Method for synthesizing of 2'-fucosyllactose

A technology of fucosyllactose and fucosyl, which is applied in the field of genetic engineering, can solve the problems of expensive glycoside donors, complex synthetic routes, and inability to achieve large-scale production, and achieve strong basic theoretical research value and social and economic benefits. The effect of reducing production costs and broad market development prospects

Inactive Publication Date: 2019-08-27
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Human milk oligosaccharides are obtained by chemical synthesis. Due to the complexity of the synthetic route and the high cost of glycoside donors, most processes still cannot achieve large-scale production, which has become an insurmountable obstacle for chemical synthesis of oligosaccharides.

Method used

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  • Method for synthesizing of 2'-fucosyllactose
  • Method for synthesizing of 2'-fucosyllactose
  • Method for synthesizing of 2'-fucosyllactose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1 Construction of recombinant Lactococcus lactis and recombinant Escherichia coli

[0067] The genes involved in the present invention can be obtained through genomic PCR amplification, or can be obtained through complete sequence synthesis according to the gene sequence. The following is an example of PCR amplification:

[0068] 1. Amplification of the target gene

[0069] (1) Extract the genomic DNA of Escherichia coli (E.coli K12), and design the following primers to amplify hexokinase gene glk, phosphomannose mutase gene manB, and mannose-1-phosphate guanylyltransferase in sequence Gene manC, GDP-mannose 4,6-dehydratase gene gmd, GDP-4-keto-6-deoxymannose 3,5-mutrotase / 4-reductase gene wcaG:

[0070]

[0071]

[0072] (2) Genomic DNA of Helicobacter pylori (Helicobacter pylori) HPAG1 was extracted, and the following primers were designed to amplify the α1,2-fucosyltransferase gene futc:

[0073] Primer name Sequence 5'-3' Restriction...

Embodiment 2

[0092] Example 2, Induced expression analysis of recombinant strains

[0093] 1. Induced expression analysis of recombinant Escherichia coli

[0094] (1) Pick the genetic engineering strain BL21 / pET-gmd that embodiment 1-3 obtains, BL21 / pET-wcaG, BL21 / pET-futc single bacterium colony transfers respectively in 5mL LB liquid medium, 37 ℃, 220r / min cultivated overnight;

[0095] (2) Transfer the bacterial solution to 50mL M9 liquid medium, and control the initial bacterial concentration OD 600 =0.1, continue to cultivate;

[0096] (3) When the strain concentration reaches OD 600 When the temperature is 0.4, add 40 μL of 0.1mol / L IPTG and induce at 16°C for 4 hours;

[0097] (4) Centrifuge at 6000r / min for 10min to collect Escherichia coli;

[0098] (5) Wash the strain twice with PBS buffer, break the cells with an ultrasonic cell breaker, centrifuge at 12000r / min for 10min at 4°C, collect the supernatant, and take a sample for SDS-PAGE analysis. The results are shown in F...

Embodiment 3

[0106] Example 3 Recombinant Engineering Strain Fermentation and Enzyme Co-catalyzed Synthesis of 2'-Fucosyllactose

[0107] (1) Pick a single colony of recombinant Lactococcus lactis NZ3900 / pNZ-glk-manB-manC and inoculate it into 5mL GM 17 In liquid culture medium, cultivate overnight at 30-37°C, 160-220r / min. Inoculate the bacterial liquid into 50mL M9 fermentation medium, and control the initial bacterial concentration OD 600 = 0.1, the OD of the strain to be 600 When = 0.4, add 20 mg / mL of substrate mannose, and add Nisin as an inducer at the same time to make the final concentration 35 ng / mL, and ferment for 40 hours at 35°C and 200 r / min. Break the cells, centrifuge, and collect the supernatant;

[0108] (2) Preparation of GDP-mannose 4,6-dehydratase, GDP-4-keto-6-deoxymannose 3,5-mutrotase / 4-reductase and α1,2-fucosyltransferase : Pick single colonies of recombinant Escherichia coli BL21 / pET-glk, BL21 / pET-manB and BL21 / pET-manC and inoculate them into 5mL LB liquid ...

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Abstract

The invention belongs to the field of genetic engineering and relates to a method for producing 2'-fucosyllactose, in particular to a method for synthesizing 2'-fucosyllactose by co-catalysis of a genetically engineered strain and enzyme. The method of the invention comprises the steps of using a recombinant strain of lactococcus lactis which efficiently expresses hexokinase, phosphomannomutase and mannose-1-phosphate guanyl transferase as a production strain, and using mannose as a substrate to synthesize GDP-mannose, and then using GDP-mannose 4,6-dehydratase, GDP-4-keto-6-deoxymannose 3,5-mutarotase / 4-reductase, alpha1,2-fucosyltransferase to catalyze GDP-mannose in vitro to synthesize the 2'-fucosyllactose, thus providing a new method for the industrial production of the 2'-fucosyllactose.

Description

[0001] Technical field: [0002] The invention belongs to the field of genetic engineering and relates to a production method of 2'-fucosyllactose, in particular to a method for co-catalyzing the synthesis of 2'-fucosyllactose by using genetic engineering strains and enzymes. [0003] Background technique: [0004] In recent years, with the rapid development of food industry and biotechnology, the development of functional oligosaccharides has become an important topic in the field of international biotechnology. industry. Human milk oligosaccharides not only have physiological effects such as anti-viral infection, enhancing immune regulation, and reducing inflammation, but also have certain preventive effects on cancer and chronic recurrent colitis. As a class of carbohydrates with a special structure, it can act as a soluble receptor analog of epithelial cells and participate in the enhancement of the non-immune defense system of breastfed infants. The content of human milk...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/00C12R1/01C12R1/19
CPCC12P19/00
Inventor 李玉何光明石爱琴王洪彬秦慧民刘夫峰刘逸寒路福平
Owner TIANJIN UNIV OF SCI & TECH
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