DD158916A1 - PROCESS FOR PREPARING BACILLUS BETA-1,3-1,4-GLUCANASE - Google Patents

Abstract

The invention relates to a process for the production of Bacillus beta-1,3-1,4-glucanase for technical, pharmaceutical and scientific purposes, when a hydrolytic cleavage of beta-glycosidic bonds is required or desired. The aim of the invention is to improve the economics of the industrial production of beta-glucanase by using a cell material with higher capacity for extracellular beta-glucanase, the production of which requires reduced labor and time for production. According to the invention, the aim is achieved by Bacillus spec. ZF-194, which is deposited in the central collection of the GDR (Central Institute for Microbiology and Experimental Therapy of the Academy of Sciences of the GDR, Jena) under the number IMET B615, is used for enzyme synthesis. The strain can be stored as Schraegagarkultur and spore suspension in saline. The spore suspension is prepared by washing away cell material from agar nutlets, treating with lysozyme, thermal inactivation, and suspension in saline and stored at 4 degrees C. It is used for the long-term storage ("preserve") and for the provision of Schraegagarkulturen. The latter are used to inoculate submersed cultures. Spores showed over a period of 15 months, Schraegagarkulturen over 6 months full performance stability. The influence of media components on the level of extracellular beta-glucanase activity was tested.

Description

2 1971

a) Tital of the invention

Method 2112 Preparation of 3acillus β-1 _s 3-1 j4 glucanase

b) Application of the invention

The invention relates to a biotechnological process producing Bacillus 2m-beta-1,3-1,4-Glucariase for technical, pharmaceutical and scientific Put * The Snsym, in the preparation ¹ VQS ITahsungsaittelu, Genußaitteln and G-etreLaksn as well as aids the FÜS Basic research and applied research are used, vvenn for different reasons, a hydrolytic cleavage of ß-glycosidic bonds is required or erminscht. The main current application of SDsyin is in the brewing industry, νιο in beer production in increasing MaSe malt replaced by Hohfrucht (z · B * by barley) ^ ird.

c) Characteristic of the known technical solutions

ß-glucanase is already produced industrially. In process variants according DL WP 69,330 and DL WP C 12 D / 190,401 Bacillus subtilis strains subtilis up to a maximum I50 Γ3 ß-glucanase / ml culture fluid synthetisierenο fluctuations in the ß-glucanase formation of the B _e Stanimes made the conservation measures described in 12 SL IP C 12 D / 190 401;

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and hydrogenation required to achieve improved hepar- eability and trouble-free production by stabilizing growth course and isazym synthesis. These proposed solutions for counteracting splitting and degeneration phenomena are laborious and time consuming.

d) Object of the invention

The object of the invention is to improve the economics of the industrial production of β-glucanase by using a cell material with higher extracellular β-glucanase production capacity, the production of which requires reduced labor and time for production.

e) Explanation of the nature of the invention

The essential characteristics of the invention are

1, a strain of Bacillus with a 3-glucanase production increased compared to previously available parent material,

2. a preservative form of the enzyme-forming Bacillus strain that is stable with respect to growth behavior and level of β-glucanase formation and that allows the production of reproducible seed material with minimum labor and time expenditure,

The possibility of producing a higher β-glucanase activity when using the Bacillus strain, without the biotechnical and technological parameters of the usual industrial process having to be significantly changed.

According to the invention, this is achieved by "Bacillus spec." ZE -194, in the central collection of the DDH (Central Institute for Microbiology and Experimental Therapy of the Academy of Sciences of the DDH ₃ Jena)

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under the number JMBS B 615 is used.

WICKED ?. B 615 grows extremely aerobic. The mean generation times at 30 - 37 ⁰ C in glucose broth media and carbohydrate-based with varying concentrations of polysaccharides and sugars amount to 60-70 min. Under test conditions are after

14 - 16 hours 10 ⁹ - 10 ¹⁰ lines / ml in the SchuttelkoIben

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bzw.V 10 - 10 cells / ml achieved in forced-air culture flasks. In emersed growth, about 90 % dex cells are lost after 5 days. The spores are stretched oval and are central.

HMD B 615 grows on the usual complex agar media as medium sized single colonies with a matte surface. In the central area, lytic effects occur, as a result of which, after prolonged incubation, flattened colonies with a wrinkled to folded surface can be observed.

The biochemical reactions of EUST B 615 (formation of acid or gas from various mono- and disaccharides as well as sugar alcohols, enzyme tests, ITratratverwertung etc.) suggest a close taxonomic relationship with the morphological group I, especially with Bacillus amyloliquefaciens, according to BEEGSI (1974). , The lack of an extracellularly stable α-amylase and differences in sensitivity or insensitivity to 20 different Bacillus phages, which can be interpreted as differences in the structure of the cell wall, argue against a direct association with this species. Compared to the species Bacillus subtilis is IMHD B 615 by acid formation from galactose, poor growth on glucose-nitrate agar, gas formation from nitrate and a less marked acidification of the medium with growth ± d G-Iucοse broth (5 8 _- 6, 9 instead of 5j4 - S ₅ 2) in addition to differences in the morphology of the colon clearly distinguishable.

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Compared with the Bacillus subtilis strains used in DL WP 69 33-0 or DL WP G 12 D / 190 401, BIST B 615 differs in its ability to degrade arabinose, xylose and galactose to acidify its ability to react in the presence of 5 %. NaCl can grow through a variety of morphologic features, through its behavior towards test phages, and through its high leaching rate.

EIHlT 3 615 produces up to 260 HS J3-glucanase / ml culture fluid under test conditions (see working examples), up to 3 Tu c neutral protease / ml culture medium.

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liquid and no detectable e6-amylase.

HIST 3 615 is non-pathogenic and releases no toxic substances.

Spore suspensions J5rfindungsgemäß prepared were stored at +4 ⁰ G · The verifications of ß-glucanase BiIdung over a period of 15 months resulted in a stable Snzymbildungsvermögen of ca, 200 Γ3 ß-glucanase / ml SuIturflüssigkeit (negative deviations of up to X3 / inl, Positive deviations up to 250 IS / ml). These spore suspensions can be used for slants cultivations which can be directly used up to δ months without loss of performance or used for planting of veryagagar subcultures without loss of power. The β-glucanase activities of such slant cultures were approximately 200 × 13 / ml (negative deviations up to 180 × 13 / ml, positive deviations up to 260 × / ml). The stability of β-glucanase formation is additionally documented by that the standard deviation of a test series was a maximum of 7 % of the associated mean value.

According to the invention, cultivation and synthesis of snotes are carried out with submerged cultivation of 33 l of 3T B 615 in media consisting of polysaccharide-containing ITatures, an inorganic nitrogen source and inorganic salts. The concentration ranges were deliberately adjusted to the usual

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These are suitable because they are suitable for large-scale fermentation and for the fermentation of fermentation. Länzymaktivität au an administrable enzyme preparation have proven. Components of the main culture medium have been varied individually and in combination. The highest extracellular ß-glucanase activities merchandise under the chosen Y he search conditions (corresponding embodiments 1-5.) After 45 - 55 hours detectable "increases in Polysaccharide prevent Aktivitätsabηahmen the fermentation existing 8-glucanase. With regard to the level of extracellular detectable β-glucanase activity, there are relatively broad polynuclear optimizations for polysaccharides, (KEE ₂ ,) Mg ⁺⁺ , Ga ⁺⁺ , SO ₄ ² ", and Gl", which is advantageous for large scale fermentations. ! The concentration of the offered as inorganic nitrogen source and as pH stabilizer (M, J pE ^ O '^ ¹³ _-?.. Are carefully matched ³ to Gesanitkonsentration of polysaccharides offered as a beta Giucanase degradation is otherwise observable excess supply of sulfate Ions led to drastic loss of activity.

f) embodiments

The indication is to be explained in more detail by the following examples:

example 1

Culture leadership

Zeliinaterial of IF ^ VIMISD B 6 ^ 5 of Schrägagarkuituren of ^ lähragar I (National Institute of immune preparations and Mhrmedien, Berlin-Weißensee, DDH) are Naeh 48 hours Bebrütxmg at 37 ⁰ G either directly or after a 2wischeapassage on Hähragar I or Gerstensehnotagar (2.5 % grist, 1 % sorrel, 0.5 % glucose, 2 % ITa ₉ HPO 3 .beta. 2 H ₂ O, 3 % agar, pH 7, O) in 50 ml Yorkulturmedium (O ₅ 5 % Orphaned bread, 0 _s 7 % Sojasciicot, '1 %

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Corn starch, 0.1 % yeast extract, 0.1 % glucose, 0.5% (M ₂ ) ₂ HPO ₄ , 0.1 % KCl, 0.05 % MgSO ₄ .7H ₂ O, pH 7.0) Vaccinated 500 ml. After 16 hours incubation at 30 ⁰ C with shaking (Hundschütteltisch, 200 U / min) cells contact removed and 50 ml of main culture medium in 5OO ml Ihindschüttelkolben with a cell number of 2-5. 10 ^ / ml inoculated. The cultivation is carried out at 30 ⁰ C with shaking. The β-glucanase activities are determined after 48 and 72 hours.

main Culture

In a main culture medium consisting of 2 % white fine meal, 1.5 % soybean meal, 2.0 % cornstarch, 1.5 JS (Μϊ ₄ ) ₂ ΗΡ0 ₄ , 0.15 % KCl, 0.05 % MgSO ₄ . 7H ₂ O, 0.02 % CaCl ₉ . 6 H ₂ O, pH 7j0 were measured after 48 hours 160-200 L3 / ml and after 72 hours 180-225 / alβ-glucanase.

Example 2 Culture guide

V / 13 in Example 1 (see 0,). The cell material for inoculation of the Yorkultur was kept on barley bread agar.

nauptkultur

As Example 1. B-glucanase activities after 48 hours 160-190 IS / ml and after 72 hours 200-260 HS / ml.

Example 3 Sulturführung

BacV amyloliquefaciens ΖΡ-194 / ΏΙΈΠ? B 615 winds on ITähragar for 15-7 days at 37 ⁰ C as an einael colony. The Yersporungsrate was 60 - 80 %, Thereafter, the cell material was distilled with sterile distilled water. washed off, with intermediate washing in sterile Aqua de st. heat inactivated twice (30 minutes in a steam pot) and the resulting

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rensuspension in saline (0.585% HACL, 0.15% KH ₂ PO _4, 0.84% Ka ₂ HPO ₄ VIa H ₂ O, 0.002% MgSO ₄ V 7 H ₂ O, pH 7.0) ⁰

stored at 4 ⁰ C. Hit her Sabragar I plates were inoculated · Sinzeikoloniea (roundish, dirty-white, matte surface, irregularly filed hand, center wrinkled) were transferred to Ger st en schrot ag ar; Further treatment as Example 1.

Hauptkuitur

As Example 1 β-glucanase activities after 48 hours 140-210 I3 / ml and, after 72 hours I70-250 IU / ml.

Example 4

cultivation

Spore recovery as in Example 3 · Additional cleaning of the spores by 30 mjja lysozyme treatment (400 / μg / ml) before the first heat inactivation »Further treatment as in Example 1.

main Culture

As Example 1. ß-glucanase activities after 48 hours 145-210 ΙΕ / ml and after 72 hours 130-240 IS / ml.

Example 5

Culture leadership

As in Examples 1 to 4.

main Culture

IjQ a main culture medium from O ₉ 5 % wheat fine meal, 0.7 % soybean meal, 1 % * corn starch, 0.2 % dry yeast, O ₅ 5 % (SH ₄ ) 2HPO ₄ , 0.1 % KGl, 0.05 % MgSO _4. 7 H ₂ O, pH 7.0, were determined after 48 hours 90-110 IU / ml and after 72 hours 120-140 IU / ml β-glucanase »

2 1971

Example 6

Eucleation management

Procedure until pre-culture as in Examples 1-4. Main culture in the laboratory fermenter "Biofer" 5 1 medium, cultivation at 30 ⁰ C and Bührung (800-900 U / min) using silicone antifoam Smulsion SLS (Wacker Chemie, Braunsehweig / BHD, 1: 10 in water), Air intake 80 l / hour.

main Culture

In medium according to example 1, ~ 120 ΙΕ / ml were measured after 20 hours, after 40 hours * / 155 13 / ml, after 60 hours »ν, 170 ΙΕ / ml and after 70 hours λΊ40 is / ml J3-glucanase«

In medium according to Example 5, the following β-glucanase activities were determined; After 20 hours' ^ 100 O / ml, after 40 hours ^ 100 I55 / ml, after 60 hours ~ 100 and after 70 hours *> 90 I3 / ml.

Claims (4)

- 9 - 2 19710 g) 3rfindungsanSprüche 1 · A method for the production of Bacillus-ß-1,3-1,4-glucanase dusch submerse cultivation and Sinzynisynthese, characterized in that Bacillus spec. ZE-194 (deposited as IM3T ITr »3 615), which is characterized by a high laxation rate on complex agar tracts in addition to a high formation of axferazeliular β-glucanase. 2 »Method according to item 1, characterized in that the storage of the strain ZF-194 takes place in the form of spores, which are stored as saline lysozyme-treated suspensions at 4 ⁰ C for prolonged periods. 3. The method according to items 1 and 2, characterized in that spores are brought to jtfähragar aua germination, wherein typically typical colonies Schrägagarkulturen be created for a period of at least S Llonaten directly to the inoculation of a powerful Subniersvorkultur or to obtain a further Schrägagarpassage , which then serves to supplement an efficient Subniersvorkultur suitable. 4 »method according to points 1 to 3, characterized in that the fientientative B-G-lucanase formation Medienzusamniensetzungen be used, the entsdenrechen the usual regulations.