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Mutant of endoglucanase, coding gene and application thereof

A technology for encoding genes and residues, applied in the field of mutants of endoglucanase, can solve the problems of inability to meet the needs of industrial production, low enzyme activity or low yield, etc., and achieve good industrial application prospects and a wide working temperature range. Effect

Active Publication Date: 2013-04-03
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, cellulase derived from thermophilic bacteria (≥70°C) often has low enzyme activity or yield, which cannot meet the needs of industrial production.

Method used

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  • Mutant of endoglucanase, coding gene and application thereof
  • Mutant of endoglucanase, coding gene and application thereof
  • Mutant of endoglucanase, coding gene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Embodiment 1, the acquisition of endoglucanase mutant and its coding gene

[0028] 1. Extract Tengchong thermophilic anaerobic bacteria (Thermoanaerobacter tengcongensis MB4) (purchased from the Bacteria Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, the strain preservation number is CGMCC No.1.2430, AS 1.2430 T =JCM11007 T ) and using it as a template, 5'-CGCGGATCCATGAATAAATGGTATGC-3' (forward) (sequence 7) and 5'-CCGCTCGAGTTACTTTCCTCTTGCCTCAC-3' (reverse) (sequence 8). The PCR amplification conditions were as follows: 4 minutes of pre-denaturation at 95°C, followed by 30 cycles of 95°C for 45s, 55°C for 30s, and 72°C for 1 min; finally, extension at 72°C for 10 minutes.

[0029] The above PCR reaction product was recovered and detected by agarose gel electrophoresis, and a band of 1143bp was obtained as a result.

[0030] 2. Obtaining of recombinant bacteria containing endoglucanase coding gene

[0031] The PCR product obtained in ...

Embodiment 2

[0044] Embodiment 2, the acquisition of endonuclease after mutation

[0045] 1. Production of endoglucanase mutants by fermentation of recombinant bacteria

[0046] 1. Express

[0047] Inoculate the single colonies of BL21(DE3) / pET28a-celA-mut(3F6) and BL21(DE3) / pET28a-celA-mut(C3-13) obtained above into the cells containing kanamycin (final concentration: 50 μg / ml) of LB liquid medium, cultured at 37°C. After 12 hours, transfer the fermentation broth to 1000ml fresh LB liquid medium according to the inoculum size of 1%, and cultivate it to OD at 37°C 600 Reach 0.6, then add sterile IPTG in the medium, make the final concentration of IPTG in the medium be 1mM to induce fermentation. After 5 hours, the fermentation was finished, and the fermentation broth was centrifuged at 5000 rpm for 10 minutes to collect the bacteria.

[0048] Bacteria resuspended in 50ml concentration is the purification buffer of 20mM (NaCl is dissolved in the Tris-HCl buffer of 20mM, the final conce...

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Abstract

The invention discloses a mutant of endoglucanase, coding gene and application thereof. A protein provided by the invention is artificially synthesized and named as celA-mut(3F-6) or celA-mut(C3-13); the protein is a protein 1) or protein 2) as below: 1) a protein consisting of amino acid residue sequence in a sequence 2 in the sequence table; 2) a protein consisting of amino acid residue sequence in a sequence 4 in the sequence table; 3) a protein obtained by replacement and / or deletion and / or addition of one or a plurality of amino acid residues on the amino acid residue of the sequence2 or a sequence 4 in the sequence table, and with endoglucanase activity and derived from 1). Experiments of the invention have proved that the mutant of endoglucanase obtained by the invention has increased vitality compared with a wild type thereof and wide operational temperature range while maintaining an optimum reaction temperature (75 DEG C), therefore the mutant of endoglucanase has good industrial application prospect.

Description

technical field [0001] The invention relates to a mutant of endoglucanase, its coding gene and application. Background technique [0002] Endoglucanase (1,4-β-D-endoglucanase, EC 3.2.1.4) can randomly act on the β-1,4-glycosidic bonds connecting glucose units in the long chain of cellulose from the inside to generate different lengths of glucose chains. If the enzyme works together with exoglucanase and β-glucosidase, the cellulose can be further degraded into glucose or other products that are easy to be further processed and converted, and finally generate high value-added biotechnology products. Therefore, endoglucanase has important application value in bioenergy, food processing, papermaking, washing and other industries. [0003] Compared with other enzyme proteins, enzyme products derived from thermophilic bacteria not only have higher temperature stability, but also often have other stress resistance properties at the same time. Therefore, the application of therm...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12N7/01C12N15/11C12P19/14
CPCY02P20/52
Inventor 马延和梁朝宁薛燕芬
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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