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Construction and expression of AusMan5A-TviCBM fusion enzyme gene

A technology for fusing enzymes and gene fragments, which is applied in the fields of genetic engineering and protein expression, can solve the problems of unreported research on design and expression, and achieve the effects of large-scale industrial production, high catalytic efficiency, and strong substrate affinity

Inactive Publication Date: 2012-06-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, although there are some literatures and patent reports on the cloning and expression of β-mannanase gene, the molecular transformation of mannanase based on rational design, especially the research on the design and expression of chimeric enzyme gene no reports

Method used

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  • Construction and expression of AusMan5A-TviCBM fusion enzyme gene
  • Construction and expression of AusMan5A-TviCBM fusion enzyme gene
  • Construction and expression of AusMan5A-TviCBM fusion enzyme gene

Examples

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Effect test

Embodiment 1

[0026] Example 1 Construction of chimeric enzyme gene and its expression plasmid

[0027]The fusion gene man5A-cbm was constructed by overlapping PCR technology, which was mainly divided into four steps: the first round of PCR was performed with AMan5A-F and AMan5A-R as primers (94°C for 4 min; 30 cycles, 94°C for 30s, 53°C for 30s, 72 ℃1min; 72℃10min) to synthesize the gene fragment of Aspergillus ussami Aus Man5A to be fused; use Tman5A-F1 and Tman5A-R as primers for the second round of PCR (94℃4min; 30 cycles, 94℃30s, 53℃30s , 72 °C for 1 min; 72 °C for 10 min) to synthesize the gene fragment of the CBM to be fused; the two rounds of PCR products were analyzed by 1% agarose gel electrophoresis, and the target bands were recovered by tapping the gel. The third round of PCR was performed at 94°C for 4 min; 10 cycles of 94°C for 30s, 52°C for 30s, 72°C for 70s; 72°C for 10min); the third round of PCR reaction solution was used as the template, AMan5A-F and Tman5A-R were used a...

Embodiment 2

[0028] Example 2 Construction, expression, product purification and activity assay of GS115 / man5A-cbm

[0029] The pPIC9K-man5A-cbm was linearized with Sal I, and electro-transformed and screened according to the Pichia expression manual to obtain a high-copy Pichia recombinant GS115 / man5A-cbm. The genetically engineered bacteria were induced to express with 1.0% methanol for 96 hours, and the mannanase activity in the fermentation broth was measured by DNS method to reach 65 IU / mL. The supernatant of the fermentation broth after centrifugation is the crude enzyme solution of the chimeric enzyme. The crude enzyme solution is concentrated by an ultrafiltration membrane with a molecular weight cut-off of 10,000 Da, and then subjected to DEAE-Sepharose Fast Flow ion exchange chromatography and Sephadex G-75 It was purified by gel filtration chromatography and detected as a single band by SDS-PAGE after purification.

[0030]

[0031]

[0032]

[0033]

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Abstract

The invention provides a method for designing and constructing AusMan5A-TviCBM fusion enzyme gene, wherein the AusMan5A-TviCBM fusion enzyme is strong in substrate affinity and high in catalyzing efficiency. The nucleotide sequence of the AusMan5A-TviCBM fusion enzyme is SEQ ID NO.1, the amino acid sequence thereof is SEQ ID NO.2, and corresponding genes thereof are named man5A-cbm. The invention further discloses a construction and a high-efficient expression and purifying method of engineering bacteria of fusion enzyme. The prepared fusion enzyme has the characteristics of strong substrate affinity and high catalyzing efficiency and has greater industrialized production protentials and economic values.

Description

technical field [0001] The invention relates to the fields of genetic engineering and protein expression, in particular to the construction and expression of the AusMan5A-TviCBM fusion enzyme gene, and the research on the properties and applications of the enzyme. Background technique [0002] β-mannanase (β-1,4-D-mannan mannohydrolase, EC 3.2.1.78) is an enzyme capable of degrading β-1,4-mannoside from the interior of homogeneous and isomannan backbones The hydrolases of bonds belong to the class of hemicellulases, which are widely present in microorganisms, plants and animals. It can be widely used in food, medicine, feed, paper, printing and dyeing and petroleum industries. In the field of food and medicine, β-mannanase can produce functional oligosaccharides after acting on mannans. This oligosaccharide can effectively reduce the level of blood sugar and cholesterol in humans and is beneficial to the probiotics in the human and animal intestines. The growth of the grou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N1/19C12N15/81C12R1/84
Inventor 邬敏辰唐存多李剑芳陈忠法赵顺阁
Owner JIANGNAN UNIV
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