Polygalacturonase mutant with high catalytic efficiency, and preparation method and application thereof

A technology of polygalactose and catalytic efficiency, applied in the field of genetic engineering and genetic engineering, can solve the problems of heavy workload of artificial mutagenesis, difficulty in obtaining target strains, low frequency of beneficial mutations, etc.

Active Publication Date: 2014-01-22
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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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 an

Method used

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  • Polygalacturonase mutant with high catalytic efficiency, and preparation method and application thereof
  • Polygalacturonase mutant with high catalytic efficiency, and preparation method and application thereof
  • Polygalacturonase mutant with high catalytic efficiency, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Cloning of High Catalytic Efficiency Polygalacturonase Mutant Encoding Gene PG63X

[0038] The present invention takes the acid polygalacturonase (its amino acid sequence as SEQ ID NO.3) derived from Penicillium sp. post expression.

[0039] SEQ ID NO.3 is as follows:

[0040] SPAAEPAEGNRLIPRGSACTYSGVNGAAAAIAGKAGCSSITLNNVAVPAGTTLDLTGLAAGTKVIFEGTTTFGHKQWVGPLISISGTNIAVSGAAGHVIDGQGARWWDGKGSNTKTNIKPKFFLAHNLKGASTITGLNIKDTPVQVFSIDSSSGLTISGVTIDN RN GDKGSL GHNTDGFDIGDSDHITITGATVYNQDDCLAINSGTNIIFSGGYCSGGHGLSIGSVGGRSNNVVDTVHISSTQVVNSQNGVRVKAVAGATGSIKGVTYQDITLSGITSQGVTIRQDYTNSGYTGNPTTQVPITGLTLNNVHGTVTSSGTDITVECGSAASCSGWTWTKVAVSGANGKADLCKNAP

[0041] Genomic DNA of Penicillium sp. was used as a template to design region-replacing primers at the loop region of the catalytically active channel of polygalacturonase, and over-lap PCR was used to amplify polygalacturonase mutants with high catalytic efficiency Encoding gene PG63X.

[0042] Table 1. High catalytic effici...

Embodiment 2

[0044] Example 2 Preparation of polygalacturonase mutants with high catalytic efficiency.

[0045] The expression vector pPIC9r was double digested (SnaB I+Not I), and the gene PG63X encoding the high catalytic efficiency polygalacturonase mutant was double digested (SnaB I+Not I), and the cut code was mature The gene fragment of the polygalacturonase mutant with high catalytic efficiency (removing the signal peptide fragment) was connected with the expression vector pPIC9r, and the recombinant plasmid pPIC9r-PG63X containing the polygalacturonase mutant gene PG63X with high catalytic efficiency was obtained and Transform Pichia pastoris GS115 to obtain recombinant yeast strain GS115 / PG63X.

[0046] 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 at 3000g for 5min, discard the supernatant, and use 100mL ...

Embodiment 3

[0048] Example 3 Activity Analysis of Recombinant High Catalytic Efficiency Polygalacturonase Mutant and Wild Type

[0049] 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. One enzyme activity unit (U) is defined as the amount of enzyme required to decompose polygalacturonic acid to generate 1 μmol D-(+)-galacturonic acid per minute under given conditions.

[0050] 2. Determination of the properties of recombinant high catalytic efficiency polygalacturonase mutant and wild type

[0051] 1, the optimal pH assay method of recombinant high catalytic efficiency polygalacturonase mutant and wild type is as follows:

[0052] The recombinant polygalacturonase mutant with high catalytic efficiency purified...

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Abstract

The invention relates to the field of genetic engineering, specifically to a polygalacturonase mutant with high catalytic efficiency and a preparation method and application thereof. According to the invention, acidic polygalacturonase originated from Penicillium sp. is used as a female parent, and molecular biological techniques are employed for domain replacement and expression of the sequence of the acidic polygalacturonase; under such a condition, specific activity of the acidic polygalacturonase is increased by 11.1 times compared with wild polygalacturonase (before mutation), catalysis efficiency of the acidic polygalacturonase is increased by 33.5 times compared with the wild polygalacturonase (before mutation), and the pH value of an optimal reaction maintains unchanged. Thus, catalysis efficiency of polygalacturonase can be substantially improved, which lays a foundation for application of polygalacturonase in industrial production fields like food, fruit and vegetable processing. The invention is of important guiding significance to improvement of catalysis efficiency of polygalacturonase and other enzymes.

Description

technical field [0001] The invention relates to the field of genetic engineering and genetic engineering, in particular, the invention relates to a polygalacturonase mutant with high catalytic efficiency and its preparation method and application. Background technique [0002] Pectin is a kind of negatively charged macromolecular polysaccharide, which is a polysaccharide chain connected by D-(+)-galacturonic acid with different degrees of esterification through α-1,4 glycosidic bonds, often with rhamnos Side chains composed of sugar, arabinose, galactose, xylose, etc., with a molecular weight between 25 and 360kDa (Jayani et al., 2005). It widely exists in higher plants, especially in the tissues of fruits and vegetables, and is the main component of the glue layer (intercellular layer) in the cells of fruit and vegetable plants. [0003] Endopolygalacturonase (EC3.2.1.15) belongs to the 28th family of glycoside hydrolase (GH28), and when acting on pectic acid, it can rando...

Claims

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Application Information

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IPC IPC(8): C12N9/26C12N15/56C12N15/81C12N1/19A23L2/04A23L2/84C12R1/80
CPCC12N9/2408C12Y302/01015
Inventor 姚斌孟昆涂涛罗会颖石鹏君黄火清王亚茹柏映国杨培龙
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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