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Bacillus subtilis capable of producing high purity 3-hydroxy butanone

A technology of Bacillus subtilis and hydroxybutanone, which is applied in the field of Bacillus subtilis strains and Bacillus subtilis, can solve the problems that the 3-hydroxybutanone process has not been widely used, difficulties, raw material sources, prices and application restrictions of products, etc.

Active Publication Date: 2007-08-15
SHANDONG FOOD & FERMENT IND RES & DESIGN INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The difference is that the enzymatic conversion process has a high yield of products, no other by-products, and the product has optical rotation, but it is difficult to obtain a large number of specific enzymes
[0005] Both chemical synthesis production and biological enzymatic conversion use diacetyl or 2,3-butanediol as raw materials, but diacetyl and 2,3-butanediol are also important synthetic fragrances rather than bulk chemical products. It is mainly produced by chemical synthesis process, so the source of raw materials, price and product application are all limited, which is also one of the main reasons why the process of chemical synthesis and biological enzymatic conversion to produce 3-hydroxybutanone has not been widely used
[0006] 3-Hydroxybutanone is an intermediate metabolite of a variety of microbial sugar metabolism, and 3-hydroxybutanone can be produced by microbial fermentation using sugar as raw material. It is a theoretical study on microbial metabolic pathways, and a few reports involving the production of 3-hydroxybutanone are mainly mentioned as by-products of diacetyl and 2,3-butanediol
There is no research report on 3-hydroxybutanone as the main target product of microorganisms, nor is there any report on the application of Bacillus subtilis strains to produce high-purity 3-hydroxybutanone

Method used

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  • Bacillus subtilis capable of producing high purity 3-hydroxy butanone
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  • Bacillus subtilis capable of producing high purity 3-hydroxy butanone

Examples

Experimental program
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Effect test

Embodiment 1

[0059] Example 1 Breeding of Bacillus subtilis producing 3-hydroxybutanone

[0060] The starting strain SFA-W15 (a strain of Bacillus subtilis that accumulates a small amount of 3-hydroxybutanone) was inoculated into a liquid seed medium, cultivated at 37°C to the mid-log phase, centrifuged to collect the bacteria, and washed with sterile saline. Times, and then add sterile saline to make a bacterial suspension so that the cell concentration is 1×10 8 -10 9 Pcs / ml.

[0061] Take 10ml of the above bacterial suspension and put it in a petri dish, place it under a 30W ultraviolet lamp, and irradiate it with an irradiation distance of 15cm. The irradiation time is 3 minutes, 5 minutes, 7 minutes, and 10 minutes. The interval sampling is appropriately diluted and coated with 0.5% LiCl. Plate, protected from light at 37°C for 1-2 days, pick a single colony to transfer to the inclined plane, culture at 37°C for 1-2 days, inoculate a shake flask containing fermentation medium after the in...

Embodiment 2

[0064] Example 2 Breeding of Bacillus subtilis CGMCC NO.1869 strain producing high-purity 3-hydroxybutanone

[0065] The mutant strain SFA-U97 screened in Example 1 was inoculated into liquid seed culture medium, cultivated at 37°C to the mid-log phase, centrifuged to collect the bacteria, washed twice with sterile saline, and then added with sterile saline Make a bacterial suspension so that the cell concentration is 1×10 8 -10 9 Pcs / ml, spare.

[0066] Preparation of nitrosoguanidine treatment solution: Weigh 20mg of nitrosoguanidine, place it in a 100ml sterile Erlenmeyer flask, add 2ml acetone to dissolve it, then add 18ml Tris buffer (pH6.0, 0.5mol / L) and mix well ,spare.

[0067] Take 10ml of the above-mentioned nitrosoguanidine treatment solution, add 10ml of bacterial suspension, incubate at 30°C and shake for 50-60 minutes, and sample once every 10 minutes. After sampling, first dilute 1000 times to terminate the reaction, then dilute appropriately, and coat the plate. In...

Embodiment 3

[0074] Example 3 Sequencing of 16S rDNA of Bacillus subtilis CGMCC NO.1869 strain

[0075] The 3-hydroxybutanone high-yielding strain SFA-H31, CGMCC NO. 1869 strain, which was selected in Example 2, was entrusted to TaKaRa Biotechnology (Dalian) Co., Ltd. for 16S rDNA sequencing.

[0076] The experimental method is: pick the slant culture in 10μl sterile water, centrifuge at 99℃ and centrifuge to take the supernatant as template, use TaKaRa 16S rDNA Bacterial Identification PCR Kit (Code No.D310), use Forward / Reverse primer2 as primer, Amplify the target fragment. Take 5 μl for agarose gel electrophoresis, use TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 (Code No.DV805A) to cut the gel to recover the target fragments, and use Seq Forward and Seq Reverse Seq Internal as primers to perform DNA sequencing on the recovered products.

[0077] Sequencing results: The 16S rDNA sequence of Bacillus subtilis CGMCC NO.1869 strain is 1468 bp in length, and the nucleotide sequence is shown...

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Abstract

The invention discloses a bacillus subtilis of high purity 3-hydroxy butanone and appliance, which is characterized by the following: the (Bacillus subtilis)SFA-H31 have been stored in China Microbe Bacterial Preserving Management Committee Common Microbe Center with keeping number as CGMCC NO.1869; this bacillus can yeast and invert amylaceum and can generate goal-product 3-hydroxy butanone without generating butanedione and 2, 3-butanediol and so on 3-hydroxy butanone coproduction compound; this bacterial strain possesses big exploiting value.

Description

Technical field [0001] The invention relates to a Bacillus subtilis strain, in particular to a Bacillus subtilis strain capable of producing high-purity 3-hydroxybutanone, and belongs to the field of biotechnology. Background technique [0002] 3-Hydroxybutanone (acetoin), also known as acetoin, acetomethylmethanol, naturally exists in many foods such as corn, grapes, cocoa, apples, bananas, cheese, meat, etc. It has a unique creamy aroma. In addition, beer Both flavor and cheese aroma are related to 3-hydroxybutanone. [0003] 3-Hydroxybutanone is a widely used and beloved edible spice, and it is a common spice variety in the world. It is mainly used as flavors such as cream, dairy, yogurt and strawberry type. Chinese national standard GB2760-86 stipulates that it is a permitted food flavor. The 80% content of 3-hydroxybutanone is commonly known as "Vinegar Om", and it is an important variety in the aroma of alcohol. 3-Hydroxybutanone is also an important chemical synthesis raw ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P7/26C12R1/125
Inventor 赵祥颖刘建军张家祥田延军韩延雷
Owner SHANDONG FOOD & FERMENT IND RES & DESIGN INST
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