Preparation method of hybrid xylanase atxb

A technology of xylanase and xylan, applied in the direction of microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve problems such as hindering the hydrolysis of enzyme molecules, and achieve the effect of strong binding ability

Inactive Publication Date: 2012-09-19
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

The xylan-binding domain of F / 10 family xylanases can bind insoluble xylan and microcrystalline cellulose, and its function is similar to the cellulose-binding domain of cellulase, but most of the G / 11 family xylanases It does not have a xylan binding domain, so when hydrolyzing natural substrates (such as wheat bran, rice and straw, etc.), there is a steric hindrance between the enzyme molecule and cellulose and lignin, which hinders the hydrolysis of the enzyme molecule ( Sunna et al., 2000)

Method used

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  • Preparation method of hybrid xylanase atxb
  • Preparation method of hybrid xylanase atxb
  • Preparation method of hybrid xylanase atxb

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: Construction of hybrid xylanase gene atxb

[0053] 1. Preparation of materials:

[0054] Genetic material: hybrid xylanase gene atx and Thermomonospora variegata xylanase A gene, located in pBS-T / atx vector and pBS-T / tfx vector respectively.

[0055] Bacterial species: Escherichia coli TOP10F' was purchased from Invitrogen. Shuttle vector: pPIC9K, purchased from Invitrogen Company, expression host bacteria: Pichia pastoris GS115, purchased from Invitrogen Company.

[0056] High-fidelity DNA polymerase, blunt-ended DNA fragment plus A kit: purchased from Shanghai Sangon Company, and various restriction enzymes and T-vectors were purchased from Promega Company.

[0057] Chemical reagents: Yeast extract and tryptone were purchased from OXOID Company, tissue culture reagents and birch xylan were purchased from Sigma Company. All other chemical reagents were of domestic analytical grade. Primer synthesis: Shanghai Sangon Company or Shanghai Boya Biotechno...

Embodiment 2

[0079] Example 2: Expression of hybrid xylanase gene atxb in Pichia pastoris

[0080] 1. Construction of pPIC9K-atxb

[0081] After sequencing verification, the pBS-T / atxb vector was EcoR 、Not Double enzyme digestion and rubber tapping to recover the target band. The recovered atxb was ligated with pPIC9K, which had also undergone double enzyme digestion, and transformed into Escherichia coli TOP10F'competent cells, picked a single colony on the LB plate containing kanamycin and cultured, and tested by PCR ( Figure 4 ) and double enzyme digestion identification ( Figure 5 ) to obtain a recombinant transformant named TOP10F' / pPIC9K-atxb, and the obtained recombinant expression plasmid was named pPIC9K-atxb.

[0082] 1.1 Double digestion of pPIC9K vector

[0083] Enzyme cutting system:

[0084] Buffer Y + / Tanqo with BSA 10× (purchased from TaKaRa Company) 2.0 μl Eco R (10 U / μl) 1.0 μl not (10 U / μl) 1.0 μl pPIC9K vec...

Embodiment 3

[0145] Example 3: Detection of Yeast Positive Transformants

[0146] 1. Genome extraction of yeast transformants

[0147] Take 1.5 ml yeast transformant liquid, centrifuge at 5000 rpm at 4°C for 5 min, and discard the supernatant;

[0148] Add 500 μl extract solution, and pipette fully to suspend;

[0149] Add 100 μl 10% SDS solution and 300 μl benzyl chloride, shake vigorously;

[0150] Incubate at 50°C for 1 hour, shake and mix once every 10 minutes;

[0151] Add 300 μl 3M NaAC (pH5.2), ice bath for 15 min;

[0152]Centrifuge at 10,000 rpm for 15 min at 4°C, and take the supernatant;

[0153] Add twice the volume of ice-cooled absolute ethanol, and precipitate for 20 min;

[0154] Centrifuge at 10,000 rpm at room temperature for 15 min, discard the supernatant, and wash once with 70% ethanol;

[0155] Naturally dry, add 100 μl TE buffer to dissolve, that is, the genome sample, and store at -20°C.

[0156] 2. PCR detection

[0157] Using the extracted yeast recombinan...

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Abstract

The invention provides a preparation method of hybrid xylanase atxb. The method enables connecting sequence of brown high temperature single spore bacterium xylanase A and a xylan binding domain to be fused at the tail end C of hybrid xylanase atx to obtain the hybrid xylanase atxb. Birchwood xylan is hydrolyzed by hybrid enzyme to obtain xylo-oligosaccharide mainly, main products are xylbiose and xylotriose, the xylbiose and the xylotriose are main components of functional xylo-oligosaccharide, and prebiotic effect of the functional xylo-oligosaccharide is remarkably higher than other xylo-oligosaccharide. In addition, a hybrid gene expression adopts a pichia pastoris expression system, and the system has the advantages of being safe, free of toxic metabolite secretion and easy to culture. Compared with an escherichia coli expression system and an animal cell expression system, the pichia pastoris expression system has remarkable advantages so as to enable fermentation production of the hybrid xylanase atxb to have remarkable advantages.

Description

[0001] technical field [0002] The invention relates to a method for transforming enzyme molecules by using genetic engineering or protein engineering technology in the field of genetic engineering to obtain a recombinant enzyme gene with strong substrate binding ability and good hydrolysis performance, and to produce recombinant xylanase. [0003] Background technique [0004] Xylanase has a wide range of industrial applications (Subramaniyan S et al . 2002). The application of xylanase in the feed industry can improve the nutritional value of feed (especially bran) and promote animal growth; in the paper and pulp industry, the use of xylanase can reduce the use of chemical bleaching agents, It is beneficial to environmental protection; in the food industry, xylanase can be used in bread making, fruit juice clarification, brewing industry and preparation of functional xylooligosaccharides. However, in these practical applications, the substrate of xylanase is not a sing...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/81C12R1/84
Inventor 翁晓燕刘明启詹仪花孙建义
Owner ZHEJIANG UNIV
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