CS204148B1 - Method of producing enzyme of alpha-amylase - Google Patents
Method of producing enzyme of alpha-amylase Download PDFInfo
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- CS204148B1 CS204148B1 CS365177A CS365177A CS204148B1 CS 204148 B1 CS204148 B1 CS 204148B1 CS 365177 A CS365177 A CS 365177A CS 365177 A CS365177 A CS 365177A CS 204148 B1 CS204148 B1 CS 204148B1
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Description
Vynález sa týká spósobu produkcie enzýmu α-amylázy pomocou kvasinkovitého organizmu Cryptococcus luteolus.The present invention relates to a process for the production of the .alpha.-amylase enzyme by the yeast Cryptococcus luteolus.
V súčásnej době sa enzým alfa-amyláza získává izoláciou z produkčného prostredia baktérie Bacillus substilis a Streptococcus aureofaciens. Produkcia alfa-amylázy je popísaná u niektorých kmeňov bývalého rodu Endomycopsis. Je to Endomycopsis capsularis (dnes Saccharomycopsis capsularis), popísaný H. Ebertovou (Folia microbiologica 11 : 422—428, 1966], dále) Endomycopsis fibuliger (dnes Saccharomycopsis fihuliger), Endomycopsis javanensis (dnes Arthroascus javanensis), Endomycopsis lindneri (dnes je to synonymum pre Saccharomycopsis fibuliger) a Endomycopsis hordei (dnes Saccharomycopsis fibuliger var, hordei), popísaná E. Matsutani, M. Sáto a F. Hatori (US pat.Currently, the enzyme alpha-amylase is obtained by isolation from the production medium of Bacillus substilis and Streptococcus aureofaciens. Alpha-amylase production is described in some strains of the former genus Endomycopsis. It is Endomycopsis capsularis (today Saccharomycopsis capsularis), described by H. Ebert (Folia microbiologica 11: 422—428, 1966], hereinafter) Endomycopsis fibuliger (today Saccharomycopsis fihuliger), Endomycopsis javanensis (today Arthroascus javanensis) is Endner synonym for Saccharomycopsis fibuliger) and Endomycopsis hordei (now Saccharomycopsis fibuliger var, hordei), described by E. Matsutani, M. Sato and F. Hatori (US Pat.
297 547) a konečne u Schwanniomyces occidentalis a Schwanniomyces alluvius. Nevýhodou spósobov produkcie a-amylázy u týchto kmeňov je poměrně nízká aktivita a-amylázy, vysoká koncentrácia zdrojov uhlíka a zložitá separácia biomasy.297 547) and finally in Schwanniomyces occidentalis and Schwanniomyces alluvius. Disadvantages of α-amylase production methods in these strains are the relatively low α-amylase activity, high concentration of carbon sources, and difficult biomass separation.
Uvedené nevýhody odstraňuje sposob produkcie α-amylázy na tekutej živnej póde, obsahujúcej zdroje asimilovatel'ného uhlíka, komplexně zdroje dusíka a minerálně živné soli, ktorého podstata spočívá v tom, že kmen Cryptococcus luteolus CCY 17-6-2b sa kultivuje na živnej pode obsahujúcej 0,8 až 3 hmot. % kukuřičného škrobu pri teplote 20 až 38 °C, po dobu 48 až 120 hodin, pri pH 4,7 až 6,8, pričom sa enzým a-amyláza získá odpařením rastovej pódy, alebo zrážaním sířanom amónnym, alebo afinitným přečištěním na sieťovanom škrobe.These disadvantages are eliminated by the method of α-amylase production on a liquid nutrient medium containing assimilable carbon sources, complex nitrogen sources and mineral nutrient salts, which is based on the fact that the Cryptococcus luteolus CCY 17-6-2b strain is cultivated on a nutrient-containing soil 0.8 to 3 wt. % corn starch at 20 to 38 ° C, for 48 to 120 hours, at pH 4.7 to 6.8, wherein the α-amylase enzyme is obtained by evaporating the growth pod, or by precipitation with ammonium sulphate, or by affinity purification on a cross-linked starch .
Uvedený kmen Cryptococcus luteolus 17-6-2b je uložený v čsl. zbierke kvasiniek a kvasinkovitých organizmov v Bratislavě.Said strain of Cryptococcus luteolus 17-6-2b is deposited in U.S. Pat. collection of yeasts and yeast organisms in Bratislava.
Póvodný kmen Cryptococcus luteolus CCY 17-6-2, ktorý je saprofytického póvodu, bol izolovaný z pódy. Bol vyšlachtený trojnásobným pasážovaním na póde pripravenej z 8 % gélu amylózy sieťovanej epichlórhydrínoin, ponorenej do syntetickej Yeast Nitrogen Base (0,6 g/100 mlj pódy pri teplote 20 až 38 °C, po dobu 48 až 120 hodin a pH 4,7 až 6,8. Nový vyšlachtený kmeň bol označený ako Oryptococcufe luteolus CCY 17^6-2b.The original strain of Cryptococcus luteolus CCY 17-6-2, which is of saprophytic origin, was isolated from the genus. It was sieved three times by passage on a broth prepared from 8% epichlorohydrin-crosslinked amylose gel immersed in synthetic Yeast Nitrogen Base (0.6 g / 100 ml of pod at 20-38 ° C for 48-120 hours and pH 4.7-7). 6.8 The new strain was designated as Oryptococcufe luteolus CCY 17 ^ 6-2b.
Kmeň Cryptococcus luteolus CCY 17-6-2b má buňky oválné až pretiahle, málo jednotné, V tekutom prostředí tvoří prstenec až sliznatý povlak, na pevnom prostředí je sliznatý a kréme vo žltastý. Nekvasí cukry. Asimiluje okrem D-glukózy ešte D-galaktózu, maltózu, sacharózu, laktózu, melibiózu, celobiózu, trehalózu, melezitózu, D-xylózu, D-manitol, L-lyzín, rafinózu. Polysacharid, vy204148 tváraný okolo buniek dává jódšrobovú reakciu. Neasimiluje ani 1-sorbózu, ani dusičňan draselný. K rastu potřebuje vitamíny v prostředí. Optimálna teplota rastu je 23 °C pri 5 °C alebo pri 42 °C nerastie. Produkuje ureázu, aktivita katalázy je vysoká, stekucuje želatinu a rýchle autolyzuje.The strain Cryptococcus luteolus CCY 17-6-2b has cells oval to elongated, little uniform. In liquid medium, the ring forms a slimy coating, on the solid environment it is slimy and the cream is yellowish. It does not ferment sugars. In addition to D-glucose, it assimilates D-galactose, maltose, sucrose, lactose, melibiosis, cellobiose, trehalose, melezitose, D-xylose, D-mannitol, L-lysine, raffinose. The polysaccharide, γ204148, formed around the cells gives a iodine-like reaction. It does not assimilate either 1-sorbose or potassium nitrate. To grow it needs vitamins in the environment. The optimum growth temperature is 23 ° C at 5 ° C or does not grow at 42 ° C. It produces urease, catalase activity is high, fluidizes gelatin and rapidly autolyses.
Výhodou sposobu produkcie a-amylázy pódia vynálezu je: poměrně vysoká aktivita α-amylázy aj pri práci s jednoduchými fermentačnými podami, o nižšej koncentrácii zdroja uhlíka ako u fermentačných pód například pre Bacillus substilis; jednoduchšia separácia biomasy po ukončení fermentačného procesu; výhodnější charakter produktu amylolytickej reakcie katalyzovanej enzýmom z Cryptococcus luteolus (obsah D-glukózy až 34 %) ako u α-amylázy Bacillus substilis; možnost využitia biomasy na krmovinárske účely.Advantages of the method of α-amylase production according to the invention are: relatively high α-amylase activity even when working with simple fermentation broths, with lower carbon source concentration than with fermentation broths, for example for Bacillus substilis; easier separation of biomass after the fermentation process; more advantageous character of the product of the amylolytic reaction catalyzed by the enzyme Cryptococcus luteolus (D-glucose content up to 34%) than that of Bacillus substilis α-amylase; possibility of using biomass for feed purposes.
Příklad 1Example 1
Z vypratého gélu připraveného zo sletovaného a(l -* 4] glukánu podlá A. O. číslo 186 059, sa vyřežu trojúhelníkové profily o hrané 1,5 cm a vložia do Petriho- misky a zalejú sa roztokom kultivačnej tekutiny Yeast Nitrogen Base (Difco), 0,657 g na 100 ml destilovanej vody. Gély sa ponoria asi do polovice svojej výšky. Po trojnásobné opakované j sterilizácii pri 100 °C v pare (1 hodina) sa očkuje na povrch gélu kultúra Cryptacoiccus luteolus CCY 17-6^2b a inkubuje sa pri 23 °C po dobu 48 hodin. Namnožená biomasa C. luteolus na stekutemoím géli (0,21 gramu) sa použila ako inokulum na produkčnú půdu (2 1), pozostávajúcu z 0,8% kukuřičného výluhu, 0,8 % kukuřičného škrobu, 0,2 % síranu amónneho a 0,3 % dihydrofosforečňanu draselného vo vodě (pHFrom the washed gel prepared from sintered α (1 - * 4) glucan according to AO No. 186 059, triangular profiles of 1.5 cm were cut and placed in a Petri dish and poured into Yeast Nitrogen Base (Difco), 0.657 culture fluid solution. g per 100 ml of distilled water The gels are immersed to about half their height After a three-fold sterilization at 100 ° C in steam (1 hour), a culture of Cryptacoiccus luteolus CCY 17-6 ^ 2b is inoculated onto the gel surface and incubated at 23 ° C. ° C for 48 hours The multiplied C. luteolus biomass on a stekutemic gel (0.21 grams) was used as an inoculum on the production soil (2 L) consisting of 0.8% corn steep liquor, 0.8% corn starch, 0 ° C. , 2% ammonium sulphate and 0,3% potassium dihydrophosphate in water (pH
6,1). Kultivácia prebiehala po dobu 85 hodin pri 25 °C. Po ukončení kultivácie sa biomasa C. luteolus odstranila centtrifugáciou (31000 gramov, 5 -min.) a supernatant sa zahustil na vákuovej odparke. Získaný surový enzým a-amyláza mal celková aktivitu 34,3 U a Specifická aktivitu 0,081 U/mg.6.1). Cultivation was carried out for 85 hours at 25 ° C. After the cultivation was complete, C. luteolus biomass was removed by centrifugation (31000 grams, 5 min) and the supernatant was concentrated in a vacuum evaporator. The obtained crude α-amylase enzyme had a total activity of 34.3 U and a specific activity of 0.081 U / mg.
P r í k 1 a d 2Example 1 a d 2
Postupovalo sa tak ako v příklade 1, s tým ro-zdielom, že kmen Cryptococcus luteolus CCY 17-6-2b sa kultivuje na živnej pode obsahujúcej 2 % kukuřičného škrobu pri teplote 20 °C po dobu 120 hodin a pH 4,7. Namiesto zahustenia vo vákuovej odparke sa enzýmový preparát spracováva ďalej afinítnou metodou za využitia sletovaného škrobu (AO č. 157 955). Získaný přečištěný enzým mal špecifickú aktivitu 11,4 U/mg. Příklad 3The procedure was as in Example 1, except that the Cryptococcus luteolus CCY 17-6-2b strain was cultured on a culture medium containing 2% corn starch at 20 ° C for 120 hours and pH 4.7. Instead of concentration in a vacuum evaporator, the enzyme preparation is further processed by the affinity method using sintered starch (AO No. 157 955). The purified enzyme obtained had a specific activity of 11.4 U / mg. Example 3
Postupovalo sa ako v přiklade 1, s tým rozdielom, že kultivácia sa prevádzala na živnej póde obsahujúcej 3 % kukuřičného škrobu pri teplote 38 °C po dobu 60 hodin a pH 6,8. Enzým a-amyláza sa získá zrážaním so síranom amónnym (680 g/1). Získaný enzýmový preparát mal špecifickú aktivitu 0,11 U/mg.The procedure was as in Example 1, except that the culture was carried out on a nutrient broth containing 3% corn starch at 38 ° C for 60 hours and pH 6.8. The .alpha.-amylase enzyme is obtained by precipitation with ammonium sulfate (680 g / l). The obtained enzyme preparation had a specific activity of 0.11 U / mg.
Význam vynálezu spočívá v použití a-amylázy v potravinárstve (pečivársky a pivovarský priemysel), příprava sirupov apod.) a v textilnom priemysle. Amylázový hydrolyzát sa používá k príprave atypických sladidiel a čistých ollgosacharidov (maltóza, maltotrióza, maltotetraóza) ako aj D-glukózy s využitím v chémii saCharidov a v klinické] praxi.The importance of the invention lies in the use of α-amylase in the food industry (baking and brewing industries), the preparation of syrups and the like) and in the textile industry. The amylase hydrolyzate is used to prepare atypical sweeteners and pure ollgosaccharides (maltose, maltotriose, maltotetraose) as well as D-glucose for use in sacharide chemistry and in clinical practice.
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