CS276074B6 - A method for preparing an ascorbate oxidase enzyme from plant cells cultured in vitro - Google Patents

Abstract

Ascorbate oxidase is isolated from plant* cells of plant species, e.g. from the families of Solanaceae, Rutaceae, Cucurbitaceae or Poaceae cultivated in vitro in the form of selected strains of undifferentiated cells, cell aggregates, differentiated organs, whole plants, embryos or tumor plant cells, derived after transformation of Agrobacteria in the form of callus or summers culture. The propagated culture is extracted with phosphate buffer pH 4.2 to 9.8 in an amount of 0.3 to 5 times the weight of fresh biomass, centrifuged, the supernatant is precipitated e.g. with ammonium sulfate at a saturation between 20 and 70% by weight. and purified by ion exchange chromatography and gel filtration.

Description

The invention ae relates to a new process for obtaining the enzyme ascorbate oxidase (L-acorbate: O ₂ oxidoreductase, E. O <1.10.3.3./ hereinafter referred to as AO) from plant cells cultured in vitro.

The current method of obtaining A0 from cucumber and squash fruits (see E. Beránková, B. Králová, Proceedings of the Institute of Chemical Technology E60, pp. 95-1'3, 1986) is disadvantageous in terms of seasonality and sudden availability of the raw material, which also contains many ballast substances. interfering with the isolation and purification of the enzyme. Fruits can only be obtained during a certain · limited · period of fruit ripeness, which does not overlap with the period of consumer ripeness. The yield depends greatly on climatic conditions and the whole harvest is threatened by infestation by phytopathogenic organisms. These factors cause fluctuations in the price of the raw material, or even its temporary complete unavailability.

The method of preparation of AO from plant cells of selected plant species, eg from the aubergine, routing, gourd or puffball family cultured in vitro in the form of selected strains of undifferentiated cells, cell aggregates, differentiated organs, whole plants, embryos or tumor plant cells derived after transformation by Agrobacteria, wherein the selected production stones are grown in the form of a surface culture on a solidified medium or in a submerged culture, optionally also using immobilization techniques. The enzyme is obtained by extraction of the biomass with phosphate buffer pH 4.2 to 9.8 in an amount of 0.3 to 5 times the weight of fresh biomass, centrifugation, the supernatant is precipitated with e.g. ammonium sulphate at a saturation of between 20 to 70% by weight, and purified ion exchange chromatography and gel filtration.

The method according to the invention enables year-round continuous production independently of climatic changes or infestation of stands by pathogenic microorganisms. Another advantage is the simplification of the isolation procedure, as it is not necessary to remove as many ballast substances as in field crops (chlorophyll, etc.).

Suitable ascorbate oxidase production organisms according to the invention are, for example, selected cultures of routa, milk thistle, tobacco, New Zealand poroporo plants and the like.

In the practice of the invention, the production organism AO is cultured on a solidified medium or in a liquid medium suitable for the production of the enzyme. Culture media with the required mineral content (eg Murashige and Skoog medium) can be used as a source of nutrients in a wide range of plant species, with the addition of suitable natural or synthetic growth regulators such as auxides and cytokinins in an optimal combination of type and ratio. As the carbon source, sucrose, glucose, lactose and the like can be used.

The culturing temperature ranges from 17 to 33 ° C, and the culturing time varies according to the conditions for achieving the maximum yield of AO in the range of 5 days to 3 months.

From the culture thus grown, AO is obtained after extraction of fresh, frozen or lyophilized biomass in the cold with a buffer solution, preferably phosphate, after mechanical or enzymatic homogenization, using 0.3 to five times the weight of fresh biomass. Preferably, a buffer solution with a pH in the range of 4.2 to 8.9 with a concentration of 0.05 to 0.3 moles of liter can be used. The crude extract obtained after centrifugation of the material thus obtained is further purified by precipitating the supernatant with, for example, ammonium sulphate at a saturation of between 20 and 70% by weight, and purifying by ion exchange chromatography and gel filtration. The yield of the enzyme can be increased by the addition of certain substances, such as ethylenediaminetetraacetic acid (EDTA), Trion X100 anodes.

The activity of AO in the extracts is determined by means of an oxygen electrode according to the procedure described by Speck A. J., Schrijver J., Schreus W. Η. P., Agr. Food Chem., 32, 352, 1984, modified according to Beránková E., Králová B., Proceedings of the Institute of Chemical Technology, E60, pp. 95-113, 1986.

The use of ascorbate oxidase is possible in particular in clinical biochemistry and in the food industry for the determination of vitamin C. In addition, AO has numerous applications when used in combination with other enzymes and colorimetric reagents, such as test or diagnostic reagents.

The following are specific examples of embodiments of the invention, which, however, do not limit the invention in any way.

CS 276 074 B6 2

Example 1

Obtaining AO from poroporo callus culture (Solanum aviculare Forst.)

Plant cell biomass containing AO was obtained by culturing GL-5 strain cells on complete agar nutrient medium according to Murashige and Skoog, with the addition of 2,4-dichlorophenoxyacetic acid as auxin and kinetin as cytokinin, at a concentration of 1x10 ⁷ mol / liter, in the dark at 28 ° C. The homogeneous callus culture was separated from the broth after reaching the stationary growth phase. Fresh biomass was homogenized in the cold with phosphate buffer and extracted according to the above procedure. The crude extract obtained contained in each sample 450 to 800 nkat of AO activity per 1 g of dry matter.

Example 2

Obtaining AO from undifferentiated callus tobacco culture (Nicotians tábacum L.)

An undifferentiated homogeneous tobacco callus culture was cultured according to Example 1, but without the addition of growth regulators. Habituated strains were obtained using appropriate selection pressures from callus culture derived from aseptically grown plants. At the end of the exponential growth phase, the TSS strain gives an AO activity of 300 nkat / 1 g of cell dry matter in the crude extract obtained as described. .

Example 3

Obtaining AO from submergedly grown milk thistle culture (Silybum marianum L.)

A submerged culture of a suspension of fine aggregates of milk thistle cells of the OP12 strain was grown in a liquid medium according to Murashige and Skoog with the addition of growth regulators ec-naphthylacetic acid and benzylaminopurine at a concentration of 10 "3 mel / l. When cultured on a culture stirrer and 5 rpm, it gives an activity yield of up to 1000 nkat / 1 g dry matter.

Example 4

AO production by transformed poroporo tumor cells (Solanum aviculare Forst.)

Tumor cultures of poroporo cells were obtained by transformation with TI plasmid after infection of aseptically grown plants with Agrobacterium tumefaciens suspensions. The selected strain A5-VR1 is grown on the medium of Example 2 without requiring the addition of exogenous growth regulators for its growth. The biomass thus obtained provides an extract with an activity of 250 nkat from one gram of dry matter.

Example 5

Obtaining AO from differentiated melon plants grown aseptically in vitro

Aseptically grown differentiated melon plants (Cucurbita melo I.) obtained from surface-sterilized seeds on agar medium with the addition of growth regulators ε-naphthylacetic acid and benzylaminopurine at a concentration of 10% _BO i / i. The highest concentration of AO is contained in the stems, but also in extracts from leaves and roots, the concentration of AO is comparable to its content in the pericarp and reaches values of 100 nkat per 1 g of fresh tissue weight.

Example 6

Obtaining AO from differentiated melon plants, aseptically submerged in vitro in liquid s-media

Aseptically grown differentiated melon plants (Cucurita melo L.) obtained from surface sterilized seeds on agar medium with the addition of growth regulators according to Example 5 (<4-naphthylacetic acid and benzylaminopurine at concentrations of 10 "and 10 ⁷ mol / l) were converted into liquid media of the same composition without the addition of agar and grown on culture stirrers or shakers (100 rpm) at 25 ° C. The highest concentration of AO is again contained as in Example 5 in the stems. The average concentration of AO in the obtained biomass is comparable and its content in the pericarp and reaches values of 100 nkat per 1 g of fresh tissue weight.

CS 276 074 B6

Example 7

Obtaining AO from differentiating poroporo culture (Solanum aviculare Forst.)

Plant cell biomass containing AO was obtained by culturing cells of the AVI7C strain on complete agar nutrient medium according to Murashige and Skoog, without the addition of auxin and cytokinin, in the light at 30 ° C. The differentiating culture (shoots) was separated from the nutrient medium after reaching the stationary growth phase. Fresh biomass was homogenized in the cold with phosphate buffer and extracted according to the above procedure. The crude extract obtained contained in each sample 250 to 1300 nkat of AO activity per 1 g of dry matter.

Example 8

Obtaining AO from callus culture of milk thistle (Silybum marianum L.)

A culture of homogeneous decaying callus of strain 0P38S derived from aseptically injured milk thistle embryo was grown on Murashige and Skoog agar medium with the addition of 10 ⁶ aol / lc growth regulators and benzylaminopurine. activity yield up to 1000 nkat / 1 g dry matter.

Example 9.

Obtaining AO from poroporo suspension culture (Solanum aviculare Porst.)

AO-containing plant cell biomass was obtained by culturing KK1N cells, selected for optimal growth in the form of a fine cell suspension, submerged in complete nutrient medium according to Murashige and Skoog, with the addition of n-naphthylacetic acid as auxin and kinetin as cytokinin at 4x10 "concentrations. ³ and 1x10 “? mol / liter, at a temperature of 26 ° C. The homogeneous suspension culture was harvested by filtration or centrifugation of the broth before the end of the linear growth phase. The medium contained 4 to 9 nkat AO in 1 ml. The cell biomass was homogenized in the cold with phosphate buffer and extracted according to the above procedure. The crude extract obtained contained in each sample 550 to 850 nkat of AO activity per 1 g of dry matter.

Example 10

Obtaining AO from embryo culture Solanum aviculare Porst. grown in a liquid medium

Plant cell biomass containing AO was obtained by growing cells of the embryogenic strain AVE03N, growing in submerged culture in complete nutrient medium according to Murashige and Skoog, with the addition of "E-naphthylacetic acid as auxin and benzylaminopurine as cytokinin, at concentrations of 4x10" ³ and 1x10 " ? mol / liter, at a light-temperature of 27 ° C, in the form of a fine suspension of cells, were transferred to a medium without growth regulators, in which differentiated embryos were formed. The culture was harvested by filtration or centrifugation of the broth before the end of the linear growth phase. The medium contained 8 to 12 nkat in 1 ml. The cell biomass was homogenized in the cold with phosphate buffer and extracted according to the above procedure. The crude extract obtained contained in each sample 800 to 1100 nkat of AO activity per 1 g of dry matter.

Example 11 ..

Obtaining AO from immobilized route culture cells (Puta graveolens L.) in batch culture in liquid medium

Rouge suspension culture cells of strain R0205 grown on Murashige and Skoog medium, with the addition of β-naphthylacetic acid as auxin and kinetin as cytokinin, at a concentration of 4x10 ** mol / liter, in the dark at 27 ° C, as a fine cell suspension , after immobilization on a calcium alginate gel, were transferred to the same medium without growth regulators and repeatedly incubated on a shaker at five day intervals. For five cycles, the bound cells secreted into the medium an average activity of 4 nkat of AO activity in 1 ml of nutrient solution.

Claims (2)

PATENT CLAIMS A process for the preparation of the enzyme ascorbate oxidase from plant biomass, characterized in that cells of selected plant species, e.g. from the aubergine, tuberous, gourd-like, puffball, cultured in vitro in the form of selected strains of undifferentiated cells, cell aggregates are used as the enzyme source. differentiated organs, whole plants, embryos or tumor plant cells derived after transformation with Agrobacterium, wherein the selected production strains are grown in the form of surface culture on solidified medium or in submerged culture, optionally also using immobilization techniques. 2. The preparation method according to item 1, characterized in that ae plant biomass obtained by in vitro cultivation is extracted with phosphate buffer pH 4.2 to 9.8 in an amount of 0.3 to 5 times the weight of fresh biomass, centrifuged, the supernatant is precipitated e.g. ammonium sulphate at a saturation of between 20 and 70% by weight, and is purified by ion exchange chromatography and gel filtration.