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Method for screening high-yield glutamic acid strains in high flux

A technology for high-yielding strains and glutamic acid, applied in the field of fermentation engineering, can solve the problems of large workload, cumbersome operation, slow screening work, etc., and achieve the effects of improving efficiency, increasing the number of colonies, and reducing the intensity of screening work.

Inactive Publication Date: 2010-06-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the need to add urea or ammonia water to maintain the pH of the fermentation broth at 7.0 during the glutamic acid shake flask fermentation process, the operation is cumbersome, the workload is heavy, and the human error is also large. The limited number of fermentation trials and the slow selection process, therefore, become a bottleneck in the selection of high-yielding strains

Method used

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  • Method for screening high-yield glutamic acid strains in high flux
  • Method for screening high-yield glutamic acid strains in high flux

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Microwell culture plate is used for the feasibility of producing glutamic acid by fermentation

[0027] The glutamic acid producing bacterium SW07-1 preserved in the laboratory has been described in the article "Breeding of thermotolerant L-glutamic acid fermenting strains" (Bioprocessing, 2009, 7(6): 74-78 ), and applied from the slant to the LB plate to activate for 24h, inoculated with a needle to pick a circle into a 250mL shake flask containing 10mL seed medium, 30 ° C, 220rpm culture for 12h, when the seeds were transferred to the fermentation medium, at 96 The 4 corners of the well microwell cell culture plate, the edge row and the middle part randomly reserved some wells as sterile blank control, and selected 11 representative positions to inoculate as the parallel samples of fermentation culture, with 8% The inoculum was inoculated into the 11 wells, each well containing 1 mL of fermentation medium. After inoculation, cover with sterile gauze and arr...

Embodiment 2

[0031] Example 2 Screening of bacterial strains after X-ray mutagenesis with resistant plate-microwell culture plate

[0032] Select plate composition: glucose 10g, peptone 10g, yeast extract 5g, beef extract 10g, NaCl 5g, sulfaguanidine 20g, ketomalonic acid 2.5g, malonic acid 16g, coumarin 2g, agar 20g, and make up to 1L , pH7.0.

[0033] Seed medium: glucose 25g, K 2 HPO 4 1.5g, MgSO 4 0.6g, FeSO 4 5mg, MnSO 4 5mg, urea 2.5g, corn steep liquor 30g, dilute to 1L, pH 7.0.

[0034] Fermentation medium: glucose 140g, K 2 HPO 4 1g, MgSO 4 6g, FeSO 4 5mg, MnSO 4 5mg, 0.05mg of thiamine, 7g of urea, 3g of corn steep liquor, dilute to 1L, pH 7.0.

[0035] The glutamic acid producing strain SW07-1 preserved in the laboratory was subjected to X-ray mutagenesis for 40 minutes to obtain a mutant strain library. Spread the mutagenized bacterial suspension onto the selection plate, culture at 30°C for 3 days, pick out a total of 89 colonies grown on the plate, and inoc...

Embodiment 3-4

[0038] Example 3-4 Screening of bacterial strains after mutagenesis with pH-tolerant plate-microwell culture plate

[0039] The glutamic acid producing strain SW07-1 preserved in the laboratory was subjected to X-ray mutagenesis for 40 minutes to obtain a mutant strain library. Spread the mutagenized bacterial suspension on a low pH gradient plate and a high pH gradient plate respectively, and culture at 30° C. for 3 days.

[0040] Among them, the low pH gradient plate is composed of the following components: glucose 10g, peptone 10g, yeast extract 5g, beef extract 10g, NaCl 5g, agar 20g, dilute to 1L, adjust the pH to 4.6-5.0 with glutamic acid after sterilization .

[0041] The high pH gradient plate consists of the following components: glucose 140g, K 2 HPO 4 1g, MgSO 4 6g, FeSO 4 5mg, MnSO 4 5mg, 0.05mg of thiamine, 7g of urea, 3g of corn steep liquor, 0.1g of bromothymol blue, 20g of agar, dilute to 1L, adjust the pH to 10.5-11.2 with NaOH after sterilization. ...

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Abstract

The invention relates to a method for screening high-yield glutamic acid strains in high flux and belongs to the technical field of fermentation engineering. The screening of high-yield strains is always the bottleneck of selecting seeds of the high-yield glutamic acid strains. A traditional screening method comprises the following steps: selecting a single colony from a slab; performing fermentation and preliminary screening on the colony through a test tube or a shake flask; and performing fermentation and second screening in the shake flask. The method improves the traditional screening, and comprises the following steps: selecting the single colony on the slab, and performing primary screening by a microporous cell cultivation plate and fermentation and second screening in the shake flask according to the characteristics of the high-yield glutamic acid strains. The method effectively increases the number of the colonies in each screening, reduces the intensity of screening work, and improves the efficiency of the whole screening work.

Description

technical field [0001] The invention relates to a fast and high-throughput screening method for glutamic acid high-yield strains based on flat plates and microporous cell culture plates, and belongs to the technical field of fermentation engineering. Background technique [0002] Glutamic acid is a traditional bulk fermentation product, the earliest amino acid produced by large-scale fermentation, and the amino acid with the largest production volume in the world. Glutamic acid is widely used in food, medicine, industry, agriculture, etc., and its monosodium salt-sodium glutamate (trade name monosodium glutamate) is an important condiment. In 2008, the output of glutamic acid in my country was about 1.6 million tons, accounting for about 75% of the global total. However, our country mainly uses glutamic acid (monosodium glutamate) as a pure condiment (85%), and other countries use glutamic acid as a pure condiment only 52%, and use it as an intermediate in chemical industry...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12R1/13
Inventor 郑璞孙志浩杜巧燕
Owner JIANGNAN UNIV