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Cellulose endonuclease gene, protein and application thereof

A cellulose endonuclease and gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problem of few cellulose endonucleases, etc., and achieve the effect of a simple and efficient production and preparation method

Active Publication Date: 2019-01-11
HUAIHUA UNIV
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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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  • Cellulose endonuclease gene, protein and application thereof
  • Cellulose endonuclease gene, protein and application thereof
  • Cellulose endonuclease gene, protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] This embodiment provides an optimized artificial chemically synthesized endocellulase gene (EG), 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 the sequence listing Shown in SEQ ID NO.2. The synthesized sequence has no sequence with a similarity of 30% in the NCBI database. It is a DNA optimized and synthesized according to the expression characteristics of Escherichia coli.

[0056] Connect the above-mentioned optimized gene into the E. coli expression vector pET28 to obtain the recombinant vector. The recombinant vector verified by sequencing above is heat-shocked and transformed into the competent cells of the E. coli expression strain, and the corresponding resistant LB plate is coated and kept at 37°C Cultivate in a constant temperature incubator for 12 hours, and screen transformants, wherein the pET28 / EG vector is constructed as figure 1 as shown, figure 1 It is a schematic d...

Embodiment 2

[0059] This embodiment provides a method for preparing protein, which specifically includes the following steps:

[0060] S1: Optimization of gene, construction of prokaryotic expression vector and transformation: Artificial chemical synthesis of the optimized mature cellulase gene was ligated to the pUC universal vector to obtain pUC / EG, pUC / EG was double-digested with BamHI and HindIII, and the obtained EG was obtained The fragment was then subcloned into the expression vector pET28 to obtain the recombinant expression vector pET28 / EG. The vector was constructed as follows: figure 1 shown. The main steps of pET28 / EG vector construction are as follows:

[0061] (1) Digest the recombinant vector pUC / EG with BamH I and Hind III to obtain the target fragment EG. The reaction system is as follows (all the endonucleases and buffers used are purchased from Dalian TAKARA Company):

[0062]

[0063] (2) Digest pET28 with BamH I and Hind III to obtain vector fragments. The reacti...

Embodiment 3

[0078] 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: 300 μg of purified recombinant endocellulase is added to the CMC-Na solution containing 1% at pH 4, and reacted for 4 hours at 40° C.; after the reaction, the sample is filtered with a 0.22 μm microporous membrane to 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 Figure 6 As shown, wherein the upper figure shows that CMC-Na does not detect the glucose peak without adding the recombinant endo-cellulase, and the lower fi...

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Abstract

The invention relates to a cellulose endocellulase gene, wherein the nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the protein encoded is shown as SEQ ID NO. 2. The invention also provides a method for efficiently expressing and purifying the recombinant protein, the purity of the purified protein is as high as 95%, and the enzyme activity of hydrolyzing cellulose to produce glucose is provided, which has important industrial value for the production of biofuel ethanol, food, feed and other industries.

Description

technical field [0001] The invention belongs to the field of biogenetic engineering, specifically relates to a cellulase gene, and also relates to a protein coded by the gene and its 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 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, cellulase c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12N15/70C12R1/19
CPCC12N9/2437C12N15/70C12Y302/01004Y02E50/10
Inventor 胡兴王晓红吴贤文李洪波
Owner HUAIHUA UNIV
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