High xylose tolerance bifunctional hemicellulose degrading enzyme, its coding gene and its preparation method

A hemicellulose and dual-function technology, applied in the field of genetic engineering, can solve the problems of low tolerance to xylose and difficulty in meeting industrial needs, and achieve the effects of reducing anti-nutritional effects, increasing fragrance, and increasing concentration

Inactive Publication Date: 2018-09-25
YUNNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This enzyme not only has the functions of arabinosidase and xylosidase, but also can maintain a high activity in a low temperature environment, but the enzyme's xylose tolerance is small, and its environmental adaptability is still difficult to meet the needs of existing industries

Method used

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  • High xylose tolerance bifunctional hemicellulose degrading enzyme, its coding gene and its preparation method
  • High xylose tolerance bifunctional hemicellulose degrading enzyme, its coding gene and its preparation method
  • High xylose tolerance bifunctional hemicellulose degrading enzyme, its coding gene and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Cloning of the gene XylRBM26

[0048] Extraction of Massilia sp.RBM26 genomic DNA: Centrifuge the bacterial liquid from the liquid culture for 2 days, add 1 mL of lysozyme, treat at 37 °C for 1 h, and then add lysate. The lysate consists of: 50mM Tris, 20mM EDTA, NaCl 500mM, 2 % SDS (w / v), pH 8.0, lysed in a water bath at 70°C for 1 h, mixed every 10 min, and centrifuged at 10,000 rpm for 5 min at 4°C. The supernatant was extracted with phenol / chloroform to remove impurity proteins, and then the supernatant was added with an equal volume of isopropanol, and after standing at room temperature for 5 min, centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was discarded, the precipitate was washed twice with 70% ethanol, dried under vacuum, dissolved in an appropriate amount of TE, and placed at -20°C for later use.

[0049]The genome of 5 μg of Massilia sp.RBM26 was fragmented into fragments of 400-600bp using the ultrasonic fragmentation instrument B...

Embodiment 2

[0051] Example 2: Preparation of recombinant bifunctional hemicellulose degrading enzyme XylRBM26

[0052] Primers for amplifying mature peptides were designed according to the sequence analysis of the bifunctional hemicellulose degrading enzyme gene XylRBM26:

[0053] XylRBM26F: ATGATCCACAACCCGATCCTGC

[0054] XylRBM26R: CAGCCCGGCTGAGGTAGGGCC

[0055] Using the genome of the strain Massilia sp.RBM26 as a template, the above-mentioned primers were used to amplify the target gene by PCR. PCR reaction parameters were pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30S, annealing at 72°C for 30S, extension at 72°C for 1min30S, a total of 20 cycles; denaturation at 94°C for 30S, annealing at 52°C for 30S, extension at 72°C for 1min30S, a total of 10 cycles; After amplification at 72°C, the extension was performed for 7 min; the temperature decreased by 1°C in each cycle from 72°C to 52°C.

[0056] The bifunctional hemicellulose degrading enzyme gene XylRBM26 was co...

Embodiment 3

[0058] Example 3 Characterization of the purified recombinant bifunctional hemicellulose degrading enzyme XylRBM26

[0059] 1. Activity analysis of purified recombinant bifunctional hemicellulose-degrading enzyme XylRBM26

[0060] The activity assay method of the recombinant bifunctional hemicellulose degrading enzyme XylRBM26 adopts the pNPX method: pNPX is dissolved in 0.1M buffer to make the final concentration 2mM. 450μL of 2mM substrate; after preheating at the reaction temperature for 5min, add 50μL of appropriately diluted enzyme solution, after 10min of reaction, add 2mL of 1M Na 2 CO 3 The reaction was terminated, and the released pNP was measured at a wavelength of 405 nm after cooling to room temperature; 1 unit of enzyme activity (U) was defined as the amount of enzyme required to decompose pNPX to generate 1 μmol of pNP per minute (the method for measuring the enzyme activity of pNP substrates is the same as that of pNPX). ). The activity of the substrates oat ...

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Abstract

The invention discloses a high-xylose-tolerance difunctional hemicellulolytic enzyme and a preparation method and application thereof. The amino acid encoding sequence of the enzyme is shown as SEQ ID NO.2 and comprises 542 amino acids, and the theoretical molecular weight of the enzyme is 61.85 kDa. The high-xylose-tolerance difunctional hemicellulolytic enzyme (XylRBM26) has the activity of beta-D-xylosidase and the activity of alpha-L-Arabinfuranosidease, the higher activity (the Ki value is 500 mM) can be kept in a high-xylose environment, the optimum pH is 6.5, and the optimum temperature is 50 DEG C; 95% or above of the activity still can be kept after the enzyme tolerates the condition at 45 DEG C for 60 min, and xylobiose, xylotriose and xylotetraose can be thoroughly hydrolyzed into xylose. The enzyme can serve as a novel enzymic preparation and can be widely applied to food, feed, energy industry and the like.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a bifunctional hemicellulose degrading enzyme with high xylose tolerance, its coding gene and its preparation method. Background technique [0002] Xylan is the main component of plant hemicellulose, and xylose is the final product of its complete degradation. The completion of the entire hydrolysis process requires the synergy of xylanases. Xylanase mainly includes: endo-1, 4-β-D-xylanase (endo-l, 4-β-D-xylanohydrolase, EC 3.2.1.8), which is mainly the main agent for randomly cutting xylan The chain generates xylooligosaccharides of different lengths; β-xylosidase (β-D-xylosidase, EC 3.2.1.37), acts on oligosaccharides and xylobiose to degrade them into xylose; and a-D-glucuronic acid glycosidase (a-D-glucatronidase, EC 3.2.1.139), acetylxylan esterase (acetylxylan esterase, EC 3.1.1.72), a-L-arabinofuranosidase (a-L-arabinofuranosidase, EC 3.2.1.55), fe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N1/21C12N1/15C12N1/19A23L29/00A23K20/189
CPCC12N9/2402C12N9/248C12Y302/01037C12Y302/01088
Inventor 许波戴利铭黄遵锡李俊俊唐湘华杨云娟
Owner YUNNAN NORMAL UNIV
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