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Cellulose excision enzyme artificial synthesis gene and protein and recombinant vector thereof

A recombinant vector, artificial synthesis technology, applied in the direction of vector, nucleic acid vector, genetic engineering, etc., can solve the problems of low recovery rate and troublesome purification steps, and achieve the effect of preventing degradation, realizing mass production, and maintaining natural activity.

Active Publication Date: 2019-12-20
HUAIHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the application number CN201811238622.2, pET32 was used as the expression vector and BL21 was used as the expression strain to express and purify exocellulase to obtain a certain amount of recombinant protein with good activity, but the proteins expressed by the above vectors were all intracellular For protein, it is necessary to crush the bacteria first when purifying, the purification steps are cumbersome and the recovery rate is low

Method used

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  • Cellulose excision enzyme artificial synthesis gene and protein and recombinant vector thereof
  • Cellulose excision enzyme artificial synthesis gene and protein and recombinant vector thereof
  • Cellulose excision enzyme artificial synthesis gene and protein and recombinant vector thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] This example provides an optimized artificially synthesized exocellulase gene with a 6×His tag at the C-terminal, the specific sequence of which is shown in SEQ ID No.1 in the sequence listing, and the protein corresponding to the gene The 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.

[0061] 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 prep...

Embodiment 2

[0064] This embodiment provides a method for preparing exocellulase protein, which specifically includes the following steps:

[0065] S1: Construction of expression vector and transformation: The sequence characteristics of the gene itself in Example 1 and the DNA sequence synthesized by yeast codon preference, that is, the DNA in SEQ ID No.1, were connected to the Pichia pastoris inducible secretory expression vector pPICZαA to obtain The recombinant vector pPICZαA-exocellulase, 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-exocellulase in the embodiment of the present invention. The main vector construction steps are preferably as follows:

[0066] (1) Use Xho I and Xba I to double digest the artificially synthesized plasmid containing the synthetic cellulase gene to obtain the target fragment. The reaction system is as follows (the exonuclease and buffer used are...

Embodiment 3

[0103] In this embodiment, the enzyme activity of the purified exocellulase 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 exocellulose to hydrolyze carboxymethylcellulose sodium (CMC-Na) to produce glucose, and hexokinase catalyzes the glucose (D-Glucose) to make It is phosphorylated 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 Measure the absorbance change of NADH at 340nm wavelength, and quantitatively detect the concentration of glucose in the sample. The specific steps and results are as follows: add 2 μl of purified exocellulase with a concentration of 1 mg / mL to 98 μl of CMC-Na containing 1% Sodium dihydrogen phosphate and citric acid buffer solution (pH=4), react at 40°C for 1 hour; take 10 μl and 2 μl...

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Abstract

The present invention relates to an artificial synthesis gene for coding cellulose excision enzymes. The gene at least contains a DNA piece with one of the following nucleotide sequences: 1) a nucleotide sequence of SEQ ID NO.1 in a sequence table; and 2) a nucleotide sequence which has more than or equal to 90% of homology with the nucleotide sequence shown in SEQ ID NO. 1 and codes a protein with a same biological function. According to the gene sequence, a recombinant vector is further constructed to transform yeasts, so that secretory expression of a recombinant cellulose excision enzyme under induction of methanol can be realized, the active recombinant cellulose excision enzyme protein with a purity higher than 95% can be obtained through nickel affinity purification, and the activeprotein has capacity of hydrolyzing cellulose and generating glucose.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and relates to an artificially synthesized exocellulase gene, its protein and a recombinant carrier. Background technique [0002] Cellulose is the most abundant natural organic matter in nature, and it can be called the largest renewable resource in the world. The annual output of crop straw alone in my country is as high as 6×10 8 ~7×10 8 tons, but these valuable cellulose resources have not been effectively utilized. Cellulose can only be utilized if it is degraded into small molecule polysaccharides or glucose. The use of cellulase produced by microorganisms to decompose cellulose is an efficient and environmentally friendly method. Cellulase belongs to the glycoside hydrolase class, which specifically catalyzes the hydrolysis of β-1,4-glucosidic bonds in cellulose chains. [0003] According to the structure of cellulase, cellulase can be divided into two categories: cellu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12N15/81C12N1/19C12R1/84
CPCC12N9/2437C12N15/815C12N2800/22C12Y302/01091
Inventor 李洪波胡兴董海丽洪樱
Owner HUAIHUA UNIV
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