Continuous endo-cellulase as well as coding gene and application thereof

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: 2021-09-24
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 o...

Method used

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  • Continuous endo-cellulase as well as coding gene and application thereof
  • Continuous endo-cellulase as well as coding gene and application thereof
  • Continuous endo-cellulase as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Cloning expression of embodiment 1 persistent endocellulase

[0027] 1. Target gene cloning

[0028] (1) Primer design

[0029] The present invention selects a gene SmCel5A predicted as an endocellulase as the 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 cellulase precursor protein encoded by it is shown in SEQ ID NO.2. Through the online analysis service of InterPro software, it is predicted that the 25 amino acids at the N-terminal of the protein are Signal peptide sequence (MRLKRKLALIGCILSLFLSSSTSTFA), the mature protein of cellulase is obtained after removing the signal peptide sequence, its amino acid sequence is shown in the sequence table SEQ ID NO.4, and the gene sequence corresponding to the mature protein encoding the cellulase is shown in the sequence table Shown in SEQ ID NO.3.

[0031] According to the sequence characteristi...

Embodiment 2

[0046] Embodiment 2 Purification of persistent endocellulase

[0047] The IPTG-induced engineering bacteria in Example 1 were centrifuged to collect the bacteria, resuspended with ultrasonic buffer, and then ultrasonically crushed. The obtained broken liquid was centrifuged at 12000g for 15min to obtain the supernatant, which was passed through Ni column affinity chromatography. For specific operations, see Protein According to the purification operation manual, the purified processive endocellulase SmCel5A can be obtained.

Embodiment 3

[0048] The property of embodiment 3 recombinant persistent endocellulase

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

[0050] Carboxymethylcellulose sodium salt, filter paper or phosphoric acid-swelled cellulose are used as substrates, and the persistent endocellulase SmCel5A of the present invention is used for reaction, and then the fiber is analyzed by detecting the amount of reducing sugar generated in the reaction system The activity of the enzyme was detected by ion chromatography or TLC to detect the species of reducing sugar. The amount of reducing sugar produced was measured by the DNS method. Protein concentration was determined by Coomassie brilliant blue detection method.

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Abstract

The invention discloses a continuous endo-cellulase as well as a coding gene and application thereof, and belongs to the technical field of bioengineering. The nucleotide sequence of the gene is shown as SEQ ID NO.3. The amino acid sequence of the continuous endo-cellulase coded by the gene is shown as SEQ ID NO.4. The continuous endo-cellulase SmCel5A is a multifunctional enzyme of a single structural domain, the molecular weight of the continuous endo-cellulase SmCel5A is less than 40 kD, heterologous expression is easy to conduct, active protein with the activity of endo-cellulase and excision-cellulase enzyme is obtained, and the problems that the growth cycle of sporocytophaga is long, enzyme components are complex, and the cellulase is not easy to directly separate and purify are effectively solved. The enzyme has wide application potential in the industries of biological energy, feed and the like.

Description

technical field [0001] The invention relates to a persistent endo-cellulase and its coding gene and application, belonging 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 resource utilization of waste, and meet the needs of sustainable development of contemporary society. [0003] The biodegradation of cellulose usually requires the synergy 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, the endocellulase acts on the non-crystalline region inside the cellulose molecule, truncates the long-chain cellulose macrom...

Claims

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Application Information

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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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