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β-Xylosidase and its application

A technology of xylosidase and xylosyl, which is applied in the application field of biotransformation and can solve the problems of large effect and poor tolerance.

Active Publication Date: 2021-01-08
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing β-xylosidases have poor tolerance to xylose, therefore, β-xylosidases with xylose tolerance play an important role in the process of degrading hemicellulose

Method used

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  • β-Xylosidase and its application
  • β-Xylosidase and its application
  • β-Xylosidase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1, Obtaining of β-xylosidases CsXyl39A and CsXyl39B and their amino acid sequences

[0061] Genomic DNA of Caldicellulosiruptor saccharolyticus (ATCC 43494 / DSM8903) was extracted and used as a template for PCR amplification with forward primer 1 and reverse primer 1 (see Table 1) to obtain β-wood The gene sequence of glycosidase CsXyl39A is shown in sequence 3. Perform PCR amplification with forward primer 2 and reverse primer 2 (see Table 1) to obtain the gene sequence of β-xylosidase CsXyl39B, as shown in sequence 4.

[0062] The PCR amplification system and conditions of the two are the same, specifically: 50 microliters of PCR amplification system includes 1 microliter of genomic DNA (15 ng / microliter), 25 microliters of 2×EASYPfu SuperMix, 1 microliter of Primers (10 micromolar) and 22 microliters of sterile ultrapure water. The PCR reaction conditions were: pre-denaturation at 95°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for...

Embodiment 2

[0067] Embodiment 2, the mensuration of β-xylosidase hydrolysis activity:

[0068] The enzyme activity was determined with p-Nitrophenyl-β-D-xylopyranoside (pNP-X) as the substrate. In 100 microliters of sodium citrate buffer solution (pH 5.0), 80 microliters of pNP-X (10 mmol / liter) and 20 microliters of different concentrations of the β-xylosidase CsXyl39A obtained in the above-mentioned Example 1 were sequentially added Or CsXyl39B, incubate in a water bath (75° C.) for 5 minutes, then add 100 microliters of 2 mol / liter sodium carbonate solution to terminate the reaction. The reaction solution was then placed in a spectrophotometer, and the absorbance was measured at 405 nm (see image 3 ). 1 millimole of p-nitrophenol is released in hydrolyzing the substrate per minute, which is defined as 1 enzyme activity unit.

[0069] Determination results: the results of the hydrolysis activity of the enzyme are as follows: image 3 It was shown that the specific enzyme activities...

Embodiment 3

[0070] Embodiment 3, the influence of organic solvent on the hydrolysis activity of β-xylosidase CsXyl39A:

[0071] The hydrolysis activity of β-xylosidase was determined with p-nitrophenol xyloside pNP-X as substrate. Add respectively the organic solvent (machine solvent is methyl alcohol, ethanol, Virahol, n-butanol, acetone or acetonitrile) that makes final concentration in the system be different content in the hydrolysis reaction system, and reaction system is in 100 microliters sodium citrate buffer solution (pH 5.0), add 80 μl of 10 mmol / L pNP-X, and then add 20 μl of CsXyl39A enzyme mixed with the above organic solvent, incubate in a water bath (75°C) for 5 minutes, and then The reaction was terminated by adding 100 microliters of sodium carbonate solution (2 mol / liter). The reaction solution was then placed in a spectrophotometer, and the absorbance was measured at 405 nm (see Figure 4 ). 1 millimole of p-nitrophenol was hydrolyzed from the substrate per minute, w...

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Abstract

The invention relates to the technical field of biology, in particular to two types of beta-xylosidase from caldicellulosiruptor saccharolyticus and application thereof in biological conversion of natural compounds containing xylosyl modification. Beta-1,4-xylosidase is beta-xylosidase CsXy139A and beta-xylosidase CsXy139B from the same gene cluster of the caldicellulosiruptor saccharolyticus. The beta-xylosidase has the advantages that proved by experiment, the CsXy139A can resist the organic solvent, and the natural compound containing the xylosyltaxol can be hydrolyzed in an organic solvent water solution with higher concentration; meanwhile, the CsXy139A is suitable for the enzyme immobilizing technique; CsXy139B has a good tolerance ability on the hydrolysis products of xylose and glucose, and the heat stability is relatively good; and the two types of beta-xylosidase can perform high-efficiency biological catalyzing on natural drugs containing xylosyl modification.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to two β-xylosidases from Caldicellulosiruptorsaccharolyticus and their application in biotransforming natural compounds containing xylosyl modifications. Background technique [0002] In the in vitro biotransformation process of organic compounds, most of the biotransformation reactions need to be carried out in organic solvents (many organic compounds are more soluble in organic solvents than water), therefore, organic solvent tolerance is a key factor for the industrialization of enzyme preparations. Important parameters and constraints of the application. β-Xylosidase is an enzyme that can hydrolyze xylooligosaccharides such as xylooligosaccharides from the non-reducing end or catalyze the hydrolysis of organic compounds containing xyloside, and the hydrolyzate is mainly xylose. Currently, most β-xylosidases have poor tolerance to organic solvents, which largely limits their applic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12N15/11C12P19/14C12P19/02
CPCC12N9/2402C12P19/02C12P19/14C12Y302/01037
Inventor 吕明李福利韩文杰阿布萨利赫
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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