Microcomputer control applied to the Vickers M85/86 microdensitometer

Summary An electronic interface for a Research Machines 380Z microcomputer and a Vickers M85/6 integrating microdensitometer is described, together with outlines of the control software. A typical applications programme, which may be written in several high-level languages, is outlined for the determination of a time course plot for enzyme activity in a tissue section. This system facilitates rapid accurate measurement and analysis of experimental data.     

Rapid continuous monitoring of enzyme activity in tissue sections: experience with the M85A and Zeiss UMSP-30 systems

We have previously described methods for the continuous monitoring of formazan deposition in tissue sections with a system based on the Vickers M85A microdensitometer. However, this instrument only allows the monitoring of a single field in each section. We have now developed a new system based on the Zeiss UMSP-30 microspectrophotometer. This machine is entirely computer controlled and by virtue of its fast-scanning stage allows the rapid (less than 0.5 s) sequential monitoring of multiple fields (up to 35) in each section. Thus a number of cell types may be studied simultaneously and work which used to take a full working day with the M85A system now can be performed in 45 min. As with the M85A the Zeiss system has full capability for data analysis (i.e. calculation of initial velocity rates, etc.). We have found that continuous monitoring of tissue sections by microdensitometry is a precise, sensitive and biochemically valid method of studying enzyme activity within the cellular matrix.     

Kinetic and morphometric measurements of enzyme reactions in tissue sections with a new instrumental setup

Summary An instrumental setup is described for the measurement of enzyme kinetics and morphometry in tissue sections. It consists of a Vickers M85 microdensitometer and computer-assisted Kontron Videoplan system. The Videoplan system consists of a minicomputer with two mini-floppy disks, a keyboard, a graphic tablet, a TV monitor and a printer/plotter. The measuring component of the M85 is linked to the minicomputer via a BCD interface, and the optical system of the M85 is coupled to a TV camera for display on the monitor screen. The enzyme-kinetic data obtained with the M85 in a specified area of the tissue section (density values as a function of reaction time) are stored in the minicomputer. The measurement process is controlled by a corresponding measuring program. Through correlation analysis (a component of the commercial software) between density values and reaction time, the initial and thus maximum enzyme activity is determined. Upon completion of the kinetic measurements, the measured area of tissue is transferred by the TV camera to the monitor, and the reaction area is described and measured with the graphic tablet in video dialogue and related to the initial enzyme activity. With the setup described, it is possible to make microdensitometric measurements of enzyme activities in a specified tissue area while morphometrically analyzing the associated reaction area. To illustrate the use of the system, enzyme-kinetic (succinate dehydrogenase) and morphometric measurements are performed in tissue sections from the proximal tubule of the rat nephron. Additional applications of the system are discussed.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Activity and operational stability of immobilized mandelonitrile lyase in methanol/water mixtures

Summary The enzyme mandelonitrile lyase was covalently immobilized on solid support materials using different methods. Immobilization on porous silica using coupling with glutaraldehyde afforded preparations with high enzyme loading (up to 9% (w/w)). The immobilized enzyme was used in a packed bed reactor for the continuous production of d-mandelonitrile from benzaldehyde and cyanide. The influence of the flow rate, pH, substrate concentrations and enzyme loading on the reaction yield and the enantiomeric purity of the product was investigated. In order to suppress the competing spontaneous reaction, the enzymatic reaction must be rapid. A flow rate of 9.5 ml/min (0.1 M benzaldehyde and 0.3 M HCN) through a 3 ml reactor afforded a 86% yield of mandelonitrile with 92% enantiomeric excess. No leakage of enzyme occurred under continuous operation. One column was used continuously for 200 h without any decrease in yield or enantiomeric purity of the product. High concentrations of benzoic acid were shown to decrease the operational stability of the system.     

Synthesis of esters using a nylon-immobilized lipase in batch and continuous reactors.

Direct esterifications using a nylon-immobilized lipase from Candida cylindracea were carried out in batch and continuous-flow reactors. The immobilized enzyme was effective in catalyzing the synthesis of ethylpropionate, isoamylpropionate, and isoamylbutyrate. With ethanol dissolved in hexane as a substrate, the maximum initial esterification rate was 0.02 mole/(h x g of immobilized protein), but the enzyme was stable only when the substrate concentrations were lower than 0.2 M. With isoamyl alcohol in hexane as a substrate, esterification rates as high as 0.085 mole/(h x g of immobilized protein) were observed and the immobilized enzyme was stable over a much broader concentration range. However, in this case, the use of a solvent, such as hexane, was not necessary for esterification, and the enzyme could be employed in equimolar acid/alcohol mixtures. A packed-bed reactor was operated successfully for the continuous synthesis of esters. The reactor was stable for long periods of time, and the steady-state performance could be accurately predicted on the basis of batch reaction experiments.     

A continuous fluorescence assay for lecithin cholesterol acyltransferase

A continuous fluorescence assay was adapted to the measurement of the phospholipase reaction of lecithin cholesterol acyltransferase (LCAT). The fluorescent phospholipid 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)aminocaproyl phosphatidylcholine (C6-NBD-PC) in micelle form reacted with LCAT to yield NBD-caproic acid, resulting in up to 5-fold increases in fluorescence in 30 min. The reaction rates were optimal in mixtures containing 0.1 M NaCl and 4 mM beta-mercaptoethanol at 37 degrees C. Apolipoprotein A-I did not activate the enzyme and bovine serum albumin bound monomeric substrate and interfered with the fluorescence assay. Under similar reaction conditions, bee venom phospholipase A2 was almost 100-fold more reactive than LCAT.     

Economically pertinent continuous amide formation by direct lipase-catalyzed amidation with ammonia

An economically pertinent process for the lipase-catalyzed synthesis of amides was developed. A continuous plug flow reactor was used. The model reaction was the production of oleamide, a lubricant and anti-slip agent, via direct Candida antarctica lipase B-catalyzed amidation of oleic acid with ammonia. Of all solvents tested, 2-methyl-2-butanol was found to respond optimally to the demands formulated in our specifications. A continuous conversion of oleic acid into oleamide of 85% was obtained. A productivity of 4.5 tons oleamide per kg of enzyme per year was calculated, indicating a contribution of enzyme to the product price of only 4%. The volumetric productivity, 100 g. L(-1). h(-1), is 4 to 100 times higher than in literature procedures. A simple crystallization procedure leads to 99% purity.     

A continuous spectrophotometric assay for the Bacillus cereus phospholipase C using a thiophosphate substrate analog: evaluation of assay conditions and chromogenic agents

A thiophosphate analog of dioctanoylphosphatidylcholine has been used as the substrate in a continuous spectrophotometric assay for the Bacillus cereus phospholipase C. The reaction has been monitored at 412 nm using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and at 324 nm using 4,4'-dithiopuridine (DTP) as the respective thiol-reactive chromogenic agents. An optimum pH 6.0 was determined for the phospholipase C-catalyzed reaction which was independent of the chromogen utilized. Although the reaction rates observed when DTP was used were increased over those seen with DTNB, the rates were insensitive to changes in the concentration of the chromogen normally used for the assay. The initial velocities were shown to be linearly dependent upon the amount of enzyme added over at least a 20-fold enzyme concentration range. The dependency of the initial velocity on the concentration of substrate showed a discontinuity at [S] = 40 microM when either DTP or DTNB was used. This was consistent with a value of 56 microM estimated for the substrate critical micelle concentration by an independent measurement. While the substrate data measured using DTP could not be fit to existing equations based on Michaelis-Menten kinetics, the data obtained using DTNB as the chromogen conformed with the model proposed by Wells for enzymes acting upon micelle-forming substrates (M. A. Wells (1974, Biochemistry 13, 2248-2257). This allowed for the estimation of monomer and micelle Michaelis-Menten parameters for the B. cereus phospholipase C-catalyzed reaction with a thiophosphate analog substrate.     

A continuous single organic phase process for the lipase catalyzed synthesis of peroxy acids increases productivity

The design of an optimal process is particularly crucial when the reactants deactivate the biocatalyst. The reaction cascades of the chemo‐enzymatic epoxidation where the intermediate peroxy acid is produced by an enzyme are still limited by enzyme inhibition and deactivation by hydrogen peroxide. To avoid additional effects caused by interfaces (aq/org) and to reduce the process limiting deactivation by the substrate hydrogen peroxide, a single‐phase concept was applied in a fed‐batch and a continuous process (stirred tank), without the commonly applied addition of a carrier solvent. The synthesis of peroxyoctanoic acid catalyzed by Candida antarctica lipase B was chosen as the model reaction. Here, the feasibility of this biocatalytic reaction in a single‐phase system was shown for the first time. The work shows the economic superiority of the continuous process compared to the fed‐batch process. Employing the fed‐batch process reaction rates up to 36 mmol h^?1 per gram_cat, and a maximum yield of 96 % was achieved, but activity dropped quickly. In contrast, continuous operation can maintain long‐term enzyme activity. For the first time, the continuous enzymatic reaction could be performed for 55 h without any loss of activity and with a space‐time yield of 154 mmol L^?1 h^?1, which is three times higher than in the fed‐batch process. The higher catalytic productivity compared to the fed‐batch process (34 vs. 18 g_Prod g^?1_cat) shows the increased enzyme stability in the continuous process.     

Development of a Polyaniline-coated Monolith Reactor for the Synthesis of Cephalexin Using Penicillin G Acylase Aggregates

A monolith reactor for the synthesis of cephalexin was developed using capillary columns. The micro channel in the monolith reactor was coated with polyaniline (PANI), and penicillin G acylase was aggregated with PANI using 0.5% of glutaraldehyde as a cross-linker. The developed monolith reactor exhibited many advantages over other enzyme reactors such as batch and continuous reactors. It showed fast enzyme reaction rates owing to the decrease in external mass transfer and internal diffusion limitations. The reactor can easily be scaled up by bundling together multiple monolith reactors, enabling a corresponding increase in feed rate. Furthermore, the monolith reactor showed good operational stability, with 95% of its original activity maintained after 48 h of continuous operation. The PANI coating on the surface of the capillary column increased the enzyme immobilization capacity and conversion was increased from 15.4% to 70.6% after PANI coating. The conversion ratio increased to approximately 70.6% with an increase in residence time and reactor length.     

A spectrophotometric method for the assay of phospholipase D activity

Phosphatidylcholine phosphatidohydrolase (EC 3.1.4.4, phospholipase D) catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline. We have developed a spectrophotometric assay for phospholipase D using choline kinase, pyruvate kinase, and lactate dehydrogenase to couple the release of choline with the oxidation of NADH. The assay was linear both with time and with enzyme concentration. The assay should prove useful for continuous monitoring of enzyme activity, determination of initial rates of reaction, and detailed kinetic studies of phospholipase D. The method is limited to analysis of purified preparations of phospholipase D lacking competing activities to the coupled system.     

Conversion of the aminocrotonate intermediate limits the rate of gamma-elimination reaction catalyzed by L-cystathionine gamma-lyase of the yeast Saccharomyces cerevisiae

L-Cystathionine gamma-lyase [EC 4.4.1.1] of Saccharomyces cerevisiae was shown to bind cofactor pyridoxal 5'-phosphate, up to 2 molecules/subunit. The association constants of the enzyme for the cofactor were estimated to be 3.67 x 10(5) M(-1) and 9.05 x 10(3) M(-1). However, the latter value was too small for the binding to play a catalytic role. Changes in the absorption spectra of the enzyme in gamma-elimination reaction mixtures with various amino acids as substrates were observed at 10 degrees C to elucidate the reaction mechanism of the enzyme. The enzyme formed a chromophore exhibiting absorption at approximately 480 nm, which is characteristic of an aminocrotonate intermediate with O-succinyl-L-homoserine, L-cystathionine, L-homoserine, or O-acetyl-L-homoserine, at rates in this order. The intermediate was consumed at much lower rates than those of formation. The order of the rates of consumption was the same as the order of the formation rates and the order of the gamma-elimination activity of the enzyme with the above-mentioned substrates. These results strongly suggested that the intermediate was essential for gamma-elimination and that the reaction was rate-limited by its conversion into the product alpha-ketobutyrate. L-Cysteine sensitively inhibited the alpha, gamma-elimination activity of the enzyme, and also retarded the formation of the chromophore when it was provided to the enzyme together with a substrate. The reason for these phenomena is discussed.     

Stability studies of immobilized enzyme stir rods

Alcohol dehydrogenase has been covalently attached to the surfaces of nylon stir rods. Several rod types have been evaluated in terms of their mixing efficiency and enzyme loading. Fluorometric monitoring of the rate of conversion of NAD to NADH serves as a measure of the reaction rate under varying conditions. The rate of reaction of the enzyme stir rods has been evaluated in terms of RPM, buffer concentration, NAD reagent concentration, and pH. The rate of reaction is seen to reach a plateau at higher stir rates, indicating a lack of diffusional hindrances. The reaction rate also begins to level off at phosphate buffer concentration of 0.1M to 0.15M. Saturating conditions are reached at an NAD concentration of 2.5mM. The optimum pH is found to be 9.0. The Stability of the covalent bond between the enzyme and the nylon has been assessed by comparing the bond strength to the energies of various disruptive forces to which the enzyme is exposed. Centrifugal, drag, and shear forces are shown to be insufficient to cause rupture of the bond. The stability to the immobilized enzyme preparation has been investigated under varying conditions of immobilization and use. No effect on activity loss was found for rotation rate or for continuous versus intermittent use. It was found that enhanced stability occurred for hydrolytic cleavage of the nylon, using nitric acid, as compared to nonhydrolytic cleavage. Hydrolytic cleavage also led to some degree of adsorption of the enzyme to the surface of the nylon. Thus, the possibility of increased stability to multipoint attachment of the enzyme is discussed. Possible cause of activity loss are discussed, as well as the extension of the enzyme stir rod to use in scale model reactor studies.     

A novel procedure for the preparation and characterization of catalytically active fatty acid synthetase immobilized on sepharose beads

A novel procedure for immobilization of enzymatically active fatty acid synthetase is presented. The enzyme is coupled to a Sepharose 4B matrix containing covalently attached antibodies which recognize, and bind specifically to, the thioesterase domain of this polyfunctional enzyme. A continuous flow system is described for assay of the immobilized enzyme. Fatty acid synthetase activity apparently is not limited by movement of substrates through the Nernst diffusion layer surrounding the matrix particles, since normal Michaelis-Menten kinetics are observed and reaction rates are independent of flow rate. The Km values for acetyl-CoA and malonyl-CoA, the pH/activity profile, and the reaction products are essentially the same as for the freely soluble enzyme, although the specific activity is lower by about 55%. The preparation and characterization of immobilized subunits of the enzyme could provide a valuable approach for studying the role of structural and functional subunit interactions in the enzyme. In addition, the immobilized enzyme offers a model for studying the properties of this enzyme in a highly structured environment such as might exist in vivo, permitting study of both physical and functional interactions of fatty acid synthetase with other lipogenic enzymes.     

Partial Purification and Characterization of Hydroxycinnamoyl-Coenzyme A:Tyramine Hydroxycinnamoyltransferase from Cell Suspension Cultures of Solanum tuberosum

A pathogen elicitor-inducible soluble acyltransferase (tyramine hydroxycinnamoyltransferase [THT], EC 2.3.1), which catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-coenzyme A (CoA) esters to tyramine in the formation of N-hydroxycinnamoyltyramine, was partially purified with a 380-fold enrichment and a 6% recovery from cell-suspension cultures of potato (Solanum tuberosum L. cv Datura). The enzyme showed specific activities of 33 mkat (kg protein)-1 (formation of feruloyltyramine). The apparent native Mr was found to be approximately 49,000. Highest activity was at pH 6.8 in K-phosphate. The isoelectric point of the enzyme was approximately pH5.2. The apparent energy of activation was calculated to be 96 kJ mol-1. The enzyme activity was stimulated more than 5-fold by 10 mM Ca2+ or Mg2+. The apparent Km values were 36 [mu]M for feruloyl-CoA and 85 and 140 [mu]M for cinnamoyl- and 4-coumaroyl-CoA, respectively. The Km value for tyramine in the presence of feruloyl-CoA was 22 [mu]M. In the presence of 4-coumaroyl-CoA, however, the Km for tyramine increased to about 230 [mu]M. The mode of action was an iso-ordered bi bi mechanism in which A, B, P, and Q equal hydroxycinnamoyl-CoA, tyramine, N-hydroxycinnamoyltyramine, and CoA, respectively. Thus, the reaction occurred in a ternary complex of the enzyme and substrates. The equilibrium constant of the reaction was determined to be 1.3 x 104. This gave a [delta]G[deg][prime] eq value of -23.5 kJ mol-1.     

Automated kinetic analysis of pyruvate kinase

An apparatus for the automatic determination of enzyme kinetics of pyruvate kinase is described. A continuous plit of velocity versus substrate concentration is obtained using quantities of enzyme and substrates comparable to manual determinations. The automated procedure offers a number of advantages over manual methods including elimination of repetitive pipetting, simpler reaction temperature regulation, reduced analysis time, and possible on-line computer analysis. The apparatus utilizes a commercially available column uv flow monitor to measure NADH/NAD changes in the coupled lactic dehydrogenase reaction at 340 nm in a continuous flow system. The optical density changes are directly related to the velocity of the enzyme-catalyzed reaction. A linear substrate gradient is generated from a density gradient maker to provide the required relationship between velocity and substrate concentration. The system is calibrated by forming a gradient from a hemoglobin solution of known concentration. The procedure has been evaluated by determination of the kinetic parameters of three of the isozymes of pyruvate kinase. Values obtained by the continuous flow method are in close agreement with those obtained by individual point determination in a recording spectrophotometer.     

An on-line method for the collection and analysis of quasi-steady-state kinetic data for enzyme-catalyzed reactions.

A special mixing device for initiating enzyme-catalyzed reactions is used to rapidly achieve an unperturbed quasi-steady state. An on-line computer is employed to sample the initial conditions, the mixing time, and concentrations that change as a function of time during this quasi-steady state phase. A statistical method for estimating initial, quasi-steady state rates from the time course of the enzyme-catalyzed reaction is described. Practical considerations for using this parameter estimation system lead to the conclusion that for the enzyme-catalyzed reaction tested, the extent overall reaction should be above .2% for high initial substrate concentrations, and above 1% for initial substrate concentrations in the range of the Michaelis constant. Application of this method to a typical enzyme-catalyzed reaction suggests that objective estimates of initial rates from a given set of concentrations and corresponding times can be obtained with a standard error in the range of 2–3%, but that reproducibility is not better than about 10%. When this procedure was used to estimate initial rates for the glycerol dehydrogenase-catalyzed oxidation of glycerol by NAD, it was found that this enzyme did not behave according to the classical “Michaelis-Menten” mechanism of enzyme action.     

The effect of fluidic conditions on the continuous-flow bioluminescent detection of ATP in a microfluidic device

The effect of fluidic conditions on the bioluminescent detection of ATP in a microfluidic device was surveyed using homemade detector system. The bioluminescent reaction of ATP was directly affected by the retention time and flow rates of the solutions in this diffusion-based mixing and reaction system due to the laminar flow in the microchannel. ATP and enzyme solutions were separately injected into the microfluidic device at different flow rates through 3 inlet ports. The ATP solution was injected through the middle port, while the enzyme solution was injected in the two remaining ports. When the ratio of ATP to enzyme solution was fixed, the optimum flow rates of enzyme, ATP, and enzyme solution was 3.5, 8.0, and 3.5 μL/min, respectively. The optimal total flow rate was 15 μL/min. The detection limit for the concentration of ATP at optimal conditions was less than 10⁻⁷ M.     

The Mycobacterium tuberculosis Ag85A is a novel diacylglycerol acyltransferase involved in lipid body formation

Mycobacterium tuberculosis accumulates large amounts of triacylglycerol (TAG) which acts as storage compounds for energy and carbon. The mycobacterial triacylglycerols stored in the form of intracellular lipid droplets are essential for long-term survival of M. tuberculosis during a dormant state. We report here that when the M. tuberculosis mycolytransferase Ag85A is overexpressed in Mycobacterium smegmatis mc(2)155, cell morphology was changed and the cells became grossly enlarged. A massive formation of lipid bodies and a change in lipid pattern was observed simultaneously. We suspected a possible role of Ag85A in the acyl lipid metabolism and discovered that the enzyme possesses acyl-CoA:diacylglycerol acyltransferase (DGAT) activity in addition to its well-known function as mycolyltransferase. Ag85A mediates the transesterification of diacylglycerol using long-chain acyl-CoA as acyl donors. The K(m) and K(cat) values for palmitoleoyl-coenzyme A were 390 μM and 55.54 min(-1) respectively. A docking model suggests that palmitoleoyl-coenzyme A and 1,2-dipalmitin occupy the same active site as trehalose 6,6'-dimycolate and trehalose 6'-monomycolate. The site-directed Ser126Ala mutation of the active site proved that this residue is involved in the catalytic activity of this enzyme. Although not proven conclusively for dormant stage of M. tuberculosis, our novel finding about the synthesis of TAGs by Ag85A strongly suggests that Ag85A may play a significant role in the formation of lipid storage bodies and thus also in the establishment and maintenance of a persistent tuberculosis infection.     

Kinetics of Encapsulated Yeast Alcohol Dehydrogenase Dispersed in an Organic Solvent

Microcapsules dispersed in organic solvents provide a suitable environment for conducting enzyme reactions involving cofactors and hydrophobic substrates. Encapsulated YADH is active and stable in cyclohexane provided the pH is adjusted appropriately. Mass transfer does not influence batch reaction rates. Conversion in a fluidized-bed reactor containing encapsulated YADH/NAD⁺ and employing cyclohexane as the continuous phase depends strongly on residence time and inlet cinnamyl alcohol concentration. However, interpretation of these results is complicated by enzyme inactivation by the product, cinnamaldehyde, and interference from residual encapsulating agents.