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Immobilization method of sucrose isomerase

A sucrose isomerase, sucrose technology, applied in the directions of isomerase, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of complex methods, high cost of carriers, and difficulty in large-scale industrial application.

Active Publication Date: 2017-02-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the methods of these immobilization methods are complicated and the cost of the carrier is high, so it is difficult for large-scale industrial application

Method used

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  • Immobilization method of sucrose isomerase
  • Immobilization method of sucrose isomerase
  • Immobilization method of sucrose isomerase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 B.brevis / pNY 326-pal I LSP build

[0035] At the 5' and 3' ends of the sucrose isomerase gene (pal I) (see SEQ ID NO.1 for the gene sequence), the restriction sites Nde I and Hind III were designed and introduced, and finally provided by Shanghai Jierui Bioengineering Co., Ltd. Synthesized to obtain pUC57-palI.

[0036] The plasmid pUC57-palI carrying pal I and the expression vector pET-24a(+) were digested with restriction endonucleases NdeI and Hind III respectively, and the target fragment was recovered and ligated to obtain the recombinant plasmid pET-24a-palI.

[0037]Using pET-24a(+)-pal I as a template, design the PCR primers shown in Table 1G1374A-For and G1374A-Rev, and use one-step PCR to perform site-directed mutation at the G1347 site of the sucrose isomerase gene, and remove the inner part of pal I by mutation The pst I site; then use primers P-For, P-Rev PCR amplification to obtain the target gene fragment palI with Pst I and Hind III restricti...

Embodiment 2

[0041] The impact of embodiment 2 shake flask culture medium on fermented liquid enzyme activity

[0042] Bacillus pumilus B.brevis / pNY 326-pal I LSP As the starting strain, after cultured in the seed medium, it was inserted into different shake flask medium:

[0043] (1) Single nitrogen source

[0044] With glucose as the carbon source, industrial yeast powder, industrial peptone, soybean peptone, beef extract, casein, cottonseed powder cake, poly-peptone, tryptone, beef peptone, angel peptone, etc. were used as nitrogen on TM medium. source, the content of each nitrogen source is 15g L -1 , the inoculum size was 1%, and the enzyme activity was determined after culturing on a shaker at 200r / min and 30°C for 48h.

[0045] (2) Compound nitrogen source

[0046] On the basis of a single nitrogen source, two better nitrogen sources were selected, mixed and prepared according to a certain ratio, the inoculum amount was 1%, and the enzyme activity was measured after culturing on...

Embodiment 3

[0060] Embodiment 3 Preparation of immobilized sucrose isomerase

[0061] (1) Bacillus pumilus (B.brevis / pNY 326-palI LSP ) is the starting strain, and the fermentation medium finally determined in Example 2 is used to ferment and prepare the sucrose isomerase enzyme liquid. After the fermentation is completed, the bacteria are removed by centrifugation to obtain the enzyme liquid.

[0062] (2) Using 4% acetic acid solution as a solvent, prepare a chitosan colloid solution with a final concentration of 2%-5%, and vacuumize to remove air bubbles.

[0063] (3) Slowly drop the fully dissolved chitosan colloid solution into 4M sodium hydroxide solution with a syringe, stir at a constant speed, and form chitosan microspheres with a diameter of about 1.5 mm. After the microspheres are completely formed, pour off the hydroxide Sodium solution, the prepared microspheres were washed with deionized water until neutral.

[0064] (4) Add the chitosan microspheres that have been washed t...

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Abstract

The invention discloses an immobilization method of sucrose isomerase, and belongs to the technical field of bioengineering. According to the method, recombinant bacillus brevis is taken as production strain to produce the sucrose isomerase; then immobilization is performed; the enzyme activity is 35.2 U / g after immobilization; the enzymatic activity recovery reaches 70.3 percent; the optimum temperature of immobilized enzyme is 40 DEG C (30 DEG C for free enzyme); and the optimum pH is 4.5 (6.0 for free enzyme); the fact indicates that the immobilized enzyme has better temperature and pH tolerance. Sucrose concentration at 600g*L <1> is taken as a substrate, and the maximum product yield can reach 87.8 percent. After conversion is continuously performed for 16 times under the optimum conversion condition, the product yield is still 87.52 percent, and the fact indicates that the immobilized enzyme has good operation stability and high isomaltulose synthesis capability. The immobilization method is simple to operate and low in cost, and provides a reference for industrial application.

Description

technical field [0001] The invention relates to a method for immobilizing sucrose isomerase, which belongs to the technical field of bioengineering. Background technique [0002] Sucrose isomerase (EC 5.4.99.11), also known as isomaltulose synthase or α-glucosyltransferase and trehalulose synthase, can catalyze sucrose to generate isomaltulose (6-O-α-D -glucopyranosyl-D-fructose) and trehalulose (1-O-α-D-glucopyranosyl-D-fructose), are the key enzymes for the production of isomaltulose by biotransformation. [0003] Due to the shortcomings of liquid enzyme preparations that cannot be reused, sucrose isomerase can be immobilized by physical and chemical methods to make it highly active, continuous, and automatic, and the product is easy to follow-up separation and purification, thereby improving product quality. , reduce production costs, and vigorously promote the production of isomaltulose. There are few studies on the immobilization of sucrose isomerase. Fabiano Jares C...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75C12N11/10C12N9/90C12P19/24C12P19/12C12R1/08
Inventor 吴敬陈晟段绪果耿梦华
Owner JIANGNAN UNIV
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