Method for preparing tagatose by using whole-cell catalysis

A whole-cell, tagatose technology, applied in biochemical equipment and methods, microorganism-based methods, microorganisms, etc., can solve problems such as inability to be widely used, high production cost of tagatose, and impact on the final price of tagatose , to achieve the effect of low pollution, high yield and low cost

Active Publication Date: 2018-05-04
天工生物科技(天津)有限公司
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  • Abstract
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AI Technical Summary

Problems solved by technology

At present, the method of biotransformation to produce tagatose that has been studied more is to use L-arabinose isomerase to catalyze the conversion of D-galactose into tagatose. However, the higher price of galactose affects the final price of tagatose, resulting in Cannot be widely used (Rhimi M, Aghajari N, Juy M, Chouayekh H, Maguin E, Haser R, Bejar S: Rational design of Bacillus stearothermophilus US100l-arabinose isomerase: Potential applications for d-tagatose production.Biochim.2009,91:650 -653. OhH-J, Kim H-J, Oh D-K:Increase in d-tagatose Production Rate by Site-directedMutagenesis of l-arabinose Isomerase from Geobacillusthermodenitrificans.Biotechnol.Lett.2006,28:145-149.Bosshart A,Hee CS, Bechtold M, Schirmer T, Panke S:Directed Divergent Evolution of a Thermostable D-Tagatose Epimerase towards Improved Activity for Two Hexose Substrates.ChemBioChem 2015,16:592-601.Men Y,Zhu Y,Zhang L,Kang Z,Izumori K,Sun Y, Ma Y: Enzymatic conversion of D-galactose to D-tagatose: Cloning, overexpression and characterization of l-arabinose isomerase from Pediococcuspentosaceus PC-5.Microbiol.Res.2014,169:171-178.)
[0004] Korean scientists invented a multi-enzyme-catalyzed method to convert fructose into tagatose, including using 6-phosphate tagatose epimerase and 6-phosphate tagatose phosphatase to convert fructose into tagatose (Oh DK , HONG SH, Lee SH: Aldolase, aldolase mutants and tagatose using the same production methods and compositions for production.WO 2015016544A1.Google Patents; 2015.), but this method is to produce 6-phosphate fructose from fructose, so ATP is needed to carry out fructose Substrate phosphorylation, resulting in high production cost of tagatose, not suitable for large-scale production

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  • Method for preparing tagatose by using whole-cell catalysis

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Embodiment 1

[0041] Embodiment 1 prepares tagatose from starch

[0042] (1) Constructed genes containing α-glucan phosphorylase gene, glucose phosphate mutase gene, glucose phosphate isomerase gene, 6-phosphate tagatose epimerase gene, 6-phosphate tagatose phosphate The prokaryotic expression plasmid of the enzyme gene; the constructed prokaryotic expression plasmid was transferred into Escherichia coli engineering bacteria to obtain recombinant strains; the recombinant strains were induced to express and produce enzymes and then permeabilized:

[0043] In this example, α-glucan phosphorylase is derived from Thermotoga maritima, and the gene number on KEGG is TM1168; glucose phosphomutase is also derived from Thermotoga maritima, and the gene number on KEGG is TM0769; The isomerase is derived from Clostridium thermocellum, and the gene number on KEGG is Cthe0217; the 6-phosphate tagatose epimerase is from Thermoanaerobacter indiensis, and the enzyme coded by the gene is numbered WP_0199072...

Embodiment 2

[0049] Embodiment 2 prepares tagatose from starch

[0050] (1) Constructed genes containing α-glucan phosphorylase gene, glucose phosphate mutase gene, glucose phosphate isomerase gene, 6-phosphate tagatose epimerase gene, 6-phosphate tagatose phosphate Prokaryotic expression plasmids of enzyme genes and starch debranching enzymes; the constructed prokaryotic expression plasmids were transformed into Escherichia coli engineering bacteria to obtain recombinant strains; the recombinant strains were induced to express and produce enzymes and then permeabilized:

[0051] In this embodiment, the starch debranching enzyme is derived from Sulfolobus tokodaii, and the number of the gene on KEGG is ST0928. Genomic DNA can be obtained from the official website of ATCC (www.atcc.org). The gene was obtained by PCR and cloned into the pET20b vector to obtain the corresponding expression vector pET20b-StIA. The plasmid was transformed into Escherichia coli expression strain BL21(DE3), and...

Embodiment 3

[0054] Embodiment 3 prepares tagatose from starch

[0055] (1) Construct the genes containing α-glucan phosphorylase gene, glucose phosphate mutase gene, glucose phosphate isomerase gene, 6-phosphate tagatose epimerase gene, 6-phosphate tagatose phosphate Prokaryotic expression plasmids of enzyme genes, starch debranching enzyme, glucan transferase, and polyphosphoglucokinase; the constructed prokaryotic expression plasmids were transformed into E. Personalization:

[0056]In this embodiment, polyphosphoglucokinase is derived from Thermobifida fusca, and the number of the gene on KEGG is Tfu1811. Glucanotransferase is derived from Thermococcus litoralis, and the gene number on KEGG is OCC_10078. Genomic DNA can be obtained from the official website of ATCC (www.atcc.org). These two genes were obtained from the corresponding genomic DNA by PCR with different primers, and cloned into the pET20b vector to obtain the corresponding expression vectors pET20b-TfuPPGK and pET20b-Tl...

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Abstract

The invention discloses a method for preparing tagatose by using whole-cell catalysis. The method comprises the following steps of transferring plasmid containing an alpha-glucan phosphorylase gene (or cellulosic polysaccharide phosphorylase and cellobiose phosphorylase genes, or a sucrose phosphorylase gene), a phosphoglucomutase gene, a glucose phosphate isomerase gene, a 6-phosphate tagatose epimerase gene, and a 6-phosphate tagatose phosphatase gene into engineered escherichia coli, so as to obtain converted bacterial strain; inducing the converted bacterial strain to produce enzyme, and permeabilizing; mixing the permeabilized bacterial strain, establishing a whole-cell enzyme catalysis reaction system with starch or starch derivative (or mixture of cellulase and cellulose or cellulose derivative, or sugarcane), and performing the whole-cell enzyme catalysis reaction, so as to obtain the tagatose. The method has the advantages that the tagatose is prepared by the whole-cell catalysis; the cost is low, the pollution is low, the yield rate is high, and the like; the method is suitable for preparing the tagatose in a scale way.

Description

technical field [0001] The invention relates to a method for preparing tagatose, in particular to a method for preparing tagatose by catalyzing whole cells, and belongs to the field of tagatose preparation. Background technique [0002] Tagatose (D-Tagatose) is a rare monosaccharide that occurs naturally, it is the ketose form of galactose, and the epimer of fructose. The sweetness properties are similar to sucrose, and the calories produced are only one-third of sucrose, so it is called a low-calorie sweetener. Tagatose has excellent nutritional properties such as low calorie value, zero glycemic index, blood sugar passivation, no caries, prebiotic effect and antioxidant activity. Natural tagatose is mainly found in dairy products such as yogurt and milk powder. Tagatose has four major functions: low energy, lowering blood sugar, improving intestinal flora and anti-caries (Oh D-K: Tagatose: properties, applications, and biotechnological processes. App. Microbiol. Biotechn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/24C12P19/18C12P19/16C12P19/14C12P19/02C12R1/19
CPCC12P19/02C12P19/14C12P19/16C12P19/18C12P19/24
Inventor 马延和孙媛霞杨建刚李运杰
Owner 天工生物科技(天津)有限公司
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