DD69330B1 - Process for the biotechnical production of a plant glucan-degrading enzyme - Google Patents

Description

a) Title of the invention

Process for the biotechnical production of a plant gluc on degrading enzyme

b) Field of application of the invention

According to the invention, a method for the biotechnical production of a plant glucan-degrading enzyme is proposed.

The enzyme can be used to degrade cereal glucan, especially glucan from barley, wheat or other vegetable origin, with β-1,3- and β-1, Ц- binding in the production of beer u, a. be used.

c) Characteristic of the known technical solutions

In addition to xylan and araban, glucan is counted among the so-called rubbers of barley. It is known that barley glucan is a glucose polymer composed of dissimilar amounts of β-1,3 and β-1, α-glycosidic bonds. The glucan molecule consists of an unbranched chain of glucose units, in which probably the ß-1,4-bonds are more common than the ß-1,3-bonds.

There are currently 3 known polysaccharides linked by β-1,3 and β-1,4 linkages. These are barley glucan, wheat glucan and lichenin. "There is conflicting information about the sequence of 1,3- or 1,4-glycosidic bonds. Another glucan, the auc

B-Glucopyranoseresten and contains ß-1,3-BiDdUDgen has been eotected in the Hefezellwaod. The synthesis of an unspecified ß-glucaos by a mutant vod Alkaligeoes faecealis has also been beschriebeo been. Comyceteo cell walls contain glucans with ß-1,3- and β-Ι, Α-glycosidic bonds, Agrobacterium. In suitable culture solutions, tumefaciens precipitates another type of glucan with β-ljE-glycosidic bonds. Furthermore, glucans with β-1,6 bonds (pustulan and luteose) are described.

Depending on the binding conditions, some specific enzymes have become known for the enzymatic degradation of some glucans. Bs was a ß ~ 1,3 ~ glucanase made from fungi. Purification and properties of Bacillus circulans β-1,3-glucanase are also reported. The substrate laminarin is cleaved by β-1,3-glucanase into glucose, laminaribiose, laminaritriosis and other oligosaccharides. Also from the gastric juice of Helix pomatia a similar enzyme could be obtained. Also reported was an isolation method of a glucanase derived from an amylase concentrate. There was no data on the recovery of the amylase concentrate.

Furthermore, suboptimal glucanase formation on the scale of iodine using a culture medium according to Güntelberg has been reported.

d) Object of the invention

The aim of the invention is to develop a biotechnical process for the production of an enzyme with high glucanase activity, in particular for the degradation of cereal glucan.

e) Presentation of the essence of the invention

The object of the present invention is to attach a glucanase-producing microorganism strain to a high glucan

1.1 »Fold of vegetative cells

Cleavage capacity to adapt and then cultivate in a suitable nutrient medium.

According to the invention, this is achieved by using as microorganism strain Bacillus subtilis COHN 1872, which no longer has the ability to reduce nitrate. The trunk has the following characteristics:

a) Morphological characteristics

Movable longitudinal rods with rounded cell ends, as single or double rods or short chains

2.5 - 6.0 χ 0.6 - 0.8 / i oval without specific localization in the sporangia not swollen positive, in the age group labile young cells homogeneous tin-

2. 3 *

4, 1.5.

Size spurs

Sporangia Graor behavior

1 · 6th Methylene blue staining

1 · 7th growth

a) on meat stock agar

b) meat bouillon does not yaw vacuolised

lush growth, colonies dull, dirty white, ribbon lobed, z.T. fissured mycelium Agar-stripe: lush colony on the surface, along the line weak growth lush growth in the form of white streaks and a delicate, slightly brittle surface covering, slight cloudiness

(c) meat bouillon and more abundant growth than meat broth pure in glucose,

thick, firm surface cuticle

d) meat broth and 7 % NaCl: very weak NaCl growth

5 % NaCl: lush growth

e) Growth to potato-rich growth, colonies slices or, slimy, moist-shiny, -kekeilen dirt-white to yellowish brown, after 3-4 days at 37 ° C bubbling (filled with mucus), colonies in the age dark brown, no folding

b) Physiological characteristics

VOGES-PKOSKAÜSB REACT K> N (acetylmethylcarbinol) +

Gelatin liquefaction +

Coagulation of milk +

Casein hydrolysis +

Catalase +

Kitrat reductase -

Gas formation in nitrate- and glucose-containing peptone-kährlösung +

Gas formation in nitrate-containing meat broth (without glucose) -

NHy formation in peptone broth +

H ₂ S formation

Indole formation -

Citrate recycling +

Growth at + 56 ° C

pH change in peptone broths (6 days at 37 ° C) 7.0 8.6 / 8.8 changes in litmus milk (10 days at 37 ° C) alkalization (pH 6.0 / 8.3)

Acid formation from carbohydrates (without gas formation):

Glucose +

Sucrose +

Mannose +

Galactose +

Arabinose +

Lactose

Maltose +

Dextrin +

Mannitol +

Salic in +

Strength +

Despite the morphological and physiological range of variation, the diagnosis of the strain used revealed a reliable association with the species Bacillus subtilis COHN 1872 and a clear differentiation from other species according to the key in Bergey's manual (Breed et al. 1957) and. that of Smith et al. AGR, Misc., Publ. No. 559) and Smith et al., 1952 ("Aerobic sporeforming bacteria," U.S. Dept. Agric., Monogram No. 16).

A sample of the production strain used according to the invention has been since. 30. 3 · 1967 in the collection of the Institute for Microbiology of the Humboldt-tmiversitat in Berlin, field office 1532 Kleinmachnow, Max-Reimann-Str. 16, under the strain collection number 3712 deposited.

The strain used in the method according to the invention for the biotechnical production of a glucan-cleaving enzyme splits in particular barley glucan, where as cleavage protein

oligosaccharides and, to a lesser extent, glucose. Furthermore, no enzymatic degradation of the laminarin could be achieved with the glucanase produced. Since barley glucan contains both β-1,3- and β-1,4-glycosidic bonds in a specific sequence and steric arrangement, the cleavage capacity of said enzyme extends to the specific chemical structure present in the cereal glucan.

The Bacillus subtilis COHN 1872, which no longer has the ability to reduce nitrate, is adapted to high cleavage ability by alternately cultivating it on solid and liquid nutrient media, optionally with or without glucan.

The production of glucanase is carried out with the above-mentioned Bacillus subtilis strain in the submerged procedure. The selected stock material is first transferred to a suitable inoculum containing meat water, Λ% peptone and 0.5 % NaCl. The shake flasks are incubated for 18 to 24 hours at 30 ° C on a debris elemase with about 220 strokes / min. Thereafter, the over inoculation of the inoculum into the respective production medium takes place ·

The process according to the invention for producing glucanase takes place aerobically under submerged conditions in shake flasks with about 10 % filling. On an industrial scale, fermentation tanks made of stainless steel with stirrers and sterile air supply are used. The production volume of carefully sterilized culture liquid can be several 10 m ^J. The nutrient solution must contain suitable sources of carbon and nitrogen, which must not be significantly damaged by the sterilization mode. The inoculation of the sterilized nutrient medium is carried out with 1 to 5 % inoculum. The cultivation temperatures are preferably between 28 and 37 ° C. As antifoams, for example, silicone oils and vegetable or animal fats or oils with good

Success be used. The culture time is measured depending on the composition of the medium to 24 to 72 hours. The amount of aeration is 0.3 to 1 l / min depending on the physiological state of the culture. At the end of the fermentation time 100 to 150 IS / ml glucanase, 3,000 to 5,000 ΙΕ / ml amylase and 1 to 2 PUcas protease are achieved.

The determination of the glucanase was carried out in accordance with the recommendations of the IUB (1961) using barley glucan as substrate (IA Preese and KG Mackenzie J. Inst. Brew. J28, 353-363, 1952) with 3,5-dinitryl salicylic acid , After completion of the fermentation, the culture solution is separated from the bacteria and Nährbodenbestandteilen, concentrated in vacuo and precipitated with organic solvents, inorganic salts or other precipitants and then dried.

f) Exemplary embodiments Example 1

Bacillus subtilis COHN 1872, which no longer has the ability to reduce nitrate, is initially flattened out on a selection agar without glucose. The characteristic single colonies with slightly folded band, shiny and light pink in color, were spread on a nutrient medium consisting of barley meal, agar, NaCl and MgSO4. The incubation of this first inoculation, which at the same time involves adaptation to the barley glucan, lasts for at least 48 hours at 30 ° C. By at least two more passages on the same glue-containing culture medium, the adaptation to glucan cleavage is further increased. The incubation period of these passages is expediently extended to several days to several weeks. Cell suspensions from the last passage are then inoculated into a liquid propagation medium containing no glucan. At this stage, it only depends on a course-time multiplication of the germs,

why this medium in addition to 0.1% strength and 0.5 % glucose. Propagation is complete after 6 to 7 hours without the high cleavage of the parent material being attenuated. This propagation step may only be repeated once more at an incubation period which may be longer than M up to 6 hours. This inbculum is then inoculated and fermented in production media containing up to 9% barley meal (high glucan concentration) and fermented.

Example 2

Pur the inventive method for the production of glucanase with the Bacillus subtilis strain according to the invention the following Nänrboden is used!

9 % Ger st se bread

3 % soybean meal

0.25 % (KV ₂ SO ₄₎

0.2 % KH ₂ PO ^ and

1.2 * Na ₂ HPO ^

tap water

pH before sterilization: 7.2

pH after sterilization: 6.5 to 6.9.

The production medium is filled to 50 ml in 500 ml ßundstehkolben, sterilized, inoculated and then placed on the shaker. The necessary inoculation amount is 1 to 2 %. The germinal density of the inoculum should suitably be 6 x 10 6 germs / ml. The shaking period is 3 days, the incubation temperature is expediently 30 ° C. During an incubation period of 3 days, in addition to a considerable amount of glucanase, namely 80 to 150 μl / ml, amylase and protease are also formed in the production medium. Amylase production is between 2,000 and 5,000 ΙΕ / ml. The amount of amylase formed is independent of the amount of glucanase formed.

- 9 Example 3

The production of glucanase takes place in a 500 l V2A steel fermentation tank with forced ventilation. The composition of the medium is as follows:

2 % barley meal

2. % potato starch

0.95% soy flour

1.5% (NH 2) 2H 2 PO 4

0.08 % sodium citrate and

various trace elements, tap water,

After sterilization at 120 ° C and 30 minutes, the H ion concentration is adjusted to pH 7.0 to 7.2. After cooling, the inoculation is carried out under sterile conditions with 2 % inoculum. Aeration during the logarithmic growth phase is 0.7 1 of air per 1 1 of fluid per minute. The stirring speed of the Ruhrers is depending on the nature of the same 160 to 420 U / min. After 36 hours, the fermentation is interrupted as the carbohydrates are used up and a shift in the pH occurs. After a culture period of 36 hours, 100 to 150 IU of glucanase, 2,000 to 3,000 of IS amylase and about 1 PUase are formed.

Example Ц

1,000 ml of culture filtrate having a ß-glucanase activity of 150.0 ΙΕ / ml and an alpha-amylase activity of 5000 XE / pil are clarified by the addition of calcium gel. The precipitation of the desired enzyme is carried out with stirring with solid (NH ^) ₂ 50 ^. The precipitant can be used in pro analysi or technical quality. The precipitation takes place at a temperature of 10 ° C to room temperature. The precipitate is dried in vacuo at a maximum temperature of 40 ° C and then homogenized.

By brief treatment of the moist preparation with acetone below 0 ° C, the drying time of the preparation is significantly reduced and achieved a brightening of the drug. The end product is powdery, non-hygroscopic and readily soluble in water.

yield

From 1 000 ml culture filtrate above activity 10 g of solid product with an activity of 9.0 IS / mg ß-glucanase and 160 IU alpha-amylase are obtained, which corresponds to a yield of about 90 % ß-glucanase and about 35 % alpha amylase.

Example 5

Precipitation with acetone

1 000 ml of culture filtrate of the above activity are added with stirring at a temperature below 0 ° C with acetone to the degree of saturation of 0.65. The pH of the culture solution is adjusted to pH 7 with phosphate buffer according to SORENSEN. The precipitation product is processed as described above.

yield

There are obtained 11.5 g of solid product having the following activity:

β-glucanase: 8.0 ΙΕ / mg = 82 %

Alpha-amylase: 300 ΙΕ / mg = 77%.

Claims (3)

invention claims Process for the biotechnical production of a plant glucan-degrading enzyme, characterized in that Bacillus subtilis СОШТ 1372, which no longer has the ability to reduce nitrate and which is deposited under the number 3712 in the strain collection of the Institute of Microbiology of the Humboldt University in Berlin , is used. 2. The method according to item 1, characterized in that the strain is adapted to a high Glucanspaltungsvertngen and that derived from the natural substrate Bacillus subtilis Stäcime are cultured alternately on solid or liquid and on the other hand glueanhaltigen or glueanfreien culture media. Method according to points 1 and 2, characterized in that said strain is used to increase glucanase glucan as an inducer in the selection well, your inoculum and / or the production medium and that as carbohydrate and glutinous sources barley meal, Wei2.kleie, Wheat or wheat flour in conjunction with starch and all glucan source of other vegetable origin.