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Preparation method for L-gulose

A technology of gulose and oxidase, which is applied in the field of rare sugar biosynthesis, can solve the problem of low production efficiency of L-gulose, and achieve the effect of high-efficiency green biosynthesis, advanced technology, and easy industrialization

Inactive Publication Date: 2018-04-20
CHINA PHARM UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although Woodyer et al. utilize NAD + Dependent mannose dehydrogenase and sorbitol can realize the biosynthesis of L-gulose, but there is still room for improvement and improvement in its synthesis efficiency
NAD + The dependent mannose dehydrogenase will generate co-product NADH in the process of catalyzing the oxidation of sorbitol, and a large amount of accumulated NADH will form a product inhibitory to mannose dehydrogenase, which will gradually reduce the production efficiency of L-gulose

Method used

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  • Preparation method for L-gulose
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  • Preparation method for L-gulose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1 Mannitol dehydrogenase and NADH oxidase activity assay method

[0019] Mannitol dehydrogenase activity assay: 0.6 μmol NAD in 1 mL reaction system + , 0.60 μmol of D-sorbitol, 0.1M Tris-HCL (pH 8.0) and 10 μl of crude enzyme solution. The absorbance change of the reaction system at a wavelength of 340 nm was detected within 1 min at 32°C. One mannitol dehydrogenase enzyme activity unit is defined as the number of micromoles of NADH produced per minute at 32°C, and the specific activity of the enzyme is defined as the number of activity units per mg of total protein.

[0020] Determination of NADH oxidase activity: 0.6 μmol NADH, 0.1M Tris-HCL (pH 8.0) (air saturated) and 10 μl crude enzyme solution were contained in 1 mL reaction system. The absorbance change of the reaction system at a wavelength of 340 nm was detected within 1 min at 32°C. One NADH oxidase enzyme activity unit is defined as the number of micromoles of NADH consumed per minute at 32°C, ...

Embodiment 2

[0021] Example 2 HPLC detection method of L-gulose (PMP derivatization method)

[0022] Take 50 μL of L-gulose standard solution or diluted reaction solution and place it in a centrifuge tube, add an equal volume of 0.6M sodium hydroxide solution, add 100 μL of 0.5M PMP methanol solution, and react at 70°C for half an hour. Take it out and let it cool naturally, add 50 μL of 0.6M hydrochloric acid to neutralize the reaction, add 1 mL of sterile water to mix and dilute. Add 0.8 mL of chloroform, shake and mix well, centrifuge at 12,000 rpm, take the water layer, filter with a 0.22 μm filter membrane, and perform HPLC analysis.

[0023] HPLC analysis method:

[0024] Chromatographic column: YMC C18 column, 150mm×4.6mm, 5μm

[0025] Mobile phase: A: ultrapure water (containing 0.1% formic acid) B: acetonitrile (containing 0.1% formic acid)

[0026] Gradient: 20% B isocratic elution for 20min

[0027] Column temperature: 40°C; Flow rate: 1ml / min; Wavelength: 245nm;

[0028] I...

Embodiment 3

[0030]Example 3 Construction of heterologous expression strains of mannitol dehydrogenase and NADH oxidase

[0031] Use Nco I and EcoR I to insert the mannitol dehydrogenase gene mdh into the multiple cloning site I of pACYCDuet-1, and Nde I and Xho I to insert the NADH oxidase gene nox into the multiple cloning site II to obtain the recombinant plasmid pACYCDuet-mdh- nox. Similarly, use Nco I and EcoR I to insert nox into multiple cloning site I, and use Nde I and Xho I to insert mdh into multiple cloning site II to obtain pACYCDuet-nox-mdh. On this basis, the two plasmids were transformed into Escherichia coli BL 21 to obtain recombinant expression strains E.coli (pACYCDuet-mdh-nox) (pMN for short) and E.coli (pACYCDuet-nox-mdh) (pNM ).

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Abstract

L-gulose is a rare aldohexose, which can be used as a synthetic precursor of vitamin C and a drug intermediate for the synthesis of nucleoside antiviral drugs, with high potential economic value. In view of the shortcomings of the existing gulose synthesis process, the present invention co-expressed NAD+-dependent mannitol dehydrogenase and NADH oxidase in Escherichia coli to construct a whole-cell biocatalyst. A green biosynthetic process for the preparation of L-gulose.

Description

technical field [0001] The invention belongs to the technical field of rare sugar biosynthesis, and specifically relates to the preparation of rare aldohexose L-gulose by co-expressing mannitol dehydrogenase and NADH oxidase Escherichia coli cells to catalyze the oxidation of sorbitol. Background technique [0002] Rare sugars are monosaccharides and their derivatives that exist in nature but are low in content. Although rare sugars are rare and difficult to obtain, different types of rare sugars have different physiological functions and can be used in food, medicine, nutrition, In many fields such as pesticides and fine chemicals, the annual global transaction volume exceeds several tons (Poonperm W, et al. J. Biosci. Bioeng., 2007, 103: 282-285; TB, et al.J.Biosci.Bioeng., 2004,97:89-94.), wherein rare sugars and their nucleosides have shown considerable application prospects in the research and development of anti-tumor drugs and anti-viruses (Li Z, et al. Beilstein. J...

Claims

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Application Information

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IPC IPC(8): C12P19/02
CPCC12P19/02
Inventor 吴旭日卞柳云修琪昆陈依军
Owner CHINA PHARM UNIV
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