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Cellulase mutant with high catalytic efficiency as well as coding genes and application thereof

A technology of cellulase and catalytic efficiency, applied in the field of genetic engineering, can solve the problems of low frequency of beneficial mutations, large blindness, and heavy workload of artificial mutagenesis, and achieve the goals of shortening transformation time, high catalytic efficiency, and broad application prospects Effect

Inactive Publication Date: 2017-08-15
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mutagenesis is divided into natural mutation and artificial mutagenesis. The probability of success of natural mutation is very small, and the workload of artificial mutagenesis is relatively large and the frequency of beneficial mutation is still low. The direction and nature of mutation are difficult to control
The blindness of the screening is large, and it is not easy to obtain the target strain

Method used

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  • Cellulase mutant with high catalytic efficiency as well as coding genes and application thereof
  • Cellulase mutant with high catalytic efficiency as well as coding genes and application thereof
  • Cellulase mutant with high catalytic efficiency as well as coding genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 High Catalytic Efficiency Cellulase Mutant Encoding Gene Bacel5 127 ,Bacel5 167 clone

[0057] Using the genomic DNA of Talaromyces emersonii CBS394.64 and Bispora antennata CBS 126.38 as templates, primers were designed at the spliced ​​region of the paternal cellulase N-terminus and the maternal cellulase C-terminus, and over-lapPCR was used to amplify the high catalytic efficiency Cellulase Mutant Encoding Gene Bacel5 127 ,Bacel5 167 .

[0058] Table 1. High catalytic efficiency cellulase mutant BaCel5 127 , BaCel5 167 Specific primers used

[0059]

[0060] a The restriction sites are underlined

Embodiment 2

[0061] Example 2 Preparation of cellulase mutants with high catalytic efficiency.

[0062] The expression vector pPIC9r was subjected to double digestion (EcoR I+Not I), and at the same time the gene Bacel5 encoding the cellulase mutant with high catalytic efficiency was 127 ,Bacel5 167 Double enzyme digestion (EcoR I+Not I), the cut gene fragment encoding the mature cellulase mutant with high catalytic efficiency (removing the signal peptide fragment) is connected with the expression vector pPIC9r, and the cellulase mutant gene containing high catalytic efficiency is obtained The recombinant plasmid pPIC9r-Bacel5 127 , pPIC9r-Bacel5 167 And transform Pichia pastoris GS115 to obtain recombinant yeast strain GS115 / Bacel5 127 ,GS115 / Bacel5 167 .

[0063] Take the GS115 strain containing the recombinant plasmid, inoculate it in a 1L Erlenmeyer flask with 300mL of BMGY medium, place it at 30°C, and culture it on a shaker at 220rpm for 48h; then centrifuge the culture solution...

Embodiment 3

[0065] Example 3 Activity Analysis of Recombinant Cellulase Mutant with High Catalytic Efficiency and Parental Wild Type

[0066] 1. DNS method: The specific method is as follows: under the given pH and temperature conditions, 1mL of the reaction system includes 100μL of appropriate diluted enzyme solution, 900μL of substrate, react for 10min, add 1.5mL of DNS to terminate the reaction, and boil for 5min. After cooling, the OD value was measured at 540 nm. Definition of cellulase activity unit: Under certain conditions, the amount of enzyme required to decompose carboxymethyl cellulose to generate 1 μmol reducing sugar per minute is 1 activity unit (U).

[0067] 2. Determination of the properties of the recombinant cellulase mutant with high catalytic efficiency and the parental wild type

[0068] 1. The optimum pH and pH stability determination methods of recombinant high catalytic efficiency cellulase mutant and female parent wild type are as follows:

[0069] The recombin...

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Abstract

The invention relates to the field of genetic engineering and in particular relates to a cellulase mutant with high catalytic efficiency as well as coding genes and application thereof. The mutant has an amino acid sequence shown as SEQ ID NO.3 or SEQ ID NO.4. Compared with an activity ratio female parent wild type, the cellulase mutant has the advantage that the catalytic efficiency and specific activity are obviously improved. The catalytic efficiency and action conditions of the cellulase can be greatly improved, and a basis is provided for application of the cellulase in the field of industrial production.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a cellulase mutant with high catalytic efficiency and its coding gene and application. Background technique [0002] Plant cell walls are mainly composed of cellulose, hemicellulose, and lignin. Cellulose is an important polysaccharide. It is the material of plant cell support material and the most abundant biomass resource in nature. The structure of cellulose is determined as β-D-glucose units connected by β-(1→4) glycosidic bonds. The resulting linear polymer has no branches in the structure, which can be degraded to glucose by cellulase. [0003] Cellulase refers to the general term for a group of enzymes that can hydrolyze glucosidic bonds and decompose cellulose into cellobiose and glucose. The hydrolysis process of cellulose mainly includes three steps: the first step is that the endo-cellulase acts on the amorphous region inside the cellulose, and then hydrolyzes the ...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/2437C12N15/815C12N2800/102
Inventor 姚斌罗会颖郑菲王亚茹苏小运黄火清柏映国王苑马锐师霞
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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