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A method for immobilizing cells containing glucose isomerase

A glucose isomerase and cell technology, applied in the field of immobilization of glucose isomerase-containing cells, can solve the problems of weakened competitiveness, high production cost of HFCS, low conversion rate of HFCS, etc., and achieve satisfactory mechanical strength and immobilization method Simple and easy-to-operate effects

Active Publication Date: 2021-02-26
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

my country's immobilized GI is completely dependent on imports, resulting in high production costs of HFCS in my country and weakened competitiveness
[0006] In the production of HFCS, the conversion rate is low, and only HFCS with a fructose concentration of 42% can be obtained. The syrup with a high fructose concentration can only be separated and concentrated

Method used

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  • A method for immobilizing cells containing glucose isomerase
  • A method for immobilizing cells containing glucose isomerase
  • A method for immobilizing cells containing glucose isomerase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Construction of Recombinant Escherichia coli E.coli BL21(DE3) / pET-28b-TEGI and Preparation and Performance Measurement of Wet Cells

[0032] The recombinant Escherichia coli E. coli BL21(DE3) / pET-28b-TEGI constructed in this laboratory is used as the production strain, and the method is as follows:

[0033] (1) Construction of recombinant bacteria: The heat-resistant GI gene TEGI of Thermoanaerobacter ethanolicus (the nucleotide sequence is shown in SEQ ID NO.1, which has been disclosed in Chinese patent 201410484596) and PGEM-T vector After ligation, it was introduced into E.coli JM109, TEGI / PGEM-T and plasmid pET28b(+)-Nit were double digested, ligated overnight with ligase, and the ligation product pET28b(+)-TEGI was introduced into the host E.coli BL21( In DE3), the recombinant Escherichia coli E. coli BL21(DE3) / pET28b(+)-TEGI was obtained.

[0034] The PGEM-T carrier connection conditions are as follows: 10 μL of the connection system is added to the PCR...

Embodiment 2

[0040] (1) Take the wet cells obtained by the method in Example 1, mix them according to the wet weight of the cells: Tris-HCl buffer solution (pH=7.5, 50mM)=1:10 (m / v), then take 10g of wet cells and add Prepare 100mL bacterial suspension in 100mL Tris-HCl buffer.

[0041] (2) Add 0.6g of diatomaceous earth to the 100mL bacterial suspension in step (1), mix evenly on a magnetic stirrer, and then add 1mL of polyethyleneimine aqueous solution with a concentration of 10% (v / v) It is 25% (v / v) glutaraldehyde aqueous solution. After cross-linking at 20°C for 2 hours, the reaction solution is suction-filtered. After the filter cake is washed with distilled water, it is extruded into long strips with an axial extruder and air-dried at room temperature. into small granules (0.5-2 mm in diameter), that is, immobilized cell granules containing GI. Under the same conditions, the order of adding polyethyleneimine and glutaraldehyde was reversed, that is, glutaraldehyde was added first, ...

Embodiment 3

[0045] (1) Take the wet thalline prepared by the method of Example 1, and mix it according to the wet weight of the thalline: Tris-HCl buffer solution (pH=7.5, 50mM)=1:10 (m / v) to obtain a bacterial suspension, that is, take 10g Wet cells were added to 100 mL Tris-HCl buffer to make 100 mL bacterial suspension.

[0046] (2) Add 0.6 g of diatomaceous earth to the 100 mL bacterial suspension in step (1), mix evenly on a magnetic stirrer, then mix with 3 mL of polyethyleneimine aqueous solution with a concentration of 10% (v / v), and then add 1.5 mL of glutaraldehyde aqueous solution with a concentration of 25% (v / v), after cross-linking at 20°C for 1.5 h, the reaction solution was filtered with suction, the filter cake was washed with distilled water, extruded into long strips with an axial extruder, and air-dried at room temperature Afterwards, small particles (0.5-2 mm in particle size) were made to obtain immobilized cells.

[0047] The residual enzyme activity test method is...

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Abstract

The invention discloses a method for immobilizing cells containing glucose isomerase. The method is to add wet bacteria obtained through fermentation and culture of recombinant genetically engineered bacteria containing glucose isomerase genes into a buffer to prepare a bacterial suspension solution; add the carrier to the bacterial suspension, stir and mix, then add polyethylenimine, then add glutaraldehyde for cross-linking, stir and cross-link at 0-30°C for 1-2 hours, filter, and wash the filter cake with distilled water Extrude into long strips with an axial extruder, air-dry at room temperature, and pulverize into granules to obtain immobilized glucose isomerase cells containing glucose isomerase; the immobilization method provided by the present invention has the advantages of low cost of immobilized materials, The operation is simple, the mechanical strength is high, and it is relatively stable under high temperature conditions. The immobilized glucose isomerase-producing recombinant E. coli whole cells prepared are used to catalyze the production of D-glucose from D-glucose under high-temperature conditions. At 85°C, the conversion The yield is as high as 54%, and after repeated use for 10 batches, it still has more than 90% of the enzyme activity, and has a good industrial application prospect.

Description

[0001] (1) Technical field [0002] The invention relates to a cell immobilization method, in particular to a cell immobilization method containing glucose isomerase. [0003] (2) Background technology [0004] Glucose isomerase (Glucose isomerase, GI), also known as xylose isomerase, can catalyze D-glucose to generate D-fructose. one of the key enzymes. HFCS is a natural food additive (sweetener) widely used in food and beverage industries. Every year, there are tens of millions of tons of HFCS produced in the world. The increasing demand for HFCS and the vigorous development of the HFCS industry have promoted the in-depth research on GI and the rapid development of immobilized GI technology. He Jiaming et al. (He Jiaming et al., application number 85100412) used a macroporous strongly basic styrene-based quaternary amino anion exchange resin to adsorb enzymes in the fermentation broth to prepare immobilized GI. Cao Longkui et al. (Cao Longkui et al., application number 200...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/14C12N11/089C12N9/92C12R1/19
Inventor 金利群郑裕国郭东京柳志强
Owner ZHEJIANG UNIV OF TECH
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