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Tagatose preparation method

A technology of tagatose and phosphate tagatose, applied in the direction of fermentation, etc., can solve the problems of not being widely used, affecting the final price of tagatose, not suitable for large-scale production, etc., and achieve the effect of high conversion rate

Active Publication Date: 2017-02-15
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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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 2015016544 A1. Google Patents; 2015.), but the production of fructose-6-phosphate from fructose requires ATP to phosphorylate fructose as a substrate , leading to high production cost of tagatose, which is not suitable for large-scale production

Method used

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Examples

Experimental program
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experiment example 1

[0060] Experimental example 1 in vitro multi-enzyme catalyzed conversion of starch into tagatose

[0061] Starch was converted into tagatose by an in vitro multi-enzyme catalytic system ( figure 1 ). These key enzymes include: (1) α-glucan phosphorylase (αGP, EC 2.4.1.1), which releases 1-phosphate glucose from the non-reducing end of starch by adding 1 phosphate; Bitase (PGM, EC 5.4.2.2), which catalyzes glucose 1-phosphate to glucose 6-phosphate; (3) glucose phosphate isomerase, which converts glucose 6-phosphate to fructose 6-phosphate; (4) tower 6-phosphate Lattose epimerase, which converts fructose-6-phosphate to tagatose-6-phosphate; (5) 6-phosphate-tagatose phosphatase, which dephosphorylates tagatose-6-phosphate to tagatose and phosphoric acid.

[0062] In the present invention, 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 KEG...

experiment example 2

[0066] Experimental example 2 in vitro multi-enzyme catalyzed conversion of starch into tagatose

[0067] The preparation of α-glucan phosphorylase, glucose phosphomutase, glucose phosphate isomerase, 6-phosphate tagatose epimerase and 6-phosphate tagatose phosphatase was the same as in Experimental Example 1.

[0068] Contain 30mM phosphate buffer (pH 7.0) in a 0.75 milliliter reaction system, the divalent magnesium ion of 5mM, the consumption of described α-glucan phosphorylase is 10U / mL, described glucose phosphomutase The dosage is 10U / mL, the dosage of the glucose phosphate isomerase is 10U / mL, the dosage of the 6-phosphate tagatose epimerase is 10U / mL, the 6-phosphate tagatose phosphate The dosage of the enzyme is 10U / mL, 100g / L of soluble starch is used to catalyze the reaction at 20°C for 24 hours.

[0069] After the reaction, the final concentration of tagatose was 16g / L, and the conversion rate was 16%.

experiment example 3

[0070] Experimental example 3 in vitro multi-enzyme catalyzed conversion of starch into tagatose

[0071] The preparation of α-glucan phosphorylase, glucose phosphomutase, glucose phosphate isomerase, 6-phosphate tagatose epimerase and 6-phosphate tagatose phosphatase was the same as in Experimental Example 1.

[0072]Contain 30mM phosphate buffer (pH 7.0) in a 0.75 milliliter reaction system, the divalent magnesium ion of 5mM, the consumption of described α-glucan phosphorylase is 10U / mL, described glucose phosphomutase The dosage is 10U / mL, the dosage of the glucose phosphate isomerase is 10U / mL, the dosage of the 6-phosphate tagatose epimerase is 10U / mL, the 6-phosphate tagatose phosphate The amount of enzyme used is 10U / mL, 100g / L soluble starch, and the catalytic reaction is carried out at 50°C for 24 hours.

[0073] After the reaction, the final concentration of tagatose was 8g / L, and the conversion rate was 8%.

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Abstract

The invention discloses a tagatose preparation method and belongs to the field of multi-enzyme catalytic preparation of tagatose. Starch, cellulose, or their derivatives, or sucrose is used as substrate, and the substrate is efficiently catalyzed in a multi-enzyme reaction system by an in-vitro multi-enzyme molecular machine into tagatose. By adding enzymes capable of accelerating hydrolysis of starch, cellulose or sucrose and enzymes making use of the byproduct glucose, material conversion rate and tagatose yield are significantly increased. The method of the invention is high in tagatose yield, the materials are cheap, the production cost is low, and the method is suitable for industrial production of tagatose.

Description

technical field [0001] The invention relates to a method for preparing tagatose, in particular to a method for converting starch or cellulose and derivatives thereof into tagatose by multi-enzyme catalysis in vitro, and belongs to the field of enzymatic preparation of tagatose. 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...

Claims

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Application Information

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IPC IPC(8): C12P19/24C12P19/18C12P19/02
CPCC12P19/02C12P19/18C12P19/24
Inventor 马延和孙媛霞其他发明人请求不公开姓名
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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