Method for producing xylobiose, and dedicated immobilized enzyme

A technology for immobilizing enzymes and xylobiose, applied in directions such as hydrolase, bacteria, etc., which are immobilized on/in organic carriers, can solve the problems of low proportion of xylobiose, and achieve easy control and solution of product composition. Environmental problems, the effect of saving enzymes

Inactive Publication Date: 2007-02-21
CHINA AGRI UNIV
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  • Summary
  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology describes how certain types of bacteria called Bacillus spores were found to produce various compounds useful in industrial applications like medicine (chemotherapy). These microorganisms contain beta glucans made up by B-1-3 linkages between galactose molecules) along their backbone structure. By breaking down these sugars they release different substances including gamma ray absorbing agents, flavorings, antibodies, etc., making them valuable ingredients for food products. Overall, this new method provides more efficient ways to extract specific chemical components needed during fermentation processes than existing methods.

Problems solved by technology

This patented technical problem addressed in this patent relates to improving the quality and quantity of beta-galactopylactone-3 galactoperohexaursapectum polysilverasteroids containing various types of sugars called lignin. Current processes involve complicated steps like extraction, isolating specific compounds, crystallizing them separately, and combining them together again.

Method used

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  • Method for producing xylobiose, and dedicated immobilized enzyme
  • Method for producing xylobiose, and dedicated immobilized enzyme
  • Method for producing xylobiose, and dedicated immobilized enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, preparation immobilized xylanase

[0042] 1. Preparation of Eupergit C 250L-Ni immobilized XynB

[0043] 1. Preparation of XynB enzyme solution

[0044] The crude enzyme solution was obtained according to the method described in the first paragraph of Example 2, Example 3, Example 4, and Example 5 of Chinese Patent ZL 02156022.6. The specific method is as follows:

[0045] (1) Amplification of xylanase gene xynB

[0046] The primers used are TM-XynB-Nco I-FWD and TM-XynB-Hind-His-REV, and their sequences are as follows:

[0047] TM-XynB-Nco I-FWD: 5'-CCATGGAAA TATTACCTTC TGTGTGAT (SEQ ID NO: 2)

[0048] TM-XynB-Hind-His-REV: 5'-AAGCTTT CTTTCTTCTA TCTTTTTCTC CA (SEQ ID NO: 3)

[0049] According to the full sequence of the xynB gene of Thermotoga maritima MSB8DSM3109 preserved in the German Microorganism Collection Center (DSM), the above-mentioned pair of primers were designed, and the primers were introduced into the pET28a (+) (Novagen, U.S.) plasmid...

Embodiment 2

[0087] Embodiment 2, the preparation of steam explosion liquid or high temperature cooking liquid

[0088] 1. Preparation of corn cob steam explosion liquid or high temperature cooking liquid

[0089] 1. Preparation of steam explosion liquid

[0090] Weigh 100g corn cob (containing 32.4% xylan), add 800mL of distilled water according to the solid-to-liquid ratio of 1:8, soak at room temperature for 12h, put it into a high-pressure tank, cover and seal, heat, and keep the temperature at 165-170°C for 30min Immediately after opening the ball valve, the material is released into the collection tank. When the temperature of the collected material drops to 60-70°C, it is sucked and filtered, and the filtrate is the steam explosion liquid. After cooling to room temperature, determine its total sugar and reducing sugar content. Among them, the content of reducing sugar was determined by Somogyi method with D-xylose as the standard. The total sugar was determined by the Orcinol-HC...

Embodiment 3

[0095] Embodiment 3, using the immobilized xylanase of embodiment 1 to continuously hydrolyze the steam explosion liquid or high-temperature cooking liquid of embodiment 2

[0096] 1. Hydrolysis of Eupergit C 250L-Ni immobilized XynB column treated with 1M phosphate buffer Example 2 Corn cob steam explosion liquid or high temperature cooking liquid

[0097] The experiment set up 3 repetitions. Each repetition added 40g wet state (prepared by 10g Eupergit C 250L) 1M phosphate buffered Eupergit C 250L-Ni immobilized XynB to a stainless steel jacketed chromatography column with an internal diameter of 15mm, the actual height of the enzyme bed was 33cm, and the connection constant flow pump and fraction collector ( Figure 5 ), equilibrated with 50mM, pH 6.2 citrate buffer for 30min. The substrate is corn cob steam explosion liquid or high-temperature cooking liquid, wherein, the total sugar content of corn cob steam explosion liquid is 22.0 mg / mL, and the reducing sugar content...

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Abstract

This invention discloses a method for preparing xylobiose and its specific immobilization enzyme. The method comprises: (1) immobilizing recombined xylanase XynB using metal-chelating epoxy carrier Eupergit C 250 L to obtain immobilization enzyme; (2) suspending the obtained immobilization enzyme in 0.5-1.25 M phosphate buffer with a pH of 5.5-8.0, and reacting at 20-35 deg.C for 12-60 h to obtained immobilization xylanase. The recombinant xylanase XynB is prepared by expressing xylanase gene (Sequence ID 1) in Escherichia coli. The obtained xylanase has such advantages as high recovery rate; high stability and good operation performance after loaded into the column, and can be used for continuous production. The method for preparing xylobiose by using xylanase is simple, and can save xylanase usage, realize separation of enzyme and hydrolyte, largely lower production cost and improve production efficiency, thus is suitable for mass production.

Description

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Claims

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Application Information

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Owner CHINA AGRI UNIV
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