Bacillus licheniformis strain, application thereof and method for producing poly-gamma-glutamic acid thereby

A technology of Bacillus licheniformis and glutamic acid, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problem of high production cost, and achieves low production cost, short fermentation period and good genetic stability. Effect

Inactive Publication Date: 2009-12-16
HEBEI WELCOME PHARMACEUTICAL CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the above method has improved the yield of poly-γ-glutamic acid, it needs to add high-concentration glycerol in the fermentation process, so its production cost is high

Method used

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  • Bacillus licheniformis strain, application thereof and method for producing poly-gamma-glutamic acid thereby
  • Bacillus licheniformis strain, application thereof and method for producing poly-gamma-glutamic acid thereby

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: Isolation and screening of bacterial strains of the present invention

[0041]Weigh 1g of high-salt environment soil sample from Yingcheng Salt Mine in Hubei into a sterilized test tube, add 9mL of sterile water, seal it with a cotton plug, shake for 2min, then put it in a water bath, heat at 80°C for 10min, and then let it stand for 30min. After cooling, draw 0.1mL supernatant, dilute 10-2, 10-3, take 0.2mL respectively, spread on LB plate, incubate at 37°C for 48h to observe the results, and select a single colony that is emulsion-like and silk-drawing on LB medium , into fresh liquid seed culture medium, and after 12 hours of shaking culture at 37°C, they were inoculated to 50

[0042] In a 250mL shake flask containing sterilized basic fermentation medium, shake at 37°C and 190r / min for 36h to complete the fermentation. The fermentation products were sampled and centrifuged to remove the bacteria, and the supernatant was precipitated with 3 times of et...

Embodiment 2

[0043] Embodiment 2: the identification of bacterial strain of the present invention

[0044] Physiological and biochemical identification

[0045] The bacterium is straight rod-shaped, Gram staining is positive, and the spores are approximately round and partial. On LB solid medium, the colonies are round, with irregular edges, hairy, and the lawn is dry, opaque, spreading, off-white. The optimum growth temperature is 30-37℃, and the suitable pH is 6.8-7.2. The contact enzyme test was positive, the nitrate utilization was positive, the citrate utilization was positive, and the gelatin liquefaction test was positive.

[0046] Identification of Bacterial Species Using 16S rRNA Gene Sequence Analysis

[0047] Genomic DNA Extraction of Bacillus licheniformis WX-02

[0048] Streak Bacillus licheniformis WX-02 on an LB plate (containing peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 15 g / L, pH 7.0) and cultivate overnight at 37°C Finally, pick a small amount of bacteri...

Embodiment 3

[0081] Embodiment 3 adopts the yield determination of the poly-γ-glutamic acid produced by bacterial strain of the present invention

[0082] Its quantitative analysis of poly-γ-glutamic acid produced by bacterial strain of the present invention is completed by HPLC, chromatographic column Agilent1100, mobile phase is 100mmol.L -1 K H 2 PO 4 Add 5.0% methanol (adjust the pH value to 2.5 with phosphoric acid) to filter, degas by ultrasonic, the ultraviolet detection wavelength is 210nm, the flow rate is 1mL / min, and glutamic acid is used as a quantitative standard.

[0083] The specific measurement steps are as follows:

[0084] Take 5 ml of fermentation centrifuge supernatant, add 3 times the volume of alcohol to precipitate PGA, and dissolve the product, add an equal volume of concentrated hydrochloric acid to hydrolyze at 110°C for 24 hours under vacuum conditions, add 10mol.L of the hydrolyzed solution after hydrolysis -1 NaOH was neutralized to constant volume, filtere...

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Abstract

The invention discloses a bacillus licheniformis strain which is preserved as bacillus licheniformis WX-02 with the preserving number of NO: M208065 and the preserving date of April 24th, 2008 and the preserving unit of CCTCC. The invention also discloses an application of the bacillus licheniformis strain and a method for producing poly-gamma-glutamic acid by using the bacillus licheniformis strain. Poly-gamma-glutamic acid products are produced by processes of seed solution preparation and liquid submerged fermentation, and the strain has stable genetic performance of the strain, high production efficiency and low production cost. When the bacillus licheniformis strain of the invention and the fermentation method thereof are used for preparing 3000L of poly-gamma-glutamic acid, the yield of a fermentation tank can reach 35.05g/l.

Description

technical field [0001] The present invention relates to bacillus and its use, in particular to the bacillus licheniformis strain and its use and its production method for poly-γ-glutamic acid. Background technique [0002] Bacillus licheniformis is a kind of facultative anaerobic saprophytic bacteria widely existing in nature. In industry, it is often used as the production bacteria of proteolytic enzymes, amylases, bioactive agents, antibiotics, etc. At present, researchers have used specific Bacillus licheniformis to produce poly-γ-glutamic acid, such as someone using Bacillus licheniformis A35 to produce poly-γ-glutamic acid (see reference 1 for details: Cheng C, Asada Y, Aaida T. Production of γ-polyglutamic acid by Bacillus licheniformi A35 underdenitrifying conditions. Agric Biol Chem, 1989, 53: 2369-2375;). However, the use of the bacterial strain to produce poly-γ-glutamic acid has the disadvantages of long fermentation period and low fermentation yield. In order ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P13/00C12R1/10
Inventor 陈守文谢萍魏雪团刘晓雷冀志霞郭威张红艳孙君伟李海涛李建波米造吉黄品奇王正品
Owner HEBEI WELCOME PHARMACEUTICAL CO LTD
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