CS206914B1 - Manufacturing process of extracellular endo-1,4-beta-xylanasis of yeast strain cryptococcus - Google Patents

Description

The present invention relates to a process for the production of extrecellular endo-1,4- β-xylanase in yeasts of the genus Cryptococcus induced by a non-metabolized synthetic inducer methyl-β-xylozide during growth on glucose, xyloha or eabharose * or during incubation of loaded biomass cells carbon source.

Β-1,4-xylanases are quite abundant in mycelial fungi and some species of bacteria (R.F.H. Dekker and G.N. Richarde Adv. CeErbohydr. Chem. Biochem. 32. (1976).

The main producers of xylan degrading enzymes are still considered to be breeds and bacteria. Such ability has been found in yeasts of the genus Cryptococcus (P. Biely, Z. Kratky, A. KockovaKratochvilova, S. Bauer) Folia Microbiol. 23, 366 (1978), which may be considered a more suitable type of microorganism for obtaining xylan degrading enzymes. Cryptococci produce (3-1,4-xylase only during growth on xylan), ie the enzyme is inductive and its formation is induced by xyloza oligosaccharides, which are formed from xylan in the culture medium.

In a study of the control of γ-oxylanase of yeasts of the genus Cryptococcus, it was found that extrecellular xylanase production can be induced during growth on conventional carbon sources such as glucose or saccharose if synthetic glycoside, methyl- (5-D-) is added to the microorganism culture. An analogy of this phenomenon has been described in the Streptomyces 3137 strain by Japanese authors (K. Nakanishi, T. Yausi and T. Kobayashi, J. Ferm. Technol. 54 801 (1976) Japan Kokai 77 61,285). effect of methylxy206 914

208 814 lozide only in the ethnational phase of growth, when the level of carbohydrates used as a carbon source drops to a level that does not cause catabolic repression of enzyme synthesis. Chromatographic analysis of the hydrolysis products of [beta] -1,4-xylanes as well as the viscosimetric method aa showed that the enzyme induced by methyl-β-D-xylozide is identical to the enzyme produced by yeast during growth on woody xylene.

The principle of the invention is that the production of the enzyme is induced by a synthetic inducer of methyl-β-D-xylopyranoside during a raid on conventional carbon sources, preferably glucose, xylose and aacherosis, or during the incubation of cells engraved on any carbon source,

Methylxylozide simulates the inducing effect of xylose oligosecharides in cells, which function as natural inducers of β-xylanase during a raid on the xylan from which they are released, penetrate into the cell and give a signal to start the production of xylanase. Without the addition of methyl oxylozide, cells cultured on glucose, xylose, or aacherosis produce about two rows less β-xylanase than on xylan when related to the per-unit of the native biomaey.

An advantage of the proposed method of producing β-xylanase in cryptococcus species is that:

- Enables the production of the enzyme on the basis of available carbon sources; hemicellulose

- the preparation of the inducer used is undemanding and rapid (boiling xylose in methanol under the catalytic action of acids and subsequent crystallization of the β-anomer of xylopyranoside),

- yeast biomaaa can be used repeatedly to produce extrecellular xylanase if and prevents infection by foreign microorganisms.

Example 1

The strain Cryptococcus Paurentii CCY 17-3-2 (MS Collection of Microorganisms, Institute of Chemical Technology, SAV) is inoculated into a liquid supplement containing 1% glucose, 0.2% quartz autolysate, 0.1% potassium dihydrogen phosphate, 0.2% ammonium sulfate and 0.1 to 0.5% methyl β-D-xyloside and cultured at 27 ° C with aeration for 4-5 days. After removal of the grown biomaa aa, a liquid containing 0.2 to 0.4 β-xylanase units in 1 ml is obtained. The unit of activity is considered to be the amount of enzyme which, in 0.05 acetate buffer, pH 5.4, at 30 ° C liberates in 0.5 ml of an incubation mixture containing 1 mg of xylan reducing saccharides equal to 1 µmol of xylose equivalents.

Example 2

The strain Cryptococcus albidus CCY 17-4-1 (MS collection of microorganisms, Institute of Chemical Technology of SAS) and cultivated in nutrient soil containing 0.67% of diacaoedium base (Difeo), 0.2% of aeparagan, 0.5% of potassium dihydrophosphate, 1 % xylose and 0.1 to 0.5% methyl-β-D-xylosin. After 4-5 days of cultivation, the biomaaa of cvaaines are centrifuged to give a liquid containing 1 ml of 0.8-1.7 units of β-xylanase, which is optimally up to 50% higher levels of enzyme produced during cell growth in xylene pdde cells. .

Example 3,209,914

Yeast biomass obtained by centrifugation of a 4 to 5 day culture of Cryptococcus albidus CCY 17-4-1 strain (MS collection of microorganisms, Institute of Chemistry, SAS) in 1 liter of medium with any carbon source (eg glucose, xyloh, sucrose) in the absence of methylxylozide in 200 ml carbohydrate-free media containing 0.5% yeast autolysate, 0.1% potassium dihydrogen phosphate, 0.2% ammonium sulfate and 0.1 to 0.5% methyl-β-D-xyloside. The suspension is incubated with shaking at 27 ° C for 10-24 hours until the cells are secreted into the environment of 0.3 to 0.6 unit / 3-xylanase per ml. The biomass obtained by centrifugation does not produce approximately the same amount of extracellular β-xylanase during repeated incubation in methylxylozide medium.

The importance of extracellular endo-1,4-β-xylanases has recently increased with cellulases with an interest in the use of wood waste as a source of carbohydrates for the preparation of microbial proteins. Xylanases, together with xylozidases, are a means of obtaining xylosis from plant hemicelluloses under mild enzyme hydrolysis conditions. In this context, the use of immobilized enzymes in a system in which xylose has been continuously produced is contemplated. This pentose is not only an advantageous carbon source for the growth of a wide variety of yeasts of interest as protein producers, but also a starting material for the preparation of xylitol, which can be used as a suitable diabetic sweetener.

Claims (1)

3 Example 3 209 914 Yeast Biomass obtained by centrifugation of 4-5 days culture of Cryptococcus alsus CCY 17-4-1 (Czechoslovak Collection of Microorganisms, Institute of Chemistry SAS in 1 liter of medium with free carbon source (e.g. glucose, xylo , sucrose) is suspended in 200 ml of carbohydrate-free medium containing 0.5% yeast auto-lysate, 0.1% potassium dihydrogen phosphate, 0.2% ammonium sulfate and 0.1 to 0.5% methyl in the absence of methylxylozide β-D-xylozide The suspension is incubated with shaking at 27 ° C for 10 to 24 hours until the cells are secreted in the medium from 0.3 to 0.6 units / 5-xylanase per ml. The importance of extracellular endo-1,4-phytolanases has recently increased with cellular interest in using wood wastes as sources of carbohydrates for microbe preparation. Xylanases together with xylosidases represent a means for obtaining xylozymes from plant hemicelluloses under mild enzyme hydrolysis conditions. In this context, the use of immobilized enzymes in a system in which the oxylose is continuously produced is contemplated. This pentose is not only a preferred carbon source for the growth of a number of industrially interesting yeasts as protein producers, but also a starting material for the preparation of xylitol, which can be used as a suitable diabetic sweetener. BACKGROUND OF THE INVENTION A method of producing extracellular endo-1,4-β-xylanase in yeast of the genus Cryptococcus, characterized in that the production of the enzyme is induced by synthetic methyl β-D-xylapyranoside inducerty during growth on conventional carbon sources, glucose, xylose and sucrose, or the incubation period of cells grown on Tubal's carbon source.