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Low-temperature beta-xylosidase mutant with improved thermal stability and specific activity and encoding gene and application thereof

A technology of xylosidase and thermal stability, applied in the field of genetic engineering and enzyme engineering, can solve the problems of poor thermal stability of low-temperature xylosidase, unfavorable application of wild-type low-temperature β-xylosidase, and low specific activity

Active Publication Date: 2020-01-17
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, due to the poor thermal stability and low specific activity of low-temperature xylosidase, the remaining activity of the wild-type strain after treatment at 40°C for 1 hour is only 1.79%, and the specific activity is 4.85U / mg. Around 50°C, which is not conducive to the application of this wild-type low-temperature β-xylosidase

Method used

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  • Low-temperature beta-xylosidase mutant with improved thermal stability and specific activity and encoding gene and application thereof
  • Low-temperature beta-xylosidase mutant with improved thermal stability and specific activity and encoding gene and application thereof
  • Low-temperature beta-xylosidase mutant with improved thermal stability and specific activity and encoding gene and application thereof

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Experimental program
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Effect test

Embodiment 1

[0090] Cloning of embodiment 1 mutant enzyme and wild enzyme expression gene

[0091] The present invention uses the low-temperature xylosidase AX543 as the parent, adopts the method of Overlap-PCR, uses the primers in Table 1 to mutate and express the low-temperature xylosidase AX543, and its gene sequence is as shown in SEQ ID NO.4 / SEQ ID NO.5 / SEQ ID NO.6 shown.

[0092] Table 1. PCR-specific primers for β-xylosidase mutants G110S, Q201R and loop2

[0093]

Embodiment 2

[0094] Expression and purification of embodiment 2 mutant enzyme and wild enzyme in Pichia pastoris

[0095] The PCR products of β-xylosidase mutants G110S, Q201R, and loop2 and the expression vector pPIC9 were subjected to double enzyme digestion (EcoRI+XholⅠ), and the cut gene fragments were connected to the vector to obtain a low-temperature protein with high thermal stability and specific activity. The recombinant plasmids of β-xylosidase mutants G110S, Q201R and loop2 were linearized and transformed into Pichia pastoris GS115 competent cells by electric shock to obtain recombinant yeast strains GS115 / G110S, GS115 / Q201R and GS115 / loop2.

[0096] Pick the monoclonal colony on the MD plate, and use a toothpick stained with bacteria to spot it on another numbered MD plate and a 96-well plate containing 1mL of BMGY medium, respectively. While culturing the MD plate at 30°C, Cultivate a 96-well plate containing 1mL of BMGY medium at 30°C and 220r / min for 48h; after 48h of cultu...

Embodiment 3

[0098] Activity Analysis of Embodiment 3 Mutant Enzyme and Wild Enzyme

[0099] One enzyme activity unit (U) is defined as the amount of enzyme required to decompose the substrate pNPX to produce 1 μmol p-nitrophenol (pNP) per minute under certain reaction conditions.

[0100] Determination of β-xylosidase activity: Add 150 μL citric acid-disodium hydrogen phosphate buffer solution to 250 μL substrate pNPX, add 100 μL enzyme solution (2U / ml) after preheating at 20°C for 2 minutes, react at 20°C for 10 minutes, add 1.5mL, 1mol / L Na 2 CO 3 Solution termination reaction, OD 405 Measure its absorbance.

[0101] Optimum temperature for β-xylosidase: 2mM pNPX as substrate, 150μL 0.2mol / L citric acid-disodium hydrogen phosphate buffer (pH6.0) was added to 250μL substrate, and the substrate and buffer The mixture was preheated in a water bath at 0°C-50°C for 2 minutes, then 100 μL of enzyme solution (2U / ml) was added, reacted at 0°C-50°C for 10 minutes, and 1.5 mL of 1M Na 2 CO ...

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Abstract

The invention relates to a low-temperature beta-xylosidase mutant with improved thermal stability and specific activity. The mutant is a low-temperature beta-xylosidase mutant G110S, and an amino acidsequence of the mutant is SEQ ID NO.1; alternatively, the mutant is a low-temperature beta-xylosidase mutant Q201R, and an amino acid sequence of the mutant is SEQ ID NO.2; and alternatively, the mutant is a low-temperature beta-xylosidase mutant loop2, and an amino acid sequence of the mutant is SEQ ID NO.3. The low-temperature beta-xylosidase mutant with improved thermal stability and specificactivity is more suitable for industrial applications, and has wider application prospects in the fields of food, feed, papermaking and the like, so that the application potential of low-temperature beta-xylosidase is expanded in food, pharmaceutical, papermaking, feed and other fields.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and enzyme engineering, in particular to a low-temperature beta-xylosidase mutant with improved thermal stability and specific activity, its coding gene and its application. Background technique [0002] Xylan is widely distributed and is the main component of hemicellulose in plant cell walls. Xylan is a kind of heterogeneous polysaccharide, the main chain is formed by β-1,4-glucosidic bonds linking xylose units, its structure is complex, and the degradation process requires a variety of glycoside hydrolases to complete; among them, β-xyloside enzyme and α-L-arabinosidase are essential for the complete degradation of xylan. [0003] β-Xylosidase (EC 3.2.1.37) is an exoglycosidase, which has the ability to hydrolyze the non-reducing end of xylan oligosaccharides into xylose. It mainly catalyzes alkyl and aryl glycosides and Hydrolyze xylobiose and xylooligosaccharides above xylobiose...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/2434C12N15/815C12Y302/01037
Inventor 李中媛张同存刘仲琦李爽赵军旗
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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