of this enzyme electrode was a pH electrode modified with an immobilized organophosphorus hydrolase (OPH) layer formed by cross-linking OPH with bovine serum albumin (BSA) and glutaradehyde. OPH catalyses the hydrolysis of organophosphorus pesticides to release protons, the concentration of which is proportional to the amount of hydrolysed substrate. The sensor signal and response time was optimized with respect to the buffer pH, ionic concentration of buffer, temperature, and units of OPH immobilized using paraoxon as substrate. The best sensitivity and response time were obtained using a sensor constructed with 500 IU of OPH and operating in pH 8.5, 1 mM HEPES buffer. Using these conditions, the biosensor was used to measure as low as 2 microM of paraoxon, ethyl parathion, methyl parathion and diazinon. The biosensor was completely stable for at least one month when stored in pH 8.5, 1 mM HEPES + 100 mM NaCl buffer at 4 degrees C.     

An enzyme electrode for acetylcholine.

A new enzyme electrode is described to measure continuously acetylcholine concentration. A coating containing active acetylcholinesterase is produced on a pH-glass electrode. The mean thickness of the coating is 50 micrometer. Optimal operational conditions with respect to buffer concentration, ionic strength, linearity, stability, sensitivity, pH of the bulk solution, and response time are studied and discussed. The use of acetylcholinesterase-containing membranes as sensors could offer several novel advantages.     

Estimation of fermentation biomass concentration by measuring oxygen uptake off-line with an oxygen electrode

Summary An indirect method for biomass determination was developed for aerobic cultivation in media containing solid substrates. This method involved off-line estimation of oxygen uptake with an oxygen electrode using a calibration factor and was applied to penicillin fermentation with Penicillium chrysogenum. The volumetric oxygen uptake rate was independent of the amount of sample sealed in the measuring cell. Storage of the sample for a period up to 2.5 h also had no influence on the rate of O₂ uptake. The specific O₂ uptake rate at 70 to 75 h fermentation time was used as the calibration factor to calculate the respiration-active part of the biomass. Specific penicillin V formation rates related to net dry weight, respiration-active biomass (X _A )and RNA content were compared. Only the penicillin V production rate related to X _A showed a similar behaviour as the penicillin V production rate related to RNA content. For this reason, it is assumed that X _A ,like RNA content, is more closely related to the metabolic state of the cells than the net dry weight. This method, therefore, provides a more suitable reference parameter for fermentation control than the generally used net dry weight.Offprint requests to: D. Siegmund     

根霉纤溶酶发酵条件的优化

研究了不同培养基对中国根霉１２号（Ｒｈｉｚｏｐｕｓ ｃｈｉｎｅｓｉｓ１２）发酵生产纤溶酶的影响,发现Ｃ／Ｎ的降低有利于产酶,同时用正产实验给出了最佳培养条件为：以麸皮水＋胰蛋白胨＋豆渣为培养基,接种量２０％,转速１７０ｒ／ｍｉｎ;用响应面设计确定了最佳培养基为４．３６６６１％麸皮水＋０．９８９７５％胰蛋白胨＋４．８９８９５％豆渣;在最优条件下,纤溶酶的发酵效价可达３．０６Ｕ／ｍｌ。发现添加金属离子     

重组毕赤酵母产青霉素G酰化酶的分批发酵动力学研究

构建了重组毕赤酵母产青霉素G酰化酶的分批发酵动力学模型。实验考察了分批发酵过程中甘油消耗、甲醇浓度、菌体浓度、溶氧、补料时间对青霉素G酰化酶活力的影响。应用Matlab软件,对菌体生长、基质消耗和产物生成方程进行最优参数估算和非线性拟合,得到相应的动力学模型。模型的计算值与实验值能较好地拟合,表明所建模型能较好反映重组毕赤酵母产青霉素G酰化酶的分批发酵过程。     

Magnetic enzyme membranes as active elements of electrochemical sensors: specific amino acid enzyme elctrodes.

The basic principle of the described magnetic enzyme electrodes is a kinetic accumulation of CO2 at the active layer electrode interface. The local pCO2 level is linked to three simultaneous phenomena: substrate diffusion in, enzyme reaction CO2 diffusion out. After a transient state there is a stationary state between the quantity of CO2 produced by the enzyme reaction and the CO2 diffusing from the active membrane to the bulk solution. Continuous determination of free amino acids in biological media is useful in biological processing, fermentation, medicine, pharmaceutical industries and biological research. No methods are presently available for any specific continuous measurement of lysine which is of nutritional importance in protein industrial syntheses; of phenylalanine and tyrosine which have to be monitored in several inborn diseases (phenylketonuria being the most important of them); of arginine and histidine which play a still imperfectly understood part in neurochemistry. The use of decarboxylase bearing membranes as sensors in such measurements could offer several novel advantages: (a) a simple device made of a currently manufactured electrode slightly modified by the use of an enzyme membrane; (b) The absence of any enzymic consumption due to the immobilization and the negligible consumption of substrate during the measurements; (c) The sensitivity which can be sharpened by a systematic study of the membrane parameters; (d) the continuous response of the electrode as long as it is in contact with the substrate solution; (e) the further feasibility as a miniature sensor. The magnetic device introduced allows obviously a convenient use of the enzyme electrode, the active part can be removed and replaced without disturbance for the pCO2 electrode itself. The enzyme electrodes are not only useful at the applied point of view but also at the fundamental point of view by allowing a direct measurement of an intra membrane concentration. The influence of simple structures on enzyme kinetics was studied with enzyme electrodes by our group, in the case of memory and oscillations obtained with enzyme systems.     

Comparative study of the enzymatic synthesis of cephalexin at high substrate concentration in aqueous and organic media using statistical model

Synthesis of cephalexin with immobilized penicillin acylase at high substrates concentration at an acyl donor to nucleophile molar ratio of 3 was comparatively evaluated in aqueous and ethylene glycol media using a statistical model. Variables under study were temperature, pH and enzyme to substrate ratio and their effects were evaluated on cephalexin yield, ratio of initial rates of cephalexin synthesis to phenylglycine methyl ester hydrolysis, volumetric and specific productivity of cephalexin synthesis, that were used as response parameters. Results obtained in both reaction media were modeled using surface of response methodology and optimal operation conditions were determined in terms of an objective function based on the above parameters. At very high substrates concentrations the use of organic co-solvents was not required to attain high yields and actually almost stoichiometric yields were obtained in a fully aqueous media with the advantages of higher productivities than in an organic co-solvent media and compliance with the principles of green chemistry.     

A cellular automata model for simulating fed-batch penicillin fermentation process

A cellular automata model to simulate penicillin fed-batch fermentation process (CAPFM) was established in this study, based on a morphologically structured dynamic penicillin production model, that is in turn based on the growth mechanism of penicillin producing microorganisms and the characteristics of penicillin fed-batch fermentation. CAPFM uses the three-dimensional cellular automata as a growth space, and a Moore-type neighborhood as the cellular neighborhood. The transition rules of CAPFM are designed based on mechanical and structural kinetic models of penicillin batch-fed fermentation processes. Every cell of CAPFM represents a single or specific number of penicillin producing microorganisms, and has various state. The simulation experimental results show that CAPFM replicates the evolutionary behavior of penicillin batch-fed fermentation processes described by the structured penicillin production kinetic model accordingly. __________ Translated from ACTA BIOPHYSICA, 2005, 21(2) [译自: 生物物理学报, 2005,21(2)]     

Enhancement of enzyme activity in supercritical carbon dioxide via changes in acid-base conditions

Enzyme performance is often impaired in supercritical carbon dioxide. We were able to enhance enzyme activity in this medium via changes in acid-base conditions by using ion-exchange materials (solid H(+)/Na(+) buffer pairs and a zeolite), which were selected on the basis of the response of an organosoluble acid-base indicator. The concentration of ion-exchange materials had an important effect on the catalytic activity of subtilisin Carlsberg cross-linked enzyme crystals (CLECs), and this was related to the protonation and hydration states of the enzyme. The buffer Na(2)CO(3)/NaHCO(3) gave the highest enhancement in enzyme activity (by a factor of 54), probably as a result of its high basicity and capacity to counteract the deleterious effect of carbonic acid to a greater extent than the other materials tested.     

On-line membrane inlet mass spectrometry for feed-back control of precursor concentration in penicillin fermentation

Summary A sampling system which enables on-line measurements of the precursor phenoxyacetic acid (POAA) in penicillin fermentation by membrane inlet mass spectrometry is presented and its capacity for feed-back regulation of POAA to a low predefined concentration in a penicillin-V fermentation over 150 hours is demonstrated. The system measures alternately filtered sample and standard solution in a measuring cycle which is shorter than the response time of membrane inlet mass spectrometry (MIMS) but sufficiently long to decide whether the concentration of POAA in the sample is higher or lower than in the standard solution at set point concentration. The decision is used for on-off regulation of the addition of POAA.     

A new amperometric enzyme electrode for alcohol determination

A new enzyme electrode for the determination of alcohols was developed by immobilizing alcohol oxidase in polvinylferrocenium matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of enzymatically generated H(2)O(2) was measured at a constant potential of +0.70 V versus SCE. The effects of substrate, buffer and enzyme concentrations, pH and temperature on the response of the electrode were investigated. The optimum pH was found to be pH 8.0 at 30 degrees C. The steady-state current of this enzyme electrode was reproducible within +/-5.0% of the relative error. The sensitivity of the enzyme electrode decreased in the following order: methanol>ethanol>n-butanol>benzyl alcohol. The linear response was observed up to 3.7 mM for methanol, 3.0 mM for ethanol, 6.2 mM for n-butanol, and 5.2 mM for benzyl alcohol. The apparent Michaelis-Menten constant (K(Mapp)) value and the activation energy, E(a), of this immobilized enzyme system were found to be 5.78 mM and 38.07 kJ/mol for methanol, respectively.     

Optimization of bagasse,nutrients and initial moisture ratios on the yield of penicillin in solid-state fermentation

An advanced solid-state fermentation (SSF) system (liquid medium absorbed on an inert support) has been applied to antibiotic production. The main components of this solid medium are: support (sugarcane bagasse), nutrients and water. The first two are solids and have to be considered to calculate the initial moisture content (IMC) of the medium. Earlier work indicated the importance of using high IMCs and concentrated media to obtain high penicillin yields in SSF. Nevertheless, the present work shows that high values of IMC or nutrients content can stimulate or inhibit penicillin production, depending on the strategy used to compensate this change (i.e. the proportions of the other two components). Conversely, increasing bagasse content always showed an inhibitory effect on the production. Since penicillin production depends on the combinations of these components, a global approach was used. The effect of the proportions of the three components on penicillin production was studied by means of a triangle of combinations and a 3D graph. It was possible to establish that high penicillin production is only obtained in a zone of low support content (10–12.5%). Surprisingly, one production maximum was observed in a zone of low moisture and high nutrients content (62 and 25.5% respectively); and another one in a zone of high moisture and a relatively low nutrients concentration (75.5 and 12.4% respectively).     

Osmotic pressure effects and intracellular accumulation of ethanol in yeast during fermentation

Summary The intracellular accumulation of ethanol in yeast and its potential effects on growth and fermentation have been topics of controversy for the past several years. The determination of intracellular ethanol based on the exclusion of [¹⁴C]sorbitol to estimate aqueous cell volume was used to examine the question of intracellular ethanol accumulation. An intracellular accumulation of ethanol inSaccharomyces cerevisiae was observed during the early stages of fermentation. However, as fermentation continued, the intracellular and extracellular concentrations of ethanol became similar. Increasing the osmotic pressure of the medium with glucose or sorbitol was observed to cause an increase in the intracellular ethanol concentration. Associated with this was a decrease in yeast growth and fermentation rates. In addition, increasing the osmotic pressure of the medium was observed to cause an increase in glycerol production. Supplementation of the media with excess peptone, yeast extract, magnesium sulfate and potassium phosphate was found to relieve the detrimental effects of high osmotic pressure. Under these conditions, though, no effect on the intracellular and extracellular ethanol distribution was observed. These results indicate that nutrient limitation, and not necessarily intracellular ethanol accumulation, plays a key role during yeast fermentations in media of high osmolarity.     

Palladium hexacyanoferrate hydrogel as a novel and simple enzyme immobilization matrix for amperometric biosensors

An amperometric glucose biosensor with glucose oxidase (GOx) immobilized into palladium hexacyanoferrate (PdHCF) hydrogel has been prepared and evaluated. The sensor was based on a two-layer configuration with biocatalytic and electrocatalytic layers separately deposited onto the electrode. To reduce the overpotential for reduction of hydrogen peroxide liberated in the enzyme catalyzed oxidation of glucose, an inner thin layer of nickel hexacyanoferrate (NiHCF) electrodeposited onto the surface of graphite electrode was used as an electrocatalyst. As an outer layer, the hydrogel of palladium hexacyanoferrate with entrapped glucose oxidase was used. Under optimal operating conditions (pH 5.0 and E = -0.075 V versus calomel (3.0 M KCl) reference electrode), sensor showed high sensitivity to glucose (0.3-1.0 microA/mM) and a response time of less than 30s. The linear response to glucose was obtained in the concentration range between 0.05 and 1.0 mM in batch analysis mode and 0-7.0 mM in FIA. During the 32 days testing period, no significant decrease in the sensor sensitivity was observed. The sensor was applied for the determination of glucose concentration in fruit juice and yoghurt drink, and the results obtained showed good correlation with results obtained by reference spectrophotometric enzyme method.