JPH08214880A - Disintegration of microbial cell - Google Patents

Abstract

PURPOSE: To sufficiently disintegrate microbial cells in a short time with low ultrasonic power without causing the destruction and oxidation of enzymes and nucleic acids by adding a sodium alkylbenzene sulfonate to an aqueous suspension containing microbial cells and subjecting the mixture to ultrasonic treatment. CONSTITUTION: Microbial cells are sufficiently disintegrated for the purpose of e.g. separation of chromosome gene and enzymes, etc., with low ultrasonic power in a short time without causing the destruction and oxidation of polymeric enzymes and nucleic acids by adding a sodium alkylbenzene sulfonate (ABS) to an aqueous suspension such as a 50mM phosphate buffer solution containing microbial cells and having a Klett value (turbidity) of 400, stirring the mixture, subjecting to shaking culture at 35 deg.C for 1hr and treating the culture liquid with ultrasonic radiations of 80W for 1hr using an ultrasonic cleaner to lower the Klett value to 15.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for destroying microorganisms. More specifically, it relates to a method for destroying microorganisms by ultrasonic treatment.

[0002]

2. Description of the Related Art Microorganisms contain various useful substances such as physiologically active substances and enzymes, as well as genes that synthesize them. When a large amount of these substances is required, a method is generally used in which the target substance is extracted by proliferating a large amount of the microorganism and then destroying it unless the microorganism releases the substance to the outside. . In addition, because the number of microbial species that release synthetic compounds outside the microorganism is small,
In most cases, the method of destroying microorganisms is used.

Methods for destroying microorganisms include chemical methods and physical methods. The chemical method is a method using an enzyme such as lysozyme, but it cannot be used for a microorganism that is not sensitive to the enzyme.

On the other hand, physical methods include ultrasonic treatment and
There are a method using a homogenizer (a method using a Brown cell homogenizer) and a method using a French press. However, the method using a homogenizer may destroy high-molecular enzymes and nucleic acids, and the method using a French press requires expensive equipment.

[0005] The ultrasonic treatment method may destroy high-molecular enzymes and nucleic acids when the ultrasonic output is high, so that the ultrasonic output must be lowered and used to destroy the bacterial cells. It takes a long time to do so, and if the processing time becomes long, there is a drawback that the enzymes and nucleic acids are oxidized by the action of radicals generated therein.

[0006]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a method for destroying microorganisms by an ultrasonic treatment method, which enables destruction in a relatively short time even when a low ultrasonic output is applied. To do.

[0007]

SUMMARY OF THE INVENTION The object of the present invention is as follows.
This is accomplished by adding sodium alkylbenzene sulfonate to an aqueous suspension containing microorganisms and sonicating to destroy the microorganisms.

The microorganisms to be destroyed may be either Gram-negative bacteria or Gram-positive bacteria. For example, a buffer solution such as 50 mM phosphate (pH 7.2) per 1 g of these wet bacterial cells. Is added at a rate of 10 ml to prepare an aqueous suspension containing microorganisms and used.

This aqueous suspension has a final concentration of about
An aqueous solution in which sodium alkylbenzenesulfonate is dissolved in an amount of 0.01 or more, preferably 0.05 to 0.1% is added, and after stirring, shaking culture at 35 ° C is performed for about 30 to
It will take about 60 minutes.

After that, a treatment with ultrasonic waves having an output of about 50 to 120 W is performed, and when the Klett value (turbidity) is reduced to evaluate the degree of destruction of microorganisms, the ultrasonic treatment for 1 hour significantly lowers the value. Can be made.

[0011]

EFFECTS OF THE INVENTION When destroying a microorganism by an ultrasonic treatment method, if a small amount of sodium alkylbenzenesulfonate is allowed to coexist therewith, the destruction can be sufficiently advanced in a short time with a low ultrasonic output. There is almost no risk of the enzymes and nucleic acids of the enzyme being destroyed or oxidized.

[0012]

Next, the present invention will be described by way of examples.

Example 1 Upon disruption of a microorganism for isolating and extracting a chromosomal gene of Bacillus sp. (Specific to the genus) that is insensitive to lysozyme, a 50 mM phosphate buffer solution (pH 6) having a Klett value of 400 containing Bacillus sp. .8) Add 5 ml of sodium alkylbenzenesulfonate aqueous solution (dissolved in distilled water) of various concentrations to 5 ml.
(Thus, the final concentration is 1/2 of the added aqueous solution). After stirring, shaking culture was carried out at 35 ° C for 1 hour. Then, ultrasonic waves with an output of 80 W were applied to the culture solution with an ultrasonic cleaner. The Klett values for the added ABS concentration (final concentration of sodium alkylbenzene sulfonate) are shown in FIG.

From these results, when ABS was added to a final concentration of 0.1%, Klet was treated with ultrasonic treatment for 1 hour.
It can be seen that the t-value can be reduced from 400 to 15. On the other hand, when ABS is not added,
The Klett value could be reduced only from 400 to 340 in 1 hour of ultrasonic treatment, and it took 4 hours to reduce this value to 15.

Example 2 When E. coli WP2 strain was treated in the same manner as in Example 1, the following change in Klett value was observed. Ultrasonic treatment ABS 0.1% ABS 0 minutes 400 400 30 minutes 15 210 60 minutes 6 163

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between added ABS concentration and Klett value in Example 1.

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[Procedure amendment]

[Submission date] September 22, 1995

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0009

[Correction method] Change

[Correction contents]

To this aqueous suspension, an aqueous solution in which sodium alkylbenzene sulfonate is dissolved in an amount such that the final concentration thereof is about 0.01% or more, preferably 0.05 to 0.1% is added. After stirring, shaking culture at 35 ° C. is performed for about 30 to 60 minutes.

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical display location C12R 1:19)

Claims (1)

[Claims] 1. A method for destroying microorganisms, which comprises adding sodium alkylbenzenesulfonate to an aqueous suspension containing the microorganisms and performing ultrasonic treatment.