The present invention concerns a process for preparation of -glucanase.

It has long been known that barley has a content of the poly- saccharide -glucan. Barley-β-glucan is structurally a poly- saccharide consisting of glucose with β-1,4- and β-1,3-bonds between the glucosidic units, where approximately 70 pph of the bonds are β-1,4-bonds and 30 pph are 0-1,3 bonds.

If barley with a high content of β-glucan is used as chicken feed, the chickens get into trouble because their digestive system is not capable of degrading the fi--glucan. It passes through the digestive system and comes out as a sticky droppings, and the chickens show symptoms of diarrhoea.

When pure barley is used instead of malted barley for brewing of beer, the wort will be viscous, which makes the filtration of the wort from the mask difficult.

The reason is that unmalted barley only has a small content of the glucanάegradable enzyme -glucanase.

The abovementioned problem can be avoided by adding -glucanase to the barley, but this is a question of price and for beer-brewing also a question of legislation.

Moreover, it is also a question of cultivating microorganisms which are particularly suited for the production of β-glucanase. It is also a question of the yield of enzymes from and the correct cultivation conditions of the micro- organism.

It is known that some microorganisms are capable of producing the enzyme β-glucanase in use for degrading the o-glucan.

UK Patent No. 1 421 127 describes a method for preparation of β-glucanase suited for degradation of ^β -glucan in barley when the microorganism Penicillium emersonii is used. It is a thermophilic microorganism with an optimum of growing at 50-54°C and where the growing is extremely low at tempe¬ ratures below 37-40°C. Below optimum conditions the fermentation will last for 7-10 days.

DD patent No. 148 891 also describes a process for prepara¬ tion of /3-glucanase, but in this process the microorganism Bacillus subtilis is used. The preparation of the enzyme is done under aerobic and submersible conditions in a free flowing nutrient medium at approx. 30 C.

DE patent application No. 2 048 237 describes a process for preparation of -glucanase by means of the microorganism Aspergillus phoenicis. This organism is not described as thermophilic, and the optimum of growing seems to be at 25-35 C when grown under aerobic conditions.

The abovementioned known processes have some drawbacks.

Firstly, the fermentation lasts too long when the thermo- philic microorganism Penicillium emersonii is used and secondly the enzyme produced by fermentation of Penicillium emersonii and Bacillus subtilis will have minimized activity at lower pH. Aspergillus phoenicis, as used in the West- German process, cannot be characterized as thermophilic at all, as its optimum of growinσ will be in the area of „

It now appears that microorganisms of the species Rhizomucor pusillus (Lindt) Schipper satisfy the purpose of the inven¬ tion because the microorganism is thermophilic, is growing fast and gives a high yield of enzymes in relation to the short time of fermentation.

Species of the said organism have been isolated from earth and from barley grain. One of these has shown to be extremely well suited and is deposited and described as CBS 551.82 at the Centralbureau voor Schimmelcultures, Holland. Further, species of Rhizomucor pusillus from the American Type Culture Collection, USA have been tested. The one best suited has the description ATCC 22074. In addition to the significance of the said organism for the good result it should also be mentioned that the composition of the nutrient medium, the support of air and the shape of the fermentation vessel are important factors in order to achieve optimal conditions during the fermentation and in this manner contribute to fulfil the purpose of the invention.

The nutrient medium

The fungi mentioned above can be cultivated by growing them on a solid medium, e.g. potatous-dextrose-agar, at 40-45 C. To produce larger quantities of β-glucanase the fungi have to be transferred to a liquefied medium. A piece of agar of approximately 2 x 2 cm 2 is cut out and homogenized in the well-known way with about 10 ml of the medium. 2 ml of the boπιθ enisat is transferred to sterile 300 ml Erlenir.eyer with 100 ml medium.

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As medium is used:

Soluble starch 5,0 g

KH ₂ 0 ₄ ^* 1,0 g

MgS0 ₄ . 7 H ₂ 0 0,88 g

CaCl ₂ 0,1 g

Trace elements-solution 1,25 ml

(NH_)-.-tartrate 3,0 g

Aqua dest. 1,0 1

with pH adjusted to 5,5.

The trace elements-solution consisted of:

H2_SO4. cone. 1/5 ml

MnS0 ₄ . H ₂ 0 1.5 g

Aσua dest, 1,0 1

The Erlenmeyers are incubated at 40 C by shaking until fair growth is achieved after 48-72 h. 300 ml of the inoculum is transferred to a 7 1 fer enter with 5 1 salt medium of the following composition:

KH ₂ P0 ₄ 2,0 g gS0 ₄ . 7 H ₂ 0 1 ,75 g

CaCl ₂ 0,2 g

Trace elements-solution 2,5 g

Water 1,0 1

The trace elements-solution has the same composition as the one described above.

The medium also contains a substrate which supplies the fungus with the necessary energy and carbon together with a nitrogen source.

Different energy and carbon sources are used during the growth, of which common potato flour, barley flour and corn¬ flour are most suitable.

The flour is added in relation to the oxygen transferring capacity of the fer enter in such a way that the culture always is under an aerobic condition.

The following may be used as suitable N-sources: K 0 ₃ ,

NH4.C1 and urea. The nitrog ³ en source is added in relation to the amount of carbon in such a way that the weight proportion between carbon and nitrogen is less than 7.

The fermenter must be equipped in a known way for adding of acid or caustic during the growth to regulate the pH in the area ot 4,0-b,0. Equipment useo for growing of Rhizomucor pusillus is sterilized beforehand, and aseptic tecniques are used in connection with the fermentation.

The support of air

It ^' will be of vital importance for the yield of enzymes that the microorganism is mixed well in the fermenter during the fermentation. It is also of importance that the air or the oxygen which is added to the fermenter is dispersed in the. nutrient medium in such a way tnat there will be aerobic conditions all over the fermenter during the fermentation.

The design of the apparatus

During the work leading to the invention it was found that the size and the shape of the fermenter is essential ^' to the yield of the enzyme. It is important tnat the supplied air or

oxygen is distributed all over the fermenter, that the mixing is effective so that the fungus is also smoothly distributed during the fermentation and does not scale on the fermenter walls and in the pipe systems and that the conditions inside the fermenter are aerobic all the time. Reference is made to Examples 1 and 5 in the following, where Example 1 describes the use of a 7 litre fermenter and Example 5 describes the use of a 300 litre fermenter.

Analysis

The enzyme activity is measured by measuring the rising amount of reducing sugar by means of photometric at 450nm. The Analysis of reducing sugar follows S. Dygert, L.H. Li, D. Florida and J.A. Thoma, as published in Analytical Bio¬ chemistry, Vol. 13 (1965) page 367-374 and described below.

A 5 ml copper-reagent is added to each of 6 eprouvettes. A 5 ml sample of enzyme, temperated at 30 C, is mixed together with 5 ml barley-β -glucan (also temperated at 30 C) in a clear eprouvette and 1 ml of the mixture is sucked and added to the first eprouvette with copper-reagent. The time is to be recorded. Then every 5 minutes during a period of 20 minutes a 1 ml sample of the barley-β -glucan mixture is sucked, which then is adoed to the idle eprouvettes with copper-reagent. Finally, 1 ml of acetate buffer is added to the sixth and last eprouvette with copper-reagent. A 5 ml neocuproinic-reagent is added to all of the eprouvettes and then all of them are placed on a boiling waterbath for 12 minutes. The eprouvettes are then cooled with cold water and 11 ml of aqua destillata is aoded to each of them. The content of reducing sugar is measured photometrically at 450 nm against the eprouvette with pure acetate buffer as blank.

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The copper-reagent mentioned above is produced by weighing 40.0 g Na ₂ CO-. and 16.0- g glycine -which are soluted in 600 ml aq. dest. When dissolved, 0.45 g CuS0 ₄ . 5 H ₂ 0 is added and the solution is adjusted to 1000 ml.

The neocuproinic-reagent mentioned above is produced by weighing 1.20 g neocuproin . HC1

(2,9-dimethyl-1 , 1O-phenanthrolinehydrochloride) which is dissolved in water and adjusted to 1000 ml.

Barley-β-glucan solution mentioned above is produced by weighing 0.50 g barley- -glucan which is dissolved in 70 ml aq. dest. by heating to 80-90°C for 20 minutes. Filtering and the filtrate is added 10 ml 0.5 M acetate buffer (pH 4.0). Diluted to 100 ml in a measuring flask and then 0.02 g NaN ₃ is added.

-The acetate buffer is produced by weighing 30.3 g acetic acid which is diluted with appr. 900 ml aq. dest. Then 2.5 g NaOH in solid form is added and the pH is regulated to pK 4.0 with 1 N NaOH. Then 0.2 g aN ₃ is added and the solution is diluted to 1000 ml.

During the test the concentration of the enzymatic solution has to be regulated so the amount of reducing sugar coming from the barley-β-glucane is in linearic proportion with the time. When necessary the enzymatic solution is diluted with 0.05 M acetate buffer. The enzyme activity is analyzed according to the abovementioned analytic method as reducing sugar with glucose as reference. One enzyme unit (EU) is defined as the amount of enzyme which is giving reducing sugar equivalent 1 .umol glucose each minute at 30°C.

The invention is according to the claims

For better understanding of the invention reference is made to the following examples:

Example 1

300 ml of a inoculum of Rhizomucor pusillus (Lindt) Schipper CBS 551.82 is added to a fermenter (7 litre) with 5 litre salt medium of the following composition:

KH ₂ P0 ₄ 2.0 g

MgSO . 7 H ₂ 0 1.75 g

CaCl ₂ 0.2 g

Trace elements solution 2.5 ml

Water 1.0 1

The solution of trace elements has the same composition as the solution described above for producing inoculum.

For foam depression Berol 374 (Berol Kemi AB, Sweden) was used. The fermentation was done at pH 5.5 with a temperature of 40 C and a stirring speed around 600 rpm. The air support was 3 vvm, i.e. 15 litre air/min. to the fermenter.

40 g/litre potato starch was used as C-source and 13.4 g/litre

NNHH^ a.CCl1 aass NN--ssoouurrccee.. TThhee ffeenrmentation time is 67 h. The enzy nme activity was 400 EU/litre.

Example 2

Rhizomucor pusillus (Lindt) Schipper CBS 551.82 was grown on 40.0 g/litre barley flour and fermented under the same conditions as mentioned in Example 1, but tne fermentation time was 45 h. The enzyme activity was measured to be 950 Eϋ/litre.

Example 3

300 ml of an inoculum with Rhizomucor pusillus (Lindt) Schipper CBS 551.82 was fermented under the same conditions as mentioned in Example 2. 7.5 g/litre urea was used as N-source. The enzyme activity was measured to be 870 Eϋ/litre,

Example 4

300 ml of an inoculum with Rhizomucor pusillus ATCC 22074 was added in a fermenter (7 litre) with 5 litre salt medium as in Example 1 and fermented under the same conditions as in Example 2. The enzyme yield was 170 EU/litre.

Example 5

300 ml of an inoculum with Rhizomucor pusillus (Lindt) Schipper CBS 551.82 was added in a fermenter (14 litre) with 10 litre salt medium and fermented as in Example 2. These 10 litres were used as inoculum in a fermenter (300 litre) with 200 litre salt medium as in Example 1. The fermentation was done in a medium with 40 g/litre barley flour and 13.4 g/litre NH.C1 at pH 4.7 and a temperature of 40°C at a stirring speed around 410 rpm. The air support was 0.17 vvm, i.e. 34 litres air/minute to the fermenter. The enzyme activity was 4800 EU/litre after 79 h and 5200 EU/litre after 92 h.

The examples show the following inventable advantages in preference to the use of known micro organisms:

Rhizomucor pusillus (Lindt) Schipper CBS 551.82 gives a high yield of enzymes even when the fermenting time is short ^' (2-3 days).

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The said micro organism is not sensitive even at high temperatures (40-50°C) and low pH (pH approx. 4.0).

The achievement of aerobic conditions all over the fermenter is highly important to get a good yield of enzyme.

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