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Preservation method of microbial cells and suspension of microbial cells

A technology of microorganisms and preservation methods, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as loss or reduction of microbial enzyme catalytic ability, and achieve the effect of reducing labor and cost

Active Publication Date: 2016-03-16
MITSUBISHI CHEM CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

That is, the catalytic ability of microbial enzymes cannot be lost or reduced due to the mixing of bacteria, corruption or lysis during storage.

Method used

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  • Preservation method of microbial cells and suspension of microbial cells
  • Preservation method of microbial cells and suspension of microbial cells
  • Preservation method of microbial cells and suspension of microbial cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 (room temperature preservation)

[0036] to cultivate

[0037] Prepare 100ml of culture medium (PH7.0) containing 2% glucose, 1% urea, 0.5% peptone, 0.3% yeast extract, and 0.05% cobalt chloride in a 500ml Erlenmeyer flask, and sterilize at 121°C in an autoclave 20 minutes. In this medium, Rhodococcus rhodochrous J-1 strain (Rhodococcus rhodochrous J-1: FERMBP-1478) having nitrile hydratase activity was inoculated, and cultured at 30° C. and 230 rpm for 66 hours.

[0038] Determination of dry bacterial mass of bacterial suspension

[0039] About 1 g of the cultured bacterial cell suspension was collected into an aluminum pan and weighed. The bacteria buffer was centrifuged at 12,000 rpm for 20 minutes, and 10 g of the supernatant was filtered with a filter membrane (Advantec, pore size: 0.45 μm), placed in an aluminum pan, and weighed. They were weighed after drying at 120° C. for 3 hours by a desiccator (manufactured by Yamato Scientific Co., Ltd., D...

Embodiment 2 and 3

[0047] Examples 2 and 3 (preserved at 30°C)

[0048] to cultivate

[0049] Culture was carried out in the same manner as in Example 1, except that a 3 L fermenter (manufactured by Takayama Seisakusho) was used instead of the 500 ml Erlenmeyer flask.

[0050] Determination of dry bacterial mass of bacterial suspension

[0051] The same method as in Example 1 was used to measure the dry thalline mass of the thalline suspension. Preparation of bacteria suspension for storage

[0052] The cultured microbial cells were treated with hollow fiber membrane modules (Kuraray Corporation, pore diameter 0.05 μm, surface area 39000m 2 ) was concentrated, washed with 0.1% sodium acrylate aqueous solution (pH7.0), concentrated again to a dry cell mass of 10.0%, and a 10.0% dry cell mass suspension was prepared (Example 2). A part of this bacterium suspension is diluted with 0.1% acrylic acid aqueous solution, and the bacterium of 5.0% (embodiment 3), 2.5% (comparative example 2), 0.2% ...

Embodiment 4 and 5

[0057] Preparation of transformant having nitrile hydratase derived from Rhodococcus rhodochrous strain M8.

[0058] (1) Chromosomal DNA M8 strain (SU1731814) prepared from Rhodococcus rhodochrous M8 strain (hereinafter referred to as M8 strain) can be obtained from the Institute of Biochemistry and Physiology of Microorganisms (Institute of Biochemistry and Physiology of Microorganisms: IBFM, Pushchino, 142290, Moscow Region, Asia) (commission number: VKPMS-926 ) purchased.

[0059] The M8 strain was cultured in 100ml of MYK (0.5% polypeptone, 0.3% Bacto yeast extract (Bacto イ ストエキス), 0.3% Bacto malt extract (Bacto molt エキス), 0.2% K 2 HPO 4 , 0.2% KH 2 PO 4 ) medium (pH 7.0), cultured with shaking at 30°C for 72 hours. The culture solution was separated by centrifugation, and the collected bacteria were suspended in 4 ml of Saline-EDTA solution (0.1 MEDTA, 0.15 M NaCl (pH 8.0)). Add 8 mg of lysozyme to the suspension, shake at 37°C for 1 to 2 hours, and then freeze at -2...

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Abstract

The present invention provides a cheap and simple microbe for replacing that of the prior art, and a preserving method thereof. The invention relates to the preserving method of microbe and is characterized in that: the microbes with nitrile hydratase activity are dispersed in a dispersion medium with a concentration that the mass of dry microbe is 4-20wt%.

Description

technical field [0001] The present invention relates to a method for stably preserving microbial cells having nitrile hydratase activity in a cell suspension at a predetermined concentration. In addition, the present invention also relates to a cell suspension containing microbial cells having nitrile hydratase activity at a predetermined concentration. Background technique [0002] Enzymes produced by microorganisms are often used as enzymes for chemical conversion reactions. In particular, amides, carboxylic acids, α-hydroxycarboxylic acids, etc., which are important in the chemical industry, can be produced inexpensively by using nitrile hydratase, nitrilase, etc. which have hydration or hydrolysis of nitrile groups. In addition, optically active carboxylic acids, amino acids, α-hydroxycarboxylic acids, etc., which are important raw materials for the production of medicines and agricultural chemicals, can also be produced by using the above-mentioned enzymes having optic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/04C12N1/00C12R1/01
Inventor 竹内雅人
Owner MITSUBISHI CHEM CORP
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