Cloning, expression and application of high temperature resistance isoamylase genes

A technology of isoamylase and high temperature resistance, which can be used in applications, genetic engineering, plant genetic improvement, etc., and can solve problems such as poor stability and heat intolerance

Inactive Publication Date: 2015-10-21
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the pullulanase or isoamylase that has been found so far has problems such as poor stability and heat resistance, which limits the application of this type of enzyme in industry

Method used

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  • Cloning, expression and application of high temperature resistance isoamylase genes
  • Cloning, expression and application of high temperature resistance isoamylase genes
  • Cloning, expression and application of high temperature resistance isoamylase genes

Examples

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Embodiment 1

[0029] The high temperature resistant isoamylase gene provided by the present invention has a DNA sequence as shown in SEQ ID NO.1, with a full length of 2151bp. The gene is obtained from the KEGG (Kyoto Encyclopedia of Genes and Genomes) database. Sulfolobus tokodaii It was obtained by screening in the genome, and its code is: ORF ST0928.

[0030] The high-temperature-resistant isoamylase encoded by the high-temperature-resistant isoamylase gene has an amino acid sequence as shown in SEQ ID NO.2, including 716 amino acids, and a molecular weight of 83.1KDa, wherein amino acids 17 to 108 of the coding sequence belong to the 48th family Carbohydrate binding module (CBM); 204-545 amino acids of the coding sequence are the catalytic domain of amylase; 546-716 of the coding sequence are unknown functional polypeptides; the enzyme was presumed to be a glycogen debranching enzyme before the application of the present invention, The present application has been verified to prove tha...

Embodiment 2

[0076] Since the main purpose of the present invention is to use the prepared high-temperature-resistant isoamylase in biofuel cells, the specific application of the high-temperature-resistant isoamylase in this example is briefly introduced as follows.

[0077] The biofuel cell used in this example is improved on the basis of the published literature method (Zhu et al. Nat. Commun., 2014, 5, 3026), and the structural principle is as follows Figure 4 shown.

[0078] The enzymatic biofuel cell designed in this embodiment is a single-pole chamber without a diaphragm type, and its structure includes: a reaction vessel, an ion exchange membrane and a reactant;

[0079] The reactants include: starch, high temperature resistant isoamylase, α-glucan phosphorylase, phosphoglucomutase, 6-phosphate glucose dehydrogenase, dihydrolipoic acid dehydrogenase, HEPES buffer, NaCl 、NAD + , Mg 2+ , Mn 2+ and electron shuttles (AQDS).

[0080] Specific physical structures such as Figure 5...

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Abstract

The invention belongs to the technical field of genetic engineering and biological energy sources, and particularly relates to cloning and expression of high temperature resistance isoamylase genes and application in enzymatic biofuel cells. The DNA sequence of amylase is shown in the SEQ ID NO.1, and the total length of the DNA sequence is 2151bp. The sequence of high temperature resistance isoamylase amino acids is shown in the SEQ ID NO.2 and includes 716 amino acids, the molecular weight is 83.1KDa, and the amino acids with the coded sequences being 17-108 are carbohydrate combined module order belonging to the 48th family; the amino acids with the coded sequences being 204-545 are the amylase catalytic structural domain. The high temperature resistance isoamylase can be used for enzymatic biofuel cells. The high temperature resistance isoamylase has the beneficial effects that the isoamylase is resistant to high temperature and stable in activity, and has the strong industrial application potential; and when the isoamylase is used for a sugar fuel cell, the starch substances can be completely hydrolyzed into glucose, so that the energy conversion efficiency is improved, and the application range of the biofuel cells is enlarged.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and bioenergy, and specifically relates to the cloning and expression of a high-temperature-resistant isoamylase gene and its application in enzyme biofuel cells. Background technique [0002] Biofuel cells are a type of green fuel cells that use organic matter as raw materials to convert chemical energy into electrical energy by using cells or enzymes. According to the type of catalyst, it can be divided into: microbial fuel cell and enzyme biofuel cell. They have the advantages of high energy conversion efficiency, good biocompatibility, wide source of raw materials, low cost and environmental protection, etc. They are widely used in medical, electronic, environmental protection and other fields important use value. However, most of the current biofuel cells have low output power density, which greatly limits their application in production. Microbial fuel cells are less efficient...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/44C12N15/70H01M8/16
CPCY02E60/50Y02P70/50
Inventor 陈红歌杨森程琨王明道张以恒
Owner HENAN AGRICULTURAL UNIVERSITY
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