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Beta-xylosidase and application thereof

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: 2017-09-22
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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  • Beta-xylosidase and application thereof
  • Beta-xylosidase and application thereof
  • Beta-xylosidase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1. Obtainment of β-xylosidase CsXyl39A and CsXyl39B and their amino acid sequences

[0061] The genomic DNA of Caldicellulosiruptor saccharolyticus (ATCC 43494 / DSM8903) was extracted and used as a template. PCR amplification was carried out 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. PCR amplification was performed 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 μl PCR amplification system includes 1 μl genomic DNA (15 ng / μl), 25 μl 2×EASYPfu SuperMix, 1 μl Primer (10 micromolar) and 22 microliters of sterilized ultrapure water. The PCR reaction conditions were: 95°C pre-denaturation for 5 minutes; 94°C denaturation for 30 seconds, 55°C annealing for 30 seconds, 72°C extension for 3.5 minutes...

Embodiment 2

[0067] Example 2. Determination 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 (pH 5.0), add 80 microliters of pNP-X (10 mmol / L) and 20 microliters of β-xylosidase CsXyl39A obtained in Example 1 above in different concentrations. Or CsXyl39B, after incubating in a water bath (75°C) for 5 minutes, add 100 μl of 2 mol / L sodium carbonate solution to stop the reaction. The reaction solution was then put into a spectrophotometer, and the absorbance value was measured at 405 nm (see image 3 ). 1 millimole of p-nitrophenol is released from the substrate by hydrolyzing the substrate per minute, which is defined as 1 unit of enzyme activity.

[0069] Measurement result: the result of enzyme hydrolysis activity is as image 3 It is shown that the specific enzyme activity of β-xylosidase CsXyl39A and CsXyl39B to pNP-X is 180 enzyme activity units / mg ...

Embodiment 3

[0070] Example 3. The effect of organic solvents on the hydrolytic activity of β-xylosidase CsXyl39A:

[0071] The hydrolytic activity of β-xylosidase was measured with pNP-X as a substrate. Add organic solvents (organic solvents of methanol, ethanol, isopropanol, n-butanol, acetone or acetonitrile) to make the final concentration of the system into the hydrolysis reaction system. The reaction system is in 100 microliters of sodium citrate buffer (pH 5.0), add 80 microliters of 10 mmol / L pNP-X, then add 20 microliters 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 moles / liter). The reaction solution is then put into a spectrophotometer, and the absorbance value is measured at 405 nm (see Figure 4 ). The hydrolysis release of 1 millimole of p-nitrophenol from the substrate per minute is defined as 1 unit of enzyme activity. The...

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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 present invention relates to the field of biotechnology, in particular to two β-xylosidases from the pyrocellulose bacterium Caldicellulosiruptorsaccharolyticus and their application in biotransformation of natural compounds containing xylose groups. 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 (compared to water, many organic compounds are more soluble in organic solvents). Therefore, the tolerance of organic solvents is whether enzyme preparations can be industrialized. Important parameters and limitations of the application. β-xylosidase is an enzyme that can hydrolyze xylo-oligosaccharides and other xylo-oligosaccharides from the non-reducing end or catalyze the hydrolysis of organic compounds containing xylosides, and the hydrolyzed product is mainly xylose. At present, most β-xylosidase enzymes have poor tolerance to...

Claims

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Application Information

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Patent Type & Authority Applications(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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