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Xylose enzyme method preparing method

A technology of enzymatic preparation and xylanase, applied in the field of enzymatic preparation of xylose and xylose preparation, can solve the problems of increasing environmental protection burden, slow growth, low concentration of substrate action, etc., and achieve important economic benefits and Social benefits, mild reaction conditions, the effect of maintaining natural quality

Inactive Publication Date: 2010-06-30
NANJING NORMAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

[0005] But the method for acid hydrolysis has multiple deficiencies, which are mainly reflected in: 1. In the crop wastes, not only cellulose, pentosan, lignin and other useful components, but also protein, starch, fat, Pectin, pigments, metal ions, inorganic salts, tannins, chemical fertilizers and pesticides, etc., cannot be removed by the existing utilization methods, and these main components are hydrolyzed together with useless impurities during high temperature and strong acid hydrolysis , will produce or release a large amount of substances that are toxic to yeast, mainly including furfural (degradation by-product), acetic acid (released by acetylated xylan), certain lignin derivatives (such as phenolic compounds), heavy metal ions, etc.
This not only causes great pollution to the environment, but also requires detoxification of the hemicellulose hydrolyzate before it is used in microbial fermentation production
2. Although dilute acid hydrolysis can reduce the poison content, the hydrolyzate often contains a large amount of non-fermentable oligosaccharides, which ultimately affects the yield of xylose and xylitol
At the same time, low-temperature hydrolysis method is adopted, not only the hydrolysis effect is poor and the speed is slow, but also high-concentration acid solution must be added to carry out hydrolysis, which brings about the problem of acid solution in the hydrolysis solution, so neutralization treatment must be carried out after hydrolysis , increasing the cost
3. Due to the many impurities in the sugar solution of acid hydrolysis, the post-treatment efficiency is low, the chemical procedures are many, the requirements for equipment are high, and the environmental protection burden is increased.
[0006] Although enzymatic hydrolysis has the advantages of high efficiency, specificity, and environmental protection, many strains with compound enzymes capable of degrading xylan hemicellulose in nature are often suitable for environments with poor nutrition and grow slowly. The cell density is low, which cannot meet the production needs; at the same time, the concentration of substrates in the enzymatic hydrolysis of xylan is low, and the efficiency and cost caused by easy pollution are low. Therefore, enzymatic hydrolysis has not been used in the production of xylose so far.

Method used

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  • Xylose enzyme method preparing method
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  • Xylose enzyme method preparing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] (1) Preparation of heat-resistant xylan degrading enzyme

[0033] Primers were designed according to the gene sequences of xylanase, xylosidase, arabinosidase and α-glucuronidase in Genbank,

[0034]Tm-xynB-N: 5'-CCGTTCCATGGACTACAGGATGTGC-3'

[0035] Tm-xynB-C: 5'-CCGCTCGAGCGGATATATCTTTTCTTCCCTT-3'

[0036] Tm-xyl-N: 5'-CATGCCATGGAACTGTACAGGGATC-3'

[0037] Tm-xyl-C: 5'-ATAAGAATGCGGCCGCCTCCTCGCAGGCTTCC-3'

[0038] Tm-aguA-N: 5'-GGAATTCCATATGAAAATATTACCTTCTGTGTTGAT-3'

[0039] Tm-aguA-C: 5'-CCC TCGAGTCTTTTCTTCTATCTTTTTCTCAG-3'

[0040] The genomic DNA of Thmotiga maritima was used as template for PCR amplification.

[0041] After inserting the PCR products of xylanase, xylose / arabinosidase and α-glucuronidase into plasmids pET-20b and pET-28a, respectively, the recombinant expression plasmids pET-20b-xynB, pET-28a-xyl and pET-28a-aguA.

[0042] 0.01 μg of recombinant expression plasmids pET-20b-xynB, pET-28a-xyl and pET-28a-aguA were electrotransformed into 50 μL ...

Embodiment 2

[0050] Embodiment 2: substantially the same as Example 1, the difference is that the xylan used is oat xylan, purchased from Sigma Company, and the ratio of xylan and multi-enzyme solution is 1: 10 (w / v), The obtained enzymolysis solution adopts HPLC detection to show that xylose content is more than twice as high as using xylanase and xylose / arabinosidase (see attached Figure 5 and Figure 4 Compare).

Embodiment 3

[0051] Embodiment 3: basically the same as Example 1, the difference is that the pretreated corn cob is used as raw material, and the specific operations are as follows:

[0052] (1) Raw material pretreatment

[0053] Take the air-dried corn cobs with a chopped diameter of about 3-20mm, soak them in clean water and then centrifuge to remove the water, add sodium hydroxide solution according to the solid-to-liquid ratio of 7-10:1, and the final concentration is 1%-3%. Or extract at high temperature, then collect the filtrate and neutralize it to slightly acidic with acid, and then use 3 times the volume of ethanol to precipitate to obtain xylan.

[0054] (2) Adding xylanase, xylose / arabinosidase and α-glucuronidase to the buffer in sequence at an addition ratio of 1:1:0.75 to prepare a multi-enzyme mixture, wherein the amount of xylanase 20U / g, xylose / arabinosidase 20U / g and α-glucuronidase 15U / g, the ratio of xylan to multi-enzyme solution is 1:4 (w / v).

[0055] (3) While st...

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Abstract

This invention involves a kind of enzymatic production method of xylose. The specific steps are pre-treat wasted crop to produce xylan or steam exploded substance; then add xylanase, bi-activity xylose / arabinosidase and alpha-glucuronidase to buffer to generate multi- enzyme mix liquid. Mixing it and adding the xylan or steam exploded substance to multi- enzyme mix liquid to perform enzymatic hydrolysis. After hydrolysis, adds ethanol to enzymatic hydrolysis liquid and further condese and crystallize to get xylose product. The release rate of xylose can reach as much as two times of using Xylanase and xylosidase so to increase the productivity. The power consuming is low. The product can keep the natural quality. At the same time the xylose produced with enzyme has no harm to microbial species, and can be used directly in biotransformation of xylitol by yeast. It is virus free and with good fermentation performance. It is benefical for the transformation from xylose to xylitol.

Description

technical field [0001] The invention relates to a preparation method of xylose, in particular to an enzymatic preparation method of xylose, and relates to the fields of food industry, clean production, enzymology, molecular biology and the like. Background technique [0002] Xylose is an aldose with five carbon atoms (CH 2 OH(CHOH) 3 CHO), white crystalline powder, sweet taste, melting point 144°C, soluble in water. As a food additive, xylose is mainly used as a sweetener for diabetics, and industrially as an auxiliary agent in printing and dyeing and leather making. With the wide application of xylitol as a therapeutic agent for diabetic patients, and the development of xylitol as a sweetener for anti-cavity chewing gum, candy, beverages, etc., the preparation technology of xylose as a raw material for xylitol production has also received much attention. There is widespread concern. [0003] Crop waste mainly includes straw, corn cob, bagasse, wheat bran, rice bran, sug...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/02
Inventor 邵蔚蓝薛业敏吴爱莲曾红艳
Owner NANJING NORMAL UNIVERSITY
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