CS206914B1 - Manufacturing process of extracellular endo-1,4-beta-xylanasis of yeast strain cryptococcus - Google Patents
Manufacturing process of extracellular endo-1,4-beta-xylanasis of yeast strain cryptococcus Download PDFInfo
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Vynález sa týká spĎsobu produkcie extrecelulárnej endo-1,4- β-xylanázy u kvasiniek rodu Cryptococcus indukovanej nemetabolizovaným syntetickým indukterom metyl- ft-D-xylozidom počas rastu na glukóze, xyloze alebo eabharóze*alebo počas inkubácie naraetenej biomasy buniek v prostředí bez Saláieho sacharidového zdroja uhlíka.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.
Ř-1,4-xylanázy sa poměrně hojné vyekytujú u myceliárnych húb a niektorých druhov baktérií (R. F. H. Dekker a G. N. Richarde Adv. CeErbohydr. Chem. Biochem. 32. (1976).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).
Za hlavné producenty xylan degradujúcich enzýmov sa dodnes povážujú plieene a baktérie. Takáto schopnost sa objevila u kvasiniek rodu Cryptococcus (P. Biely, Z. Krátký, A. KockováKratochvílová, S. Bauer) Folia Microbiol. 23, 366 (1978), ktoré sa dajú považovat za vhodnější typ mikroorganizmu na získanie xylan degradujúcich enzýmov. Cryptococci produkujú (3-1,4-xylázu iba počas rastu na xylane, teda enzým je induktívny a jeho tvorbu indukujú oligosacharidy xylozy, ktoré sa v živnom prostředí z xylanu tvoria.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.
Pri štúdiu regulácie ^-xylanázy kvasiniek rodu Cryptococcus sa zistilo, že produkcie extrecelulárnej xylanázy sa dá indukovat i počas rastu na běžných zdrojoch uhlíka ako je glukóza alebo sacharo'za, ak sa ku kultúre mikroorganizmu přidá syntetický glykozid, metyl- (5-D-xylopyranozid. Analógiu tohto javu popísali u kmeňa Streptomyces 3137 japonakí autoři (K. Nakanishi, T. Yausi a T. Kobayashi, J. Ferm. Technol. 54 801 (1976) Japan Kokai 77 61,285). U kvasiniek rodu Cryptococcus dochádza k uplatneniu indukčného účinku metylxy206 914In 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 lozidu až v etacionárnej fáze rastu, kedy poklesne hladina sacharidov použitých ako zdroj uhlíka na úroveň, ktorá neapdeobuje katabolickú repreaiu syntézy enzýmu. Chromatografickou analýzou produktov hydrolýzy /3—1,4-xylanov ako i viskozimetrickou metodou aa dokázalo, že enzým indukovaný metyl- β-D-xylozidom je totožný s enzýmom, ktorý produkujú kvasinky počaa rastu na drevnom xylene.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.
Podstata vynálezu spočívá v tom, že produkcie enzýmu sa indukuje syntetickým induktorom metyl- β-D-xylopyranozidlom počae raetu na běžných zdrojoch uhlíka, výhodné glukóze, xylóze a aacheroze alebo počae inkubácie buniek vyraetených na 1’ubovolnom zdroji uhlíka,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,
Metylxylozid simuluje v buňkách indukčný účinok oligosecharidov xylózy, ktoré majú funkciu prírodných induktorov β-xylanázy počae raetu na xylane, z ktorého sa uvol’ňujú, prenikajú do buňky a dávajú signál pre zehájenie produkcie xylanázy. Bez pridania metylxylozidu buňky kultivované na glukóze, xylóze alebo aacheroze produkujú zhruba o dva rady menej β-xylanázy ako na xyláne ak vztiahneme jej množstvo na výhovú jednotku naraatenej biomaey.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.
Výhodě navrhovaného spčsObu produkcie β-xylanázy u kvaeiniek rodu Cryptococcus je, že:An advantage of the proposed method of producing β-xylanase in cryptococcus species is that:
- umožňuje produkciu enzýmu na báze dostupných zdrojov uhlíka, nevyžaduje teda izoláciu xylenu, resp. hemiceluloz,- Enables the production of the enzyme on the basis of available carbon sources; hemicellulose
- prípreva použitého induktora je nenáročná a rýchla (varenie xylózy v metanole za katalytického účinku kyselin a nasledovná kryátalizácia β-anoméru xylopyranozidu),- 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),
- biomaaa kvasiniek sa dá na produkciu extrecelulárnej xylanázy použit opakované, pokia! aa zabráni infekcii cudzími mikroorganizmami.- yeast biomaaa can be used repeatedly to produce extrecellular xylanase if and prevents infection by foreign microorganisms.
Příklad 1Example 1
Kmen Cryptococcus Paurentii CCY 17-3-2 (čs. zbierka mikroorganizmev, Chemický ústav SÁV) sa zaočkuje do tekutej pfldy obeahujúcej 1 % glukózy, 0,2 % kvaeničného autolyzátu, 0,1 % dihydrofosforečňanu draselného, 0,2 % síranu amonného a 0,1 až 0,5 % metyl- β-D-xylozidu a kultivuje sa pri 27 °C za aerácie po dobu 4 až 5 dní. Po pdstránenl narastenej biomaey aa získá tekutina, ktorá obsahuje v 1 ml 0,2 až 0,4 jednotiek β-xylanázy. Za jednotku aktivity je považované také množstvo enzýmu, ktoré v 0,05 acetátovom pufri, pH 5,4, pri 30 °C uvolní v 0,5 ml inkuvačnej zmesi obeahujúcej 1 mg xylanu redukujúce sacharidy rovné 1 umolu ekvivalentov xylózy.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.
Příklad 2Example 2
Kmeň Cryptococcus albidus CCY 17-4-1 (čs. zbierka mikroorganizmov, Chemický ústav SAV) aa pestuje v živnej pdde obeahujúcej 0,67 % kvaaničnej duaíkatej bázy (Difeo), 0,2 % aeparagánu, 0,5 % dihydrofosforečňanu draselného, 1 % xylózy a 0,1 až 0,5 % metyl-β-D-xyloziůu. Po 4-5 dňovej kultivácii sa biomaaa kvaainiek odstředí a získá sa tekutina obsahujúca v 1 ml 0,8 až 1,7 jednotiek β-xylanázy, čo je v optimálnom případe až o 50 % vyáéia hladina enzýmu vyprodukovaná počas rastu buniek v pdde siř xylenem.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. .
Příklad 3 209 914Example 3,209,914
Biomasa kvasiniek získaná centrifugáciou 4 až 5 dnovej kultúry kmeňa Cryptococcus albidus CCY 17-4-1 (čs. zbierka mikroorganizmov, Chemický ústav SAV) v 1 litri média s libovolným zdrojom uhlíka (napr. glukóza, xyloze, sacharóza) v nepřítomnosti metylxylozidu se suspenduje v 200 ml média bez sacharidového zdroja uhlíka obsahujúce 0,5 % kvasničného autolyzátu, 0,1 % dihydrofosforečnanu draselného, 0,2 % síranu amo'nneho a 0,1 až 0,5 % metyl- β-D-xylozidu. Suspenzie sa inkubuje za trepania pri 27 °C 10 až 24 hodin, kým buňky nenasekretujú do prostredia 0,3 až 0,6 jednotky /3-xylanázy na 1 ml. Biomasa získaná centrifugáciou neprodukuje poSas opakovanej inkubácie v médiu s metylxylozidom zhruba rovneké množstvo extracelulárnej β-xylanázy.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.
Význam extracelulárnych endo-1,4- fó-xylanáz v poslednom období rastie spolu s celulázami so záujmom o využitie dřevených odpadov ako zdrojov sacharidov na přípravu mikrobiálnych bielkovín. Xylanázy spolu s xylozidázami predstavujú prostriedok na získanie xylozy z rastlinných hemicelulóz za miernych podmienok enzýmovej hydrolýzy. V tejto súvislosti sa uvažuje o použití imobilizovaných enzýmov v systéme, v ktorom ba sa kontinuálně vyrábala xylóza. Táto pentóza představuje nielen výhodný zdroj uhlika pre rast celého radu priemyeelne zaujímavých kvasiniek ako producentov bielkovín, ale i východisková surovinu pre přípravu xylitolu, ktorý sa dá využit ako vhodné diabetické sladidlo.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.
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