Escherichia.coli with high L-aspartase yield and application thereof

A technology of Escherichia coli and aspartase, applied in the direction of bacteria, microorganism-based methods, microorganisms, etc., can solve the problems of short half-life of immobilized cell method and complicated immobilization operation, and achieve high conversion efficiency Effect

Active Publication Date: 2012-07-11
YANTAI HENGYUAN BIOENGINEERING CO LTD
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0039] The half-life of the immobilized cell method is short (about half a month) and the immobilization operation is more complicated

Method used

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  • Escherichia.coli with high L-aspartase yield and application thereof
  • Escherichia.coli with high L-aspartase yield and application thereof
  • Escherichia.coli with high L-aspartase yield and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Inoculate the strains on the preservation slant medium, culture at 37°C±1°C for 24 hours, store at 4°C, and subculture once every 2-3 months.

[0065] ②Strain activation

[0066] Take a ring of preserved strains from the slant, transfer to the production slant, and cultivate at 37°C±1°C for 15-20 hours.

[0067] ③ strain mutagenesis

[0068] Transfer the plate-isolated single colony culture of the starting strain Escherichia coli HY to the production liquid medium, and cultivate it at 37° C. for 15-20 hours. Take 5ml of vigorously growing culture, centrifuge at 4000rpm for 15 minutes, pour off the supernatant, wash the pelleted cells with normal saline for 2-3 times, suspend the bacterial pellet with 50ml of pH7.0 phosphate buffer solution, and dilute to about 10 7 cells / ml, spare.

[0069] Add nitrosoguanidine acetone solution to the test tube containing the above bacterial suspension to make the final concentration reach 200 μg / ml, mix well and keep warm at 37°C f...

Embodiment 2

[0075] ⑥2 tons of seed tank cultivation

[0076] Combine 20 bottles of seed culture solution under aseptic conditions into 5L seed solution, and put it into a 2-ton fermenter with a liquid volume of 1.8m 3 Cultivate at 37°C±1°C for about 12 hours, and measure the changes in OD value and pH value of Escherichia coli.

[0077] After repeated mutagenesis and selection, a strain of Escherichia coli HY-05C with high L-aspartase production was obtained, and the activity of its shake-flask fermentation biotransformation enzyme (L-aspartase) could reach 4200U / ml; 2-ton tank production-scale fermentation biotransformation enzyme activity can reach 4500U / ml; it is preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee, and the preservation number is: CGMCC No.5450.

[0078] Embodiment 2: assay method

[0079] ① Biotransformation enzyme (L-aspartase) activity assay method:

[0080] Definition of enzyme activity: at 45°C and pH 7.5...

Embodiment 3

[0107] ① Shake flask seed liquid culture

[0108] Get one ring of the Escherichia coli bacterial classification of the good embodiment 1 of activation, inoculate in the shake flask culture medium (1000ml Erlenmeyer flask, filling volume is 250ml, 20 bottles). Shaker culture conditions are as follows: the rotating speed is 90 rpm, 37°C±1°C for 12h. Measure the biotransformation enzyme activity in the primary seed liquid by the method of embodiment 2 and reach 4200U / ml;

[0109] ② 2 tons of seed tank cultivation

[0110] Combine 20 bottles of seed culture solution under aseptic conditions into 5L seed solution, and put it into a 2-ton fermenter with a liquid volume of 1.8m 3 Cultivate at 37°C±1°C for about 12 hours, and measure the changes in OD value and pH value of Escherichia coli. According to the method for embodiment 2, biotransformation enzyme activity reaches 4500U / ml in secondary seed liquid;

[0111] ③Transfer the cultivated 2 tons of secondary seed liquid into 20m...

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Abstract

The invention provides escherichia.coli with high L-aspartase yield. The collection number of the escherichia.coli collected in the China General Microbiological Culture Collection Center is CGMCC No.5450. The activity of a shake flask-fermented biotransformation enzyme (L-aspartase) can be up to 4,200 U / ml; the activity of a biotransformation enzyme fermented with a 2-ton tank can be up to 4,500 U / ml, which is increased by over 30 percent in comparison to the activity of a fermentation strain; and fumaric acid is biologically transformed directly and efficiently by using free cells with high transformation activities to generate L-aspartase; and when the concentration of fumaric acid is 18.5-20.0 percent, the biological transformation time of a 20-ton tank is shortened by one third in comparison to strain deriving time, and is 6-8 hours, the transformation rate is over 97.5 percent, and the transformation efficiency is higher that of the same level.

Description

technical field [0001] The invention relates to the technical field of biotransformation, in particular to an Escherichia coli with high L-aspartase production and application thereof. Background technique [0002] L-Aspartic acid is an amino acid widely used in the food industry, chemical product intermediates and pharmaceutical industries. [0003] (1) Used in synthetic sweeteners [0004] L-Aspartic acid is the main raw material for the synthesis of aspartame. [0005] Aspartame is a new type of sweetener, which is produced by esterifying L-phenylalanine with methanol and then condensation and amidation with L-aspartic acid. Traditional sugars are high in calories and can easily cause cardiovascular disease, obesity and other diseases that endanger human health. With the deepening of people's understanding of the harmful effects of sugars, people put the focus of research on finding new sweeteners , Aspartame has become a new type of healthy sweetener widely praised. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P13/20C12R1/19
Inventor 马玉岳姜国政姜增妍马秀亮杨振平李虹李金霞姚粟赵洪坤王志雪刘彩凤陈玲玲高琳赵庆军魏善玺
Owner YANTAI HENGYUAN BIOENGINEERING CO LTD
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