High-activity poria cocos cellulose incision enzyme gene and protein and recombinant vector thereof

A cellulose endonuclease and recombinant vector technology, applied in genetic engineering, plant gene improvement, recombinant DNA technology, etc., can solve the problems of target product inactivation, low protein yield, and troublesome purification steps, and achieve strong biological activity. , reduce costs, and achieve the effect of mass production

Active Publication Date: 2020-01-03
HUAIHUA UNIV
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  • 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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  • High-activity poria cocos cellulose incision enzyme gene and protein and recombinant vector thereof
  • High-activity poria cocos cellulose incision enzyme gene and protein and recombinant vector thereof
  • High-activity poria cocos cellulose incision enzyme gene and protein and recombinant vector thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] This example provides an optimized artificially synthesized Poria cellulose endonuclease gene with a 6×His tag at the C-terminal. The specific sequence is shown in SEQ ID No. 1 in the sequence listing. The gene corresponding to The protein sequence is shown as SEQ ID No. 2 in the Sequence Listing. The optimized DNA sequence was aligned by NCBI, and there was no obvious similarity to the natural sequence.

[0059] Yeast is a unicellular lower eukaryote. Compared with insect expression system and mammalian expression system, yeast expression system has both advantages. At the same time, yeast expression system has the characteristics of simple operation, low cost, and large-scale fermentation. Therefore, it is an ideal production and preparation tool for recombinant eukaryotic protein. Yeast expression products have been widely used in the food industry for a long time, do not produce toxins, and have good safety. They have been identified as safe organisms, and their ex...

Embodiment 2

[0062] The present embodiment provides a method for preparing Poria cocos cellulose endonuclease protein, which specifically includes the following steps:

[0063] S1: Construction of expression vector and transformation: The DNA sequence containing the restriction enzyme cleavage site synthesized by the gene itself sequence characteristics of Example 1 and the yeast codon preference, that is, the DNA shown in SEQ ID No. 1, was subjected to Xho I and Xba After I, it was connected to the Pichia pastoris constitutive secretory expression vector pGAPZαA to obtain the recombinant vector pGAPZαA-Poria cellulose endonuclease. The vector construction is as follows figure 1 shown, figure 1 This is a schematic diagram of the construction of the eukaryotic expression vector pGAPZαA-Poria cellulose endonuclease in the embodiment of the present invention. The main vector construction steps are preferably as follows:

[0064] (1) Use Xho I and Xba I double enzyme to cut the plasmid conta...

Embodiment 3

[0096] In this example, the enzyme activity of the purified soluble Poria cocos cellulose endonuclease is detected, and the specific steps and results are as follows:

[0097]Using p-nitrophenyl-β-D-glucopyranoside as a substrate and p-nitrophenol as a standard product, the recombinant Poria cocos endocellulase that was purified, concentrated and freeze-dried in the step S7 of Example 2 was freeze-dried The powder was redissolved in normal saline, and the protein concentration was adjusted to 1mg / ml before enzyme activity detection.

[0098] (1) Drawing of standard curve: Take 1 g / L glucose and dilute it to 800, 400, 200, 100, 50, 25 and 0 μg / ml with 20 mM citrate buffer solution at pH 4.0, respectively. Take 10 μl of each of the above dilutions, add them to 90 μl of sodium dihydrogen phosphate containing 1% CMC-Na and citrate buffer (pH=4), and react at 40°C for 1 hour; take 50 μl of the reacted sample and 2 μl of Concentration of 10mmol / L NAD and ATP mixture, add water to t...

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Abstract

The invention relates to a high-activity cellulose incision enzyme artificially synthesized gene. The gene is shown as a nucleotide sequence of SEQ ID NO.1 in a sequence table; or the gene is as shownin the sequence of a biological function protein with 90 or above homology with the nucleotide sequence and same coding. According to the high-activity cellulose incision enzyme artificially synthesized gene, such as the nucleotide sequence of SEQ ID NO.1 in the sequence table, a pichia pastoris constitutive expression plasmid pGAPZalpha can be used as a carrier, and high-level recombinant secretory expression of a target protein in pichia pastoris host bacteria X33 can be realized. Meanwhile, screened high-level expression converters can realize high-level serection of a recombinant proteinby using high-density fermentation of an inorganic salt, and novel high-activity recombinant cellulose incision enzyme protein sterling can be obtained through simple nickel affinity chromatography purification. The recombinant protein has the high ability of hydrolyzing cellulose substances and generating glucose, and has important application value in aspects such as biological energy sources, fodder and food industries.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, and relates to a novel high-activity cellulose endonuclease gene and encoded protein derived from Poria cocos, and a recombinant vector constructed by the gene. Background technique [0002] Cellulase widely exists in natural organisms, such as bacteria, fungi, plants, animals and many other organisms can produce cellulase. These raw materials can be utilized by adding cellulase produced by living organisms to cellulose raw materials, so how to mass-produce cellulase has become the main bottleneck restricting the utilization of cellulose. With the development of biotechnology, the use of genetic engineering to develop cellulase resources with high activity and high yield has attracted more and more attention from scholars at home and abroad. At present, great progress has been made in the cloning and expression of cellulase genes, but there are few reports on the successfu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12N15/81
CPCC12N9/2437C12N15/815C12Y302/01004
Inventor 胡兴董海丽李洪波
Owner HUAIHUA UNIV
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