High-throughput screening method of guanosine production strains

A technology for producing strains and screening methods, applied in the field of fermentation engineering, can solve the problems of ineffectiveness, large workload, long screening period, etc., and achieve the effect of high-efficiency screening methods

Active Publication Date: 2015-06-17
STAR LAKE BIOSCI CO INC ZHAOQING GUANGDONG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the screening method of traditional microbial strains is a heavy work, the workload is large, the screening period is long, and it is often useless, the target strains cannot be obtained, and the blindness of screening is very high.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0010] Cultivate the guanosine-producing bacterial strain to the logarithmic growth phase, and collect the thalline by centrifugation to make a bacterial suspension (about 10 8 1), add the chemical mutagen nitrosoguanidine NTG, the concentration is 3mg / ml, the concentration of NTG solution and bacterial suspension is 0.6mg / ml, treat at 37°C for 30min, and use potassium phosphate solution (0.1mol / L, pH 7.0) was centrifuged and washed 3 times, after centrifugation, the plates were coated with different dilutions and cultured at 35°C to obtain a total of 480 single colonies, which are colonies formed by a single bacterium.

[0011] A single colony was inserted into a 96-well plate containing liquid medium, and cultured with shaking at 37° C. at 200 rpm for 36 hours.

[0012] Centrifuge using a microplate centrifuge to obtain the supernatant. After diluting the supernatant in each well of the 96-well plate by 10 times, the same volume was sequentially transferred to another 96-w...

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Abstract

The invention discloses a high-throughput screening method of guanosine production strains, comprising the following steps of: (1) inoculating the guanosine production strains to be screened into a microtiter plate I containing a culture medium, and carrying out shaking culture; (2) centrifugalizing the microtiter plate I, and removing thalli to obtain supernate; and (3) diluting the supernatant supernate of the microtiter plate I, then sequentially transferring to a microtiter plate II, measuring a light absorption value by using a full-waveband enzyme-labeling instrument, and screening out the guanosine production strains. The high-throughput screening method integrates the effects of microtiter plate culture, fast enzyme-labeling instrument detection, a continuous sample injector and a volley rifle, achieves the handling capacity of more than 200 samples / day everyone and provides a simple, convenient and efficient screening method for the screening of the guanosine production strains.

Description

technical field [0001] The invention belongs to the technical field of fermentation engineering, and in particular relates to a breeding method for a microbial strain producing guanosine. Background technique [0002] Guanosine, also known as guanosine, its trade name is GR, also known as 9-13-D-ribofuranosylguanine. It has a wide range of uses and is an important intermediate of food and pharmaceutical products. It can be used to synthesize food flavor enhancer disodium 5'-guanylate, taste nucleotide disodium and nucleoside antiviral drugs such as ribavirin , Acyclovir, etc., are also the main raw materials for the manufacture of drugs such as sodium guanosine triphosphate. Someone selected mutant strains resistant to guanine or guanine structural analogs when breeding guanosine-producing bacteria, and found that the strains that lost GMP reductase mainly accumulated guanosine. Watanabe et al. in Japan cloned the key enzyme PRPP transamidase gene in the guanosine biosynth...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/00
Inventor 徐达刘剑郑明英黄秀旭黄永嫦
Owner STAR LAKE BIOSCI CO INC ZHAOQING GUANGDONG
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