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Acid-proof and high-temperature resistant alpha-amylase and production thereof

An amylase and high temperature-resistant technology, which is applied in the field of acid-resistant and high-temperature-resistant alpha-amylase and its preparation, can solve the problems of application limitation, acid-resistance and high-temperature resistance of alpha-amylase cannot be taken into account at the same time, and achieves a wide pH applicable range , the effect of good acid stability

Inactive Publication Date: 2008-09-03
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the above situation, the present invention solves the problem that the existing α-amylase cannot take into account both acid resistance and high temperature resistance, 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 an alpha-amylase mutant capable of both high temperature resistance and acid resistance and a preparation method thereof

Method used

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  • Acid-proof and high-temperature resistant alpha-amylase and production thereof
  • Acid-proof and high-temperature resistant alpha-amylase and production thereof
  • Acid-proof and high-temperature resistant alpha-amylase and production thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1: Amplification of the Precursor Alpha-Amylase Gene

[0033] Chromosomal DNA of Bacillus licheniformis [China Industrial Microorganism Collection Center (CICC) 10181] was extracted. Design the following primers (the primers are commissioned to be synthesized by Shanghai Bioengineering Co., Ltd.):

[0034] Upstream primer F1: 5'-AGGATCCCTTGAAGAAGTGAAGAAGCAGAGAGG

[0035] Downstream primer R1: 5'-AAAAGCTTCCTGAGGGCTGATGATGACACTTTG

[0036] The upstream primer F15' end contains a BamHI restriction site, and the downstream primer R15' end contains a HindIII restriction site. Perform PCR amplification using the chromosomal DNA of Bacillus licheniformis 020401 as a template, and mix the components in a sterilized thin-walled centrifuge tube in the following order: Use 50L amplification system: ddH 2 O 41.5L, 10×buffer 5L, dNTP (2.5mmol / L each) 1L, upstream primer F1 (20mol / L) 0.5L, downstream primer R1 (20mol / L) 0.5L, DNA template 1L, TaqDNA polymerase 0.5L . The...

Embodiment 2

[0037] Example 2: Site-directed mutagenesis of precursor alpha-amylases

[0038] The mutation diagram of α-amylase is shown in image 3 . Design overlapping primers as follows:

[0039] Overlap Primer A: 5'-GAGAACACCGCATTAAAGCCTGGAC-3'

[0040] Overlap Primer B: 5'-GTCCAGGCTTTAATGCGGTGTTCTC-3'

[0041] Overlapping primer C: 5'-AAGCATCCGTTGAAAGCGGTTACAT-3'

[0042] Overlapping primer D: 5'-ATGTAACCGCTTTCAACGGATGCTT-3'

[0043] Overlapping primer A is complementary to overlapping primer B, and overlapping primer C is complementary to overlapping primer D. Overlapping primers A and B contained a mutation at amino acid 134, while overlapping primers C and D contained a mutation at amino acid 320. Use the recombinant plasmid pUCA as a template for PCR amplification, and mix the components in a sterilized thin-walled centrifuge tube in the following order: PCR1: use 50 μL amplification system, ddH 2 O 38.5 μL, 10× buffer 5 μL, dNTP (2.5 mmol / L each) 2 μL, upstream primer F1 (...

Embodiment 3

[0044] Embodiment 3: Preparation of expression vector

[0045] Escherichia coli JM109 strain carrying the plasmid pBCH (purchased from Treasure Biotech Co., Ltd.) was inoculated in LB medium containing ampicillin (50 μg / mL), and cultured overnight at 37° C. with shaking. Transfer 1.5 mL of bacterial liquid into a microcentrifuge tube, centrifuge at 12,000 rpm for 30 seconds to collect bacterial cells, discard the supernatant, and empty the residual liquid. Resuspend the pellet in 100 μL of pre-cooled solution I (50 mmol sucrose, 25 mmol Tris, 10 mmol EDTA, pH 8.0), and mix well. Add 200 μL of newly prepared solution II (0.2mol NaOH, 1% SDS), cap the tube tightly, shake gently, and place it on ice for 1-2 minutes until the liquid becomes clear. Add 150 μL of pre-cooled solution III (3 mol potassium acetate, pH 4.8), gently rotate the centrifuge tube to mix solution III evenly in the viscous bacterial lysate, and place it on ice for 3-5 minutes. Centrifuge at 12,000 rpm for 5 ...

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Abstract

A fire-resistant and acid-proof alpha-amylase and its production are disclosed. The process is carried out by separating precursor-alpha-amylase gene from lichenized bacillosporin, mutating for L134 and S320 amino acid residues and high-efficient expressing alpha-amylase mutant in bacterium. It achieves better safety, expression and acid stability, and more yield.

Description

technical field [0001] The invention belongs to site-directed mutation α-amylase and its preparation method by using recombinant PCR technology, and relates to acid-resistant and high-temperature-resistant mutant α-amylase mutated at a specific site and its preparation technology. The obtained α-amylase has good acid stability and can complete its functional activity under the process conditions required in production. Background technique [0002] α-amylase (α-1,4-glucan-glucanhydrolase EC 3.2.1.1) is also known as liquefying amylase. It can arbitrarily cut α-1,4 bonds from the inside of starch molecules to produce maltodextrin with relatively small molecular weight. Under the action of α-amylase, the starch molecules degrade rapidly, the viscosity decreases, and the liquefaction is completed. α-amylase has considerable commercial value and is widely used in starch deep processing industry, alcohol industry, beer industry, citric acid industry, monosodium glutamate and st...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/28C12N15/56C12N15/63
Inventor 路福平杜连祥蔡恒陈忠军
Owner TIANJIN UNIV OF SCI & TECH
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