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Glucoside hydrolase family 61 protein gene, protein thereof and preparation method of protein

A technology of glycoside hydrolase and family, which is applied in the field of glycoside hydrolase family 61 protein gene and its protein and preparation, which can solve the problems of low hydrolysis efficiency, long production cycle and high production cost

Active Publication Date: 2019-03-08
HUNAN BUTIAN PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the industrial preparation of cellulase in my country is still in the research and development stage, and the production of cellulase still has problems such as low cellulase activity, high production cost, and long production cycle, which limit the application of cellulase.
Among them, the inefficient hydrolysis of cellulose by cellulase is one of the main bottlenecks in the industrial production of cellulosic ethanol (fuel ethanol produced by converting lignocellulose), and it is also an important topic of concern.

Method used

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  • Glucoside hydrolase family 61 protein gene, protein thereof and preparation method of protein
  • Glucoside hydrolase family 61 protein gene, protein thereof and preparation method of protein
  • Glucoside hydrolase family 61 protein gene, protein thereof and preparation method of protein

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Embodiment 1

[0046] This embodiment provides an optimized artificially synthesized glycoside hydrolase family 61 protein gene, the specific sequence is shown in SEQ ID NO.1 in the sequence listing, and the protein sequence corresponding to the gene is shown in SEQ ID NO in the sequence listing .2 shown. The sequence provided by the present invention has no sequence with a similarity of 70% in the NCBI database. It is based on the characteristics of Escherichia coli expression such as codon preference, preventing complex DNA structures from affecting transcription efficiency, ensuring reasonable GC content, Select a DNA sequence among many sequences that are artificially optimized and synthesized with the characteristics of suitable restriction site, ideal expression tag and termination signal. The sequence described in the present invention and the highly homologous DNA sequence have higher expression of soluble target protein in Escherichia coli than other sequences.

[0047] Connect the...

Embodiment 2

[0060] This embodiment provides a method for preparing GH61 active protein, which specifically includes the following steps:

[0061] 1) Expression and extraction of soluble recombinant glycoside hydrolase family 61 protein: the transformant of the E. coli expression strain containing the recombinant vector pET26 / GH61 screened in Example 1 was cultured in liquid LB medium at 37°C until the OD600 was 0.4 , then add IPTG with a concentration of 0, 0.1, 0.2 and 0.5mM, respectively, and induce at 18°C ​​for 24 hours. The supernatant was used to obtain the recombinant glycoside hydrolase family 61 protein, and the SDS-PAGE results were as follows: figure 2 shown.

[0062] 2) Purification of recombinant glycoside hydrolase family 61 protein: expand the Escherichia coli recombinant transformant of pET26 / GH61 vector in liquid LB medium, then induce with 0.1mM IPTG at 18°C ​​for 20-24 hours, collect The thalline of the expressed bacterium after induction of expression was resuspende...

Embodiment 3

[0072]The invention uses high-performance liquid chromatography to detect the ability of sodium carboxymethylcellulose (CMC-Na) to be hydrolyzed by an endonuclease to produce a small amount of glucose to determine the enzyme activity. The specific method is as follows: add 500 μg of the purified recombinant glycoside hydrolase family 61 protein to a 1% CMC-Na solution at pH 4, and react at 40°C for 4 hours; after the reaction, filter the sample with a 0.22 μm microporous membrane to sample vials for liquid chromatography analysis. The liquid phase method is as follows: chromatographic column: Agilent amino column, 250×4.6mm, 5μm; mobile phase: acetonitrile:water=70:30 (volume ratio), flow rate: 1.0mL / min, injection volume: 10uL, column temperature : 35°C, detector: differential refractive index detector. HPLC results such as Image 6 As shown, wherein the upper figure a is that CMC-Na does not detect the glucose peak without adding the recombinant endo-cellulase, and the low...

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Abstract

The invention relates to a glucoside hydrolase family 61 protein gene. The nucleotide sequence of the glucoside hydrolase family 61 protein gene is shown as SEQ ID No. 1. The invention relates to a protein of the glucoside hydrolase family 61 protein gene, and the amino acid sequence of the protein is shown as SEQ ID NO. 2. The glucoside hydrolase family 61 protein gene can further utilize pET26 plasmids to structure recombinant plasmids and converts the recombinants plasmids into Escherichia coli expression strains, thereby achieving soluble expression of expression products. The invention also provides preparation and purification methods of the protein. The preparation and purification methods can help obtain recombinant glucoside hydrolase family 61 proteins with a degree of purity upto higher than 95%; the prepared recombinant glucoside hydrolase family 61 proteins have enzymatic activity for hydrolyzing sodium carboxymethylcellulose to produce glucose, belong to glucoside hydrolase family 61 enzymes and can be matched with other types of cellulases during different types of application, preferably, be combined with one or more beta-glucosidases or endoglucanases.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and relates to a glycoside hydrolase family 61 protein gene, the protein and a preparation method thereof. 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 and is currently the most abundant renewable resource on earth. 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 belongs to glycoside hydrolase, which is a general term for a class of enzymes that specifically catalyze the hydrolysis of β-1,4-glycosidic bonds in cellulose chains. It is a highly active biocatalyst that can decompose cellulose to produce glucose. According to the structure of cellulase, cellu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21C12P19/02C12P19/14C12R1/19
CPCC12N9/2437C12N15/70C12P19/02C12P19/14
Inventor 胡兴王晓红岳芬芳李洪波
Owner HUNAN BUTIAN PHARMA
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