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Acid resistant medium temperature alpha-amylase and preparation method thereof

An amylase and medium-temperature technology, applied in the field of acid-resistant and medium-temperature α-amylase and its preparation, can solve the problems of α-amylase acid resistance and temperature adaptability can not be taken into account at the same time, application limitations, etc., to achieve industrial application, wide Applicable pH range, effect of good acid stability

Inactive Publication Date: 2011-11-30
GUANGXI ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the above situation, the present invention solves the problem that the existing α-amylase cannot take into account the acid resistance and temperature adaptability at the same time, so that the application is limited; by adopting recombinant DNA technology, a site-directed mutation precursor α-amylase is provided to improve its characteristics , obtained acid-resistant and temperature-resistant alpha-amylase mutant with improved activity and preparation method thereof

Method used

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  • Acid resistant medium temperature alpha-amylase and preparation method thereof
  • Acid resistant medium temperature alpha-amylase and preparation method thereof
  • Acid resistant medium temperature alpha-amylase and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Amplification of the precursor α-amylase gene

[0047] Extract the chromosomal DNA of Bacillus subtilis [purchased from China Industrial Microorganism Collection and Management Center (CICC 10028)]. Based on the Bacillus subtilis amylase gene reported in Genbank, the following primers were designed (the primers were synthesized by Shanghai Bioengineering Co., Ltd.):

[0048] Upstream primer F1: 5’-GCGC GAATTC GGAAACTGCAAACAAATCGAATGAGGTG-3' (SEQ ID NO: 1)

[0049] Downstream primer R1: 5’-CGCG AAGCTT ATGCGGAAGATAACCATTCAAACCGG-3' (SEQ ID NO: 2)

[0050] The 5'end of the upstream primer F1 contains an EcoRI restriction site (underlined), and the 5'end of the downstream primer contains a Hind III restriction site (underlined). Use Bacillus subtilis chromosomal DNA as a template for PCR amplification, and mix the components in a sterile thin-walled centrifuge tube in the following order: 50μL amplification system: ddH 2 O 41.5μL, 10×buffer 5μL, dNTP (2.5mmol / L) 1μL, ...

Embodiment 2

[0056] Example 2: Site-directed mutagenesis of precursor α-amylase

[0057] The mutation diagram of α-amylase is shown in figure 1 , Design complementary primers containing mutant amino acid residues as follows:

[0058] Primer 1: 5'-TTGGTCGGACGGATGGGACATCACT-3' (SEQ ID NO: 4)

[0059] Primer 2: 5'-AGTGATGTCCCATCCGTCCGACCAA-3' (SEQ ID NO: 5)

[0060] Primer 3: 5'-GGGCCATAGAAGATACCGGATCATG-3' (SEQ ID NO: 6)

[0061] Primer 4: 5'-CATGATCCGGTATCTTCTATGGCCC-3' (SEQ ID NO: 7)

[0062] Primer 1 and primer 2 are complementary, and primer 3 and primer 4 are complementary. Primers 1 and 2 contain a mutation to amino acid 223, and primers 3 and 4 contain a mutation to amino acid 558. The recombinant plasmid pET22-ACN7 (constructed in Example 1) was used as a template for PCR amplification, and the components were mixed in a thin-walled centrifuge tube in the following order: PCR1: 50μL system, ddH 2 O38.5μL, 10×buffer 5μL, dNTP (2.5mmol / L) 2μL, primer 1 (20μmol / L) 2μL, primer 2 (20μmol / L) 2μL, D...

Embodiment 3

[0066] Example 3: Construction of mutant α-amylase expression vector

[0067] The Bacillus subtilis expression vector uses pWB980 [purchased from American Biogenetic Sciences, Inc], and the following primers are designed:

[0068] Upstream primer F2: 5’-CGC GGATCC AGGAAACTGCAAACAAATCGAATGAGGTG-3' (SEQ ID NO: 9)

[0069] Downstream primer R2: 5’-CGCG GCATGC ATGCGGAAGATAACCATTCAAACCGG-3' (SEQ ID NO: 10)

[0070] The F25' end of the upstream primer contains a BamH I restriction site (underlined), and the 5'end of the downstream primer contains a Sph I restriction site (underlined). Use the recombinant expression vector pETCN7-2 as a template for PCR amplification, and mix the components in a sterile thin-walled centrifuge tube in the following order: 50μL amplification system: ddH 2 O 41.5μL, 10×buffer 5μL, dNTP (2.5mmol / L) 1μL, upstream and downstream primer 2 (20μmol / L) each 0.5μL, DNA template 1μL, TaqDNA polymerase 0.5μL. Amplification conditions: 94℃ 2min, 1 cycle: 94℃40s, 58℃, 4...

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Abstract

The invention discloses acidproof medium-temperature alpha-amylase and a preparation method thereof. The preparation method comprises the following steps of: A1, amplifying a precursor alpha-amylase gene; A2, performing site-directed mutagenesis on the precursor alpha-amylase; A3, constructing a mutant alpha-amylase expression vector; and A4, transforming bacillus subtilis by using the expression vector. A recombinant strain obtained by the method can be used for industrialized production of an acidproof alpha-amylase mutant. The required acidproof alpha-amylase mutant can be obtained by the following steps of: culturing recombinant cells in a liquid culture medium with selection pressure without induced expression, precipitating supernatant by using ammonium sulfate, dialyzing a precipitate, desalting, adding an allyl dextran S300 gel column, and eluting by using eluent.

Description

Technical field [0001] The invention relates to an acid-resistant mid-temperature alpha-amylase and a preparation method thereof, and belongs to the field of biotechnology. Background technique [0002] α-Amylase (α-1,4-glucanhydrolase EC3.2.1.1), also known as liquefying amylase. It can arbitrarily cut α-1,4 glycosidic bonds from the inside of starch molecules to produce maltodextrin, glucose and other oligosaccharides with smaller molecular weights. Under the action of α-amylase, the molecules of starch are rapidly degraded and the viscosity decreases, and the liquefaction is completed. It is widely used in starch sugar processing industry, textile industry, paper industry, pharmaceutical industry, wine industry and fuel ethanol production, and has considerable commercial value. At present, in industrial production, α-amylase is generally produced on a large scale by microbial fermentation. These microorganisms include Bacillus subtilis, Bacillus licheniformis, Bacillus amylo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N15/75C12N9/28
Inventor 王青艳朱婧黄日波申乃坤秦艳谢能中王成华廖思明黎贞崇陈东
Owner GUANGXI ACAD OF SCI
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