CS262239B1 - Demodulator arrangement for amplitude-modulated pulse - Google Patents

Description

262238 fungi of the genus Aspergillus. Glucose oxidase and catalase enzymes are located in microbial cells together in so-called electrotro-condensation microsomes - peroxisomes. This localization of both enzymes in the cell's intracellular structure is of great importance, since glucose oxidase-generated hydrogen peroxide is immediately "in situ" catalysed into oxygen and water. If there were no photosynthesis of both enzyme-catalyzed biochemical reactions, then the hydrogen peroxide, which was gaining and accumulating, would irreversibly denature its own enzyme for a very short period of time, generating it, resulting in cell death.

Thus, by intact, especially microbial, cells containing both of these enzymes, D-gluconate, respectively, can be prepared by a single enzymatic biotransformation of the aqueous D-glucose solution. its salts, since the emerging D-gluconic acid must be continuously neutralized by the alkali solution during the reaction to avoid a strong reduction in the pH of the environment, which would reduce the pH or the pH of the environment. stopping the enzyme re-action. A second possibility is to prepare the D-gluconate from the D-glucose by submersing one-time cultivation of the production microorganisms in the fermentation tank. It is understandable that the enzymatic conversion of D-glucose can also be carried out in different purities of both soluble isolated enzymes in a single manner.

However, these processes are not very economical, since a new batch of cells is always needed to prepare D-gluconate from D-glucose. In rare cases, the intact microbial mycelium can be used repeatedly, it being understood that the degree of theoretical substrate conversion to the desired product decreases depending on the number of repeated cycles of use, since the intact mycelium is mechanically destroyed by the shear in the stirred reactor and the enzymes are The subtle structures of the cell release the aqueous phases, solubilize and the specific enzyme activity decreases. Another complicating factor, in terms of the enzymatic preparation of D-gluconate from D-glucose, is the quantitative ratio of glucose oxidase and catalase in the production of microbial cells. V čs. author's certificate No. 247 531 describes the high-producing mutant strain Aspergillus foetidus var. pallidus AN 166, synthesizing high levels of glucose oxidase. The specific activity of glucose oxidase in this strain is 7 to 10-fold higher compared to other industry-leading microbial strains - producers of this enzyme.

However, the production of catalase enzyme is quantitatively comparable with other strains.

This strain also synthesizes the β-D-fructofuranosidase-invertase enzyme (EC 3.2.1.26), which allowed the production of a fermentation process for the production of either D-gluconate from D-glucose by MS. No. 250,277 or, in particular, the fermentation process for producing D-gluconate in addition to D-fructose from sucrose and intermediates thereof. Author's Certificate No. 259 439.

However, the content of invertase in the cells of this production strain is also not exceedingly attainable, but is sufficient for the single fermentation production of D-gluconate in addition to D-fructose from sucrose.

In the world of professional and patent literature, including Czechoslovakia, efforts have been made for more than 15 years to economize the production of O-gluconate based on its production of D-glucose by various means of mobilization of glucose oxidase and catalase cells. activity or these enzymes pre-isolated and subsequently mobilized by a variety of immunization methods. These immobilized biocatalysts, whether based on immobilized enzyme or cells, have the advantage of using immobilized biocatalysts many times repeatedly or continuously for the production of D-gluconate from D-glucose over the above-mentioned fermentation processes.

The glucose oxidase enzyme is highly unstable at pH above 7.0. The pH-dependent stability of this enzyme is significantly enhanced by cross-linking protein molecules with glutaraldehyde (Solomon asp., Biopolymers, 16, 1837, 1977). Glucose zadididase is also inhibited and inactivated by hydrogen peroxide, the reduced form of this enzyme being inactivated 100 times faster than the oxidized form [Kleppe, Biochemistry, 5, 139, 1966]. Greenfield et al. (Anal. Biochem. 65, 109, 1975) therefore co-immobilized catalase together with glucose oxidation to particles of water-insoluble carrier. Karube and sp. (Biotechnol. Bioeng. 19, 1233, 1977) immobilized glucose oxidase on the surface of their own microbial mycelium, Aspergillus niger, by glucaldehyde in the presence of albumin, and finally Hertmeier and Döppner (Biotechnol. Lett. 5, 743, 1983) colmobilized of catalase on the surface of Aspergillus niger production mycelium (containing glucose oxidase in cells) by crosslinking the molecular catalase with glutaraldehyde in the presence of al-bumin. For example, patent literature discloses various methods of immobilizing enzymes of glucose oxidase and catalase to particles in water-insoluble carriers of synthetic or natural origin of organic or inorganic nature, which are disclosed in NSRF 2 021 149 and DOS 2 214 442. čs. the Certificate No. 214 096, No. 209 743 no. 234 059. In these cases, the combination of sorption of these enzymes into water-insoluble particles and their subsequent covalent crosslinking with glutaraldehyde is involved, or

Claims (1)

202233 S covalently bond a mixture of these enzymes in a variety of chemically activated particles of various carriers. In es. No. 231,656 discloses covalent crosslinking of a mixture of both of these with reactive water-soluble polymers. If immobilization of microbial cells, especially imperfect fungi with glucose-oxidase activity, occurs, then the immobilization and biocatalysts based on whole cross-linked and permeabilized cells, respectively. po-let is described in MS. author's certificate no. 209 265 and the method of immobilization and biocatalysts with these activities based on cellular aggregates formed by reactive water-soluble polymers in MS. The cited literature and patent data have led to the development of various immobilized biocatalysts either on the basis of immobilized glucose oxidase and catalase enzymes or on the basis of mobilized cells with these enzyme activities, with the aim of enzyme converting D-glucose to D-gluconate. However, these biocatalysts either exhibited low specific activity, or exhibited high specific activity with a high degree of stabilization of these enzymatic activities, but from the point of view of industrial use, the achieved half-life did not permit a realistic assumption of D-gluconate D-glucose production. The operating half-lives of these biocatalysts range from 10 to 20 cycles of repeated use of the same biocatalyst, with the increasing initial concentration of the substrate (D-glucose) decreasing the operating half-life, as higher levels of hydrogen peroxide are generated, which glucose oxidase is irreversibly inactivated if a huge excess of catalase is not co-amplified. The present invention, in order to overcome the known complications, consists in the fact that even key enzymes, glucose oxidase and catalase, co-immobilize to each other in a covalently cross-linked network free of water-insoluble carriers together with cell fragments and peroxisomes. microorganisms in such a ratio and form that the resulting hydrogen peroxide is in situ, without diffusion of the barrier, and at any point of the biocatalyst particle (both on the surface and nearer to the center of the particle), is converted into water and molecular sieve, with the eventual and simultaneous by co-imbibing other enzymes, in particular aglucose isomerase invertase, in such a ratio that the resulting mixed biocatalyst can be used not only to prepare D-gluconate from D-glucose but i. D-gluconate in addition to D-fructose from sucrose, . D-glucose. The present invention relates to a mixed mobilized cellular and enzyme biocatalyst for converting D-glucose to D-gluconate and converting sucrose to D-gluconate and D-fructose according to MS. author's testimony 2. 231 530 which is obtainable by the present invention or by autolysed mixtures of cells of the production microorganisms or of these pumped or autolysed vegetable or animal tissues comprising the glucose oxidase, catalase and the enzyme enzymes. . invertase and / or glucose isomerase, or whole cells and / or subcellular particles, especially peroxisomes and cell walls, do not possess any of these enzyme activities, or these enzymes are soluble, technical, or of varying degrees of purity isolated from different biological sources , in particular from the production cells of railroad organisms, their treatment with an aqueous solution of glutaraldehyde and / or polyethylenimine or proteins of microbial, plant or animal origin and an aqueous solution of glu-taraldehyde, separation and mechanicalization of the resulting product and its by sequentially suspending it in cooled organic solvents unlimitedly miscible with water, re-separating and drying, and finally washing with water or buffer. The present invention also relates to a process for the production of a mixed cell and enzyme biocatalyst comprising adding to the continuously stirred reaction mixture a pH of 6.0 to 8.0, preferably 6.5 to 7.5, after pH adjustment. the aqueous solution of the aldehyde within its concentration range of 0.1 to 10 .mu.m volume / volume, or the aqueous solution of polyethyleneimine or aqueous protein solution followed by the aqueous solution of glutaraldehyde, adjusting the pH of the reaction mixture again within the range and stirring the mixture continuously after 1 hour or 5 hours, the particles are separated, mechanically homogenized and treated with cooled acetone or ethanol with stirring and / or mixed with water, separated again and dried for 1 to 24 hours at 10 to 40 hours. C: C, then the biocatalyst is suspended in water or buffer, decanted several times and washed, allowed to swell and separate. Glucose oxidase activity measurement (hereinafter referred to as GOD) was performed by monitoring oxygen depletion from the reaction mixture with an oxygen cell at the beginning of oxidation of the D-glucose solution during continuous mixing. The unit of GOD activity (U) is that amount of enzyme that corresponds to the consumption of one gmol of oxygen per minute in a 0.03 M citrate buffer pH 5.8 at 30 ° C under the conditions of the reaction kinetics. Measurement of catalase activity (hereinafter KAT) was performed by manganometric titration of hydrogen peroxide in the course of continuous stirring of the reaction mixture. One unit of KAT activity (U) is that amount of enzyme that corresponds to the hydrolysis of one mole of H 2 O 2 per minute in 0.1 M phosphate buffer pH 7.1 at 20 ° C.