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

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

Active Publication Date: 2014-04-09
YANTAI HENGYUAN BIOENGINEERING CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0038] Although the immobilized enzyme has the advantages of reusability, easy purification and high utilization rate, its disadvantages are also obvious, such as the high price of biological enzymes, long production cycle, large investment in equipment, etc.
[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

[0062] Example 1: Breeding of strains

[0063] ①Bacteria preservation

[0064] The strains were connected to the preservation slant medium, cultured at 37°C±1°C for 24 hours, preserved at 4°C, and passaged every 2-3 months.

[0065] ②Strain activation

[0066] Take a ring of preserved bacteria on the slope, transfer it to the production slope, and incubate at 37°C±1°C for 15-20 hours.

[0067] ③Strain mutation

[0068] The plate-separated single colony culture of the starting strain Escherichia coli HY was transferred to the production liquid medium and cultured at 37°C for 15-20 hours. Take 5ml of the vigorously growing culture, centrifuge at 4000rpm for 15 minutes, pour out the supernatant, wash the pelleted cells with saline 2-3 times, suspend the bacterial pellet with 50ml of pH 7.0 phosphate buffer solution, and dilute to approximately 10 7 Cells / ml, spare.

[0069] Add the nitrosoguanidine acetone solution to the test tube containing the above bacterial suspension to make the fina...

Embodiment 2

[0073] ⑤First-level seed cultivation

[0074] Take a loop of activated bacteria and inoculate it in a shake flask culture medium (1000ml Erlenmeyer flask, 250ml liquid volume, 20 bottles). The conditions of the shaking table culture are: rotation speed is 90 rpm, 37°C±1°C for 12h.

[0075] ⑥ 2 tons of seed pot cultivation

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

[0077] After repeated mutagenesis and breeding, a strain of Escherichia coli HY-05C with high L-aspartase production was obtained, and the activity of the shake-flask fermentation bioconvertase (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 Microbial Cultu...

Embodiment 3

[0105] Example 3: Biotransformation to prepare L-aspartic acid

[0106] Using the L-aspartase produced by Escherichia coli in Example 1 to catalyze the conversion of fumaric acid and ammonia into L-aspartic acid includes the following steps:

[0107] ① Shake flask seed liquid culture

[0108] Take a loop of the activated Escherichia coli strain of Example 1 and inoculate it in a shake flask culture medium (1000ml Erlenmeyer flask, 250ml liquid volume, 20 bottles). The culture conditions of the shaking table are: rotation speed is 90 rpm, 37°C±1°C for 12h. According to the method of Example 2, the biotransformation enzyme activity in the primary seed liquid was determined to reach 4200 U / ml;

[0109] ②2 tons of seed pot cultivation

[0110] Combine 20 bottles of seed culture solution under aseptic conditions into 5L seed solution, and connect it to a 2 ton fermenter with a volume of 1.8m 3 Incubate at 37°C±1°C for about 12 hours, and determine the changes in the OD value and pH value o...

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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 present invention relates to the technical field of biotransformation, in particular to a strain of Escherichia coli with high L-aspartase production and its application. Background technique [0002] L-Aspartic acid is an amino acid widely used in the food industry, chemical product intermediates and the pharmaceutical industry. [0003] (1) For 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 esterification of L-phenylalanine with methanol and then condensation and amidation with L-aspartic acid. Traditional sugars are high in calories and easily cause cardiovascular diseases, obesity and other diseases that endanger human health. As people's understanding of the harmful effects of sugars has deepened, people have focused their research on finding new sweeteners. , Aspartame has become a new and healthy sweetener that is widely respec...

Claims

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Application Information

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Patent Type & Authority Patents(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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