Genetic engineering bacteria used for high efficient production of mycose

A technology of genetically engineered bacteria and trehalose, applied in the fields of genetic engineering and enzyme catalysis, can solve the problems of high enzyme cost, long conversion time, and insufficient enzyme activity, and achieve the effects of low enzyme cost, short conversion time, and simple process operation

Active Publication Date: 2013-12-25
SHANGHAI RES & DEV CENT OF INDAL BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When the starch concentration reaches above 20%, the conversion rate of trehalose is up to less than 80%, and the maltooligosaccharide-based trehalose hydrolase from this source has strong amylase activity, which is easy to cause the loss of raw materials
CN1504567A also uses Escherichia coli genetic engineering bacteria to express these two enzymes respectively, and the fermentation enzyme activities are respectively: MTSase60U / mL fermentation broth, MTHase250U / mL fermentation broth, at 30% starch concentration, 2U MTSase / g starch, 10U MTHase / g starch Under certain conditions, the conversion period is as long as 64 hours. After separation and purification, the yield of trehalose is only 50%.
[0007] So far, the dual-enzyme route has realized the industrialized production of trehalose, but there are still the following problems and shortcomings: 1. The enzyme activity produced by the original enzyme-producing bacteria is relatively low, the fermentation period is very long, and the energy consumption Large, so that the fermentation cost is very high, and the production efficiency is low; second, the strains producing these two enzymes by the recombinant bacteria need to be fermented separately, and then after measuring the enzyme activity, the enzyme is put into the reaction according to an appropriate ratio, which increases the difficulty of operation; , The fermentation enzyme activity of existing strains is not high enough, resulting in higher enzyme costs, or longer conversion time

Method used

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  • Genetic engineering bacteria used for high efficient production of mycose
  • Genetic engineering bacteria used for high efficient production of mycose
  • Genetic engineering bacteria used for high efficient production of mycose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Knockout of treA and malQ genes on the chromosome genome of Escherichia coli BL21 (DE3)

[0034] 1.1. Experimental route

[0035] In the present invention, the host bacteria Escherichia coli BL21 (DE3) is genetically engineered, and the trehalase gene (treA) and malt glucosylase gene (malQ) located on the chromosome genome are knocked out. The gene is knocked out step by step, and the knockout of the two genes is carried out according to the following method:

[0036] The first step: the construction of the resistance gene targeting fragment;

[0037] The second step: replace the treA gene or malQ gene with the resistance gene targeting fragment;

[0038] Step 3: Eliminate the resistance gene.

[0039] 1.2. Construction of treA resistance gene targeting fragment

[0040] Synthetic primers P1 and P2:

[0041] P1: 5'-AACGCCGCTGGCTGGCTAAT-3' (SEQ ID NO: 1);

[0042] P2: 5'-TATCTTCGGCAACCACGTAT-3' (SEQ ID NO: 2).

[0043] Prepare a PCR reaction solution c...

Embodiment 2

[0058] Example 2 , Construction of (Escherichia coli) BL21(DE3)MTSaseMTHase

[0059] The whole gene synthesis is derived from the maltooligosaccharide-based trehalose synthase gene MTSase and the maltooligosaccharide-based trehalose hydrolase gene MTHase ​​from Arthrobacter, Sulfolobus, Brevibacterium, Mycobacterium, subcloned into expression plasmid vectors (such as pTrc99a and pET24a, etc.), the plasmid vector containing the polynucleotide sequence encoding MTSase and the plasmid vector containing the polynucleotide sequence encoding MTHase ​​were obtained respectively. Then use two kinds of suitable restriction endonucleases to digest the plasmid vector mixture containing the MTSase polynucleotide sequence encoding various sources, and use the same restriction enzyme to digest the plasmid vector containing the MTHase ​​polynucleotide sequence encoding various sources Plasmid carrier mixture, obtain the fragment containing the MTSase polynucleotide sequence and the fragmen...

Embodiment 3

[0061] Example 3 , Using CCTCC NO: M2013237 to produce trehalose

[0062] Insert the CCTCC NO: M2013237 strain into a shaker flask containing 50mL of seed medium at a volume ratio of 1‰, and culture it at 37°C and 200 rpm for 12 hours, when OD600=1~2 stop the culture, put it into a shake flask with 1L of fermentation medium, culture it at 37°C and 200 rpm for 3 hours, lower the culture temperature to 28°C, and add isopropyl-β- D-thiogalactopyranoside, continue to cultivate for 20 hours, and finish the fermentation. The bacterial cells in the fermentation broth were collected by centrifugation, and the wet weight was 30g / L. 2 times the deionized water was added according to the mass, and after mixing evenly, the cells were broken by an ultrasonic cell breaker to obtain 90ml cell breakage solution.

[0063] Add cornstarch milk (starch mass concentration: 31%) at pH 5.3 to 5.6, add high-temperature amylase (Liquozyme supra, purchased from Novozymes) at an amount of 250 mL / ton ...

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Abstract

The invention discloses a genetic engineering bacteria used for high efficient production of mycose, and a method used for producing mycose by using the genetic engineering bacteria. Preservation number of the genetic engineering bacteria is CCTCC No.M2013237. The method comprises following steps: A) the genetic engineering bacteria is subjected to fermentation cultivation, and bacterial cells are collected; B) the bacterial cells are broken, MTSase and MTHase are released so as to obtain a water solution containing MTSase and MTHase; and C) amylose or straight-chain dextrin is converted into mycose by using the water solution containing MTSase and MTHase. The genetic engineering escherichia coli is capable of converting amylose or straight-chain dextrin into mycose efficiently; the conversion rate is more than 85%; enzyme cost is low; conversion time is short; technology operation is simple; and large-scaled industrialized application prospect of the method is promising.

Description

technical field [0001] The invention belongs to the field of genetic engineering and enzyme catalysis, and in particular relates to a genetically engineered bacterium for efficiently producing trehalose and a method for efficiently producing trehalose by using the engineered bacterium. Background technique [0002] Trehalose is a non-reducing disaccharide composed of two glucopyranose linked by α,α-1,1-glycosidic bonds. It exists widely in animals, plants and microorganisms in nature, but the content is very low. Trehalose is colorless and odorless, has strong thermal stability, acid stability and chemical stability, and has a strong stabilizing effect on proteins, nucleic acids, cell membranes, etc., and is usually produced in large quantities under environmental stress conditions. Even organisms play a protective role. Trehalose is an effective protective agent, which can protect enzymes, proteins, biofilms, biomedical preparations and even organs to be transplanted. It c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P19/24C12P19/14C12P19/12C12R1/19
Inventor 陶荣盛原犇犇朱傅赟沈青杨晟蒋宇
Owner SHANGHAI RES & DEV CENT OF INDAL BIOTECH
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