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Poria cocos cellulose endonuclease gene as well as expression vector and protein thereof

A cellulose endonuclease and Poria cocos technology is applied in genetic engineering, plant genetic improvement, introduction of foreign genetic material using vectors, etc. It can solve the problems of low protein yield, inactivation of target products, troublesome purification steps, etc. Biological activity, mass production, and load reduction effects

Active Publication Date: 2019-12-13
HUNAN BUTIAN PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the application numbers 201811240264.9 and 201811281667.8, pET28 and pET32 were used as vectors to express and purify endocellulase in the Escherichia coli system to obtain a certain amount of recombinant protein with good activity, but the proteins expressed by the above two vectors were both Intracellular protein, the bacteria need to be broken first when purifying, the purification steps are cumbersome and the recovery rate is low
Moreover, expression in E. coli may cause toxicity due to the presence of LPS, so it is often necessary to analyze and determine the toxicity of the expressed purified product
Finally, obtaining high-purity proteins often requires multi-step purification operations. The more purification steps, the lower the protein yield and the more likely it will lead to the inactivation of the target product

Method used

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  • Poria cocos cellulose endonuclease gene as well as expression vector and protein thereof
  • Poria cocos cellulose endonuclease gene as well as expression vector and protein thereof
  • Poria cocos cellulose endonuclease gene as well as expression vector and protein thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] This example provides an optimized artificially synthesized Poria cocos endocellulase gene with a 6×His tag at the C-terminus. The specific sequence is shown in SEQ ID No.1 in the sequence listing, and the gene corresponds to The protein sequence is shown as SEQ ID No.2 in the sequence listing. The optimized DNA sequences were compared by NCBI, and there was no obvious similarity.

[0058] The methanolic yeast expression system is the most widely used yeast expression system, and the exogenous gene expression system using Pichia pastoris (Pichia Pasteur yeast) as the host has developed the most rapidly in recent years and is also the most widely used. Yeast is a single-cell lower eukaryote. Compared with insect expression system and mammalian expression system, yeast expression system has the advantages of both. At the same time, yeast expression system has the characteristics of simple operation, low cost, and large-scale fermentation. Therefore, it is an ideal prepar...

Embodiment 2

[0061] The present embodiment provides a kind of method for preparing Poria cocos endocellulase protein, specifically comprises the following steps:

[0062] S1: Construction of expression vector and transformation: link the sequence characteristics of the gene itself in Example 1 and the DNA sequence synthesized by yeast codon preference, that is, the DNA shown in SEQ ID No.1, to the Pichia pastoris-inducible secretory expression vector pPICZαA, Obtain the recombinant vector pPICZαA-Poria cocos cellulase, the vector construction is as follows figure 1 as shown, figure 1 It is a schematic diagram of the construction of the eukaryotic expression vector pPICZαA-Poria cocos endocellulase in the embodiment of the present invention. The main vector construction steps are preferably as follows:

[0063] (1) Use Xho I and Xba I to double digest the artificially synthesized plasmid containing the synthetic Poria cocos cellulase gene to obtain the target fragment. The reaction system...

Embodiment 3

[0103] In this embodiment, the enzyme activity of the purified soluble poria cocos endocellulase is detected, and the specific steps and results are as follows:

[0104] The present invention uses the glucose hexokinase (HK) method to measure the ability of poria cocos cellulose endoenzyme to hydrolyze carboxymethylcellulose sodium (CMC-Na) to produce glucose, and hexokinase catalyzes glucose (D-Glucose) in the presence of ATP Phosphorylate it to generate glucose-6 phosphate (G-6-P), G-6-P and coenzyme NAD generate NADH and 6-phosphogluconic acid under the action of glucose-6-phosphate dehydrogenase, which can be measured by spectrophotometry The absorbance change of NADH at 340nm wavelength was determined by the method, and the concentration of glucose in the sample was quantitatively detected. The specific steps and results are as follows: 2μl of purified Poria cocos cellulose with a concentration of 1mg / mL was added to 98μl of 1% CMC-Na Sodium dihydrogen phosphate and citri...

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Abstract

The invention relates to an artificially synthetic gene for coding poria cocos cellulose endonuclease. The gene at least contains a DNA segment of one of the following nucleotide sequence: 1) a nucleotide sequence in SEQ ID NO.1 in a sequence table; and 2) a nucleotide sequence with 90% homology or above and coding the same biological functional protein with the nucleotide sequence in SEQ ID NO.1.Reconstruction of recombinant vectors and yeast conversion are further performed according to the gene sequence, secretary expression of the recombinant poria cocos cellulose endonuclease under induction of methanol can be realized, purification is performed by ammonium sulfate precipitation and a DEAE ion exchange column, the recombinant poria cocos cellulose endonuclease with purity higher than95% can be obtained, the enzyme has very high capacity for degrading cellulose components and producing glucose, and recombinase with high activity can be provided for cellulose decomposition and application to production of feed, textile, food and biological energy sources.

Description

technical field [0001] The invention belongs to the technical field of biomolecular cloning, and relates to a gene encoding poria cocos endocellulose and its expression carrier and protein. Background technique [0002] Cellulose is a polysaccharide composed of glucose with β-1,4 glycosidic bonds, and is an important component of plant cell walls. Cellulose has the most content in plants, accounting for about 1 / 2 of the dry weight of plants. It is the largest renewable natural resource in nature. It is a long-chain macromolecule composed of 2000-10000 glucose molecules. Cellulose is a very abundant resource in nature, such as hemp, wheat straw, straw, bagasse, etc., are all rich sources of cellulose. At present, the utilization rate of cellulose is still very low, how to improve the utilization rate of cellulose is still a world-class topic. The efficient utilization of cellulose is inseparable from the enzymes related to the degradation of cellulose. Cellulase complex is...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/42C12N15/81C12R1/84
CPCC12N9/2437C12N15/815C12Y302/01004
Inventor 李洪波胡兴
Owner HUNAN BUTIAN PHARMA
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