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Heat-resisting beta-1, 3-1, 4-dextranase and encoding gene thereof

A coding gene and gene technology, applied in the field of β-1, can solve the problems of large differences in glucanase activity and achieve the effects of high expression level, heat resistance characteristics of expression level, and great potential for popularization and application

Inactive Publication Date: 2010-07-14
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

From the analysis of patents published at home and abroad, there are few patents on the fungal β-1,3-1,4-glucanase gene, and the glucanase activity in different expression systems is also quite different

Method used

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  • Heat-resisting beta-1, 3-1, 4-dextranase and encoding gene thereof
  • Heat-resisting beta-1, 3-1, 4-dextranase and encoding gene thereof
  • Heat-resisting beta-1, 3-1, 4-dextranase and encoding gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1, Discovery of β-1,3-1,4-glucanase gene

[0043] 1. PCR amplification of the conserved region of the glucanase gene genome

[0044] With the genomic DNA of Paecilomyces thermophila J18 (CAS Microbiology Institute Culture Collection Center, preservation number CGMCC 6885) as a template, according to the amino acid sequence of the filamentous fungal β-glucanase reported by GeneBank, Using the online software Block Maker( http: / / blocks.fhcrc.org / blocks / blockmkr / make blocks.html ) to search for the conserved region, and then use the online primer design software CODEHOP (http: / / blocks.fhcrc.org / codehop.html) to design the degenerate primer PtLic16CP1 (forward): 5′-CGGCGAGATCGACATCATHGARGGNGT-3′, PtLic16CP2 (reverse) : 5'-GCCCAGTCGCGCARAANGTNRRTC-3'. PCR reaction mixture (50 μL): water 37.25 μL; 10×Ex Taq buffer, 2.5 μL; 2.5 mmol / L dNTPs, 4 μL; primers (10 μmol / L), each 2.5 μL; DNA template (50-100ng), 1 μL; Taq (5U / μL), 0.25 μL. PCR reaction conditions: 94°C f...

Embodiment 2

[0047] Embodiment 2, expression of recombinant β-1,3-1,4-glucanase gene

[0048] 1. Construction of recombinant bacteria

[0049] The complete open reading frame (ORF) encoded protein sequence was analyzed by SignalP 3.0, and there was a signal peptide with a possible cleavage site between amino acids 18 and 19 (AAA-YH). Based on this, primers were designed to remove the stop codon of the original gene. The two pairs of primers were: PtLic16ASnaBIF: 5′-TACGTATATCATCTTGTTGACGACTACG-3′ (including SnaBI restriction site); PtLic16AAvr II R: 5′- CCTAGG TTAGGGTGCGTACACGCGGAG-3' (containing the Avr II restriction site) was amplified by PCR using the above primers and the cDNA of Paecilomyces thermophila (Paecilomyces thermophila) J18 as a template. Agarose gel electrophoresis of PCR amplification products figure 2 After being recovered by the gel kit, it was cloned into the pMD-18T vector by TA cloning, transformed into Escherichia coli JM109 by heat shock method, and the recomb...

Embodiment 3

[0074] Embodiment 3, beta-1,3-1, the property of 4-glucanase

[0075] 1. Optimal pH of β-1,3-1,4-glucanase

[0076] The purified protein in Example 2 was dissolved in 4 different 50mM buffer systems (Citric-Na 2 HPO 4 , pH 2.5-5.5; MES, pH 5.0-6.5; phosphate buffer, pH 6.5-8.5; Glycine-NaOH, pH 8.5-11.0), and then measure the enzyme activity at 70°C (the enzyme activity determination method is the same as step 2) , taking the highest point of enzyme activity as 100% for plotting. The results show that the optimum pH of recombinant β-1,3-1,4-glucanase is 7.0 ( Figure 6 ).

[0077] 2. Optimum reaction temperature of β-1,3-1,4-glucanase

[0078] The purified protein in Example 2 was appropriately diluted in 50mM MES buffer solution with pH 7.0, and then the activity of β-1,3-1,4-glucanase was determined according to the above method at different temperatures of 40-100°C. Enzyme activity (the enzyme activity assay method is the same as step 2). The highest point of enzyme ...

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Abstract

The invention discloses a beta-1, 3-1, 4-dextranase and an encoding gene thereof. The beta-1, 3-1, 4-dextranase provided by the invention is protein in 1 or 2 or 3 as follows: 1. the protein which is formed from an amino acid sequence shown in 19th-314th bit of a sequence 2 in a sequence table; 2. the protein which is formed from the amino acid sequence shown in the sequence 2 in the sequence table; and 3. the protein which is formed from an amino acid residue sequence of the sequence 2 in the sequence table through the substitution and / or deletion and / or addition of one or more amino acid residues, has the activity of the beta-1, 3-1, 4-dextranase and is derived from 1. A recombined pichia formed by leading the encoding gene of the protein into pichia also belongs to the protecting range of the invention. Experiments prove that the highest enzyme activity of a recombined pichia fermented liquid supernatant can be 55,300U / mL, and crude protein is 9.1g / L. The optimum reaction temperature of the beta-1, 3-1, 4-dextranase of the invention is 70 DEG C, and the optimum pH value is 7.0. The beta-1, 3-1, 4-dextranase has great generalization and application potential in production.

Description

technical field [0001] The present invention relates to beta-1,3-1,4-glucanase and its coding gene and high-efficiency expression. Background technique [0002] Dextran is a class of polymers linked by glucose through different glycosidic bonds, and is the most abundant polysaccharide in nature. β-1,3-1,4-D-glucan is a naturally synthesized polysaccharide, which belongs to the soluble structural non-starch in plant cell walls together with arabinoxylan, pentosan, cellulose, and pectin polysaccharides. Most grains contain β-1,3-1,4-D-glucan, and the highest content in barley is 5-8%, which mainly exists in the cell wall of barley milk. β-1,3-1,4-D-glucan is a linear structure formed by glucose in the β configuration through β-1,3- and β-1,4-mixed bonds, containing about 70% β- 1,4-bond and 30% β-1,3-bond, their molecular weight and configuration vary with barley varieties, natural environment, growth stage and other factors. [0003] According to the specificity of hydrol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/63C12N15/81C12N5/10C12N1/19C12R1/84
Inventor 闫巧娟华承伟江正强唐艳斌李一男
Owner CHINA AGRI UNIV
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