A preparation method and application of immobilized cells for tagatose production

A technology for immobilizing cells and tagatose, applied in the production and preparation of tagatose, and in the field of bioengineering, can solve the problems of uneven particle size of immobilized enzymes, loss of immobilization efficiency of enzymes, cumbersome production steps, etc., and achieve simple immobilization Chemical process, which is beneficial to separation and purification, and simplifies the effect of production and preparation process

Active Publication Date: 2021-10-12
天津怡和生物科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the problems existing in the existing methods for preparing tagatose from immobilized cells, such as cumbersome production steps of enzymes, loss of enzymes caused by permeabilization treatment and reduction of immobilization efficiency, uneven particle size of immobilized enzymes prepared, etc. , the purpose of the present invention is to provide a method for producing tagatose by immobilized cells, simplify the production steps of enzymes in the production process of tagatose, simplify the separation and purification of products and enzymes in the production process of tagatose, and realize the circulation of multiple enzymes use, reduce the production cost of tagatose, and realize the industrialized production of tagatose

Method used

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  • A preparation method and application of immobilized cells for tagatose production
  • A preparation method and application of immobilized cells for tagatose production
  • A preparation method and application of immobilized cells for tagatose production

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1: Preparation of whole cells of Bacillus subtilis

[0036] Pick and express thermostable α-glucan phosphorylase gene, express thermostable glucose phosphate mutase gene, express thermostable glucose phosphate isomerase gene, express thermostable 6-phosphate tagatose epimerase gene, Bacillus subtilis recombinant engineered bacteria expressing the heat-resistant 6-phosphate tagatose phosphatase gene (the starting bacterium is SCK6, see CN112342179B), and inoculated in LB medium respectively, and cultured overnight at 37°C with shaking. The culture was transferred to LB medium at an inoculum size of 1%, and cultured with shaking at 37°C overnight to obtain Bacillus subtilis fermentation broth expressing thermostable α-glucan phosphorylase and Bacillus subtilis expressing thermostable glucose phosphotransmutation Fermentation broth of Bacillus subtilis expressing thermostable glucose phosphate isomerase, fermentation broth of Bacillus subtilis expressing thermostab...

Embodiment 2

[0037] Embodiment 2: Production of tagatose by immobilized Bacillus subtilis

[0038] According to the OD600 ratio of 1:1:1:1:1, the Bacillus subtilis fermentation broth expressing thermostable α-glucan phosphorylase prepared in Example 1 and the Bacillus subtilis expressing thermostable glucose phosphomutase were fermented Bacillus subtilis fermentation broth expressing thermostable glucose phosphate isomerase, Bacillus subtilis fermentation broth expressing thermostable 6-phosphate tagatose epimerase, expressing thermostable 6-phosphate tagatose phosphatase The Bacillus subtilis fermentation broth was mixed so that OD 600 = 100, 1% w / v montmorillonite was added to the bacterial suspension, and stirred evenly. Subsequently, 0.5% w / v polyethyleneimine aqueous solution with a molecular weight of 10000 was added to flocculate at room temperature. Then, 2% v / v glutaraldehyde aqueous solution was added to crosslink for 2 h at room temperature. The filter cake is obtained after v...

Embodiment 3

[0040] Embodiment 3: Production of tagatose by immobilized Bacillus subtilis

[0041] According to the OD600 ratio of 1:1:1:1:1, the Bacillus subtilis fermentation broth expressing thermostable α-glucan phosphorylase prepared in Example 1 and the Bacillus subtilis expressing thermostable glucose phosphomutase were fermented Bacillus subtilis fermentation broth expressing thermostable glucose phosphate isomerase, Bacillus subtilis fermentation broth expressing thermostable 6-phosphate tagatose epimerase, expressing thermostable 6-phosphate tagatose phosphatase The Bacillus subtilis fermentation broth was mixed so that OD 600 = 100, 5% w / v diatomaceous earth was added to the bacterial suspension, and stirred evenly. Subsequently, 0.1% w / v polyethyleneimine aqueous solution with a molecular weight of 70000 was added to flocculate at room temperature. Then, 1% v / v glutaraldehyde aqueous solution was added to crosslink for 2 h at room temperature. The filter cake is obtained afte...

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Abstract

The invention discloses a method for preparing immobilized cells for producing tagatose and a method for producing tagatose. Wherein, when the immobilized Bacillus subtilis of the present invention continuously catalyzes the reaction, the initial product yield can reach up to 75%, and after 65 batches of continuous catalysis, the product yield can still maintain 61%. When immobilized Escherichia coli continuously catalyzes the reaction, the initial product yield can reach up to 74%, and after 65 batches of continuous catalysis, the product yield can still maintain 60%. The invention simplifies the separation and purification steps of enzymes required in the production of tagatose, improves the recycling rate of the enzymes, and realizes the recycling of the enzymes. The method of the invention has the advantages of easy product separation, simple production process, low cost and the like.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to the field of production and preparation of tagatose. Background technique [0002] Tagatose is a rare monosaccharide that occurs naturally and is the ketose form of galactose, the epimer of fructose. The sweetness properties of tagatose are similar to that of sucrose, providing very fresh and pure sweetness, but with only one-third of the calories of sucrose, it is called a low-calorie sweetener. Studies have shown that tagatose has important physiological functional properties such as low calorie, low glycemic index, anti-caries, anti-oxidation, prebiotics, improvement of intestinal function, immune regulation, and drug precursors, and can be widely used in food, beverages, medicine, It has great economic value in fields such as health care (Oh D-K: Tagatose: properties, applications, and biotechnological processes. App. Microbiol. Biotechnol. 2007, 76:1-8). [0003] Ti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/14C12N11/10C12N11/089C12N11/087C12N11/082C12P19/02C12R1/19C12R1/125
CPCC12N11/14C12N11/10C12N11/089C12N11/087C12N11/082C12P19/02
Inventor 马延和石婷韩平平
Owner 天津怡和生物科技有限责任公司
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