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Method for preparing xylo-oligosaccharide and xylose with genetic engineering co-immobilized xylan degradation enzyme

A xylan degrading enzyme and genetic engineering technology, applied in the field of preparation of xylooligosaccharides and xylose, can solve the problems of limited application, weak binding force, etc., achieve good results, simplify the production process, and save production costs

Inactive Publication Date: 2012-08-15
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the currently used ChBD tag is derived from the normal temperature bacterium Bacillus circulus, which has a weak binding force to the chitin matrix under high temperature conditions, which limits its application in high temperature enzyme immobilization

Method used

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  • Method for preparing xylo-oligosaccharide and xylose with genetic engineering co-immobilized xylan degradation enzyme
  • Method for preparing xylo-oligosaccharide and xylose with genetic engineering co-immobilized xylan degradation enzyme
  • Method for preparing xylo-oligosaccharide and xylose with genetic engineering co-immobilized xylan degradation enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] (1) Construction of recombinant expression vector pET-20b-ChBD: Primer 1:

[0037] 5'-GCATCCAGGCACATACCAGCCTGGTGGGTTGGGAACCGCCGAACGTG

[0038] CCG GCACTGTGGCAGCTGCAGTGAGATCCGGCTGCTAAC-3' (SEQ ID NO. 1);

[0039] Primer 2:

[0040] 5'-ATTTGTAGGTGATACCGTTGTAGGTCACCAGATCACCGATTTTGTAGT

[0041] AG GTGTTGCTTGCCCATTCTCGAGTGCGGCCGCAA 3' (SEQ ID NO. 2);

[0042] Using the plasmid pET-20b (Novagen) as a template, use the above primers 1 and 2 (Shanghai Sangong) as primers for reverse PCR amplification, and introduce the coding when designing the primers

[0043] Whole gene of EWASNTYYKIGDLVTYNGITYKCIQAHTSLVGWEPPNVPALWQLQ fragment. Amplification conditions:

[0044]

[0045] Amplification conditions: 95°C for 5min, 30 cycles (94°C for 40s, 55°C for 40s, 72°C for 3min and 40s), 72°C for 10min. The PCR product was recovered and purified using QIAquick Gel Extraction kit (QIAGEN Company), treated with phosphorylase, and ligated overnight at 16°C with T4 DNA ligase (TAKARA C...

Embodiment 2

[0058] It is basically the same as Example 1, except that the α-glucuronidase is Bacillus stearothermophilus α-glucuronidase, and the hydrolysis temperature is 65°C.

Embodiment 3

[0060] It is basically the same as Example 1, except that the raw material pretreatment method and the usage amount of enzymes, the specific operations are as follows:

[0061](1) Weigh 10 kg of sawdust, soak in water, squeeze dry and discard the water, control the water content to 30%, then cook at a high temperature of 1.5Mpa for 10 minutes, then suddenly reduce the pressure, cool, and centrifuge to obtain the steam explosion liquid.

[0062] (2) While stirring, add the co-immobilized xylanase and α-glucuronidase solution into the steam explosion solution, the enzyme dosage is 15U / g, keep it warm at 80°C for 2 hours, and recover and immobilize by filtration after hydrolysis The enzyme is used for the preparation of xylose in the next batch. Add 4 times the volume of ethanol to the obtained filtrate and stir evenly, then centrifuge to take the supernatant, keep it at 75-80°C for evaporation and concentration, then cool to 60°C, and spray dry to obtain low Xylan products.

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Abstract

The invention discloses a method for preparing xylo-oligosaccharide and xylose with genetic engineering co-immobilized xylan degradation enzyme. The method comprises the following steps of: firstly, applying an affinity label which has high bonding strength at high temperature and is efficient and stable to the xylan degradation enzyme by a gene recombination technology and then co-expressing, and preparing the genetic engineering co-immobilized xylan degradation enzyme by specifically combining with an immobilized carrier material; and then hydrolyzing a xylan solution or a steam explosion liquid of agricultural and forestry waste by utilizing the co-immobilized xylan degradation enzyme, thereby obtaining xylo-oligosaccharide and xylose. Through the method, one-step method production of multi-enzyme preparation is realized, multi-enzyme purification and immobilization are synchronous, the separation and recycling of products and repeated using of enzyme are coupled, and the stability of enzyme to temperature and pH is improved; and the synergistic effect of the series of xylan degradation enzymes is achieved, the catalytic efficiency is high, the reaction by-products are less, the yield and the purity are high, an the used carrier is polysaccharide which is inert, cheap and easy to obtain, has good biocompatibility, and is especially suitable for the fields of food, medicine and the like.

Description

technical field [0001] The invention relates to a method for preparing xylo-oligosaccharides and xylose by using genetic engineering co-immobilized xylan degrading enzymes, in particular to using the prepared genetic engineering co-immobilized xylan degrading enzymes as a catalyst, and then acting on The xylan solution is subjected to an enzymatic hydrolysis reaction to prepare xylooligosaccharides and xylose. Background technique [0002] Xylooligosaccharides, also known as xylooligosaccharides, are composed of 2 to 7 xyloses connected by β-1,4-glycosidic bonds, mainly xylobiose and xylotriose. Various studies have shown that xylooligosaccharides, especially xylobiose, have significant bifidobacteria proliferation ability, non-digestibility, no dental caries and promote the absorption of calcium by the human body. Therefore, xylooligosaccharides are used as A new type of functional oligosaccharide has become one of the hot spots in the research and development of the food ...

Claims

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Application Information

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IPC IPC(8): C12P19/14C12P19/02C12N11/10
Inventor 薛业敏武溪溪张静静
Owner NANJING NORMAL UNIVERSITY
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