High-throughput screening method for efficient urea utilizing strains

A high-throughput, strain-based technology, applied in the field of metabolic engineering, can solve the problems of high dilution factor, narrow detection range, and poor agreement of recovery rate, and achieve the effect of simplifying the screening process, shortening the screening period, and reducing the content.

Inactive Publication Date: 2016-02-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Yet (Wang Liyuan, 2010) bibliographical report shows, diacetyl monoxime spectrophotometry time-consuming many, the disadvantages such as high dilution factor; , The required sample volume is large, the boiling water bath will cause the reaction system to be easily lost, and the data fluctuates greatly, the recovery rate is poor, the detection range is narrow, and it is not suitable for large-scale detection, etc.
[0005] In summary, the current natural screening is difficult and the screening efficiency is low, and the existing urea detection methods have shortcomings such as poor accuracy, stability and precision, and cannot achieve high-throughput screening of strains with high urea utilization

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: standard curve drawing

[0036] Gradient dilution of the standard urea solution is to dilute the standard solution to 0.1mg / L, 0.3mg / L, 0.5mg / L, 1.0mg / L, 1.5mg / L, 3.0mg / L, 4.5mg / L, 5.0mg / L L, 6.0mg / L, 8.0mg / L, 10.0mg / L, 20mg / L, 30mg / L, then the sample (standard urea solution), diacetylmonoxime thiamine and iron-phosphoric acid solution with a volume ratio of 4 : 1:5 mix, react in a PCR instrument at 100°C for 10 minutes, then cool down to room temperature for detection.

[0037] The maximum absorption peak is determined to be 525nm by full-wavelength scanning of a microplate reader, and the abscissa is the urea concentration, and the OD is 525 The absorption value is the ordinate, and the standard curve y=45.45x+0.062 (R 2 =0.999), the standard recovery was measured in yellow rice wine, the recovery rate was: 98.2%~101.18%, and the average recovery rate was 99.47%.

[0038] At the same time, the results of this method are basically consistent with those...

Embodiment 2

[0039] Embodiment 2: Screening of high-yield strains

[0040] (1) Inoculate the strain to be screened on the primary screening medium in which urea is the only nitrogen source, and select 400 strains with relatively large colonies as the primary screening strain;

[0041] (2) Inoculate the primary screened strains into the seed medium in 96 deep-well plates, and culture them in a 900r / min 30°C high-throughput deep-well plate constant temperature oscillator for 48 hours to obtain seed liquid, and then inoculate the seed liquid with 10% The amount was inoculated into a 48 deep-well plate, 900r / min, 30°C fermentation culture for 72h, after the fermentation culture was completed, the deep-well plate was centrifuged at 3500r / min for 10min.

[0042](3) Take the supernatant or the diluted supernatant as the sample to be tested; add 40 μL of the sample to be tested, 50 μL of iron-phosphate solution and 10 μL of diacetylmonoxime thiamine solution to a PCR plate (96-well plate), react ...

Embodiment 3

[0044] Embodiment 3: method reliability and verification of high-yield bacterial strains

[0045] (1) Method reliability verification: according to the sample concentration to be tested obtained in Example 2, randomly select 20 samples with concentration of 0.3-0.5 mg / L, 20 samples with 1.0-3.0 mg / L, 8.0 mg / L-10.0mg / L sample 20, then use HPLC and enzyme coupling reaction method to detect respectively the urea concentration in the fermentation supernatant of 60 strains of bacteria, the result finds, the detection result of the inventive method and HPLC or enzyme coupling reaction method The coupling reaction method is basically the same, and the bacterial strains with relatively high urea concentration detected by this method are also relatively high when detected by the enzyme coupling reaction method. It shows that the method of the present invention has strong reliability.

[0046] (2) Take 5 strains corresponding to the wells with the smallest urea concentration obtained i...

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Abstract

The invention discloses a high-throughput screening method for efficient urea utilizing strains, and belongs to the field of metabolic engineering. The method comprises the steps that after flat-plate preliminary screening is performed on strains to be screened, seed liquid is prepared through a deep hole plate and then fermented and cultured to obtain fermented supernatant, 40 muL of the supernatant to be detected, 50 muL of an iron-phosphoric acid solution and 10 muL of diacetyl monoxime thiosemicarbazide liquid are added in a PCR plate to be subjected to a reaction for 10 min in a PCR instrument at 100 DEG C, an OD525 absorption value is determined through a microplate reader, the concentration x of urea in a sample to be detected is calculated according to a formula y=45.45x+0.062, R2=0.999, then the result that the urea concentration is lower is obtained, and the strains with the high urea utilizing capacity are obtained. According to the method, the detection cost of a single sample is decreased by nearly 95p percent, and meanwhile the advantages of being short in detection time, high in efficiency and accuracy, capable of simultaneously detecting multiple samples and the like are achieved.

Description

technical field [0001] The invention relates to a high-throughput screening method for high-efficiency urea utilization strains, belonging to the field of metabolic engineering. Background technique [0002] Urea, also known as carbamide, is the diamide of carbonic acid, and its molecular formula is H 2 NCONH 2 (CO(NH 2 ) 2 , is a key substance in nitrogen metabolism and the urea cycle. In addition, in sake and rice wine, urea produced by the metabolism of Saccharomyces cerevisiae is the most important precursor of EC (urethane). During the fermentation process, urea in yeast cells is mainly produced by the metabolism of arginine. When there is no yeast preference nitrogen source (glutamine, asparagine, etc.) in the fermentation medium, urea can be further degraded into carbon dioxide and ammonia by ureohydrolase. However, the expression of ureohydrolase was strongly inhibited when the yeast-preferred nitrogen source was present. This phenomenon is regulated by the Ni...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/58C12Q1/04C12R1/865
Inventor 周景文陈坚程艳李江华堵国成
Owner JIANGNAN UNIV
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