Glycoside hydrolase family 7 protein gene and its encoded protein and application

A glycoside hydrolase, family technology, applied in the fields of application, sugar production, genetic engineering, etc., can solve few problems such as endocellulase and achieve good enzyme activity

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

Currently commercially available cellulase is a mixture of various enzymes, and there are few related products and technologies that use genetic engineering to obtain the highest purity and high activity endocellulase

Method used

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  • Glycoside hydrolase family 7 protein gene and its encoded protein and application
  • Glycoside hydrolase family 7 protein gene and its encoded protein and application
  • Glycoside hydrolase family 7 protein gene and its encoded protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043]This example provides an optimized artificially synthesized glycoside hydrolase family 7 protein gene (GH7), the specific sequence is shown in SEQ ID NO.1 in the sequence listing, and the protein amino acid sequence corresponding to the gene is shown in the sequence listing Shown in SEQID NO.2. The synthesized sequence has no sequence with a similarity of 70% in the NCBI database. It is optimized and synthesized according to the characteristics of E. coli expression.

[0044] Connect the above-mentioned optimized natural gene into the E. coli expression vector pET28 to obtain the recombinant vector. The above-mentioned recombinant vector verified by sequencing is transformed into the competent cells of the E. coli expression strain by heat shock, and the corresponding resistant LB plate is coated, 37 Cultivate in a constant temperature incubator for 12 hours, and screen transformants, wherein the pET28 / GH7 vector is constructed as follows: figure 1 as shown, figure 1 I...

Embodiment 2

[0055] This embodiment provides a method for glycoside hydrolase family 7 protein, which specifically includes the following steps:

[0056] S1: Transform the recombinant vector pET28 / GH7 into Escherichia coli TOP10 strain, then extract the recombinant vector pET28 / GH7 from TOP10; transfer the recombinant vector pET28 / GH7 into the host cell E. The transformants of E. coli expressing strains containing the recombinant vector pET28 / GH7 were screened on the Kan-resistant LB plate, and the recombinant transformants were verified by PCR.

[0057] S2: Expression and extraction of soluble recombinant glycoside hydrolase family 7 protein: the Escherichia coli recombinant transformants containing the pET28 / GH7 vector of the optimized artificially synthesized gene were cultured in liquid LB medium at 37°C until OD 600 Then add IPTG with a concentration of 0, 0.1, and 0.5mM, respectively, and induce at 18°C ​​for 24 hours. After the induction, the collected bacteria are ultrasonically cr...

Embodiment 3

[0066] (1) The present invention adopts the glucose hexokinase (HK) method to measure the ability of glycoside hydrolase family 7 proteolysis carboxymethylcellulose sodium (CMC-Na) to produce glucose, and hexokinase catalyzes glucose (D in the presence of ATP) -Glucose) to 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 The absorbance change of NADH at 340nm wavelength was measured by spectrophotometry, and the concentration of glucose in the sample was quantitatively detected. The specific steps and results were as follows: 2 μl of purified Trx-GH7 with a concentration of 1 mg / mL was added to 98 μl of 1% CMC- Sodium dihydrogen phosphate of Na and citric acid buffer solution (pH = 4), react at 40°C for 1 hour; take 10 μl and 2 μl of the reacted sample with a concentration of 10 mmol / L of the mixture of NAD and ATP, and add water to the total...

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Abstract

The present invention relates to a glycoside hydrolase family 7 protein gene, the nucleotide sequence of the glycoside hydrolase family 7 protein gene is shown in SEQ ID No.1. It also relates to the protein encoded by the gene, the amino acid sequence of which is shown in SEQ ID NO.2. The present invention designs a new gene sequence capable of expressing the glycoside hydrolase family 7 protein, and further utilizes the pET28 vector to construct a recombinant plasmid and transform it into an E. coli expression strain to realize the soluble expression of the expression product and obtain a large amount of soluble form of Recombinant glycoside hydrolase family 7 protein; in the expression system of the present invention, the recombinant glycoside hydrolase family 7 protein can be folded in an appropriate manner and maintain the natural conformation, and the prepared protein has the ability to hydrolyze sodium carboxymethyl cellulose to produce glucose It has good enzyme activity, and has good enzyme activity under the condition of pH3.5‑5.0, and has a certain activation effect in the presence of copper ions.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and relates to a glycoside hydrolase family 7 protein gene and its coded protein and application. Background technique [0002] Cellulose is a polysaccharide composed of glucose with β-1,4 glycosidic bonds. It is an important component of plant cell walls. Plants produce the most abundant and cheapest cellulose resources on the earth through photosynthesis. According to data, the world The annual plant body production is as high as 150 billion tons of dry matter, of which the total amount of cellulose and hemicellulose is 85 billion tons. Since cellulose has a water-insoluble, highly crystalline structure and is surrounded by a lignin layer, it is quite difficult to hydrolyze it into usable glucose, so it has not been well utilized so far. With the rapid growth of the world's population, in order to solve the growing food and energy crisis, the utilization of cellulose resources h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12N9/42C12N15/70C12N1/21C12P19/14C12P19/02
CPCC12N9/2437C12N15/70C12P19/02C12P19/14C13K1/02
Inventor 胡兴李洪波王晓红
Owner HUNAN BUTIAN PHARMA
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