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Preparation method and application of amylase mutant with high specific activity and strong ability to degrade raw starch

A mutant, amylase technology, applied in the fields of genetic engineering and genetic engineering, can solve the problems of large workload, blindness and low activity of artificial mutagenesis, shorten the time for modification of enzymatic properties, and has a strong ability to degrade raw starch. , the effect of broad application prospects

Active Publication Date: 2020-05-22
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

Fungal α-amylase is mainly derived from Penicillium and Aspergillus, but its own activity is low, and it cannot meet the industrial needs, and the heterologous expression of amylase is still a problem, especially the heterologous expression of Pichia pastoris. This article is The amylase gene obtained from Penicillium T.leycettanus JCM12802 cut off CBM20 as the wild type and performed heterologous expression, and obtained high specific activity strains through site-directed mutation. Mutation, screening and enzyme molecular improvement to obtain
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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  • Preparation method and application of amylase mutant with high specific activity and strong ability to degrade raw starch
  • Preparation method and application of amylase mutant with high specific activity and strong ability to degrade raw starch
  • Preparation method and application of amylase mutant with high specific activity and strong ability to degrade raw starch

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Experimental program
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Effect test

Embodiment 1

[0046] Cloning of embodiment 1 high specific activity amylase mutant coding gene L223F

[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 high specific activity amylase mutant L223F

[0049]

Embodiment 2

[0050] The preparation of embodiment 2 high specific activity amylase mutants

[0051] Using the Fast Mutagenesis System of Beijing Quanshijin Biotechnology Co., Ltd., the recombinant plasmid pPIC9-amy6 was amplified by specific point mutation to obtain the high specific activity amylase mutant plasmid pPIC9-L223F and transformed into Pichia pastoris GS115 to obtain the recombinant yeast strain GS115 / L223F.

[0052]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 every 12 hours to keep the concentration of methanol in the bacterial solution at 0.5%, and take the supernatant for enzyme activity detection. ...

Embodiment 3

[0054] Example 3 Activity Analysis of Recombinant High Specific Activity 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 the recombinant high specific activity amylase mutant and the wild type of the mother

[0057] 1. The optimal pH determination method of the recombinant high specific activity amylase mutant and the female parent wild type is as follows:

[0058] The recombinant h...

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Abstract

The invention provides a high-specific-activity amylase mutant with good ability to degrade raw starch 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 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 invention relates to the field of genetic engineering and genetic engineering, in particular to a method for preparing a high specific activity amylase mutant 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 molecules from non-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/30C12N15/81C12N1/19C12R1/84
CPCC12N9/242C12N15/815C12N2800/102C12Y302/01001
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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