CS217313B1 - New bacterial strain bacillus amyloliquefaciens - Google Patents

Abstract

New bacterial strain Bacillus amyloliquefaciens High CCM 3502 alpha-amylase production. It is either batch or continuous at pH 5.5 and 25 - High: 35 ° C. The formation of the enzyme is accompanied proteazy formation.

Description

The invention relates to a novel production bacterial bacterial strain Bacillus and amylo liquefaciens, which is distinguished by a high production of the exocellular enzyme t-amylase.

A characteristic feature of this new strain is the ability to grow on starch substrates supplemented with organic and inorganic nitrogen while producing the enzyme γ-amylase. The production properties (abilities) of the strain remain fully maintained in both submersal batch and continuous cultivation where maximum expression of its gene type occurs.

Kaen BaciHus amyloliquefaciens was obtained by controlled selection of the starting strain of Bacillus amyloliquefaciens (which was isolated from the soil) under continuous culture conditions. The possibility of genetic instabilnoeti culture under continuous cultivation conditions is known. The mutant overgrowth theory is developed practically in parallel with the development of the theory of continuous cultivation. The culture of the starting strain from which the new strain according to the invention was obtained, which in the chemostat disintegrated into low and high production motants.

The strain of the invention was obtained under conditions of continuous culture on media, the composition of which allowed the suppression of the non-producing mutant from the initial population. Synthetic soil (carbon source glucose, nitrogen source NHI or NO 3) stimulated the production of low - production mutants, while the selection pressure of natural medium (carbon source starch, nitrogen source com - steep liquor) influenced the multiplication of the high - production mutant. In addition to the composition of the medium, the dilution rate D (h @ -1) used in the chemostat culture was an important factor in controlled selection. At low maturation rates (up to D = 0.15 h “· * ·) nutrient limitation also occurred in the natural medium, and population composition did not improve in favor of high-production mutants.

To obtain high-production mutants, the following conditions were required: natural soil and aging rate ranging from 0.18 to 0.30 hrs. bacterial ohromosome on a special plasmid. This plasmid DNA was never detected in the cells of the parent control strain. The increase for α-amylase induction is simultaneously controlled by both chromosomal and newly obtained plasmid DNA. The dependence of the selection on the growth rate (under chemostat conditions, the growth rate of the maturation rate) was confirmed by studying the growth rates of high and non-production mutants under defined conditions. They were found to have different maximum growth rates (/ ^u max), which appears to be due to the different amino acid claims of the two mutants. The lower growth rate of the production mutant is due to the fact that in addition to its own reproduction, it also synthesizes exocellular protein and therefore has a higher need for substrate. It follows that without the use of well-defined maturation rates of a particular composite medium and current genetic analysis, it would be impossible to obtain a high-production mutant and characterize its physiological and genetic differences.

The characteristics of the new bacterial strain Bacillus amyloliquefaciens, whose culture pattern is included in the Czechoslovak collection of microorganisms at the Faculty of Science of J. E. Purkyně, Brno under the number CCU 3502, are also given.

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(a) Morphological observations

1. Vegetative cells of Bacillus and amyloliquefaciene are Gram-positive moving rods, electric to cylindrical in shape.

2. On liquid soils it grows predominantly as single sticks and their pairs, rarely in chains.

3. Apor formation: one cell does not form more than one centrally located endospore. Disputes are / thermoresistant.

4. Growth on solid soils: colonies on agar are round to irregular, mostly translucent to whitish. The colony profile is regular and flat. The surface is mostly smooth, but may be rough on the agar agar. In older colonies, signs of pigmentation are observed that turn the colonies weakly green.

b) Physiological characteristics

1. A strain cultured aerobically on glucose forms acid and catalase.

2. Temperature, strain optimum is between 25 - 35 ° C. The temperature maximum is between 45-55 ° C.

3. Active growth takes place at pH 5.5 - 8.5 ·

4. The stem reduces NO ^ ”to NC ^ ^- ·

5. On starch substrates, the strain forms a significant amount of extracellular enzyme. -amylases. The production of this enzyme is accompanied by the formation of another extracellular enzyme, protease.

6. The strain produces bacteriocin inhibiting the growth of Bacillus aubtilis 168 strains and derivatives derived therefrom.

c) Genetic characteristics

1. The strain produces a restriction endonuclease which has been identified as a restriction endonuclease identical in size and quantity to the restriction DNA restriction DNA fragments BamHI, which was found only in Bacillus amyloliquefaciene strains.

2. The bacterial strain Bacillus amyloliquefaciens CCM 3502 differs from Bacillus amyloliquefaciens (Bergey's Manual of Determinative Bacteriology, Eight Edition, RE Buchanan and Wilkins Company, Baltimore 1974) by carrying plasmid DNA having a molecular weight of 3.4 Udal. typically breaks down.

Claims (1)

SUBJECT OF THE INVENTION The bacterial strain Bacillus aayloliquefaciens CCM 3502 for the production of the enzyme γ-amylase in a batch or continuous process. Printed by Moravian Printing Works, plant 12, Leninova 21, Olomouc