Effect of metal ion concentration on a biological reactor

Saccharomyces cerevisiae (yeast) cells were employed as a source of alcohol dehydrogenase in the NAD(+)-to-NADH reaction. The cells were immobilized in calcium alginate monofilament fibers and used in a biological reactor. The alginate could not be heat sterilized since temperatures above 80 degrees C caused the polymer chains to degrade. The same proved true for the high pH necessary for the reaction, but the alginate strength was increased by Ba(2+) solution treatment. X-ray probe analysis showed that about 30% of the Ca(2+) sites exchanged with the Ba(2+) ions. The Ba(2+) ions (as well as the Ca(2+) ions) permeabilized the cells and increased the reaction rate. Long term trials showed that Ba(2+) ions were slowly elutriated from the fiber biocatalyst, causing a drop in reaction rate. The trend certainly was reversible as far as the fiber was concerned. It is assumed that the permeabilization of the cells by the Ba(2+) ions was a reversible process.     

固定化啤酒酵母细胞催化有机硅酮不对称还原

研究了固定化啤酒酵母细胞催化三甲基硅乙酮不对称还原反应,系统探讨了振荡速度、底物浓度、固定化细胞浓度、pH值和反应温度对反应速度、产率和产物光学纯度的影响。结果表明,上述因素对固定化啤酒酵母细胞催化三甲基硅乙酮不对称还原反应均有较显著的影响。振荡速度以150r/min为宜,底物浓度和固定化细胞浓度分别为14mmol/L和0.15g/mL较佳,适宜的pH值为7.3,最佳反应温度为25℃～30℃。在该优化反应条件下,反应最大产率和产物的光学纯度分别高达84.9%和90.2%ee。     

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.     

Immobilized enzymes: electrokinetic effects on reaction rates in a porous medium

The effect of the internal diffusion and electrical surface charge on the overall rate of a reaction catalyzed by an enzyme immobilized on a porous medium are examined. Effectiveness factors have been calculated which compare the global reaction rate to that existing in the absence of the internal diffusion and/or the electrical field. The surface charge, assumed to arise from the dissociation equilibria of the acidic and basic surface groups of the enzyme, generates an electrical double layer at the pore surface. The double-layer potential is governed by the Poisson-Boltzmann equation. It is shown that the diffusion potential can be characterized by a modulus which depends upon the surface reaction rate, the charges and diffusivities of the substrate and products, the ionic strength, and the pore dimensions. The flux of a charged species in the pore occurs under the influences of the concentration gradient and the electrical potential gradient. The governing equations are solved by an iterative numerical method. The effects of pH, enzyme concentration, and substrate concentration on the rates of two different hydrolysis reactions catalyzed by immobilized papain are examined. The release of H(+) in one of the reactions causes the lowering of internal pH, and also a constancy of the internal pH when the external pH in creases beyond a certain value. The latter reaction also shows a maximum in the reaction rate with respect to enzyme concentration. The reaction not involving H(+) as a product shows a maximum in the reaction rate with respect to external pH, but a monotonic increase in the reaction rate as the enzyme concentration increases.     

聚乙烯醇-明胶复合膜固定化重组大肠杆菌NAD激酶的研究

为提高烟酰胺腺嘌呤二核苷酸(NAD)激酶的稳定性,采用复合膜对NAD激酶进行固定化研究。选用聚乙烯醇(PVA)、聚乳酸(PLA)、海藻酸钠(SA)和明胶(GEL)膜材料固定化NAD激酶。通过单因素实验确定最佳固定化条件为:PVA∶GEL为4∶1,加酶量为0.6 mL,固定化时间为6h,固定化温度为35℃,此时酶活力回收率达到最高值84%。固定化酶酶学性质分析结果表明,与游离酶进行比较,固定化后NAD激酶的最适温度由50℃提高至55℃,最适pH由8.0降至7.0,NAD激酶的热稳定性和pH稳定性均得到显著提高,但固定化酶的亲和力降低。固定化NAD激酶重复利用6次后,酶活性依然可维持初始酶活性的75%以上,表明聚乙烯醇-明胶复合膜固定化酶具有良好的操作稳定性。     

Regeneration of NAD(P)H by immobilized whole cells of Clostridium butyricum under hydrogen high pressure

Immobilized whole cells of Clostridium butyricum reduced both NAD(+) and NADP(+) in the presence of hydrogen at a pressure of 100 atm. The NAD(+) and NADP(+) reduction activities were 4.45 and 4.30 U/g dry cells, respectively [U = NAD(P)H regenerated, mu mol/min]. The amount of NADH regenerated by immobilized cells increased with increasing hydrogen pressure above 10 atm. Immobilized cells (6 mg dry cells) of Cl. butyricum completely converted NAD(+) (6.4 mumole) to NADH for 5 h, whereas only 60% of NAD(+) were reduced by free cells. Immobilized cells retained 89% activity after the 5-h reactions were repeated 4 times. L-Alanine was continuously produced at the rate of 12.8 mumol/min g dry cells from hydrogen, ammonium, and pyruvate with immobilized Cl. butyricum-alanine dehydrogenase.     

Continuous production of NADP by immobilized Achromobacter aceris cells.

Several microorganisms having higher nicotinamide adenine dinucleotide kinase (NAD kinase, EC 2.7.1.23) activity were immobilized into polyacrylamide gel lattices. The enzyme activity field by immobilization was highest in Achromobacter aceris AKU 0120. By the incubation of the immobilized A. aceris cells at pH 4.0, the NAD kinase activity increased and the adenosine triphosphate (ATP)-degradation activity disappeared completely. Enzymatic properties of the immobilized A. aceris cells were investigated and compared with those of intact cells. The optimal pH and the optimal temperature of immobilized cells were the same as those of intact cells. Immobilized cell NAD kinase was more stable than that of intact cells. The operational half-life of immobilized cells was 20 days when the substrate solution was passed through a column packed with immobilized cells at a flow rate which gives a space velocity (SV) of 0.1 hr-1 at 37 degrees C. On the other hand, the half-life of the intact cells was only 6 hr.     

Continuous production ofL-malic acid by immobilizedBrevibacterium ammoniagenes cells

Summary Continuous production ofL-malic acid from fumaric acid using immobilized microbial cells was investigated. Several microorganisms having fumarase activity were immobilized into a polyacrylamide gel lattice. Among the microorganisms tested, immobilizedBrevibacterium ammoniagenes IAM 1645 showed the highest enzyme activity, but produced an unwanted by-product, succinic acid. Conditions for suppression of this side reaction were investigated, and bile extract treatment of immobilized cells was found to be effective.The bile extract treatment of immobilized cells also resulted in a marked increase of reaction rate forL-malic acid formation.No difference was observed between the native enzyme and immobilized cells in optimal pH and temperature of the enzyme reaction.The effect of temperature on the reaction rate and the stability of fumarase activity of an immobilized cell column were investigated under conditions of continuous enzyme reaction. The decay of enzyme activity during continuous enzyme reaction was expressed by an exponential relationship. Half-life of the fumarase activity of the immobilized cell column at 37°C was calculated to be 52.5 days.Presented at the Annual Meeting of the Society of Fermentation Technology, Japan, Osaka, Japan, October 30, 1975.     

An Affinity Chromatographic Reactor for Highly Efficient Turnover of Dissociable Cofactors

A conjugated enzyme system of alcool dehydrogenase and lactate dehydrogenase was immobilized in an ultrafiltration hollow fiber tube, which was inserted in a fine nylon tube to form a hollow-fiber-capillary reactor. To this reactor, the substrates, pyruvate and ethanol, were supplied continuously. The necessary cofactor, NAD, was supplied as a pulse for a short time. The retention time of NAD in the reactor, estimated from the response curve of lactate produced, was much longer than those of the other substrates and products because of the strong adsorption of NAD to the immobilized enzymes through affinity. Therefore, the reactor could produce lactate from pyruvate for a long time without any more NAD. As a typical case, when the enzyme concentration is sufficiently high, the estimated retention time of NAD was 50 times as long as those of other materials so that the NAD turnover obtained was 412,000. The effects of NAD pulse concentration and the immobilized enzyme concentration on the retention time of NAD and NAD turnover were investigated experimentally and theoretically.     

Mouse intracellular immunoglobulin M. Structure and identification of a free thiol group

1. The formation of the non-enzymic adduct of NAD(+) and sulphite was investigated. In agreement with others we conclude that the dianion of sulphite adds to NAD(+). 2. The formation of ternary complexes of either lactate dehydrogenase or malate dehydrogenase with NAD(+) and sulphite was investigated. The u.v. spectrum of the NAD-sulphite adduct was the same whether free or enzyme-bound at either pH6 or pH8. This suggests that the free and enzyme-bound adducts have a similar electronic structure. 3. The effect of pH on the concentration of NAD-sulphite bound to both enzymes was measured in a new titration apparatus. Unlike the non-enzymic adduct (where the stability change with pH simply reflects HSO(3) (-)=SO(3) (2-)+H(+)), the enzyme-bound adduct showed a bell-shaped pH-stability curve, which indicated that an enzyme side chain of pK=6.2 must be protonated for the complex to form. Since the adduct does not bind to the enzyme when histidine-195 of lactate dehydrogenase is ethoxycarbonylated we conclude that the protein group involved is histidine-195. 4. The pH-dependence of the formation of a ternary complex of lactate dehydrogenase, NAD(+) and oxalate suggested that an enzyme group is protonated when this complex forms. 5. The rate at which NAD(+) binds to lactate dehydrogenase and malate dehydrogenase was measured by trapping the enzyme-bound NAD(+) by rapid reaction with sulphite. The rate of NAD(+) dissociation from the enzymes was calculated from the bimolecular association kinetic constant and from the equilibrium binding constant and was in both cases much faster than the forward V(max.). No kinetic evidence was found that suggested that there were interactions between protein subunits on binding NAD(+).     

NADH production from NAD+ with a formate dehydrogenase system involving immobilized cells of a methylotrophic Arthrobacter strain.

A convenient and economical method of NADH production from NAD+ has been established using a formate dehydrogenase system involving immobilized cells of a methanol-utilizing bacterium. Arthrobacter sp, KM62. Four kinds of cell entrapment were studied. An immobilized cell preparation showing a high NADH production activity was obtained by entrapment in a kappa-carrageenan gel lattice. The NADH-producing activity of the immobilized cells was investigated under various conditions. The NADH-producing activity was evoked on the addition of Triton X-100 to the reaction mixture. The conditions for the continuous production of NADH with an immobilized cell column were also investigated. When a reaction mixture containing 10 mumol (6.63 mg) ml-1 NAD+ was passed through the column (1.2 x 20 cm) containing 1.62 g (as dry weight) of immobilized cells, at a space velocity of 0.125 at 35 degrees C, complete conversion was attained.     

Inhibition of porcine kidney betaine aldehyde dehydrogenase by hydrogen peroxide

Renal hyperosmotic conditions may produce reactive oxygen species, which could have a deleterious effect on the enzymes involved in osmoregulation. Hydrogen peroxide was used to provoke oxidative stress in the environment of betaine aldehyde dehydrogenase in vitro. Enzyme activity was reduced as hydrogen peroxide concentration was increased. Over 50% of the enzyme activity was lost at 100 μM hydrogen peroxide at two temperatures tested. At pH 8.0, under physiological ionic strength conditions, peroxide inhibited the enzyme. Initial velocity assays of betaine aldehyde dehydrogenase in the presence of hydrogen peroxide (0-200 μM) showed noncompetitive inhibition with respect to NAD(+) or to betaine aldehyde at saturating concentrations of the other substrate at pH 7.0 or 8.0. Inhibition data showed that apparent V(max) decreased 40% and 26% under betaine aldehyde and NAD(+) saturating concentrations at pH 8.0, while at pH 7.0 V(max) decreased 40% and 29% at betaine aldehyde and NAD(+) saturating concentrations. There was little change in apparent Km(NAD) at either pH, while Km(BA) increased at pH 7.0. K(i) values at pH 8 and 7 were calculated. Our results suggest that porcine kidney betaine aldehyde dehydrogenase could be inhibited by hydrogen peroxide in vivo, thus compromising the synthesis of glycine betaine.     

Kinetic behavior of immobilized Penicillin acylase

Penicillin acylase has been immobilized to carboxymethylcellulose and to the resin Amberlite XAD7. The reaction kinetics of the enzyme were affected by both intrinsic (molecular) and microenvironmental effects. The Michaelis constant for the enzyme increased after immobilization as a result of an intrinsic effect of the reagent, glutaraldehyde, used for enzyme immobilization. Microenvironmental effects were of two types: diffusional limitation of access of substrate and a reaction-generated pH depression in the support particles. This depression of internal pH was observed in all the preparations and could be reduced by addition of pH buffering salts to reactor. An adsorbed pH-indicating dyc was used to determine the surface and internal pH of particles of XAD7–penicillin acylase under various reaction conditions. The extent of diffusional rate limitation in XAD7–penicillin acylase was related to the penetration depth of protein into the porous support particles. The penetration depth of protein and thus the diffusional limitation of the reaction rate could be controlled by the conditions of preparation of the immobilized enzyme. A staining technique was used to observe the location of the protein.     

The kinetics of the reversible inhibition of heart lactate dehydrogenase through the formation of the enzyme–oxidized nicotinamide–adenine dinucleotide–pyruvate compound

The inhibition of lactate dehydrogenase at high pyruvate concentration was studied in three ways. First, a rapid decrease in the rate of the enzyme reaction was observed; secondly, the rate of formation of a pyruvate-NAD(+) compound was followed by the change in E(325); thirdly, the rate of quenching of the protein fluorescence was measured. The data obtained at pH6.0 at different temperatures and ionic strengths as functions of pyruvate, NAD(+) and enzyme concentrations show that the extent of inhibition can be correlated with the reversible formation of a compound between pyruvate and enzyme-bound NAD(+). It is suggested that the detailed kinetic analysis of the formation of this abortive ternary compound will give pertinent information about properties of the enzyme-NAD(+) compound involved in the normal catalytic process.     

Tagatose production by immobilized recombinant Escherichia coli cells containing Geobacillus stearothermophilus l-arabinose isomerase mutant in a packed-bed bioreactor

Using immobilized recombinant Escherichia coli cells containing Geobacillus stearothermophilus l-arabinose isomerase mutant (Gali 152), we found that the galactose isomerization reaction was maximal at 70 degrees C and pH 7.0. Manganese ion enhanced galactose isomerization to tagatose. The immobilized cells were most stable at 60 degrees C and pH 7.0. The cell and substrate concentrations and dilution rate were optimal at 34 g/L, 300 g/L, and 0.05 h(-1), respectively. Under the optimum conditions, the immobilized cell reactor with Mn2+ produced an average of 59 g/L tagatose with a productivity of 2.9 g/L.h and a conversion yield of 19.5% for the first 20 days. The operational stability of immobilized cells with Mn2+ was demonstrated, and their half-life for tagatose production was 34 days. Tagatose production was compared for free and immobilized enzymes and free and immobilized cells using the same mass of cells. Immobilized cells produced the highest tagatose concentration, indicating that cell immobilization was more efficient for tagatose production than enzyme immobilization.     

Immobilized enzymatic fluorescence capillary biosensor for determination of sulfated bile acid in urine

The authors have proposed an immobilized enzymatic fluorescence capillary biosensor (SBAs-IE-FCBS) for the determination of sulfated bile acids (SBAs). The reaction principle of the biosensor is that under the catalysis of the bile acid sulfate sulfatase (BSS) and beta-hydroxysteroid dehydrogenase (beta-HSD) immobilized on inner surface of a medical capillary, SBAs desulfates to 3beta-hydroxyl bile acids, then the latter reacts with nicotinamide adenine dinucleotide (NAD(+)), and is converted into 3-ketosteroid; meanwhile, NAD(+) is converted to reduced nicotinamide adenine dinucleotide (NADH). NADH continuously reacts with 1-methoxy-5-methylphenazinium methyl sulfate (1-MPMS) and is converted into NAD(+) circularly and 1-MPMSH(2). Finally resazurin is reduced into resorufin by 1-MPMSH(2), the formed resorufin (lambda(ex)/lambda(em): 540 nm/580 nm) is used for quantifying the concentration of SBAs. Optimized conditions being suitable with the biosensor are as follows: the concentrations of BSS and beta-HSD used for the immobilization all are 5 kUL(-1); the concentrations of 1-MPMS and resazurin all are 25 micromolL(-1); the concentrations of Tris-HCl buffer and NAD(+) are 100 and 400 micromolL(-1), respectively; total volume of the enzyme, reagent and sample is only 18 microL per time for determining; the reaction temperature is 37 degrees C; the reaction time is 15min. The concentration of SBAs is directly proportional to the fluorescence intensity of the biosensor measured from 0.5 to 5.0 micromolL(-1). The relative standard deviation is less than 3.4%, and the detection limit was 0.16 micromolL(-1). The recoveries are in the range 95.5-106%. This SBA-IE-FCBS can be used for quantifying SBAs in urine to diagnose and judge hepatobiliary diseases, etc.     

Topography, purification and characterization of thyroidal NAD+ glycohydrolase

Subcellular fractionation of bovine thyroid tissue by differential pelleting and isopycnic gradient centrifugation in a zonal rotor indicated that NAD(+) glycohydrolase is predominantly located and rather uniformly distributed in the plasma membrane. Comparison of NAD(+) glycohydrolase activities of intact thyroid tissue slices, functional rat thyroid cells in culture (FRT(l)) and their respective homogenates indicated that most if not all of the enzyme (catalytic site) is accessible to extracellular NAD(+). The reaction product nicotinamide was predominantly recovered from the extracellular medium. The diazonium salt of sulphanilic acid, not penetrating into intact cells, was able to decrease the activity of intact thyroid tissue slices to the same extent as in the homogenate. Under the same conditions this reagent almost completely abolished NAD(+) glycohydrolase activity associated with intact thyroid cells in culture. The triazine dye Cibacron Blue F3GA and its high-M(r) derivative Blue Dextran respectively completely eliminated or caused a severe depression in the NAD(+) glycohydrolase activity of FRT(l) cells. The enzyme could be readily solubilized from bovine thyroid membranes by detergent extraction, and was further purified by gel filtration and affinity chromatography on Blue Sepharose CL-6B. The overall procedure resulted in a 1940-fold purification (specific activity 77.6mumol of nicotinamide released/h per mg). The purified enzyme displays a K(m) of 0.40mm for beta-NAD(+), a broad pH optimum around pH7.2 (0.1 m-potassium phosphate buffer) and an apparent M(r) of 120000. Nicotinamide is an inhibitor (K(i) 1.9mm) of the non-competitive type. The second reaction product ADP-ribose acts as a competitive inhibitor (K(i) 2.7mm). The purified enzyme splits beta-NAD(+), beta-NADP(+), beta-NADH and alpha-NAD(+) at rates in the relative proportions 1:0.75:<0.02:<0.02 and exhibits transglycosidase (pyridine-base exchange) activity. Anionic phospholipids such as phosphatidylinositol and phosphatidylserine inhibit the partially purified enzyme. A stimulating effect was observed upon the addition of histones.     

Reactions of d-glyceraldehyde 3-phosphate dehydrogenase facilitated by oxidized nicotinamide–adenine dinucleotide

Transient kinetic methods have been used to study the influence of NAD(+) on the rate of elementary processes of the reversible oxidative phosphorylation of d-glyceraldehyde 3-phosphate catalysed by d-glyceraldehyde 3-phosphate dehydrogenase. In the pH range 5-8 NAD(+) is bound to the enzyme during the following elementary processes of the mechanism: phosphorolysis of the acyl-enzyme, its formation from 1,3-diphosphoglycerate and the enzyme and the formation and breakdown of the glyceraldehyde 3-phosphate-enzyme complex. The rates of these four elementary processes only equal or exceed the turnover rate of the enzyme when NAD(+) is bound and are as much as 10(4) times the rates in the absence of NAD(+). Autocatalysis of the reductive dephosphorylation of 1,3-diphosphoglycerate occurs when glyceraldehyde 3-phosphate release is rate determining because NAD(+) is a reaction product. An important feature of the enzyme mechanism is that the negative-free-energy change of a chemical reaction, acyl-enzyme formation, is linked in a simple way to the positive-free-energy change of a dissociation reaction, NAD(+) release.     

Purification and characterization of aminopropionaldehyde dehydrogenase from Arthrobacter sp. TMP-1

Aminopropionaldehyde dehydrogenase was purified to apparent homogeneity from 1,3-diaminopropane-grown cells of Arthrobacter sp. TMP-1. The native molecular mass and the subunit molecular mass of the enzyme were approximately 20,5000 and 52,000, respectively, suggesting that the enzyme is a tetramer of identical subunits. The apparent Michaelis constant (K(m)) for 1,3-diaminopropane was approximately 3 microM. The enzyme equally used both NAD(+) and NADP(+) as coenzymes. The apparent K(m) values for NAD(+) and NADP(+) were 255 microM and 108 microM, respectively. The maximum reaction rates (V(max)) for NAD(+) and NADP(+) were 102 and 83.3 micromol min(-1) mg(-1), respectively. Some tested aliphatic aldehydes and aromatic aldehydes were inert as substrates. The optimum pH was 8.0-8.5. The enzyme was sensitive to sulfhydryl group-modifying reagents.     

根瘤菌BK-20固定化细胞生产L(+)-酒石酸

顺式环氧琥珀酸水解酶(CESH)是根瘤菌BK-20生产L(+)-酒石酸的关键酶。为提高其生产效率和生产稳定性,首先优化根瘤菌BK-20的产酶条件,然后利用固定化细胞连续生产L(+)-酒石酸。结果显示,优化后游离细胞酶活达(3 498.0±142.6)U/g,较优化前提高643%。固定化细胞酶活达(2 817.2±226.7)U/g,其最适包埋剂、菌体浓度和凝胶浓度分别为海藻酸钠,10%(W/V)和1.5%(W/V)。固定化细胞连续反应10批后,其形状和酶活均无明显改变,单批次转化率达98%以上,具有良好的生产稳定性。