Method for fermentation production of D-tagatose by saccharomyces cerevisiae

A technology of Saccharomyces cerevisiae and tagatose, applied in the field of synthetic biology, can solve the problems of chemical pollutants, difficult separation, expensive raw materials, etc., and achieve the effect of low raw material cost, simple product purification process, and easy control

Inactive Publication Date: 2018-11-09
嘉兴欣贝莱生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the synthesis methods of D-tagatose are mainly chemical catalysis synthesis method and biotransformation method. The chemical catalysis synthesis method uses galactose as raw material, through metal 2 ) and an alkali metal catalyst (usually CaCl 2 ) catalyzes galactose to generate D-tagatose-Ca complex, and then releases tagatose from the complex through acid neutralization. This method has the advantages of expensive raw materials, chemical pollutants, and many by-products under alkaline conditions. Difficult to separate and many other disadvantages
The biotransformation method uses L-arabinose isomerase to convert galactose into tagatose, but the same reaction has the disadvantages of expensive raw materials, low conversion efficiency, and difficult product separation, making it difficult to realize large-scale industrial production.

Method used

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  • Method for fermentation production of D-tagatose by saccharomyces cerevisiae
  • Method for fermentation production of D-tagatose by saccharomyces cerevisiae

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Experimental program
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Effect test

Embodiment 1

[0024] Example 1 Plasmid construction method.

[0025] This patent studies the amino acid sequence of the corresponding gene and the codon preference of Saccharomyces cerevisiae, optimizes the nucleic acid sequence of the gene, and sends it to a commercial gene synthesis company to synthesize the gene. According to the gene sequence of GalE, according to the attached figure 2 Plasmid Y33-GalE was constructed according to the plasmid map in , and a homology arm of about 20 bp was designed between the corresponding fragments. The fragments were connected with a commercial kit (one-step cloning kit) and then transformed into Saccharomyces cerevisiae cells. Single clones were picked on the type plate to extract the genome, and PCR verification was performed to obtain engineering strains for subsequent induced expression.

Embodiment 2

[0026] Embodiment 2 conversion process.

[0027] 1) First pick positive single clones and culture them overnight at 30°C, 220rpm for 12h in the corresponding deficient medium;

[0028] 2) detect the value of OD600 of the bacterial liquid cultivated overnight with a spectrophotometer;

[0029] 3) Transfer the bacterial solution with an initial OD600 value of 0.2OD to 50 mL of fresh YPAD medium;

[0030] 4) Activate for 4-5 hours to increase the value of Saccharomyces cerevisiae for two generations so that the OD600 value of the bacterial liquid is 0.8-0.9, collect the bacterial cells by centrifugation at 3600rpm for 5min, and use 25mL ddH 2 O washed twice to fully wash off the culture medium;

[0031] 5) Resuspend the centrifuged bacteria into a sterile 1.5mL centrifuge tube with 1mL sterile water in a clean bench;

[0032] 6) Then centrifuge at 12000rpm for 30s to collect the bacteria;

[0033] 7) Resuspend the bacterial cells after the previous step of centrifugation in 1...

Embodiment 3

[0043] Embodiment 3 catalytic synthesis tagatose

[0044] Add the corresponding substrate fructose, react at 35-36°C, 220-250rpm for 24-36h, collect the produced D-tagatose, separate and purify the product tagatose, the liquid medium is LB medium, and its mass fraction group Prepare: 1% tryptone, 0.5% yeast extract, 1% NaCl, adjust the pH value to 7.5 with NaOH, and sterilize at 121°C for 20 minutes for later use.

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Abstract

The invention relates to a method for fermentation production of D-tagatose by saccharomyces cerevisiae including the following steps: codons for optimizing gene GalE according to codon preference ofsaccharomyces cerevisiae are built in the saccharomyces cerevisiae expression vector YCplac33 to produce a vector 1, wherein, the expression vector YCplac33 carries ampicillin resistance gene of escherichia coli and a selection marker URA3 gene of saccharomyces cerevisiae; the above recombined carrier 1 is transferred to a wild type saccharomyces cerevisiae W303, wherein, positive monoclonal genesare selected pursuant to the selection marker URA3 of the saccharomyces cerevisiae and the positive monoclonal genes are cultured for extraction of a genome of the yeast to be further verified by PCRto produce a final fermentation strain; the fermentation strain is cultured under a certain condition till bacteria hits a certain concentration; corresponding substrate fructose is added to the strain for a 5-day reaction under 35-36 DEG C and 220-250 rpm to produce D-tagatose, and the D-tagatose is collected, separated and purified. The process has separation and purification procedures only inthe final step of production, and the product purification process is simple.

Description

technical field [0001] The invention relates to a method for fermenting and producing D-tagatose by Saccharomyces cerevisiae, belonging to the field of synthetic biology. Background technique [0002] Rare sugar is a type of monosaccharide (the smallest unit of sugar) and sugar alcohol that exists in nature but is very small. Compared with glucose and fructose that exist in large quantities in nature, it is very rare, and there are about 50 types. Its taste is similar to sucrose, but it has the advantages of low calorie, high stability, coordinated sweetness, no hygroscopicity, no cariogenicity, and high tolerance. It can make up for the shortcomings of traditional sweeteners and is widely used in health food, In infant formula, dairy products, beverages and other foods, it plays an important role in improving the diet of special populations. There are many benefits of rare sugar, such as reducing the formation of toxic substances (such as endotoxin, ammonia, etc.), protect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/61C12P19/02C12R1/865
CPCC12N9/90C12N15/81C12N2800/22C12P19/02C12Y501/03002
Inventor 夏文豪陈贤情蒿飞杨月王筱王文
Owner 嘉兴欣贝莱生物科技有限公司
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