CS204129B1 - Method of producing cellulolytic enzymes - Google Patents

Method of producing cellulolytic enzymes Download PDF

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CS204129B1
CS204129B1 CS13077A CS13077A CS204129B1 CS 204129 B1 CS204129 B1 CS 204129B1 CS 13077 A CS13077 A CS 13077A CS 13077 A CS13077 A CS 13077A CS 204129 B1 CS204129 B1 CS 204129B1
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ccy
basidiotrichosporon
strains
glucan
cellulolyticum
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CS13077A
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Czech (cs)
Slovak (sk)
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Anna Kockova-Kratochvilova
Juraj Zemek
Ludovit Kuniak
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Kockova Kratochvilova Anna
Juraj Zemek
Ludovit Kuniak
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(54) Spdsob produkcie celulolytických enzýmov(54) Method of producing cellulolytic enzymes

Vynález sa týká spósobu produkcie celulolytických enzýmov štepiacich celulózovú zložku lignin·—celulózových komplexov kvasinkovitými orgariizmami rodu Basidiotrichosporon.The invention relates to a method for producing cellulolytic enzymes that cleave the cellulosic component of lignin-cellulose complexes by yeast-like organisms of the genus Basidiotrichosporon.

Schopnost štepenia celulózy v prečistenom stave alebo v natívnom stave, vo formě lignin—-celulózového komplexu β (1 -» 4) glukán glukánohydrolázu EC. 3. 2. 1. 4, resp. schopnost produkovat tento enzým nie je vlastnosťou rodu Basidiotrichosporon ako celku. Pri použití gélovej metody testovania mikroorganizmov na ceiulázovú aktivitu (AO č. 183 169) sa zistila výrazná produkcia /3-glukán glukánohydrolázy u kmeňov zastupujúcich dva druhy rodu Basidiotrichosporon, a to B. pullulans a B. cellulolyticum.The ability to cleave cellulose in the purified state or in the native state, in the form of the lignin-cellulose complex β (1 → 4) glucan glucanohydrolase EC. 3. 2. 1. 4, resp. the ability to produce this enzyme is not a property of the genus Basidiotrichosporon as a whole. Using the gel method for testing microorganisms for cellulase activity (AO No. 183 169), significant production of β-glucan glucanohydrolase was found in strains representing two species of the genus Basidiotrichosporon, B. pullulans and B. cellulolyticum.

Žiadny z uvedených druhov nie je uvedený v literatúre ako producent enzýmov štepiacich celulózovú zložku lignin—celulózového komplexu.None of these species is reported in the literature as a producer of enzymes that cleave the cellulose component of the lignin-cellulose complex.

Podstata vynálezu spočívá v tom, že nasledovných 10 kmeňov rodu Basidiotrichosporon·.The principle of the invention is that the following 10 strains of the genus Basidiotrichosporon.

Basidiotrichosporon pullulans:Basidiotrichosporon pullulans:

CCY 30-1-1 a CCY 30-1-1 bCCY 30-1-1 and CCY 30-1-1 p

CCY 30-1-2 CCY 30-1-3CCY 30-1-3 CCY 30-1-3

Basidiotrichosporon cellulolyticum:Basidiotrichosporon cellulolyticum:

CCY 30-5-1 CCY 30-5-3 CCY 30-5-4 CCY 30-13-1 CCY 30-5-11 CCY 30-5-12 jednotlivo alebo ich zmesi kultiváciou na živnej pode minerálneho zloženia obsahujúcej síran amónny, gel hydroxyetylcelulózy a /3(1 -> 4) glukán ako induktor enzýmu pri teplote 20 až 40 °C, po dobu 48 až 110 hodin pri pH 4,5 až 6,8 produkuje komplexný celulázový systém /3-glukán glukánohydrolázu a /3-glukozidázu, ktorý sa získá v surovou stave odpařením rastovej pódy, připadne v čiastočne prečistenom stave zrážaním sířanom amonným, připadne zrážaním organickým rozpúšťadlom s výhodou etanolom.CCY 30-5-1 CCY 30-5-3 CCY 30-5-4 CCY 30-13-1 CCY 30-5-11 CCY 30-5-12 individually or mixtures thereof by cultivation on a nutrient medium containing ammonium sulphate, hydroxyethylcellulose α / 3 (1-> 4) glucan gel as enzyme inducer at 20 to 40 ° C, for 48 to 110 hours at pH 4.5 to 6.8 produces a complex cellulase system of β-glucan glucan hydrolase and / 3 Glucosidase, which is obtained in the raw state by evaporation of the growth medium, is optionally in a partially purified state by precipitation with ammonium sulphate or by precipitation with an organic solvent, preferably ethanol.

Všetky tieto kmene produkujú ureázu a ich biomasa dává s pufrovým roztokem diazofarbiva Fast Blue B červené zafarbenie, čo svědčí, že tieto kmene patria do oddelenia Basidiomycetes. Okrem typových kultúrAll these strains produce urease and their biomass gives a red color with the buffer solution of Fast Blue B diazo dye, indicating that these strains belong to the Basidiomycetes compartment. Excluding type cultures

204 1 2 9 (CCY 30-1-1, a, b, Btr. pullulans CCY 30-13-1 Btr. ínkin) bolí všetky kmene izolované z odpadových v8d drevospracujúceho a papierenského priemyslu. Kmene CCY 30-1-2 a CCY 30-1-3, CCY 30-5-11 a CCY 30-5-12 bolí izolované najnovšie v ČSSR.204 1 2 9 (CCY 30-1-1, a, b, Btr. Pullulans CCY 30-13-1 Btr. Inkin) hurt all strains isolated from waste wood and paper industry waste. Strains CCY 30-1-2 and CCY 30-1-3, CCY 30-5-11 and CCY 30-5-12 hurt isolated recently in Czechoslovakia.

Obidva druhy sa vzájomné odlišujú tým, že Btr. pullulans nevyužívá laktózu, trehalózu, ribózu ani xylózu, zatial' čo kmene Btr. cellulolyticum áno. Obidla druhy sa líšia produkciou xylanáz: Btt. pullulans xylanázy neprodukuje, zatial' čo Btr. cellulolyticum áno. Kmene druhu Btr. pullulans tvoria bohato artrospóry, kmene Btr. cellulolyticum len sporadicky.Both species differ from each other in that Btr. pullulans do not utilize lactose, trehalose, ribose or xylose, while strains of Btr. cellulolyticum yes. The grain species differ in the production of xylanases: Btt. pullulans do not produce xylanases while Btr. cellulolyticum yes. Strains of Btr. pullulans are rich in arthospores, strains of Btr. cellulolyticum only sporadically.

Btr. cellulolyticum sa od Btr. cutaneum (CCY 30-5-10- typová kultura a CCY 30-7-1 Btr. jirovecii — typová kultúraj líši aslmiláciou galaktózy, sacharózy, xylózy, strukturou polysacharidu, a prítomnosťou xylanáz a celuláz. Btr. cellulolyticum sa liší aj od iného susedného druhu Btr. inositophilum (CCY 30-5-5, -6 a -7], ktorého kmene sú schopné asimilovat inozitol.BTR. cellulolyticum is from Btr. cutaneum (CCY 30-5-10- type culture and CCY 30-7-1 Btr. jirovecii - type culture differs by the assimilation of galactose, sucrose, xylose, polysaccharide structure, and the presence of xylanases and cellulases. Btr. cellulolyticum also differs from another neighboring Inositophilum species (CCY 30-5-5, -6 and -7), whose strains are capable of assimilating inositol.

Použité kmene rodu Basidiotrichosporon sú uložené v Ceskoslovenskej zbierke kvasiniek a kvasinkovitých organizmov, Bratislava.The strains of the genus Basidiotrichosporon used are stored in the Czechoslovak Collection of Yeasts and Yeast Organisms, Bratislava.

Praktické využitie uvedených kmeňov spočívá v tom, že produkujú do kultivačného média enzýmy β-glukán glukánohydrolázu a /3-glukozidázu, ktoré sú jednoduchou metodou izolovatelné, pričom vznikajú z makromolekulárneho substrátu typu /í(l -* 4] glukánu oligoeacharidy typu celobiózy, ceilotriózy, celotetraózy a D-glukóza. Enzýmy uvedeného typu sú využitelné v procese sacharifikácie celulózy a v základnom výskume. Ďalšia možnost využitia tejto vlastnosti uvedených kmeňov rodu Basidiotrichosporon je v hydrolytickom rozklade dřeveného odpadu za vzniku enzýmatického hydrolyzátu priamo utilizovaného nahromaděním nutričně hodnotnej bielkoviny kvasinkovitých Basidiotrichosporon využitelných v krmovinárstve. Kvasinková forma Basidiotrichosporon umožňuje použitie ako centrifugačných tak aj filtračných separačných metód k oddeleniu nakultivovanej biomasy.The practical use of these strains consists in the production of β-glucan glucanohydrolase and β-glucosidase enzymes in the culture medium, which can be isolated by a simple method, resulting from a cell type macromolecular substrate of β (1 - * 4) glucan of the cellobiose type, ceilotriosis Enzymes of this type are useful in the process of saccharification of cellulose and in basic research Another possibility of utilizing this property of said strains of the genus Basidiotrichosporon is in the hydrolytic decomposition of wood waste to form an enzymatic hydrolyzate directly utilized by the accumulation of nutritionally valuable yeasts The yeast form of Basidiotrichosporon allows the use of both centrifugation and filtration separation methods to separate cultured biomass.

PřikladlEXAMPLE

Z gelu žo sieťovanej hydroxyetylcelulózy (A. O. č. 183 169) sa vyrežú trojúhelníkové profily o straně 4 cm, vložia sa do Petrlho misky a zalejú sa roztokom kultivačného média Yeast Nitrogen Base (Oxoid) s gélom hydroxyetylcelulózy ako jediným zdrojom uhlíka, s přísadou induktora (3(1 - 4) glukánu (1 mg/100 ml), tak aby pol hrůbky gélu vyčnievalo nad hladinu kultivačného média. Po trojnásobnej sterelizácii pri 100 °C po dobu 1 hodiny sa očkuje na povrch gélu kultúra Basidiotrichosporon pullulans CCY 30-1-2 a kultivuje sa pri 28 °C po dobu 48 hodin. Stekutený gél spolu s pomnoženou biomasou B. pullulans (0,42 gj sa použil ako inokulum na produkčnú pódu (2 1) s 1 °/o kukuřičného výluhu, 0,18 % hydroxyetylcelulózy, 0,2 % síranu amónneho a 0,2 % dihydrofosforečnan draselný (pH produkčněj pódy bolo upravené na 5;9). Kultivácia prebiehala po dobu 96 hodin pri 28 °C. Po ukončení kultivácie sa biomasa odcentrlfugovala (1500 g, 10 min.) a supernatant sa zahustil na odparke. Získal sa surový enzymový preparát s celulózovou aktivitou 66 U a specifickou aktivitou 0,78 U/mg. Příklad 2Triangular profiles of 4 cm side are cut from the gel of cross-linked hydroxyethylcellulose (AO No. 183 169), placed in a Petrl dish and embedded with a solution of Yeast Nitrogen Base (Oxoid) with hydroxyethylcellulose gel as the sole carbon source, with an inducer ( 3 (1 - 4) glucan (1 mg / 100 ml), so that half the depth of the gel protrudes above the culture medium After three-fold sterilization at 100 ° C for 1 hour, a culture of Basidiotrichosporon pullulans CCY 30-1- 2 and cultured at 28 [deg.] C. for 48 hours Liquid gel together with expanded B. pullulans biomass (0.42 gj was used as inoculum on a production platform (2 L) with 1% corn extract, 0.18% hydroxyethylcellulose, 0.2% ammonium sulfate and 0.2% potassium dihydrophosphate (pH of the production stage was adjusted to 5.9) The cultivation was carried out for 96 hours at 28 [deg.] C. After the cultivation, the biomass was centrifuged (1500 g, 10 min). . ) and the supernatant was concentrated in a evaporator to give a crude enzyme preparation with a cellulose activity of 66 U and a specific activity of 0.78 U / mg. Example 2

Rovnako, ako v příklade 1, s tým rozdielom, že namiesto BasldiiotriehoeporOn :piullulans sa použil kmeň Bassidiotrichosporon cellulolyticum CCY 40-5-4. Výsledný surový enzýmový preparát mal aktivitu 94 U a špecifickú aktivitu 0,71 U/mg.As in Example 1, except that the strain Basidiotrichosporon cellulolyticum CCY 40-5-4 was used instead of BasldiiotriehoeporOn: piullulans. The resulting crude enzyme preparation had an activity of 94 U and a specific activity of 0.71 U / mg.

P r í k 1 a d 3EXAMPLE 1 a d 3

Rovnako, ako uvedené v příklade 2, s tým rozdielom, že namiesto kukuřičného výluhu a hydroxyetylcelulózy sa ako zdroj uhlíka v kultivačnej póde použije odpadná vata z hydroxyetylcelulózy (50 g/1).As in Example 2, with the exception that corn leach and hydroxyethylcellulose instead of hydroxyethylcellulose waste cotton (50 g / l) is used as the carbon source in the culture medium.

Claims (1)

PREDMETSUBJECT Spósob produkcie celulolytických enzýmov vyznačujúci sa tým, že 10 nasledovných kmeňov rodu Basidiotrichosporon: Basidiotrichosporon pullulans:Method for producing cellulolytic enzymes, characterized in that the following 10 strains of the genus Basidiotrichosporon: Basidiotrichosporon pullulans: CCY 30-1-1 a CCY 30-1-1 b CCY 30-1-2 CCY 30-1-3CCY 30-1-1 and CCY 30-1-1 b CCY 30-1-2 CCY 30-1-3 Basidiotrichosporon cellulolyticum:Basidiotrichosporon cellulolyticum: CCY 30-5-1 CCY 30-5-3 CCY 30-5-4 CCY 30-13-1CCY 30-5-1 CCY 30-5-3 VYNÁLEZUINVENTION CCY 30-5-11 CCY 30-5-12 jednotlivo alebo v zmesi sa kultivuje naživnej půdě minerálneho zloženia obsahujúce-j síran amonný, gél hydroxyetylcelulózy a /3(1 -> 4) glukán ako induktor enzýmu pri teplote 20 až 40 °C, po dobu 48 až 110 hodin pri pH 4,5 až 6,8 a vyprodukovaný komplexný celulózový systém /3-glukán glukánohydrolázy a /3-glukozidázy sa získá v surovom stave odpařením rastovej půdy, připadne v čiastočne prečistenom stave zrážaním sxranom amonným připadne zrážaním organickým rozpúšťadlom s výhodou etanolom.CCY 30-5-11 CCY 30-5-12 singly or in a mixture is cultivated in a nutrient medium of mineral composition containing ammonium sulphate, hydroxyethylcellulose gel and / 3 (1 -> 4) glucan as enzyme inducer at 20 to 40 ° C , for 48 to 110 hours at pH 4.5 to 6.8 and the complex cellulose system produced by [beta] -glucan glucanohydrolase and [beta] -glucosidase is obtained in the raw state by evaporation of the growth soil or partially purified by ammonium sulphate precipitation or precipitation an organic solvent, preferably ethanol.
CS13077A 1977-01-10 1977-01-10 Method of producing cellulolytic enzymes CS204129B1 (en)

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