DK165597B - PROCEDURE FOR PROMOTING SEPARATION OF AMYLOLYTIC ENZYMES FROM BACTERIA - Google Patents

Description

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DK 165597 B

The invention relates to a process for promoting the secretion of amylolytic enzymes (amylases and pullulanases) from thermophiles, said enzymes forming anaerobic bacteria.

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The formation of amylolytic, that is, starch-degrading enzymes is known. Thus, according to WO-A-86.01 831, Clostridium thermohydrosulfuricum ATCC 33 223 is grown portionwise on a culture medium containing 0.5% soluble starch in batch culture, thereby producing amylase, pullulanase and glucoamylase, but these enzymes is not excreted in the culture medium, see also Appl. Envir. Microbiol., 49 (1985) 1168-1173. According to WO-A-86.01 832, Clostridium thermosulfurogenes ATCC 33 743 = DSM 2 229 15 is grown portionwise in a culture medium with 0.5% soluble starch to form amylase, pullulanase and glucoamylase as amylolytic enzymes, but only the former enzyme is present in the culture medium. .

It is an object of the present invention to provide a process by which amylolytic enzymes in the cultivation of thermophilic, said enzymes forming anaerobic bacteria are highly secreted in the culture medium.

A method of this kind has the advantage that cells 25 do not have to disintegrate and that the enzymes do not have to be separated from cell degradation residues and disruptive components from the culture medium.

The invention thus relates to a process for promoting the secretion of amylolytic enzymes from thermophiles, said enzymes forming anaerobic bacteria, in a culture medium containing carbon sources, nitrogen sources, mineral salts and any vitamins, which process is characterized by the characterizing part of claim 1. ne.

DK 165597 B

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Those skilled in the art will be familiar with the isolation and cultivation of thermophilic anaerobic bacteria that form amylolytic enzymes. Useful strains are found in the genus Clostridium, e.g. the species Cl. thermosulfurogenesis such as ATCC 33 743 =

DSM 2 229, species Cl. thermohydrosulfuricum such as ATCC

33,223, DSM 567, DSM 2,247 and DSM 2,355, the species Cl. Ther mosaccharolyticum, such as DSM 571 and DSM 572, and the species Cl. spec. DSM 3,896; in the genus Thermoanaerobacter, e.g. species Th. ethanolicus, such as DSM 2,246, in the genus Thermobacteroides, e.g. species Th. acetoethylicus such as DSM

2,359, and in the genus Thermoana aerobicum, e.g. species Th. brockii, such as 1 457.

The microorganism Costridium spec. DSM 3896 exhibits the following characteristics: Growth: - it forms terminal spores 20

Cell morphology: - cells from the exponential phase on a growth medium with 0.5% starch are actively mobile and remain gram-negative 25 - the cells are in the form of small rods, and simply in short chains consisting of 2-3 cells or in filamentous chains 30 - sporulation occurs in the late stationary phase with galactose or lactose as energy and carbon source - sporulating cells have a size of 4-9 microns x 0.3-0.4 microns, the vegetative cells have a size 35 of 1.5-5 microns x 0.5 microns

DK 165597 B

3 - The fine structure of the cell wall exhibits a hexagonal pattern

Carbohydrate degradation: The growth of various carbohydrates was studied at 60 ° C for 20 hours. As soon as the optical density reached the maximum value, the pH value was measured. A pH of 5.9 - 5.2 was considered to be slightly acidic and a pH below 5.210 as highly acidic. The following table shows the results for some special carbohydrates.

TABLE

15 Growth of Clostridium spec. DSM 3896 on various carbohydrates

Carbohydrates investigated 20 Amygdalin a

Arabinose a

Esculin a

Galactose a

Lactose and 25 mannitol w

Melizitosis Melibiosis a

Pyruvate +, -

Raffinose and 30 Rhamnose +

Ribose a

Pectin +, -

Xylan a a: strong acid; w: slightly acidic; -: no growth; +, -: weak growth; + good growth but no acid.

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The microorganism Clostridium spec. DSM 3896 needs neither liver extract nor tryptone to grow.

Other biochemical properties: 5 - Clostridium spec. DSM 3896 is catalase negative and produces neither indole, acetylmethylcarbinol nor ammonia from arginine 10 - no growth in the presence of Tween 80.

Clostridium spec. In particular, DSM 3896 differs from related microorganisms in that yeast extract and / or tryptone must not be present in the growth medium.

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The microorganism is deposited in the German Collection of Microorganisms and Cell Cultures (DSM) from which it is publicly available.

The term "higher saccharide" in the present context stands for saccharides other than monosaccharides. By higher saccharides is meant e.g. polysaccharide such as starch, dextrin, dextran or pullulan, and oligosaccharides such as trisaccharide, e.g. maltotriose, or disaccharides, e.g. maltose, and mixtures thereof.

The person skilled in the art can himself choose suitable concentrations of the higher saccharide, a suitable pH value and a suitable flow rate (dilution rate). The process 30 according to the invention is carried out at a pH of 4-8 and with a flow rate of 0.01 to 0.4 hour -1.

The process is carried out at a concentration of the higher saccharide of 0.1 to 3%, preferably 0.2 to 1.5% and especially 0.5 to 1.2% (w / v).

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In choosing a suitable temperature, the skilled artisan will aim for the temperature minimum to result in too slow cell growth and that temperature maximum may lead to protein denaturation and cell killing.

In the following, the invention is explained by means of the drawing which shows the following:

FIG. 1-3: The effect of starch concentrations on--10 amylase and pullulanase formation and excretion in continuous culture. The experiments were performed with Clostridium spec. DSM 3,896 at a pH of 5.9 and a temperature of -160 C. The flow rate was 0.075 hours. The symbols used have the following meaning: Δ extracellular enzyme concentration (unit / liter) A cell bound enzyme concentration (E / liter) o intracellular enzyme concentration (E / liter) 20 · total enzyme concentration (E / liter) • optical density at 580 nm □ —o residual starch (%) d— □ reducing sugar (%) Q acetate (mM) 25 ethanol (mM) + lactate ( mM)

FIG. 4-6: The influence of pH on α-amylase, pulululanase and α-glucosidase formation and excretion in continuous culture. The experiments were performed with Clostridium spec. DSM 3,896 in the presence of 1% starch at 60 ° C. The flow rate was 0.075 hours. The symbols were as indicated for FIG. 1-3.

FIG. 7-9: Influence of the flow rate on e-amylase, pullulanase and o-glucosidase formation and excretion in continuous culture. The experiments were performed with Clostridium spec. DSM 3,896 in the presence of 1% starch at a pH of 5.9 and a temperature of 60 ° C. The symbols are as shown for FIG. 1-3.

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FIG. 10-11: The effect of starch concentration on glucoamylase and pullulanase formation and excretion in continuous culture. The experiments were performed with Clostridium thermohydrosulfuricum DSM 567 at a pH of 6.5 and a temperature of 60 ° C. The flow rate 10 was 0.05 hour-1. The symbols are as shown for FIG. 1- 3.

FIG. 12-13: The effect of pH on glucoamylase and pullulanase formation and excretion in continuous culture. The experiments were performed with Clostridium thermohydrosulfuricum DSM 567 in the presence of 0.5% starch at 60 ° C. Flow rate was 0.05 hours \

The symbols are as shown for FIG. 1-3.

FIG. 14-15: The influence of the flow rate on glucoamylase and pullulanase formation and excretion in continuous culture. The experiments were performed with Clostridium thermohydrosulfuricum DSM 567 in the presence of 0.5% starch at a pH of 6.5 and a temperature of 25 ° C. The symbols are as shown for FIG. 1-3.

Example 1 (Figs. 1-9) The continuous procedure was performed with Clostridium spec. DSM 3,896 (EM 1) and with a defined medium having the following composition (% w / v): 0.68 KH2 PO4, 0.1 (NH4) 2SO4, 0.016 MgCl2-6H20, 0.0012 CoC12.6H20 and 0 , 05 cysteine.HCl. In addition, per liter of medium, 35 ml of a vitamin solution and a tracer solution were added. The pH was 6-, 5. The medium defined was supplemented with the saccharides listed in Table 1.

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The experiments were performed in 2 liter or 5 liter fermentors. with a culture medium of 1-2 liters at 60 ° C. The cultures were kept anaerobic with a continuous stream of nitrogen. The nitrogen was sterilized, allowing it to flow through a sterile cotton filter. The cultures were stirred at 150 rpm. The pH was measured with a glass electrode and maintained at the set value, adding 2 N KOH automatically. After obtaining the stable phase (at least 6 volume changes), 10 ml samples were taken at 24 to 10 m intervals and the activity of the amylolytic enzyme as well as its localization was examined. Furthermore, the fermentation products, substrate concentration, pH and OD at 580 nm were measured.

15 The influence of the saccharide concentration, the influence of the pH value and the influence of the flow rate on the secretion of the amylolytic enzyme in the culture medium is shown in fig. 1-9 in the case of starch as a substrate.

The influence of different saccharides on the secretion of amylolytic enzymes is shown in Table 1.

Example 2 (Figures 10-15) 25

Example 1 was repeated with Clostridium thermohydrosulphuricum DSM 567 with starch as a substrate. The medium contains the following constituents (% w / v): 0.25 trypton, 0.1 yeast extract, 0.33 KH 2 PO 2, 0.016 MgC 2, 0.0012 CoC 2 .eH 2 O, 0.05 cysteine .HCl. In addition, per liter of medium was added 1 ml of a vitamin solution and a tracer solution.

The influence of the saccharide concentration, the influence of the pH value and the influence of the flow rate on the secretion of amylolytic enzymes (glucoamylase and pullulanase) in the culture medium are shown in fig. 10-15.

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Example 3

Example 2 was repeated with the following microorganisms and with starch as a substrate: 5

Clostridium thermosulfurogenes DSM 2 229 Clostridium thermohydrosulfuricum DSM 2 247 Clostridium thermohydrosulfuricum DSM 2 355 Clostridium thermosaccharolyticum DSM 571 10 Clostridium thermosaccharolyticum DSM 572 Thermoanaerobacter ethanolicum DSM 2 246 that the secretion of amylolytic enzymes can be promoted by limiting the higher saccharide as carbon source at the values of pH and flow rate indicated in Example 1.

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Claims (5)

A process for promoting the secretion of amylolytic enzymes from thermophiles, said enzymes forming anaerobic bacteria in a culture medium containing carbon sources, nitrogen sources, mineral salts and possibly vitamins, characterized in that the process is carried out continuously at a pH value. of 4-8 and with a flow rate of 0.01-0.4 hours ”*, and that as a carbon source a higher saccharide is used at a concentration of 0.1-3% (w / v). Process according to claim 1, characterized in that as a higher saccharide a polysaccharide such as starch, dextrin, dextran or pullulan, or an oligosaccharide such as a trisaccharide, e.g. mal-totriose, or a disaccharide, e.g. maltose, or mixtures thereof. 20 Process according to claim 1 or 2, characterized in that the process is carried out at a concentration of higher saccharide of 0.2 to 1.5 and in particular 0.5 to 1.2% (w / v). 25 Process according to any one of the preceding claims, characterized in that the process is carried out at a pH of 5.0 to 7.5 and in particular 5.5 to 7.0. 30 Process according to any one of the preceding claims, characterized in that the process is carried out at a flow rate of 0.02 to 0.3, preferably 0.02 to 0.2 and particularly preferably 0.03 to 0.15 hour. "*.