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A kind of amylase mutant preparation method and its application

An amylase and mutant technology, applied in the fields of genetic engineering and genetic engineering, can solve the problems of large blindness, low frequency of beneficial mutations, large workload of artificial mutagenesis, etc., and achieves high specific activity, broad application prospects, and shortened transformation. effect of time

Active Publication Date: 2020-01-21
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 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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  • A kind of amylase mutant preparation method and its application
  • A kind of amylase mutant preparation method and its application
  • A kind of amylase mutant preparation method and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Cloning of embodiment 1 amylase mutant encoding gene E87K

[0047] Using the recombinant plasmid pPIC9-amy6 of the gene amy6 cloned from T. leycettanus JCM12802 as a template, wherein the amino acid sequence encoded by amy6 is shown in SEQ ID NO: 1, primers were designed according to the instructions of the Fast Mutagenesis System, and then amplified.

[0048] (7) Table 1. Specific primers used for amylase mutant E87K

[0049]

Embodiment 2

[0050] The preparation of embodiment 2 amylase mutants

[0051] The recombinant plasmid pPIC9-amy6 was amplified using the Fast Mutagenesis System of Beijing Quanshijin Company to obtain the amylase mutant plasmid pPIC9-E87K and transformed into Pichia pastoris GS115 to obtain the recombinant yeast strain GS115 / E87K.

[0052] Take the GS115 strain containing the recombinant plasmid, inoculate it in a 1L Erlenmeyer flask with 300mL of BMGY medium, and culture it on a shaker at 30°C and 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 every 12 hours to keep the concentration of methanol in the bacterial solution at 0.5%, and take the supernatant for enzyme activity detection.

[0053] The optimum pH of the recombinant amylase mutant was 4.5, which was consistent with that of ...

Embodiment 3

[0054] Example 3 Activity Analysis of Recombinant Amylase Mutant and Female Parent Wild Type

[0055] 1. DNS method: The specific method is as follows: under the given pH and temperature conditions, 1mL of reaction system includes 100μL of appropriate diluted enzyme solution, 900μL of substrate, react for 30min, 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 amylase activity unit: under the condition of 60°C and pH 4.5, the amount of enzyme required to catalyze the hydrolysis of the substrate to release 1 μmol of reducing sugar per minute is an enzyme activity unit.

[0056] 2. Determination of the properties of recombinant amylase mutants and maternal wild type

[0057] 1. The optimal pH determination method of the recombinant amylase mutant and the parental wild type is as follows:

[0058] The recombinant amylase mutant purified in Example 2 and the wild type were subjected to enzymatic reacti...

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Abstract

The invention provides a high-specific-activity high-catalytic-efficiency amylase mutant and a preparation method and application thereof and relates to the field of gene engineering. Acid amylase derived from Talaromyces leycettanus JCM12802 is used as a female parent, and the wild type is subjected to site-directed mutagenesis by means of molecular biological techniques. Under the modification condition, the amylase mutant has significantly higher catalytic efficiency and specific activity than the wild (before mutation) female parent. By using the scheme, it is possible to greatly increase the specific activity and acting conditions of amylase, and basis is provided for the amylase in industrial production field. The scheme is of important guidance for increasing the catalytic efficiency and specific activity of amylase and other enzymes.

Description

technical field [0001] The present invention relates to the field of genetic engineering and genetic engineering, in particular, the present invention relates to a method for preparing mutant amylase and its application. Background technique [0002] Amylase is a biocatalyst with a wide range of uses, which can be used in bread making industry, starch saccharification and liquefaction, textile desizing, paper making, cleaning agent industry, chemistry, clinical medicine analysis and pharmaceutical industry, etc. The amylase family includes alpha-amylases, beta-amylases and glucoamylases. α-amylase is an endonuclease, which cuts the α-1,4 glycosidic bonds inside the starch molecule in an irregular manner, and makes the starch generate dextrin and oligosaccharides. It is a calcium ion-dependent enzyme. β-amylase sequentially cuts maltose from the non-reducing end, which is an exonuclease. Glucoamylase is an exonuclease acting on α-1,4 glycosidic bonds, cleaving glucose molec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/30C12N15/81C12N1/19C12R1/84
CPCC12N9/242C12Y302/01001
Inventor 姚斌罗会颖张多多涂涛王苑黄火清苏小运柏映国王亚茹
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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