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Extremely-heat-resistant beta-mannosidase gene as well as expression protein and application thereof

A mannosidase, gene expression technology, applied in application, genetic engineering, plant genetic improvement and other directions, to achieve the effects of strong thermal stability, strong heat resistance, and efficient enzymatic hydrolysis ability

Inactive Publication Date: 2013-03-20
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the β-mannosidases reported in the world are mainly derived from some fungi (such as Aspergillus niger) and some normal temperature and mesophilic bacteria, while β-mannosidases from extremely thermotolerant bacteria and archaea are rarely reported.

Method used

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  • Extremely-heat-resistant beta-mannosidase gene as well as expression protein and application thereof
  • Extremely-heat-resistant beta-mannosidase gene as well as expression protein and application thereof
  • Extremely-heat-resistant beta-mannosidase gene as well as expression protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Mannosidase gene Tth man B preparation of

[0025] It can be prepared artificially as follows: Tth man B genes, can also be Thermotoga thermarum The total DNA of DSM5069 (DSMz, German) was used as a template to obtain by conventional PCR (primers man1 and 2 in Example 2 were used as primers).

[0026] The mannosidase gene of the present embodiment Tth man B Synthesized by Shanghai Jierui Bioengineering Co., Ltd., the gene sequence is shown in SEQ NO:1.

Embodiment 2

[0027] Example 2 Mannosidase gene Tth man B subclones of

[0028] Use the following primers to PCR amplify the mannosidase gene among the embodiment 1:

[0029] man1: 5'-GGAATTCCATATGGATTTCCTGCTGGGTATTAACT-3';

[0030] man2: 5'-CCGCTCGAGGAAGTTCAGCAGCTTATACTCTTTC-3'.

[0031] When the above primers were synthesized, man1 introduced the Nde I restriction site, and man2 introduced the Xho I restriction site.

[0032] PCR reaction system: 1μL T. thermarum Synthetic DNA, 1 μL man1, 1 μL man2, 25 μL Premix ExTaq, 22 μL ultrapure water.

[0033] PCR reaction conditions: denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 52°C for 30 sec, extension at 72°C for 3 min, 30 cycles; extension at 72°C for 10 min; incubation at 4°C.

[0034] The yield and specificity of the PCR products were detected by 1% agarose gel electrophoresis, and purified with a PCR product recovery kit (BIOMIGA, Shanghai).

Embodiment 3

[0035] Example 3 Recombinant cloning, expression vector pET-20b- Tth man B build and verify

[0036] The purified PCR product (prepared in Example 2) and pET-20b (Novagen) were double-digested with Nde I and Xho I respectively, and agarose electrophoresis was used to recover the enzyme-digested PCR and large vector fragments. After the gel-tapping recovery, the target fragment and the carrier were concentrated and resuspended in 8 μL sterile water, 1 μL 10× Ligase Buffer and 1 μL Ligase were added, and ligated overnight at 16°C. Transform Escherichia coli pET-20b with the ligation reaction product, and then spread it on a petri dish containing 100 μg / mL Amp (ampicillin), and incubate at 37°C for 10-12h.

[0037] Pick multiple single colonies from the transformation plate, and use BIOMIGA's plasmid mini-extraction kit to extract the plasmid. The obtained plasmid was verified by double enzyme digestion and the obtained recombinant plasmid was sequenced. The sequencing result...

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Abstract

The invention discloses an extremely-heat-resistant beta-mannosidase gene as well as an expression protein and an application thereof, wherein the DNA (deoxyribonucleic acid) sequence of the extremely-heat-resistant beta-mannosidase gene is as shown in SEQ NO: 1, and the amino acid sequence of the extremely-heat-resistant beta-mannosidase expressed by the extremely-heat-resistant beta-mannosidase gene is as shown in SEQ NO: 2. The extremely-heat-resistant beta-mannosidase has extremely strong heat-resistant performance and the characteristic of high activity in a neutral pH condition, and the enzymatic activity is the highest and the specific enzyme activity achieves 144.6 U / mg in the conditions of 85 DEG C and a pH of 5.5; and the protease has a high enzyme activity at a temperature ranging from 70 to 95 DEG C and a pH ranging from 5.0 to 7.5. The mannosidase is extremely high in heat-resistant performance, and the residual enzyme activity is about 70% in the case that the mannosidase is heat-insulated for 2h at 80 DEG C. Via the characteristics aforementioned, the mannosidase expressed by the extremely-heat-resistant beta-mannosidase gene disclosed by the invention has more advantages than the existing mannosidase, is suitable for degradation for hemicellulose in the conditions of a high temperature of greater than 80 DEG C and a slightly acidic pH, and has a potential industrial application value.

Description

technical field [0001] The invention belongs to the field of genetic engineering technology and biomass utilization, and specifically relates to an extremely resistant β-mannosidase gene, its expressed protein and its application. Background technique [0002] Polyglucomannan is another important hemicellulose besides xylan plant polysaccharides. Widely present in plants such as coconut, konjac flour, seaweed and coniferous wood. Mannanase and mannosidase are the most critical hydrolytic enzymes in the polyglucomannose hemicellulase system. The former hydrolyzes mannan into manna by randomly cutting the β-1,4-glucosidic bond of mannan Oligosaccharides, which degrade mannan oligosaccharides or mannans with a certain degree of polymerization into mannose mannose, are an important glyconutrient, widely distributed in body fluids and tissues, and play an important role in regulating the immune system and increasing wound healing It plays an important role in physiological regu...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/42C12N15/63C12N1/21C12N15/11C12P19/14C12P19/02C12R1/01
Inventor 王飞时号黄颖娟李迅邓若冰
Owner NANJING FORESTRY UNIV
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