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Preparation method and application of co-immobilized double enzymes

A technology of phosphorylase and mutase, applied in the field of enzyme engineering, to achieve the effects of simple preparation process, cost saving and good storage stability

Pending Publication Date: 2021-04-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the research on immobilizing α-glucan phosphorylase and phosphoglucomutase and using it to prepare G6P has not been solved yet. If a method for immobilizing the two enzymes more efficiently can be found, it will be useful for the preparation of G6P. of great benefit

Method used

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  • Preparation method and application of co-immobilized double enzymes
  • Preparation method and application of co-immobilized double enzymes
  • Preparation method and application of co-immobilized double enzymes

Examples

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Embodiment 1

[0024] Example 1: Preparation of α-glucan phosphorylase and phosphoglucomutase

[0025] Construction of recombinant bacteria: the nucleotide sequence of the α-glucan phosphorylase coding gene shown in SEQ ID NO.1 and the coding gene of phosphoglucan mutase shown in SEQ ID NO.2 are connected respectively To the pET-22b(+) vector, it was transformed into E.coli BL21 host cells, and recombinant bacteria expressing the corresponding enzymes were constructed.

[0026] The recombinant bacteria were inoculated into the seed medium containing 50 μM ampicillin, and cultured at a constant temperature in a shaker at 37°C and 200r / min for 12 hours, and then inoculated into the fermentation medium with an inoculation amount of 2%, and shaken at 37°C and 200r / min. Cultivate at constant temperature in the bed until the absorbance of the bacterial solution at 600nm reaches 0.6–0.8. Then add 0.5mM IPTG for induction for 6h, collect the fermentation broth and centrifuge at 4°C, 8000r / min for 1...

Embodiment 2

[0027] Embodiment 2: Enzyme activity assay of α-glucan phosphorylase and phosphoglucomutase

[0028] (1) α-glucan phosphorylase enzyme activity assay

[0029] The activity of α-glucan phosphorylase was determined by double enzyme coupling method. The total volume of the enzyme reaction is 1 mL, and the substrate is maltodextrin. 1mL phosphate buffer (50mM, pH 7.0) contains 5g / L maltodextrin, 5mM MgCl 2 and 1U / mL phosphoglucan mutase, add 10 μg α-glucan phosphorylase before the reaction starts, and react at 70°C for 10 minutes. Centrifuge at 12 000r / min for 5min. The detection method of product 6-phosphate glucose (G6P) is as follows: take 0.1mL reaction liquid and mix with 3mL HEPES buffer ((100mM, pH 7.0), contain 2mM MgCl in the buffer 2 , 0.2mM NAD + And 0.7U glucose 6-phosphate dehydrogenase (G6PDH), reacted at 30°C for 10min, and measured the absorbance at 340nm.

[0030] (2) Determination of phosphoglucomutase enzyme activity

[0031] The activity determination of...

Embodiment 3

[0034] Example 3: Preparation of metal organic framework material ZIF-8

[0035] Add 5 mL of zinc acetate solution with a concentration of 20 mM into an equal volume of 2-methylimidazole solution with a concentration of 80 mM, and stir for 30 min at a constant speed on a magnetic stirrer with a rotation speed of 200 r / min at 25 °C. After standing for 1 h, centrifuge at 8 000 r / min for 15 min, and wash the collected precipitate with deionized water. The obtained samples were freeze-dried to obtain ZIF-8 nanocomposites, and the morphology of ZIF-8 was observed with a cold field emission scanning electron microscope. The result is as figure 1 As shown, ZIF-8 has a typical rhombic dodecahedral structure with an average diameter of about 300nm.

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Abstract

The invention discloses a preparation method and application of co-immobilized double enzymes, and belongs to the field of enzyme engineering. After the co-immobilized alpha-glucan phosphorylase and phosphoglucomutase provided by the invention are repeatedly operated for 8 times, the activity of the co-immobilized alpha-glucan phosphorylase and phosphoglucomutase is still maintained to be about 60.99% of the original activity; and moreover, after the co-immobilized enzymes are stored at 4 DEG C for 25 days, the relative enzyme activity of the co-immobilized enzymes is maintained to be 80% or above, and the relative enzyme activity of free enzymes is reduced to 50% or below. Therefore, the obtained co-immobilized alpha-glucan phosphorylase and phosphoglucomutase have important industrial application value and scientific research value.

Description

technical field [0001] The invention relates to a preparation method and application of co-immobilized double enzymes, belonging to the field of enzyme engineering. Background technique [0002] Glucose 6-phosphate (G6P) serves as a useful biosynthetic intermediate involved in glycolysis, starch and glycogen biosynthesis pathways. After being catalyzed by glucose 6-phosphate isomerase, G6P can be converted into fructose 6-phosphate, which is an important precursor for the production of oligosaccharides such as D-Tagatose. [0003] D-tagatose is a natural and rare hexose sugar found only in small amounts in fruit, cocoa and dairy products. D-tagatose has many health benefits, for example, it has the function of lowering blood sugar and anti-cancer, which helps to prevent cardiovascular disease, stroke and other vascular diseases; and its sweetness is similar to sucrose but the calories are three times that of sucrose One of them, so it can be used as a potential functional ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14C12P19/24C12P19/02
CPCC12N11/14C12N9/1051C12N9/90C12P19/24C12P19/02C12Y204/01001C12Y504/02002
Inventor 江波张金秀张涛陈静静代艺伟
Owner JIANGNAN UNIV
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