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A kind of heat-resistant chaetomium polygalacturonase mutant and its coding gene and application

A technology of polygalactose and coding genes, which is applied in the field of genetic engineering and can solve problems such as poor thermal stability, increased production costs, and easy inactivation

Active Publication Date: 2018-01-30
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

Enzyme preparations for feed usually need to go through the processing of plasmids (transient high temperature), while polygalacturonase belongs to medium and low temperature enzymes (mostly used for clarification and extraction of fruit juice), and its poor thermal stability not only limits its application range, but also is easy to Inactivation, increasing production costs

Method used

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  • A kind of heat-resistant chaetomium polygalacturonase mutant and its coding gene and application
  • A kind of heat-resistant chaetomium polygalacturonase mutant and its coding gene and application
  • A kind of heat-resistant chaetomium polygalacturonase mutant and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Site-directed mutagenesis

[0056] Carry out homology modeling on polygalacturonase PG8fn (Tu et al., 2013), and design the mutation site as the 244th aspartic acid is mutated to alanine and the 299th aspartic acid Mutated to arginine. The primers used in the design are shown in Table 1:

[0057] Table 1. The mutant-specific primers

[0058]

Embodiment 2

[0059] Preparation of mutants described in Example 2.

[0060] The expression vector pPIC9r was subjected to double enzyme digestion (SnaB I+Not I), and simultaneously the gene ASA encoding the mutant was double enzyme digested (SnaB I+Not I), and the gene fragment encoding the mature mutant was cut ( Remove the signal peptide fragment) and connect with the expression vector pPIC9r to obtain the recombinant plasmid pPIC9r-ASA containing the mutant gene ASA and transform it into Pichia pastoris GS115 to obtain the recombinant yeast strain GS115 / ASA.

[0061] 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 of 0.5% methanol for precipitation. The BMMY medium was resuspended, and placed again at 30°C, 220rpm to induce culture. Add 0.5 mL of methanol ...

Embodiment 3

[0064] Activity analysis of embodiment 3 mutant and wild type

[0065] 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.

[0066] 2. Determination of the properties of mutants and wild types

[0067] 1. The optimal pH determination method of mutant and wild type is as follows:

[0068] The mutant enzyme purified in Example 2 and the wild type were subjected to enzymatic reactions at different pHs to determine their optimum pH. The substrate polygalacturonic acid was tested for polygalacturonase activity in 0.1mol / L ...

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Abstract

The invention relates to the field of genetic engineering, in particular to a high temperature resistant chaetomium polygalacturonase mutant and an encoding gene and an application thereof. Polygalacturonase from achaetomium sp.xz8 is used as a female parent, and the polygalacturonase mutant with improved heat stability is obtained through rational design and molecular biological technique. Related amino acid mutation is combined mutation (ASA, Asp244Ala / Asp299Arg) of two points Asp244Ala and Asp299Arg in each mutant sequence. Optimum temperature, T50 and T1 / 2 of the mutant are improved in comparison with those of wild type enzyme. The optimum temperature is increased from 45 DEG C to 55 DEG C, T50 is increased from 56 DEG C to 73 DEG C, half-life period T1 / 2 at 50 DEG C is increased from 1.5h to 10h, half-life period T1 / 2 at 55 DEG C is increased from 33min to 78min, and Tm value is increased from 43.8 DEG C to 54.1 DEG C. Besides, the mutant keeps enzyme catalytic activity which is equal to that of the wild type enzyme. Application demands in fields of feedstuff, foodstuff and the like can be met.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a high temperature-resistant chaetomium polygalacturonase mutant, its coding gene and its application. Background technique [0002] There are quite a lot of non-starch polysaccharides (Non-starch Polysaccharides, NSPs) in feed, which is a general term for all carbohydrates in plant tissues except starch, which are composed of four parts: cellulose, hemicellulose, pectin and resistant starch. NSPs not only have no nutritional value for monogastric animals, but also become anti-nutritional factors in the intestinal tract, which can bind a large amount of water in the intestinal tract of animals, increase the viscosity of chyme, affect the activity of digestive enzymes, and stimulate the compensatory function of the digestive tract. Increased, leading to excessive reproduction of intestinal harmful bacteria, reducing the intestinal absorption of nutrients. As a non-starch polysa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/26C12N15/56C12N15/81C12N1/19A23K20/189C12R1/85
Inventor 姚斌罗会颖涂涛孟昆王亚茹石鹏君柏映国黄火清苏小运王苑马锐师霞
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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