DD273069A1 - PROCESS FOR PREPARING ENDO-1,4-BETA GLUCANASE FROM ERWINIA CAROTOVORA - Google Patents

Abstract

The invention relates to a microbial process for the production of an endo-1,4-b-glucanase by submerged fermentation. The enzyme leads to a cleavage of the b-glycosidic bonds of cellulose and can therefore be used alone or in combination with suitable pactinases and hemicellulases for the enzymatic hydrolysis of vegetable biomass for a subsequent fermentation of the low molecular weight building blocks. For this purpose, a strain of the bacterium Erwinia carotovora with a corresponding b-glucanase-forming capacity in a cost-effective culture medium, mainly from grain ashes and soybean meal, with a small addition of mineral salts, supplemented by pectin and molasses concentrate, in a fermenter of conventional design 13- Cultivated for 18 h. As an essential factor of a further increase in the enzyme yield, fermentation conditions adapted specifically to the bacterial strain were identified which are preferably in a reduction of the concentration of the geloest-O2 in the culture medium with the beginning of the logarithmic growth phase.

Description

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Field of application of the invention

The invention relates to a microbial fermentation process for the production of an endo-1,4-ß-glucanaso (hereinafter called ß-glucanase) on an industrial scale using a strain of the bacterium Erwinia carotovora. The enzyme can be used alone or in combination with pectinases for the enzymatic hydrolysis of plant biomass for a subsequent fermentation of the low-molecular-weight building blocks. The main fields of application are thus in the distillery industry for the digestion of cereal raw materials to increase ethanol yields, in agriculture for the enzymatic pre-digestion of green fodder to optimize lactic acid fermentation in silage production and as a biochemical in basic research.

Characteristic of the known state of the art

For the industrial production of enzymes which cleave the cellulose, which mainly consists of β- (1,4) -glycosidic bonds, fungal enzyme producers such as Trichoderma, Penicillium and Aspergillus spec are generally used. Efficient strains synthesize in the Rego! the enzymes required for complete degradation of ZePulose, such as:

Fndo-1,4-.beta.-glucanases (1.4 (1.3; 1: 1-.beta.-D-glucan M-glucanohydrolase, EC 3.2.1.4), exoglucanases (1,4-.beta.-D-glucan cellobiohydrolase, EC 3.2.1.91) and ß-glucosidases (ß-D-glucoside glucohydrolase, EC 3.2.1.21), The disadvantage of these processes for enzyme production, however, consists in the relatively high technological technological effort for mushroom cultivation due to special demands on the fermentation technology and process control as well as high In contrast, substrate cultivation and long fermentation times, which entail an increased risk of contamination, make the processes relatively costly, while the cultivation of bacteria for enzyme production is considerably less demanding: utilization of phytopathogenic bacterial enzyme formers, plant pectin substances, as well as celluloses have been fully hydrolyzed, has not yet been considered for the extraction of cellulytic enzymes on an industrial scale.

Object of the invention

The object of the invention is to develop a process for the production of an endo-1,4-ß-glucanase on an industrial scale in a relatively inexpensive fermentation process with a strain of the bacterium Erwinia carotovora.

Daring the essence of the invention The invention has for its object to provide an endo-1,4-ß-glucanase, which is inexpensive with conventional Fermentation plants can be produced in the submerged process. The enzyme leads to a hydrolytic cleavage of the

ß-glycosidic bonds of cellulosic constituents of plant cell walls and should therefore be used for the digestion of plant biomass for subsequent fermentation processes.

The object is achieved in that a strain of the bacterium Erwinia carotovora - deposited in the Depository for microorganisms in the ZIMET Jena, with the registration number, on oinem predominantly Getreidenaßschlempe, soybean meal and minor additions of mineral salts existing medium (pH 7.0 to 7.5, preferably

but 7.2) at a temperature of 20-30 ⁰ C, but preferably 25 ° C, over a period of 14 to 18 hours, preferably 15 hours is cultivated.

As low has been found that the culture medium at least the following complex components and inorganic Additives in the respective concentration cereal sludge lOOml / l, soybean meal 10g / l, (NH ₄ I ₂ HPO ₄ , 1,800g / l, KNO ₃ 0,750g / l, MgSO ₄ χ 7H ₂ O 0.150 g / l, Na ₂ SO x 10 K ₂ O 0.200 g / l, CaCl ₂ 0.050 g / l, Ai ₂ (SO ₄ I ₃ x 18H ₂ O 0.050 g / l ₎ was found,

that an addition of molasses concentrate in the amount of 5,000y / l to max. 10 g / l leads to an increase in the ß-glucanase production and that Poktin up to 1 g / l or pectin complex additives allow a further increase in enzyme yields.

The media are heat sterilized, adjusted to the required pH and then the confusion of the Main culture of the pre-cultures (bacterial density 4-5 χ 10 ⁹ bacteria / ml) of the drawn Leistungsmojmo, the Composition of the media for the preculture corresponds to the production media. The main culture in the Production culture medium is made in conventional, provided with aeration device and agitator fermentors. For optimum control and regulation of the fermentation process, possibilities for measuring and registering the fermentation process should be provided Content of GeIoSt-O ₂ (pO ₂ ) and the hydrogen ion concentration (cH) in the culture medium and the CC ₂ - and possibly also Oj content of the exhaust air to be present. Decisive for a high production of ß-glucanase is the reduction of the concentration of GeIoSt-O ₂ in the culture medium with Start of logarithmic growth phase at £ 50%. The most favorable time of the termination of the fermentation can be by the decrease of the concentration of CO ₂ in the exhaust air as well as

be determined by the increase in the pH. For the production on an industrial scale it is expedient, after one

Fermentation time of anfänglijh 15 hours to harvest 80% of the enzyme-containing culture fluid and by frischos medium to

replace (fed-batch culture). In the following, this process is repeated at intervals of 8 to 10 hours, since in this

Time has already reached the required ß-glucanase activity in Kulturfilirat. The further workup of the bacterial suspension is carried out in the usual manner, d. H. The bacteria are made by centrifugation

separated and the culture filtrate is concentrated by ultrafiltration or other conventional temperature-saving method. The concentrate can be used as such or stored at temperatures below freezing point, or permanent preparations are obtained by spray-drying or lyophilization. Erwinia 1,4-β-glucanase is still effective at pH 3.0 and reaches its maximum at pH 5.0-6.0. The temperature optimum of the enzyme is included

Exemplary embodiments Determination of enzyme activity Cellulase (endo-1,4-β-glucanase, E.C. 3.2.1.4.) Substrate: carboxymethylcellulose; Na-SaIz (from Serva) 0.5% in Sörensen's phosphate buffer, pH 5.1 Methodology: reduction of viscosity (viscometer to Ostwald)

1 U = the waste Viscous did 25% to 3O ⁰ C (= CMCI activity)

I.beta.-glucanase product ion on a laboratory scale

preculture

The preculture was applied as a shake culture in the 100 ml Erlenrneyer flask with 25 ml of culture medium. The inoculation of the preculture is carried out directly from stock cultures on Schrägagar tube, but can also with Bacterial suspensions from frozen foods stored at -35 ⁰ C done. The cultivation was at 25 ⁰ C.

made and completed after 15 hours. In the culture filtrate a CMCI activity of 55.60 UmI ' ^{1 was} detectable. The amount of bacterial suspension needed to inoculate the major culture is Vm of the volume of the major culture.

main Culture Type of fermenter: Laboratory fermentor LF2 (VEB Mytron) Useful volume: 2.51 Medium (in gl '') mineral salts

(NHO ₂ HPO ₄ - 1.800, KNO ₃ -0.7500, Na ₂ SO ₄ x 10H ₂ O-0.200, MgSO ₄ χ 7H ₂ O-0.150, CaCl ₂ -0.050, Ai ₂ (SO ₄ I ₃ x 18H ₂ O-

org. additions

Soybean meal - 15,000, cereal slices - 1 W) ₁ OO, pectin - 1,000, sucrose - 2,000 Sterilization: 20min to 120 ° C

After sterilization adjust cH to pH 7.2 with NaOH.

Inoculum: 50ml of a preculture of the strain Fermentation temp .: 25'C Agitator: reduced from 800 to 600 rpm after 2.6 h Air supply: 3Ol h " ¹ with further reduction to set the required oxygen partial pressure The further course of the parameters of the fermentation process i3t can be seen in Fig. 1. A = β-glucanase activity (CMCI act.) B = concentration of GeIOSt-O ₂ in the culture medium C = concentration of CO ₂ in the exhaust air (respiration) D = concentration of O ₂ in the exhaust air E = concentration of hydrogen ions After a fermentation time of only 13 hours, a CMCI activity in the culture filtrate of 90.6 OU ml ^{-1 was} detectable

to stop the fermentation, here after 24 hours, there was no further increase in activity.

2. Influence individual »media components on the β-glucanste product

The culture was carried out as a shaking culture in Erlenmeyer flask. A Mii.eralsa'zmedium (composition Example 1) was

supplemented by additives in the variants a, b, c, d.

organic additives abcd

ingl. " ¹

soybean meal 10.00 10.00 10.00 10.00 mash - 100.00 100.00 100.00 molasses - - 10.00 10.00 pectin - - - 1.00

CMCI Actual Rel. (%) 100 106 141

Claims (6)

1. A process for the production of a Erwinia-IAß-glucanase, characterized in that by cultivating a strain of the bacterium Erwinia carotovora - deposited at the depository for microorganisms at ZIMET, Jena, under the registration number ZIMET on a culture medium adapted for this microorganism for the hydrolytic cleavage of the β-glycosidic bonds of celluloses is produced. 2. The method according to item 1, characterized in that the fermentation is stopped after a period of 13 to 18 hours. 3. The method according to item 1 and 2, characterized in that after a fermentation time of initially 15 hours, 80% of the enzyme-containing culture liquid is harvested and replaced by fresh medium and that this process is repeated thereafter at intervals of 8 to 10 hours. 4. The method according to item 1 to 3, characterized in that the pre- and main crop cereal slurries, a by-product of the distillery industry is used as essential media components, supplemented by molasses and soybean meal and small amounts of pectin and mineral salts. 5. The method according to item 1 to 4, characterized in that an addition of pectin and molasses concentrate leads to an increase of the ß-glucanase production. 6. The method according to item 1 to 5, characterized in that the concentration of GeIoSt-O _{2 is} reduced with the beginning of the logarithmic growth phase to levels below 50% to the fermentation termination.