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Endoglucanase gene, endoglucanase, and preparation method and application of endoglucanase

An endoglucanase and gene technology, applied in the field of genetic engineering, can solve problems such as low enzymatic activity of endoglucanase, and achieve the effects of rich source pathways, good application prospects, industrial value, and high-efficiency expression

Pending Publication Date: 2020-11-06
深圳润康生态环境股份有限公司
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  • Abstract
  • Description
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Problems solved by technology

[0004] The purpose of the present invention is to provide endoglucanase gene macel and corresponding recombinant expression vector, recombinant expression strain, and endoglucanase MaCel and its preparation method and application, aiming to solve the problem of existing thermophilic fungus The technical problem of the low enzymatic activity of the endoglucanase of Melanocarpus albomyces

Method used

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  • Endoglucanase gene, endoglucanase, and preparation method and application of endoglucanase
  • Endoglucanase gene, endoglucanase, and preparation method and application of endoglucanase
  • Endoglucanase gene, endoglucanase, and preparation method and application of endoglucanase

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preparation example Construction

[0044] Correspondingly, the embodiment of the present invention also provides a preparation method of endoglucanase MaCel, which comprises the following steps:

[0045] S1. Provide endoglucanase gene macel, expression vector and expression strain;

[0046] S2. Amplify the endoglucanase gene macel, and connect the obtained amplified product to an expression vector to obtain a recombinant expression vector;

[0047] S3. Transforming the recombinant expression vector into an expression strain to obtain a recombinant expression strain;

[0048] S4. Cultivate the recombinant expression strain to obtain endoglucanase MaCel;

[0049] Wherein, the nucleotide sequence of the endoglucanase gene macel is shown in SEQ ID NO:1.

[0050] In the preparation method of the endoglucanase MaCel provided in the embodiment of the present invention, the corresponding recombinant expression vector and recombinant expression strain are obtained by amplifying the endoglucanase gene macel in a hetero...

Embodiment 1

[0078] This embodiment provides an endoglucanase gene sequence analysis and codon optimization process, including the following steps:

[0079] According to the NCBI database accession number (GenBank: AJ515703.1) reported in the literature, the gene sequence of the endoglucanase derived from the thermophilic fungus Melanocarpus albomyces was obtained, and the endoglucanase gene sequence was optimized with reference to the codon preference of Pichia pastoris, and the endoglucanase was obtained. Glucanase macel gene. Compared with the original gene, the optimized endoglucanase macel gene reduces rare codons (such as converting the low-frequency codon CGG encoding arginine to high-frequency codon AGA, and the low-frequency codon CCG encoding proline converted into high-frequency codons CCA, etc.); the optimized endoglucanase macel gene expression adaptation index increased from 0.51 to 0.79; the optimized endoglucanase macel gene GC content was adjusted from 68% to 50% . Compa...

Embodiment 2

[0081] This embodiment provides a construction process of a recombinant expression vector containing endoglucanase gene macel, specifically as follows:

[0082] (1) Design a pair of primers MaCel-F and MaCel-R according to the sequence of endoglucanase gene macel, its nucleotide sequence is as shown in SEQ ID NO:3-4, is used for amplifying endoglucan Carbohydrase gene macel1 (macel1 is macel with the nucleotide sequence encoding the signal peptide removed);

[0083] (2) The endoglucanase gene macel1 was obtained by PCR amplification, and the expression vector pP ICZαA and the endoglucanase gene macel1 were digested overnight with restriction endonucleases EcoRI and XbaI respectively, and the expression The vector pPICZαA and the endoglucanase gene macel1 were purified and recovered for ligation reaction;

[0084] (3) Transfer the ligation reaction product into Escherichia coli Top10 by the heat shock method, and verify the recombinant transformant by bacterial liquid PCR;

...

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Abstract

The invention belongs to the technical field of genetic engineering, and specifically relates to an endoglucanase gene, endoglucanase, and a preparation method and application of the endoglucanase. Anucleotide sequence of the endoglucanase gene macel is as shown in SEQ ID NO: 1, and the endoglucanase gene macel is obtained by the comprehensive optimization of a sequence (GenBank: AJ515703.1) of endoglucanase derived from thermophilic fungus Melanocarpus albomyces. An amino acid sequence of the endoglucanase MaCel is as shown in SEQ ID NO: 2, and the endoglucanase MaCel not only enriches typesand sources of endoglucanase, but also has relatively high enzyme activity and heat resistance, also shows good stability in a relatively wide pH range, and has good application prospects and industrial value.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to an endoglucanase gene macel, a corresponding recombinant expression vector, a recombinant expression strain, an endoglucanase MaCel and a preparation method and application thereof. Background technique [0002] Cellulase is a complex enzyme system capable of degrading cellulose into glucose. Cellulase can be divided into endoglucanase, exoglucanase and β-glucosidase according to the mode of action on the substrate. In the cellulase system, endoglucanase plays an important role. During the hydrolysis of cellulose, endoglucanase randomly cleaves the β-1,4 glycosidic bonds inside the cellulose to form small fragments of cellulosic polysaccharides, which provide energy for exoglucanase and β-glucosidase. Acting substrate. Because endoglucanase plays an important role in the process of cellulose hydrolysis, it has wide application potential in the fields of ...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/42C12N15/81C12N1/19C12P19/14C12R1/84
CPCC12N9/2434C12N15/815C12P19/14C12Y302/01004C12N2800/22
Inventor 王建荣祝木金余思王平孟建军刘小慧曹革
Owner 深圳润康生态环境股份有限公司
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