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Double-enzyme catalytic system for preparing L-tagatose and application

A catalytic system and tagatose technology, applied in the field of bioengineering, can solve the problems of affecting the catalytic efficiency of enzymes, increasing the difficulty of L-tagatose separation and purification, and increasing the cost of L-tagatose synthesis, so as to reduce the difficulty of purification, Pollution reduction and cost reduction effects

Pending Publication Date: 2021-09-03
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the whole-cell method cannot effectively synthesize L-tagatose with a higher concentration of D-galactitol as a substrate, and when the concentration of local galactitol dehydrogenase in recombinant cells is high, the coenzyme NAD inside the cell + The self-regeneration process cannot effectively meet the large amount of NAD in the process of cell catalytic reaction + demand, which in turn affects the yield of L-tagatose
[0004] Based on Rhodobacter sphaericus D ( Rhodobacter sphaeroides D ) The cell-free biocatalytic method of galactitol dehydrogenase GatDH is not hindered by the bacterial cell wall, and can provide cofactors externally, which is more suitable for the large-scale production of L-tagatose, but it still needs to be continuously added during the production process expensive coenzyme NAD +
Although the above system can use lactate dehydrogenase as NAD + Regenerates the enzyme and catalyzes the conversion of NADH to NAD using lactic acid as a co-substrate + , but the addition of lactic acid and the large amount of by-product pyruvate formed in the reaction may greatly affect the catalytic efficiency of the enzyme, and it will also increase the difficulty of separation and purification of the product L-tagatose, thereby greatly increasing the synthesis cost of L-tagatose

Method used

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  • Double-enzyme catalytic system for preparing L-tagatose and application
  • Double-enzyme catalytic system for preparing L-tagatose and application
  • Double-enzyme catalytic system for preparing L-tagatose and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] In this embodiment, the water-based NADH oxidase (SMNOX) derived from the deformation Streptococcus was expressed, and the specific operation steps are as follows.

[0027] Escherichia coli BL21 (DE3) strain with SMNOX recombinant plasmid (E. coli BL21 (DE3) strain is commercialized by Shanghai Freight Biological Engineering Co., Ltd. Reference Literature LI FL, SU WB, TAO QL, et al.EXpression, Biochemical Characterization, And Mutation of a Water Formingnadh: fmn oxidoreductase from Lactobacillus Rhamnosus [J]. Enzyme Andmicrobial Technology, 2020, 134: 109464) Turning with 50 μg · ml -1 The lb medium of kanamycin was overnight, and then expanded in the same 50 μg · ml. -1 Culture in the lb medium of kanamycin. Among them, 50 μg · ml -1 The ratio of lb medium of kanamycin is sodium chloride: trypsin: yeast extract = 1: 1: 0.5, sodium chloride, trypsin and kanamycin are purchased in China Pharmaceutical Group Chemical Reagent Co., Ltd. Yeast extracts were purchased in the l...

Embodiment 2

[0034]In this embodiment, the galactosyl sugar alcohol dehydrogenase (GATDH) of spherical Slim Fungus was expressed, and the specific operation steps are as follows.

[0035] Escherichia coli BL21 (DE3) strain with GATDH recombinant plasmid (E. coli BL21 (DE3) strain is purchased in Shanghai Freight Biological Engineering Co., Ltd., the construction method refers to the literature Zhu Y, LIU CY, CAI S, ET Al. Cloning, Expression and Characterization of a Highly Active Alcohol Dehydrogenase forProduction of Ethyl (S) -4-Chloro-3-hydroxybutyrate [J] Indian Journal ofMicrobiology, 2019, 59 (2):.. 225-233) were plated in 50 μg · mL -1 The 5 ml lb medium of kanamycin was cultured overnight, which was then expanded in the same 50 μg · ml. -1 Culture is carried out in 200 ml lb medium of kanamycin.

[0036] When the bacterial liquid reached 0.9 at 600 nm, it was 0.01 mm, and the final concentration of 0.01 mm was added as an inducer, at 15 ℃ Lower induction 5 h. The high-speed refrigerat...

Embodiment 3

[0040] In this example, the bisase catalytic system is utilized to produce L-Taggosaccharide, and the L-Taggose is measured by cysteine-carbazole to produce production.

[0041] Weigh a certain amount of D-galactitol using pH 9.0 TRIS-HCl buffer to dissolve it, and configure it 100 mm. The reaction system is added to the neutralitol dehydrogenase liquid and NADH oxidase solution such that the final concentration of the latexal alcohol dehydrogenase in the reaction system is 1 U / mL, NADH oxidase liquid in the reaction system. The concentration was 2 U / mL, then 1 mm NAD + covic enzyme factor was added in the above reaction system, and its optimal reaction temperature was 35 ° C, the optimum reaction pH was 9.0, reacted at 26 h at magnetic stirring, and cysteine- Carbazole was measured to determine L-Taggosaccharide.

[0042] The L-Taggose is used to generate production and measurement methods as follows:

[0043] (1) Drawing of standard curves:

[0044] Preparation solution: For...

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Abstract

The invention provides a double-enzyme catalytic system for preparing L-tagatose and an application, and belongs to the technical field of biological engineering. In the invention, the water-producing NADH oxidase from streptococcus mutans and the galactitol dehydrogenase from rhodococcus sphaeroides are coupled to construct a multi-enzyme catalytic system for preparing the L-tagatose, and the multi-enzyme catalytic system is low in production cost, free of other byproducts and low in purification difficulty.

Description

Technical field [0001] The invention belongs to the field of biological engineering, and in particular to a bisase catalytic system for preparing L-Taggosaccharide. Background technique [0002] L-Taggosa (V-Taggosteo enantiomer) is an ideal sweetener and a flavor, which can be used as a variety of important compounds such as L-deoxide-galactamine and 1, 2 Synthetic raw materials of 3,4-di-isopropyl-furanose have broad application prospects in the food industry. However, the synthesis cost of L-Taggose is high, low yield, and it is difficult to carry out large-scale applications. [0003] At present, L-Taggose is prepared by chemical synthesis and biosynthesis. The chemical synthesis method is low, and the generated by-products and the protection of functional groups are very complicated, and it is difficult to further apply to Industrial production of L-Taggosa. The biosynthesis method mainly uses allocyte (expression of galactyl alcohol dehydrogenase) or purified galactol dehyd...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N9/02C12P19/02
CPCC12N9/0006C12N9/0036C12P19/02C12Y101/01016C12Y106/99003
Inventor 苏文彬范小蔓黄佳滢李学勇张业旺
Owner JIANGSU UNIV
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