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

An amylase and mutant 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 enzymatic property modification, and have strong ability to degrade raw starch. , the effect of broad application prospects

Active Publication Date: 2017-09-26
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 high-specific-activity amylase mutant with good ability to degrade raw starch
  • Preparation method and application of high-specific-activity amylase mutant with good ability to degrade raw starch
  • Preparation method and application of high-specific-activity amylase mutant with good ability to degrade raw starch

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Cloning of L223F encoding gene of high specific activity amylase mutant

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

[0048] (7) Table 1. Specific primers used in high specific activity amylase mutant L223F

[0049]

Embodiment 2

[0050] Example 2 Preparation of mutants of high specific activity amylase

[0051] The Fast Mutagenesis System of Beijing Quanshijin Biotechnology Co., Ltd. was used to perform specific point mutation amplification on the recombinant plasmid pPIC9-amy6 to obtain the high specific activity amylase mutant plasmid pPIC9-L223F and transform it 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 of 300mL BMGY medium, place it in a 30℃, 220rpm shaker for 48h; then centrifuge the culture solution at 3000g for 5min, discard the supernatant, and use 100mL for precipitation with 0.5% methanol The BMMY medium was resuspended and placed again at 30°C and 220 rpm to induce culture. Add 0.5 mL of methanol every 12 hours to keep the methanol concentration in the bacterial solution at 0.5%, while taking the supernatant for enzyme activity detection.

[0053] The opti...

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, a 1 mL reaction system includes 100 μL of appropriate diluted enzyme solution, 900 μL of substrate, and reacts for 30 minutes. Add 1.5 mL of DNS to terminate the reaction, and boil for 5 minutes. Measure the OD value at 540nm after cooling. 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 substrates and release 1 μmol of reducing sugars per minute is one unit of enzyme activity.

[0056] 2. Determination of the properties of recombinant high specific activity amylase mutant and female parent wild type

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

[0058] The recombinant high specif...

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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 fields 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 very versatile biocatalyst, which can be used in breadmaking industry, starch saccharification and liquefaction, textile desizing, papermaking, detergent industry, chemistry, clinical medicine analysis and pharmaceutical industry, etc. The amylase family includes alpha-amylase, beta-amylase and glucoamylase. Alpha-amylase is an endonuclease, which cuts the alpha-1,4 glycosidic bonds in the starch molecule in a random manner, and the starch produces dextrin and oligosaccharides. It is a calcium ion-dependent enzyme. β-amylase cuts maltose sequentially from the non-reducing end, which is an exonuclease. Glucoamylase is an exonuclease that acts on α-1,4 glycosidic bonds to cut glucose molecules from the non-reducing sugar ends. [0003...

Claims

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Application Information

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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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