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Preparation method of sucrose isomerase

A sucrose isomerase and seed technology, applied in the field of enzyme engineering, can solve the problems of water use, affecting the quality of isomaltulose products, increasing production costs and the like

Inactive Publication Date: 2014-01-08
李宪臻
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the conversion products of these sucrose isomerases also contain 2 to 7% by-products such as glucose and fructose, which seriously affect the quality of isomaltulose products, and must be separated and removed in the post-extraction process
In the production process, complex separation and purification processes such as ion exchange resins must be used to remove these impurities, resulting in a substantial increase in production costs and a large amount of water, which is an industrialization issue worth considering

Method used

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  • Preparation method of sucrose isomerase
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Examples

Experimental program
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Effect test

Embodiment 1

[0031] Pick 1 ring of bacteria from the slant of Klebsiella CCTCC M2013153 stored in the refrigerator at 4°C, inoculate it into a triangular flask containing 25 mL of seed medium, and activate it for 12 hours (30°C, 150 rpm) , according to the inoculum amount of 3%, transferred to the Erlenmeyer flask containing 50mL of fresh seed culture medium, after 4.5 hours of culture in a shaking table at 30°C (150 rpm), inoculate the above seed culture solution according to the inoculum amount of 5%. In a Erlenmeyer flask containing 250 mL of fermentation medium, shake and cultivate for 10 hours at 30° C. in a shaker at 150 rpm. Take the above fermentation broth, centrifuge at 4°C and 10,000g for 25 minutes, wash the cells with deionized water three times, and suspend in 1 / 4 of the original volume of 20mM phosphoric acid-citric acid buffer solution (pH7.0) , ultrasonically crushed in an ice-water bath for 30 minutes, centrifuged at 4°C and 10,000 g for 15 minutes, washed 3 times with wa...

Embodiment 2

[0036] According to the method described in Example 1, the cell wall fusion sucrose isomerase solution was prepared, and the ratio of the enzyme solution to the sucrose solution was 1:9, the sucrose isomerase was mixed with 20wt% sucrose solution, and placed in a 30°C water bath for 6 hours to convert sucrose to isomaltulose. Confirmation by HPLC analysis ( figure 2 ), the sucrose conversion solution contained 88.39% isomaltulose and 11.61% trehalulose.

[0037] Preparation of 20wt% sucrose solution: Weigh 20g of sucrose, dissolve it in 70mL of 20mM phosphoric acid-citric acid buffer solution (pH7.0), and make up to 100mL.

Embodiment 3

[0039] The non-ultrasonic disrupted cell suspension, cell wall fused sucrose isomerase solution, purified sucrose isomerase solution and GST fused sucrose isomerase solution were mixed with 20 wt% sucrose solution at a ratio of 1:10. After being incubated in a water bath at 30° C. for 6 hours, the reaction was stopped, and the ratios of various sugar components in the sucrose conversion liquid were analyzed by HPLC.

[0040] Table 1 The product composition of sucrose conversion catalyzed by different forms of sucrose isomerase (%)

[0041] Existing form of enzyme

[0042] Compared with the product of cell wall fusion sucrose isomerase, in the conversion product of pure enzyme, the ratio of glucose / fructose is significantly increased, and has no obvious difference from the ratio of other similar enzymes (12.5-13%). When a GST fusion protein was linked to the N-terminus of sucrose isomerase, the glucose / fructose ratio in the sucrose conversion product decreased (8.35%)...

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Abstract

The invention relates to a preparation method of a cell wall fusion sucrose isomerase, and is characterized in that ultrasonic disruption technology is employed, and a cell wall fusion enzyme with sucrose isomerase activity is prepared and used as a catalyst to convert sucrose into isomaltulose. The method comprises the following steps: 1) performing bacterial activation of sucrose isomerase producing bacteria in a seed medium; 2) transferring the activated bacteria to a fermentation medium for culture so as to produce a sucrose isomerase; 3) performing ultrasonic disruption of the sucrose isomerase producing bacteria, collecting fragmented cells to obtain a cell wall fusion sucrose isomerase. When sucrose conversion is catalyzed by the sucrose isomerase prepared in the invention, the sucrose conversion rate is 99.8 wt%, the proportion of isomaltulose in the conversion product is 88.39%, and the proportion of trehalulose is 11.61%.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering, and in particular relates to a preparation method of sucrose isomerase. Background technique [0002] The chemical name of isomaltulose is α-D-glucopyranoside-1,6-D-fructofuranose, which has similar sweetness properties to sucrose, no peculiar smell, and 50% of the sweetness of sucrose. Isomaltulose not only has non-cariogenic properties, but also can prevent the formation of insoluble glucan to prevent dental caries. Since the human body itself cannot digest isomaltulose, it can only enter the blood and be absorbed by the human body after being slowly decomposed by intestinal microorganisms. Therefore, the release rate of simple sugar in the blood after consumption is slower than that of sucrose and does not stimulate insulin secretion, so it is beneficial Diabetes prevention and treatment. Most bacteria and yeast cannot utilize isomaltulose. When isomaltulose is used in the product...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12R1/22
CPCC12N9/90C12Y504/99011
Inventor 李宪臻
Owner 李宪臻
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