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A kind of persistent endocellulase and its coding gene and application

An endocellulase, sustainable technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of difficult to meet industrial production, high cost, lack of efficient hydrolysis capacity, etc., to achieve wide application potential, improve transformation rate effect

Active Publication Date: 2022-05-24
DALIAN POLYTECHNIC UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The main barrier to the biotransformation of cellulosic raw materials is the lack of cellulase with high-efficiency hydrolysis ability. It often requires the synergistic action of multiple enzymes to degrade cellulose into fermentable small molecular sugars, which leads to very high cost of enzymes and is difficult to produce. Meet the needs of industrial production

Method used

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  • A kind of persistent endocellulase and its coding gene and application
  • A kind of persistent endocellulase and its coding gene and application
  • A kind of persistent endocellulase and its coding gene and application

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1 Cloning and expression of persistent endocellulase

[0027] 1. Target gene cloning

[0028] (1) Primer design

[0029] The present invention selects a gene SmCel5A predicted to be an endocellulase as a research object, and its nucleotide sequence is SEQ ID NO.1.

[0030] The gene sequence of SEQ ID NO.1 is expected to encode 334 amino acids, and the amino acid sequence of the encoded cellulase precursor protein is shown in SEQ ID NO.2. Through the online analysis service of InterPro software, the predicted 25 amino acids at the N-terminal of the protein are Signal peptide sequence (MRLKRKLALIGCILSLFLSSTSTFA), after removing this signal peptide sequence, the mature protein of cellulase is obtained, its amino acid sequence is shown in SEQ ID NO. shown in SEQ ID NO.3.

[0031] According to the sequence characteristics of SEQ ID NO.1, as well as the signal peptide sequence and mature protein sequence characteristics of the expressed protein corresponding to the...

Embodiment 2

[0046] Example 2 Purification of Persistent Endo-Cellulase

[0047] The engineered bacteria induced by IPTG in Example 1 were centrifuged to collect bacterial cells, resuspended with ultrasonic buffer, and then ultrasonically disrupted. The resulting fragmented liquid was centrifuged at 12,000 g for 15 min to obtain a supernatant, which was subjected to Ni column affinity chromatography. For specific operations, see Protein According to the purification operation manual, the purified continuous endo-cellulase SmCel5A can be obtained.

Embodiment 3

[0048] Example 3 Properties of recombinant persistent endocellulase

[0049] (1) Cellulase activity of hydrolyzing sodium carboxymethyl cellulose, filter paper and phosphoric acid swollen cellulose

[0050] Using sodium carboxymethyl cellulose, filter paper or phosphoric acid swollen cellulose as a substrate, the continuous endo-cellulase SmCel5A of the present invention is used to react, and then the amount of reducing sugar generated in the reaction system is detected to analyze the fiber The activity of the enzyme is determined by ion chromatography or TLC to detect the species of reducing sugars. The amount of reducing sugar produced was measured by the DNS method. The protein concentration was determined by Coomassie brilliant blue detection method.

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Abstract

The invention discloses a persistent endo-cellulase, its coding gene and application, and belongs to the technical field of bioengineering. A gene, the nucleotide sequence of which is shown in SEQ ID NO.3. The processive endo-cellulase encoded by the gene, the amino acid sequence of the processive endo-cellulase is shown in SEQ ID NO.4. The processive endocellulase SmCel5A of the present invention is a multifunctional enzyme with a single domain, its molecular weight is less than 40kD, it is easy to carry out heterologous expression, and obtains the activity with endocellulase and exocellulase enzyme activity The protein effectively solves the problem that it is not easy to directly separate and purify the cellulase due to the relatively long growth period of the sporophage cellulophage and the complex enzyme components. The enzyme has wide application potential in bioenergy, feed and other industries.

Description

technical field [0001] The invention relates to a persistent endo-cellulase and its encoding gene and application, and belongs to the technical field of bioengineering. Background technique [0002] Cellulose is the most abundant carbohydrate resource on earth and the most important alternative raw material for the biomanufacturing industry. The development and utilization of cellulosic raw materials to produce bioenergy and various useful chemicals can alleviate the energy crisis to a certain extent, realize the utilization of waste resources, and meet the needs of sustainable development of contemporary society. [0003] The biodegradation of cellulose usually requires the synergistic action of three enzymes, including endocellulase (Endoglucanase, EC3.2.1.4), exocellulase (Cellobiohydrolase, EC3.2.1.91) and β-glucosidase (β-glucosidase, EC 3.2.1.21). Among them, endo-cellulase acts on the internal non-crystalline region of cellulose molecules, truncating long-chain cell...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12N9/42C12N1/21C12P19/14C12P19/12C12P19/02C12P19/00C12R1/19
CPCC12N9/2437C12P19/14C12P19/12C12P19/02C12P19/00C12Y302/01004C12Y302/01091
Inventor 陈晓艺赵鑫王辛李宪臻
Owner DALIAN POLYTECHNIC UNIVERSITY
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