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A kind of synthetic gene of endocellulase and its expression vector and protein

A cellulose endonuclease and gene technology, applied in genetic engineering, plant genetic improvement, introduction of foreign genetic material using vectors, etc., can solve the problems of low protein yield, troublesome purification steps, inactivation of target products, etc. Biological activity, enabling mass production, mitigating effects of toxic effects

Active Publication Date: 2021-02-09
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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  • A kind of synthetic gene of endocellulase and its expression vector and protein
  • A kind of synthetic gene of endocellulase and its expression vector and protein
  • A kind of synthetic gene of endocellulase and its expression vector and protein

Examples

Experimental program
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Effect test

Embodiment 1

[0063] This example provides an optimized artificially synthesized endocellulase gene with a 6×His tag at the C-terminus, 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 the sequence SEQ ID No.2 in the sequence listing. The optimized DNA sequences were compared by NCBI, and there was no obvious similarity.

[0064] 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...

Embodiment 2

[0067] This embodiment provides a method for preparing an endocellulase gene, which specifically includes the following steps:

[0068] S1: Construction of expression vector and transformation: link the DNA shown in SEQ ID No.1 in the DNA sequence list synthesized from the sequence characteristics of the gene itself and yeast codon preference in Example 1 to Pichia pastoris inducible secretory expression vector pPICZαA , to obtain the recombinant vector pPICZαA-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-endocellulase in the embodiment of the present invention. The main vector construction steps are preferably as follows:

[0069] (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 endonuclease and buffer used were all p...

Embodiment 3

[0108] In this embodiment, the enzyme activity of the purified soluble endocellulase is detected, and the specific steps and results are as follows:

[0109] The present invention adopts the glucose hexokinase (HK) method to measure the ability of endocellulase to hydrolyze carboxymethylcellulose sodium (CMC-Na) to produce glucose, and hexokinase catalyzes 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 endocellulase 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...

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Abstract

The present invention relates to an artificially synthesized gene encoding endocellulase, the gene at least contains a DNA piece of one of the following nucleotide sequences: 1) the nucleotide sequence of SEQ ID No.1 in the sequence table; 2) a combination of The nucleotide sequence shown in SEQ ID No.1 has more than 90% homology and encodes a nucleotide sequence of the same biological function protein. According to the gene sequence of the present invention, further constructing a recombinant vector and transforming yeast can realize the secretion and expression of the recombinant cellulase induced by methanol, and through nickel affinity purification, the recombinant cellulase with a purity higher than 95% can be obtained. Dicer, which has a strong ability to degrade cellulose components and produce glucose, will provide a highly active recombinant enzyme for decomposing cellulose and applying it to the production of feed, textile, food and bioenergy.

Description

technical field [0001] The invention belongs to the technical field of biomolecular cloning, and relates to an artificially synthesized gene of endocellulase and its expression carrier and protein. Background technique [0002] Cellulose is a macromolecular polysaccharide composed of n D-glucopyranose chains linked by β-1,4 glycosidic bonds. Through the association of hydrogen bonds, fiber bundles are formed, which are divided into crystalline regions according to molecular density. And the amorphous region, in which the natural cellulose is mainly crystalline cellulose. Cellulose is an important component of plant cell walls and is currently the most abundant renewable resource on earth. Cellulose conversion technology can be used to produce biofuel and its processed products, but the utilization rate of cellulose is still very low, how to improve the utilization rate of cellulose is still a world-class topic. [0003] The efficient utilization of cellulose is inseparable...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/2437C12N15/815
Inventor 李洪波李露露胡兴
Owner HUNAN BUTIAN PHARMA
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