Characteristics of yeast invertase immobilized on porous cellulose beads.

Invertase from Candida utilis was immobilized on porous cellulose beads by an ionic-quanidino bond. The immobilized invertase showed optimum activity between pH 4.0 and 5.4, while the free enzyme had a sharp optimum at pH 4.1. Both temperature profiles were fairly similar up to 55 degrees C. However, above this temperature the immobilized enzyme was more stable than the free enzyme. From the temperature data, the activation energies were found to be 7,322 and 4,052 cal/g mol for the free and the immobilized enzyme, respectively. Candida invertase shows characteristics of substrate inhibition. Both the Km and Ki for the free and the immobilized enzymes were determined. The apparent Ki for the immobilized invertase was much higher than the Ki of the free enzyme, suggesting a diffusion effect. Immobilized invertase molecules deep in the pores only see sucrose concentrations much less than the bulk concentrations. Immobilization, thus, offers certain processing advantages in this regard.     

Properties of invertase immobilized on the poly(ethylene-co-vinyl alcohol) hollow fiber membrane

Invertase was ionically immobilized on the poly(ethylene-co-vinyl alcohol) hollow fiber inside surface, which was aminoacetalized with 2-dimethylaminoacetaldehyde dimethyl acetal. Immobilization and enzyme reaction were carried out by letting the respective solutions pass or circulate through the inside of the hollow fiber, and the activity of invertase was determined by the amount of glucose produced enzymatically from sucrose. Immobilization conditions were examined with respect to the enzyme concentration and to the time, and consequently the preferable conditions at room temperature were found to be 5 mug/mL of enzyme concentration and 4 h of immobilization time. Under those conditions the immobilization yield and the ratio of the activity of the immobilized invertase to that of the native one were 89 and 80%, respectively. For both repeating and continuous usages, the activity fell to ca. 60% of the initial activity in the early stage and after that almost kept that value. The apparent Michaelis constant K(m) (') for the immobilized invertase decreased with increasing the flow rate of the substrate solution, to be close to the value for the native one. Furthermore, the possibility of the separation of the enzymatically formed glucose from the reaction mixture through the hollow fiber membrane was preliminarily examined.     

An enzyme rate equation for the overall rate of reaction of gel-immobilized glucose oxidase particles under buffered conditions. I. Pseudo-one substrate conditions

The overall rate of reaction of buffered gel-immobilized glucose oxidase particles is described by means of an enzyme rate equation which relates the overall reaction rate of a particle to the free solution characteristics of the enzyme, the effective diffusivity of the limiting substrate in the gel, the characteristic particle size, and the limiting substrate concentration adjacent to the gel surface. This equation accounts quantitatively for the limitation of the overall rate of reaction by substrate diffusion, and it is used to illustrate the influence of the system parameters, i. e., particle size, enzyme concentration, and pH, on the extent of the diffusional resistance associated with gel-immobilized glucose oxidase particles. The enzyme rate equation is generally applicable to those enzymes whose kinetics approximately follow Michaelis-Menten form when in free solution.     

Kinetics of biphasic growth of yeast in continuous and fed-batch cultures

The influence of dilution rate on the production of biomass, ethanol, and invertase in an aerobic culture of Saccharomyces carlsbergensis was studied in a glucose-limited chemostat culture. A kinetic model was developed to analyze the biphasic growth of yeast on both the glucose remaining and the ethanol produced in the culture. The model assumes a double effect where glucose regulates the flux of glucose catabolism (respiration and aerobic fermentation) and the ethanol utilization in yeast cells. The model could successfully demonstrate the experimental results of a chemostat culture featuring the monotonic decrease of biomass concentration with an increase of dilution rate higher than 0.2 hr^?1 as well as the maximum ethanol concentration at a particular dilution rate around 0.5 hr^?1. Some supplementary data were collected from an ethanol-limited aerobic chemostat culture and a glucose-limited anaerobic chemostat culture to use in the model calculation. Some parametric constants of cell growth, ethanol production, and invertase formation were determined in batch cultures under aerobic and anaerobic states as summarized in a table in comparison with the chemostat data. Using the constants, a prediction of the optimal control of a glucose fed-batch yeast culture was conducted in connection with an experiment for harvesting a high yield of yeast cells with high invertase activity.     

Preparation of some immobilized linked enzyme systems and their use in the automated determination of disaccharides

1. Glucose oxidase (EC 1.1.3.4), amyloglucosidase (EC 3.2.1.3), invertase (EC 3.2.1.26) and beta-galactosidase (EC 3.2.1.23) were covalently attached via glutaraldehyde to the inside surface of nylon tube. 2. The linked enzyme system, comprising invertase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of sucrose. 3. The linked enzyme system, comprising beta-galactosidase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of lactose. 4. The linked enzyme system, comprising amyloglucosidase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of maltose. 5. Mixtures of glucose oxidase and amyloglucosidase were immobilized within the same piece of nylon tube and used for the automated determination of maltose. 6. Mixtures of glucose oxidase and invertase were immobilized within the same piece of nylon tube and used for the automated determination of sucrose.     

Chemically surface modified gel (CSMG): an excellent enzyme-immobilization matrix for industrial processes

Invertase from S. cerevisiae has been immobilized on porous silica matrix, formed using sol-gel chemistry, with surface area of approximately 650 m(2)/g. The co-condensation of silica sol with 3-aminopropyl(triethoxy)silane produced an amino-chemically surface modified silica gel (N-CSMG) with a very high ligand loading of 3.6 mmol/g SiO(2); significantly higher than commercially available matrices. Surface amine groups were activated with glutaraldehyde to produce GA-N-CSMG, and invertase covalently attached by the aldehyde. Invertase was used as a model enzyme to measure the immobilizing character of the GA-N-CSMG material. Using an optimized immobilization protocol, a very high loading of 723 mg invertase per gram GA-N-CSMG is obtained; 3-200-fold higher than values published in literature. The reproducible, immobilized activity of 246,000 U/g GA-N-CSMG is also greater than any other in literature. Immobilized invertase showed almost 99% retention of free enzyme activity and no loss in catalytic efficiency. The apparent kinetic parameters K(M) and V(M) were determined using the Michealis-Menten kinetic model. K(M) of the free invertase was 1.5 times greater than that of the immobilized invertase--indicating a higher substrate affinity of the immobilized invertase. These findings show considerable promise for this material as an immobilization matrix in industrial processes.     

Covalent binding of enzymes to synthetic membranes containing acrylamide units, using formaldehyde

Synthetic membranes containing 10% acrylamide units were subjected to activation with formaldehyde at pH 7.5 and 45 degrees C. Trypsin, invertase, and urease were bound to this activated membrane and the kinetic properties of immobilized enzymes were studied. The permeability of the membrane for distilled water manifests certain differences depending on the enzyme bound. The membranes with immobilized enzymes stored at 4 degrees C in a moist state showed no change in their activity for 6 months. The membrane with immobilized invertase has preserved its activity even after 20 operations with 2% sucrose solution at 25 degrees C. The proposed method of binding enzymes to synthetic membranes containing acrylamide groups, through the introduction of N-hydroxymethyl groups, possesses several advantages with respect to the activation of the membrane in a one-step reaction with cheap and accessible reagent, high operative stability of the immobilized enzymes, no danger of bacterial rotting, and long shelf life of the membrane.     

Immobilization of invertase onto poly(3-methylthienyl methacrylate)/poly(3-thiopheneacetic acid) matrix

A novel immobilization matrix, poly(3-methylthienyl methacrylate)–poly(3-thiopheneacetic acid) (PMTM–PTAA), was synthesized and used for the covalent immobilization of Saccharomyces cerevisiae invertase to produce invert sugar. The immobilization resulted in 87% immobilization efficiency. Optimum conditions for activity were not affected by immobilization and the optimum pH and temperature for both free and immobilized enzyme were found to be 4.5 and 55 °C, respectively. However, immobilized invertase was more stable at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined using the Lineweaver–Burk plot. The K_m values were 35 and 38 mM for free and immobilized enzyme, respectively. The V_max values were 29 and 24 mg glucose/mg enzyme min for free and immobilized enzyme, respectively. Immobilized enzyme could be used for the production of glucose and fructose from sucrose since it retained almost all the initial activity for a month in storage and retained the whole activity in repeated 50 batch reactions.     

Biochemical and immunological properties of alkaline invertase isolated from sprouting soybean hypocotyls.

Alkaline invertase from sprouting soybean (Glycine max) hypocotyls was purified to apparent electrophoretic homogeneity by consecutive use of DEAE-cellulose, green 19 dye, and Cibacron blue 3GA dye affinity chromatography. This protocol produced about a 100-fold purification with about a 11% yield. The purified protein had a specific activity of 48 mumol of glucose produced mg-1 protein min-1 (pH 7.0) and showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) (58 kDa) and in native PAGE, as indicated by both protein and activity staining. The native enzyme molecular mass was about 240 kDa, suggesting a homotetrameric structure. The purified enzyme exhibited hyperbolic saturation kinetics with a Km (sucrose) near 10 mM and the enzyme did not utilize raffinose, maltose, lactose, or cellibose as a substrate. Impure alkaline invertase preparations, which contained acid invertase activity, on contrast, showed biphasic curves versus sucrose concentration. Combining equal activities of purified alkaline invertase with acid invertase resulted in a biphasic response, but there was a transition to hyperbolic saturation kinetics when the activity ratio, alkaline: acid invertase, was increased above unity. Alkaline invertase activity was inhibited by HgCl2, pridoxal phosphate, and Tris with respective Ki values near 2 microM, 5 microM, and 4 mM. Glycoprotein staining (periodic acid-Schiff method) was negative and alkaline invertase did not bind to two immobilized lectins, concanavalin A and wheat germ agglutinin; hence, the enzyme apparently is not a glycoprotein. The purified alkaline invertase, and a purified soybean acid invertase, was used to raise rabbit polyclonal antibodies. The alkaline invertase antibody preparation was specific for alkaline invertase and cross-reacted with alkaline invertases from other plants. Neither purified soybean alkaline invertases nor the crude enzyme from several plants cross-reacted with the soybean acid invertase antibody.     

宁夏枸杞果实糖积累和蔗糖代谢相关酶活性的关系

通过对枸杞果实发育过程中果实生长模式、蔗糖、果糖、葡萄糖和淀粉含量及糖代谢相关酶活性的测定,研究了宁夏枸杞果实生长发育过程中糖的代谢积累与相关酶活性的关系.结果表明:(1)宁夏枸杞果实发育呈双S"曲线,果实主要以积累己糖为主.(2)蔗糖磷酸合成酶(SPS)活性在果实发育初期处于下降的趋势,在花后19d开始上升,果实转色后又逐渐下降;蔗糖合成酶(SS)活性总体表现为SS分解方向的活性大于SS合成方向的活性,说明枸杞果实发育过程中,SS的活性主要以分解方向的为主;酸性转化酶(AI)和中性转化酶(NI)的活性随果实发育呈上升趋势,但在果实成熟后期有所下降,且AI和NI活性高于合成酶类的活性,较高的转化酶类活性促进了果实内部己糖的积累.(3)在枸杞果实生长发育中,葡萄糖和果糖含量与AI和NI均呈极显著正相关,而与其它酶不具有相关性.说明AI和NI在宁夏枸杞果实的糖代谢中起着主要的调控作用.     

Mass transfer analysis for immobilized cells of Lactococcus lactis sp. using both simulations and in-situ pH measurements

Mathematical modeling and in-situ pH measurements were used to characterize the effects of the microenvironment on alginate gel beads immobilized cells of Lactococcus lactis. Mass transfer limitations led to a progressive pH acidification within gel beads which determined both the cell distribution and the cellular activity of entrapped cells. The dynamics of the system is discussed in relation to the overall activity of the immobilized cell reactor.     

利用天然载体固定化活性干酵母细胞的研究

利用天然载体——蛋清固定化市售活性干酵母细胞,再经戊二醛进行共价交联而制备的具有高转化酶活力的新型粒状固定化生物催化剂,菌体包埋量大,活力回收高,机械性能好;特别是固定化生物催化剂经冷冻后,形成了均匀的多孔状颗粒,而酶活力基本不变,机械性能增强之特性．活性干酵母固定化后,其动力学特性表现为：K’m明显增大,热稳定性大大提高．于最适条件下,连续批次搅拌反应达两个月,凝胶颗粒无细胞渗漏,表现出相当稳定的酶活力．     

Enzyme immobilized in a packed-bed reactor: kinetic parameters and mass transfer effects.

To describe axial dispersion, particle film mass transfer, intraparticle diffusion, and the chemical reaction of the substrate for enzymes immobilized in porous particles in packed columns, we have developed mathematical models for first- and zero-order limits of Michaelis-Menten kinetics. Steady-state solutions were derived for both long and short column boundary conditions and for plug flow. Theory was compared to experiments by hydrolysis of sucrose catalyzed by invertase bound to porous glass particles. Steady-state conversions were measured for a range of flow rates. Pulse response experiments with inert packing were used to determine values of bed void fraction and particle porosity.     

Immobilization and stabilization of invertase on Cajanus cajan lectin support

Use of lectins as ligands for the immobilization and stabilization of glycoenzymes has immense application in enzyme research and industry. But their widespread use could be limited by the high cost of their production. In the present study preparation of a novel and inexpensive lectin support for use in the immobilization of glycoenzymes containing mannose or glucose residues in their carbohydrate moiety has been described. Cajanus cajan lectin (CCL) coupled covalently to cyanogen bromide activated Seralose 4B could readily bind enzymes such as invertase, glucoamylase and glucose oxidase. The immobilized and glutaraldehyde crosslinked preparations of invertase exhibited high resistance to inactivation upon exposure to enhanced temperature, pH, denaturants and proteolysis. Binding of invertase to CCL-Seralose was however found to be readily reversible in the presence of 1.0 M methyl alpha-D mannopyranoside. In a laboratory scale column reactor the CCL-Seralose bound invertase was stable for a month and retained more than 80% of its initial activity even after 60 days of storage at 4 degrees C. CCL-Seralose bound invertase exhibited marked stability towards temperature, pH changes and denaturants suggesting its potential to be used as an excellent support for the immobilization of other glycoenzymes as well.     

Effect of molecular mobility on coupled enzymatic reactions involving cofactor regeneration using nanoparticle-attached enzymes

Cofactor-dependent multi-step enzymatic reactions generally require dynamic interactions among cofactor, enzyme and substrate molecules. Maintaining such molecular interactions can be quite challenging especially when the catalysts are tethered to solid state supports for heterogeneous catalysis for either biosynthesis or biosensing. The current work examines the effects of the pattern of immobilization, which presumably impacts molecular interactions on the surface of solid supports, on the reaction kinetics of a multienzymic system including glutamate dehydrogenase, glucose dehydrogenase and cofactor NAD(H). Interestingly, particle collision due to Brownian motion of nanoparticles successfully enabled the coupled reactions involving a regeneration cycle of NAD(H) even when the enzymes and cofactor were immobilized separately onto superparamagnetic nanoparticles (124 nm). The impact of particle motion and collision was evident in that the overall reaction rate was increased by over 100% by applying a moderate alternating magnetic field (500 Hz, 17 Gs), or using additional spacers, both of which could improve the mobility of the immobilized catalysts. We further observed that integrated immobilization, which allowed the cofactor to be placed in the molecular vicinity of enzymes on the same nanoparticles, could enhance the reaction rate by 1.8 fold. These results demonstrated the feasibility in manipulating molecular interactions among immobilized catalyst components by using nanoscale fabrication for efficient multienzymic biosynthesis.     

Catalytic behaviors of enzymes attached to nanoparticles: the effect of particle mobility

Nanoparticles provide an ideal remedy to the usually contradictory issues encountered in the optimization of immobilized enzymes: minimum diffusional limitation, maximum surface area per unit mass, and high effective enzyme loading. In addition to the promising performance features, the unique solution behaviors of the nanoparticles also point to a transitional region between the heterogeneous (with immobilized enzymes) and homogeneous (with soluble free enzymes) catalysis. The particle mobility, which is related to particle size and solution viscosity through Stokes-Einstein equation, may impact the reaction kinetics according to the collision theory. The mobility-activity relationship was examined through experimental studies and theoretical modeling in the present work. Polystyrene particles with diameters ranging from 110-1000 nm were prepared. A model enzyme, alpha-chymotrypsin, was covalently attached to the nanoparticles up to 6.6 wt%. The collision theory model was found feasible in correlating the catalytic activities of particles to particle size and solution viscosity. Changes in the size of particles and the viscosity of reaction media, which all affect the mobility of the enzyme catalyst, evidently altered the intrinsic activity of the particle-attached enzyme. Compared to K(M), k(cat) appeared to be less sensitive to particle size and viscosity.     

Characterization of wall-bound invertase isoforms of Picea abies cells and regulation by ectomycorrhizal fungi

In culture, the ectomycorrhiza-forming fungi Amanita muscaria (Pers. ex Fries) Hock. and Hebeloma crustuliniforme (Bull. ex Fries) Quel. only grow on media with glucose or fructose but not with sucrose as sole carbohydrate source. This is due to their lack of wall-bound invertase activity. Therefore, utilization of sucrose by the fungi within a mycorrhizal association is believed to depend on the wall-bound invertase activity of the host. This enzyme activity was studied in the apoplast of suspension cultured cells of Picea abies (L.) Karst. An ionically and a tightly wall-bound isoform of acid invertase were found that function as β-d -fructofuranoside-fructohydrolases (EC 3.2.1.26). The ionically bound enzyme could be easily released from walls of intact cells with buffer of high ionic strength. In its native form, the ionically bound invertase isoform is a monomeric protein with a molecular mass of 61 kDa, as determined by gel filtration and SDS-PAGE. Glycoprotein nature of the enzyme was demonstrated with antibodies directed against the digoxigenin-labeled protein. The K_m values of both enzymes for sucrose, their natural substrate, are relatively high (ionically bound invertase K_m= 16 mM, tightly bound invertase K_m= 8.6 mM). Activity of both wall-bound invertase isoforms strongly depends on the apoplastic pH. They have a narrow pH-optimum and exhibit highest activity at pH 4.5. with elevated activity between pH 4.5 and 6.0. Furthermore, fructose acts as competitive inhibitor of both isoforms, whereas glucose is not inhibitory. Unloading of sucrose from host cells to the apoplastic interface of the Hartig net in ectomycorrhizae appears to depend on the rate of hydrolysis by the wall-bound invertase of the host. Since the activity of the plant invertase depends on the actual pH value and the fructose concentration in the mycorrhizal interface, we suggest that the fungus can actively influence the activity of the plant invertase by acidification of the cell wall and by fructose uptake. Thus, the fungus itself can regulate its own supply of glucose and fructose.     

Preparation of immobilized enzymes by N-vinylpyrrolidone and the general properties of the glucoamylase gel

Immobilized glucoamylase, invertase, and β-galactosidase were prepared by using N-vinylpyrrolidone monomer (VP) under γ-ray irradiation. The enzyme-VP solutions were gelled by irradiation with 2.9 Mrad and the added enzymes were almost completely entrapped. Activity losses on entrapping were 55% for the VP-glucoamylase gel, and more than 90% in the case of VP-invertase and VP-β-galactosidase gels. No leakage of enzyme from these gels could be detected within 1 hr. The VP-glucoamylase gel was capable of hydrolyzing dextrin (mol wt 10,400) to glucose and the glucose equivalent was equal to that obtain able with native enzyme. The optimum temperature, heat stability, pH activity curve, and pH stability of VP-glucoamylase gel were slightly inferior to those of native enzyme, while Km was a little larger than that of native enzyme.     

Characteristics of unbuffered gel-immobilized urease particles. II. Overall rate of reaction

The overall rates of reaction of unbuffered gel-immobilized urease particles have been investigated with the aid of a packed-bed differential recycle reactor. Both substrate and enzyme concentrations have received attention. Cylindrical gel particles contained within impermeable tubelets were used to provide the physical strength necessary for the packed-bed arrangement and a one dimensional diffusion path to aid understanding of the complex interactions between substrate and product diffusion, and their effect on the reactions taking place. The experimental data have been interpreted with the aid of an enzyme rate equation (ERE) which relates the free solution characteristics of the enzyme to the conditions within a diffusion limited particle. The internal hydrogen ion profiles have been accommodated by a lumped parameter, the apparent pH (pH_app). Two methods have been suggested for the calculation of pH_app and the loss of activity on particle preparation, these methods are based on the use of the ERE in conjunction with experimental data.     

Kinetics of ligand binding to receptor immobilized in a polymer matrix, as detected with an evanescent wave biosensor. I. A computer simulation of the influence of mass transport.

The influence of mass transport on ligand binding to receptor immobilized in a polymer matrix, as detected with an evanescent wave biosensor, was investigated. A one-dimensional computer model for the mass transport of ligand between the bulk solution and the polymer gel and within the gel was employed, and the influence of the diffusion coefficient, the partition coefficient, the thickness of the matrix, and the distribution of immobilized receptor were studied for a variety of conditions. Under conditions that may apply to many published experimental studies, diffusion within the matrix was found to decrease the overall ligand transport significantly. For relatively slow reactions, small spatial gradients of free and bound ligand in the gel are found, whereas for relatively rapid reactions strong inhomogeneities of ligand within the gel occur before establishment of equilibrium. Several types of deviations from ideal pseudo-first-order binding progress curves are described that resemble those of published experimental data. Extremely transport limited reactions can in some cases be fitted with apparently ideal binding progress curves, although with apparent reaction rates that are much lower than the true reaction rates. Nevertheless, the ratio of the apparent rate constants can be semiquantitatively consistent with the true equilibrium constant. Apparently "cooperative" binding can result from high chemical on rates at high receptor saturation. Dissociation in the presence of transport limitation was found to be well described empirically by a single or a double exponential, with both apparent rate constants considerably lower than the intrinsic chemical rate constant. Transport limitations in the gel can introduce many generally unknown factors into the binding progress curve. The simulations suggest that unexpected deviations from ideal binding progress curves may be due to highly transport influenced binding kinetics. The use of a thinner polymer matrix could significantly increase the range of detectable rate constants.