CS259090B1 - Method of galactose's,xylose's and fluorinated starch's controlled biosynthesis with non-specifically labelled carbon's radioisotopes - Google Patents

Abstract

Fluoride starch biosynthesis and xy-. radioactive carbon beds are not known. Radioactive labeled galactose biosynthesis Carbon uptake has been carried out so far by cultivating the lining of Iridaea, whose disadvantages are slow growth and low recovery (2 µl), low specific activity and uneconomical use of valuable 14co2. A method for controlling biosynthesis according to the invention is carried out by one-time cultivation of daughter cells of synchronous algal culture Porphyridium free of mucilage in of pure radioactive CO 2 and v nutrient solution without nitrogenous compounds (temperature 24 - 28 ° C, surface illumination) culture vessels 60 - 80 iYm ~ 2 PAR, environment: a mixture of nitrogen gas and radioactive CO2). A high yield of floride is achieved starch, glactose and xylose, as well i 'their high specific activity. The manufacturing process is characterized by simplicity and high washing machine safety.

Description

The present invention relates to a method for controlling biosynthesis, galactose, xylose and Florida starch, not specifically labeled with the radio-14 carbon isotope (C) in a synchronous culture of red cervix

Porphyridium, grown under photoautotrophic conditions.

Techniques for the production of Florida starch and xylose (radiolabeled) are not known in the literature. A procedure for the preparation of radiolabelled galactose by biosynthesis is described by Bane et al. /4.B1ol.Chem. 204: 169-173 (1953)].

For the biosynthesis of radioactive CO ₂ in this work, the insect red irides of the genus Iridaea were used, the disadvantage of which is slow growth and low yield of the desired product, since galactose constitutes only% of all organic matter. The process is characterized by an uneconomic use of valuable CO 2, which is incorporated into all of its end products in photosynthesis and has a low specific activity, not exceeding 100 µcmg. galactose.

These disadvantages are overcome by the controlled biosynthesis process according to the invention, which consists in carrying out a single cultivation of daughter cells of synchronous algae of the genus Porphyridum for 32 to 38 hours in a nitrogen-free mineral nutrient solution at 24-28 ° C in a nitrogen gas mixture environment. and radioactive carbon dioxide in a culture vessel surface lighting from 60 to 80 Wm "^ FAR, while at the beginning of the production culture was removed mucilaginous substance precipitated during the pre-cultivation .12» environment not di r oa kT and VNI it dioxide C0 ₂ cells

- 2 algae into a nutrient solution containing nitrogen compounds.

The storage intracellular polyeacharide of the Porphyridium species is Florida starch, which, due to its chemical structure, is a very suitable substrate for enzymatic processing by amylases. In addition to the production of florid starch, the red ores used during metabolism secrete mucilaginous substances into the nutrient solution, the main part of which is acidic polysaccharides which, after hydrolysis, yield 0 and L galactose, D-xylose and D-glucose.

The amount of tears forms 10-20% of the dry mass of algae cells under normal culture conditions; the proportion of carbohydrates in the dry matter of extracellular tears generally does not exceed 30%. The amount of Florida starch is most often in the range of 20 to 20% dry cell mass.

The tear synthesis of said carbohydrates takes place in an environment where the sole carbon source is ¹⁴ CO ₂ . For the cultivation, the daughter cells of a synchronous population of a selected strain of algae of the unicellular red algae Pprphyridiura, converted into a mineral nutrient solution without nitrogen compounds, are used. Removal of nitrogen ions from the nutrient solution results in the arrest of proteosynthosis, causing the accumulation of Florida starch in the cells and an increased rate of synthesis of the mucilages secreted into the nutrient solution outside the cells simultaneously with an increase in galactose and xylose content relative to glucose.

Achieving a high specific starch activity is conditioned by the use of a synchronous culture, i.e., in which all cells are at the same stage of the life cycle. Synchronization is performed by alternating the light and dark periods. The youngest cells with the lowest amount of Florida starch are used for production cultivation because they were consumed in energy-intensive metabollic 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 culture is one of the conditions for its high specific activity at the end of the production cycle. Removal of mucilaginous coatings before incorporation of labeled carbon into cell structures and into newly formed mucilage results in a high specific activity of ellzic saccharides.

Advantages of the proposed method of controlled cultivation of algae according to the invention are in particular:

· »3 Μ

- in high yield of florlde starch, unless the controlled production cultivation takes place in the cells of its exclusive synthesis, so that its amount at the end of the production cycle reaches 50-60% dry algal cell density,

- in the increased yield of extrecellular polysaccharides, which, after cultivation, produce 25% dry cell spores *,

- advantageous representation of individual carbohydrates in these elizeeh,

- _e vyeoká specific activity products caused by florideováho starch, characterized in that the TFA oo its controlled during cultivation is increased about 7 times. For extrecellular polysaccharides, high specific activity is achieved by their de novo synthesis to ¹⁴ CO ₂ ,

- in the simplicity and safety of manufacturing operations,

- the high profitability of the production of said compounds.

The following is an example of a controlled blosynthase method of the invention.

Example

(e) Pre-production cultivation

The pre-cultivation and the actual production cultivation are carried out in the cultivation equipment described in the art. AO 197 918, A selected culture of the Porphyridlum genus kept in test tubes on agar media enriched in a mineral nutrient solution at 15 ° C and an irradiance of 10 Wm ² FAR is used to cultivate ee. FAR means photoeyntetically effective radiation measured by a spectrally non-selective sensor, sensitive only in the region of 400-700 nm.

The ee culture from agar is converted into a liquid nutrient solution of the following composition:

Compound: Concentration _a mgl ** ^ KC1 Potassium chloride 4 010 NaCl sodium chloride 3 130 KŇO ₃ potassium nitrate 620 MgSO ₄ .7H ₂ 0 potassium sulphate 1 250 k ₂ hpo ₄ Potassium hydrogen phosphate 330 Ca (NO _x ) _? .4H _? 0 calcium nitrate 85 KI potassium iodide 25 KBr potassium bromide 25 ^C 10 ^H 12 ^N 2 ° 8 ^FeNa * ^aq * ferric-eodium chelatonate 18.5 ^H 3 ^B0 4 boric acid 3.1 MnSO ₄ .4H ₂ 0 manganese sulphate 1,2 CoSO ₄ .7H ₂ 0 Cobalt sulphate 1.4 CuSO ₄ .5H ₂ 0 copper sulfate 1,2 ZnSO ₄ .7H ₂ 0 Zinc sulphate 1.4 ammonium molybdate 1,8

In said nutrient solution, the culture maintains as a storage under continuous illumination of 25 Wm ~ and a temperature of 24 ° C and an optical density of seaweed suspension ^A 750 ^{B of} 0.200-0.400 and a concentration of ¹² c ° ₂ in air mixture corresponding to 2% dissolved COg in suspension eyelashes. The optical density Α _{? 5θ} j is measured at wavelength

750 nm in a 5 mm cell on a photometer. If we multiply the optical value *

density coefficient 2, we get approximate value of dry mass of algae.

A portion of the suspension is withdrawn from the stock culture to prepare a synchronous population, which is obtained by exposing the culture alternately to irradiation and dark periods at 10 * 14 h intervals at a culture temperature of 24 ° C and irradiating the culture vessel surface with < 80 Wm * ² FAR. The actual cultivation of the synchronous algae takes place in a flow chemostatic mode at a dilution rate D of 0.06 h ^-1 .

The scaling rate 0 is the ratio of the total culture volume;

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

It is equal to the specific growth rate of a steady-state flow culture, i.e. D e ^ u. This stabilization allows perfectly stabilization of the cultivation conditions and automation of the cultivation process. In steady, fully synchronous culture, the mean population irradiation, which affects the rates of biochemical processes in the algal population, is constant throughout the light period of the cycle; the optical density of the pre-production culture ee is in the range! Α ₇₅ θ »0.270 - 0.280.

(b) Production cultivation

An hour before the end of the dark period of the pre-synchronous cycle, the production photoreactor is filled with 1000 ml of inorganic solution, which differs from the pre-production nutrient buffer by removing KNO ₃ and Ca (NO ₃ ) ₂ .4H ₂ 0 is replaced by CaCl ₂ to maintain the amount of Ca ⁺⁺ in the solution. The treated nutrient solution is freed from all physically dissolved 3 * ² CO2 by heating to 100 ° C. From the gaseous space of the culture apparatus, air and replacements are expelled! with nitrogen gas, whereupon the enclosure is partially evacuated (to about 500 tons). Then, the nutrient solution in the bioreactor saturated with ¹⁴ C0 ", which arises in vyviječl dosage of 1 N HClO ^ to a suspension of Ba C03 · equilibrated in the nutrient solution the dissolved C0 ₂ atmosphere containing 2% C0 _2, occurs after about 30 minutes with vigorous bubbling. During the treatment of the nutrient solution ae, the population of the daughter cells of the synchronous culture is centrifuged from the pre-production culture. The supernatant with the extracellular tear substances is removed. The algae sediment is washed with the nutrient solution to be cultivated and centrifuged again. Blasting is carried out for 10 min. at 4000 g and 5 ° C. After the second centrifuge, the sediment of the daughter cells is gently shaken and introduced as a thick inoculum into a culture cylinder with said optimized nutrient solution.

The actual production cultivation takes place once for 36 hours. In this time, the mass of cells has tripled! and the amount of starch is increased from 20% to 55-60% dry cell mass. It generates as many eluting substances outside the cells, which represent about 25% of the dry mass of the cells.

- 6 Production culture conditions:

Initial optical density of suspension A _75Q · 0.500, culture temperature 24 ° C, partial pressure ¹⁴ C0 ₂ 0,0 0.02 at., Irradiator

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

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

The supernatant ee is mixed in a ratio of 1: 1 to 2 β 96% ethanol.

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

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

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<o Ό • rl IN « X O « • >. r-l O and O £ rl O u * -v U rl> 1 X H • o β »a. e > - · Ό • rl O U N (0 rl X rl O O <8 ω glucose O 00 * LIMB ΙΌ » ot CM ΙΌ. rl 16.9 β N *O Her and r-l « O> O • ’Μ- O • O H O • rl CM CM » 00 ΙΌ © N * 0 rl > X O 00 * Φ CM cn rl M CM ΙΌ CM • 0 » IO £ O O Ό X Ή O V «. Q E H XQ tt. rl 1 r-l • σ> β $ rl cn CM O ΙΌ * ♦. IO it m O 00 it Dry knots extracel. mg.a _-— · * _-_, - _ O CM * 10 WHAT 0) ΙΌ rl O ΙΌ CM cells ng.l ^"1 rl O in ΙΌ β 5 00 CM O O rl 8 H H © O © e> β <rt Rl * X »r-1 O 3 X She O (0 <0 rl cS it ΙΌ

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 ·