CS259090B1 - A method for the controlled biosynthesis of galactose, xylose, and non-specifically carbonated starch-labeled starch. - Google Patents

Abstract

The biosynthesis of fluoride starch and xylose labeled with radioactive carbon is not known. The biosynthesis of galactose labeled with radioactive carbon isotope has so far been carried out by cultivating the stele red algae of the genus Iridaea, the disadvantages of which are slow growth and low yield (2 > í), low specific activity and uneconomical use of valuable 14co2. The method of controlling biosynthesis according to the invention is carried out by a single cultivation of daughter cells of a synchronous culture of algae of the genus Porphyridium free from slime substances in an environment of pure radioactive CO2 and in a nutrient solution without nitrogen compounds (temperature 24 - 28 ° C, illumination of the surface of the culture vessel 60 - 80 μm~2 PAR, environment: mixture of gaseous nitrogen and radioactive CO2). A high yield of fluoride starch, galactose and xylose is achieved, as well as their high specific activity. The production process is characterized by simplicity and high safety of the washer.

Description

The invention relates to a method of controlling the biosynthesis of galactose, xylose and floride starch, non-specifically labeled with the radio14 carbon isotope /C/ in a synchronous culture of the genus Redbud.

Porphyridium, grown under photoautotrophic conditions.

The procedures for the production of radiocarbon-labeled starch and xylose are not known from the literature. The procedure for the preparation of radiocarbon-labeled galactose by biosynthesis is described in the work of Bean et al. /4.B1ol.Chem. 204: 169-173, 1953/.

For the biosynthesis of radioactive C0 _2, the stele of the genus Iridaea was used in this work, the disadvantage of which is slow growth and low yield of the desired product, since galactose constitutes only % of all organic matter. The procedure is characterized by uneconomical 14 utilization of valuable CO.,, which is incorporated into all their final products during photosynthesis, and low specific activity, not exceeding 100 uc.mg. of galactose.

These disadvantages are eliminated by the method of controlled biosynthesis according to the invention, the essence of which is that a single cultivation of daughter cells of a synchronous culture of algae of the genus Porphyridum is carried out for 32 to 38 hours in a mineral nutrient solution without nitrogen compounds at a temperature of 24-28°C in an environment of a mixture of gaseous nitrogen and radioactive carbon dioxide under illumination of the surface of the culture vessel with 60-80 Wm"^ FAR, while at the beginning of the production cultivation, slime substances secreted during the pre-production cultivation .12» in an environment of non-radioactive carbon dioxide C0 ₂ by the cells are removed

- 2 algae in a nutrient solution containing nitrogen compounds.

The storage intracellular polysaccharide of the red fungus of the genus Porphyridium is floride starch, which is, due to its chemical structure, a very suitable substrate for enzymatic processing by amylases. In addition to the production of floride starch, the used red fungi secrete mucilage substances into the nutrient solution during metabolism, the main share of which are acidic polysaccharides, which after hydrolysis yield 0 and L galactose, D-xylose and D-glucose.

The amount of slzs under usual cultivation conditions constitutes 10-20% of the dry mass of algal cells; the proportion of carbohydrates in the dry mass of extracellular slzs usually does not exceed 30%. The amount of floride starch most often ranges from 20 to 20% of the dry mass of cells.

The lacrimal synthesis of the above-mentioned carbohydrates takes place in an environment where the sole source of carbon is ¹⁴ C0 ₂ . Daughter cells of a synchronous population of a selected strain of unicellular red algae Pprphyrídiura are used for cultivation, transferred to a mineral nutrient solution without nitrogen compounds. Removal of nitrogen ions from the nutrient solution results in the cessation of proteosynthesis, which causes the accumulation of floride starch in the cells and an increased rate of synthesis of slimes secreted into the nutrient solution outside the cells, simultaneously with an increase in the content of galactose and xylose in relation to glucose.

Achieving a high specific activity of starch is conditioned by the use of a synchronous culture, i.e. one in which all cells are in the same stage of the life cycle. Synchronization is carried out by the method of alternating periods of light and darkness. For production cultivation, the youngest cells are used, in which the amount of floride starch is the lowest, since it was consumed in energy-intensive metabolic processes during cell division in the dark period of the previous synchronous cycle. The low initial amount of starch at the beginning of the production cultivation is one of the conditions for its high specific activity at the end of the production cycle. Removal of the slime envelopes before incorporation of labeled carbon into cell structures and into the newly formed slime results in achieving a high specific activity of ellagic carbohydrates.

The advantages of the proposed method of controlled algae cultivation according to the invention consist mainly of:

·» 3 M

- in a high yield of floride starch, because during controlled production cultivation its exclusive synthesis takes place in the cells, so that its amount at the end of the production cycle reaches 50-60% of the dry mass of algae cells,

- in increased yield of extracellular polysaccharides, which after the end of cultivation reach 25% of the dry mass of cells*,

- in the preferential representation of individual carbohydrates in these elixirs,

- _it increases the specific activity of the products caused by florid starch, by increasing its amount approximately 7 times during controlled cultivation. In the case of extracellular polysaccharides, high specific activity is achieved by their de novo synthesis on ¹⁴ C0 ₂ ,

- in the simplicity and safety of production operations,

- in the high profitability of the production of the said compounds.

An example of a controlled blo synthase process according to the invention is given below.

Example

e) Pre-production cultivation

Preparatory cultivation and actual production cultivation are carried out in a cultivation device described in Czechoslovak Patent Application No. AO 197 918. For cultivation, a selected culture of algae of the genus Porphyridlum is used, kept in test tubes on agar media enriched with mineral nutrient solution at temperatures of 15°C and irradiance of 10 Wm- ² FAR. FAR means photosynthetically effective radiation measured by a spectrally non-selective sensor, sensitive only in the range of 400-700 nm.

The ee culture from agar is transferred to a liquid nutrient solution of this composition:

Compound: Concentration _and mgl**^ KC1 potassium chloride 4 010 NaCl sodium chloride 3 130 HORSE ₃ potassium nitrate 620 MgS0 ₄ .7H ₂ 0 potassium sulfate 1 250 to ₂ hpo ₄ potassium hydrogen phosphate 330 Ca(N0 _x ) _? .4H _? 0 calcium nitrate 85 CI potassium iodide 25 KBr potassium bromide 25 ^C 10 ^H 12 ^N 2°8 ^FeNa * ^aq * ferric chelate 18.5 ^H 3 ^B0 4 boric acid 3.1 MnS0 ₄ .4H ₂ 0 manganese sulfate 1.2 CoS0 ₄ .7H ₂ 0 cobalt sulfate 1.4 CuS0 ₄ .5H ₂ 0 copper sulfate 1.2 ZnS0 ₄ .7H ₂ 0 zinc sulfate 1.4 ammonium molybdate 1.8

In the above nutrient solution, the culture is maintained as a stock under constant illumination of 25 Wm~ and a temperature of 24 C and an optical density of the algal suspension ^A 750 ^B 0.200 - 0.400 and such a concentration of ¹² c° ₂ in a mixture with air that corresponds to 2% of dissolved COg in the algal suspension. The optical density Α _?5θ is measured at a wavelength

750 nm in a 5 mm cuvette on a photometer. Multiply the optical value *

density by a coefficient of 2, we obtain an approximate value of the dry mass of algae.

A portion of the suspension is removed from the stock culture to prepare a synchronous population, which is obtained by exposing the culture to alternating periods of light and darkness at intervals of 10 to 14 hours at a cultivation temperature of 24°C and an irradiance of the surface of the culture vessel of 80 ^Wm2 FAR. The actual cultivation of synchronous algae is carried out in a flow chemostatic mode at a settling rate of D of 0.06 ^h1 .

The growth rate 0 expresses the proportion of the total culture volume;

which is continuously replaced by nutrient solution per unit of time.

In a steady flow culture, it is equal to the specific growth rate, i.e. D e^u. This method allows perfectly stabilizing the cultivation conditions and automating the cultivation process. In a steady, fully synchronous culture, the average population irradiance, which affects the rates of biochemical processes in the algae population, is constant throughout the entire light period of the cycle; the optical density of the pre-production culture ee ranges from Α ₇₅ θ » 0.270 - 0.280.

b) Production cultivation

One hour before the end of the dark period of the preparatory synchronous cycle, the production photoreactor is filled with 1000 ml of an inorganic solution, which differs from the nutrient solution of the pre-production cultivation in that KNO ₃ is removed from it and Ca(N0 ₃ ) ₂ .4H ₂ 0 is replaced by CaCl ₂ so that the amount of Ca ⁺⁺ in the solution remains unchanged. The nutrient solution thus prepared is freed from all physically dissolved 3* ² CO2 by heating to 100°C. The air is displaced from the gaseous space of the cultivation apparatus and replaced with nitrogen gas, after which this enclosed space is partially evacuated (to approximately 500 torr). Then the nutrient solution in the bioreactor is saturated with ¹⁴ C0«, which is formed in the developer by dosing 1 N HCIO^ into the Ba C03 suspension. The equilibrium state in the nutrient solution of dissolved C0 ₂ with an atmosphere containing 2% C0 ₂ occurs after about 30 minutes of intensive bubbling. During the preparation of the nutrient solution, the daughter cell population of the synchronous culture from the pre-production cultivation is centrifuged. The supernatant with extracellular substances is removed. The algae sediment is washed with the nutrient solution intended for production cultivation and centrifuged again. The centrifugation is carried out for 10 min. at 4000 g and a temperature of 5®C. After the second centrifugation, the daughter cell sediment is gently shaken and, in the form of a dense inoculum, is introduced into the culture cylinder with the specified optimized nutrient solution.

The actual production cultivation takes place once for 36 hours. During this time, the cell mass triples! and the amount of starch increases from 20% to 55-60% of the dry cell mass. It creates so many iron substances outside the cells that they represent about 25% of the dry cell mass.

- 6 Production cultivation conditions:

Initial optical density of the suspension A _75Q · 0.500, cultivation temperature 24°C, partial pressure ¹⁴ C0 ₂ « 0.02 at., irradiated

E^ 50 Wm” ² , after 8 hours of cultivation ee E^ increases to 80 Wb ² ,

After the cultivation is completed, the algae suspension is cooled to 2°C and centrifuged at this temperature.

Mix the supernatant ee in a ratio of 1 : 1 to 2 β with 96% ethanol.

After shaking, a white precipitate of lysate is obtained, which after washing serves as the starting material for the production of labeled galactose, xylose and glucose. From the biomass of the cells, by their disintegration and gradient centrifugation, florid starch is obtained as the final product.

The production of individual carbohydrates during production cultivation is shown in the table.

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Claims (1)

Object of the invention A method for the controlled biosynthesis of galactose, xylose and ftoride starch not specifically labeled with a carbon radioisotope, characterized in that it is carried out a single cultivation of daughter cells of synchronous algae culture of the genus Porphyridium for 32-38 hours in a nitrogen-free mineral nutrient solution at 24-28 ° C in the mixture of gaseous nitrogen gas and radioactive carbon dioxide while illuminating the surface of the culture vessel 60-80 Wm ² FAR, at the beginning of the production cultivation, removing the mucilages formed during the pre-production cultivation in the medium! of non-radioactive ¹² CO ₂ carbon dioxide by sparging cells into a nutrient solution containing nitrogen compounds ·