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Alpha-amylase BaAmy mutant capable of improving specific activity, and encoding gene and application thereof

A technology of amylase and mutants, applied in the field of genetic engineering, can solve the problems of restricting industrial applications, high production costs, and low specific activity

Active Publication Date: 2017-05-31
GUANGDONG VTR BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The α-amylase of Bacillus acididiosius is referred to as BaAmy for short. Although it has good acid stability, its specific activity is low and its production cost is high, which limits its industrial application.

Method used

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  • Alpha-amylase BaAmy mutant capable of improving specific activity, and encoding gene and application thereof
  • Alpha-amylase BaAmy mutant capable of improving specific activity, and encoding gene and application thereof
  • Alpha-amylase BaAmy mutant capable of improving specific activity, and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1. Cloning of acid alpha-amylase from Bacillus acidicola

[0025] Bacillus acidococcus was inserted into LB medium, and after 24 hours of culture, its genomic DNA was extracted. Two primers (R: 5'-ATGAATTCAACGGCACCATGATGCAGTAT-3' and F: 5'-GCTCTAGACTAAACCGAACCACCATTGACTTTG-3') were designed according to the reported sequence of Bacillus acidococcus alpha-amylase (Genebank: JN680873) for amplification of acidococcus spores Bacillus alpha-amylase gene. The amplified PCR product is purified and recovered, and ligated to the expression vector pPICzαA to obtain the expression vector pPICzαA-BaAmy.

Embodiment 2

[0026] Example 2. Site-directed mutation

[0027] Using the above-mentioned pPICzαA-BaAmy as a template, the corresponding primers are used for PCR amplification. The specific amplification reaction system is as follows:

[0028] Q5 High Fidelity Taq Enzyme MIX23uL Corresponding mutant primer1uL Corresponding mutant primer1uL pPICzαA-BaAmy(20ng)2uL Add water to50uL

[0029] The reaction procedure is as follows:

[0030]

[0031] Agarose electrophoresis is used to detect the PCR amplification results, and the PCR products are purified and recovered. The original plasmid was decomposed with the restriction enzyme DpnI, and the decomposed product was transferred into E. coli Top10 by heat shock method, the recombinant transformant was verified by bacterial liquid PCR, and the plasmid of the correct transformant was extracted and sequenced to confirm The corresponding mutant. The sequenced mutants were linearized with SacI and transferred to Pichia pastoris X33.

Embodiment 3

[0032] Example 3. High-throughput screening of high specific activity mutant strains

[0033] The yeast recombinant transformants in Example 2 were picked one by one with a toothpick to a 24-well plate, 1 mL of BMGY medium was added to each well, cultured at 30° C., 220 rpm for about 24 hours, and centrifuged to remove the supernatant. Then add 1.6mL BMMY medium to induce culture. After culturing for 24 hours, the supernatant was collected by centrifugation, and 200 μL of the above supernatant was taken out to a 96-well plate for α-amylase activity determination. The detection of α-amylase enzyme activity is carried out according to the national standard of the People's Republic of China "GB / T 24401-2009".

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Abstract

The invention relates to the field of gene engineering, in particular to an alpha-amylase BaAmy mutant capable of improving specific activity, and an encoding gene and application thereof. The mutant is a mutant of acidic alpha-amylase BaAmy with an amino acid sequence as shown in SEQ ID NO.8, wherein mutation sites are any one or more of the 37th, 85th, 191st, 241st, 279th, 291st, 319th and / or 333rd amino acid. The specific activity of the mutated alpha-amylase is increased by 45 to 100 percent relative to the specific activity of the original alpha-amylase, and foundation is laid for industrialized application of acidic bacillus acidic alpha-amylase.

Description

Technical field [0001] The invention relates to the field of genetic engineering, in particular to an alpha-amylase BaAmy mutant with increased specific activity and its coding gene and application. Background technique [0002] α-Amylase can cut α-1,4 glycosidic bonds randomly from the inside of starch molecules across α-1,6 bonds that cannot cut branch points. As an important hydrolytic enzyme, α-amylase is widely used in food processing, alcohol brewing, pharmacy, textile desizing, papermaking, washing liquid and feed and other fields. [0003] At present, the optimal pH values ​​of commercial α-amylase and saccharification enzymes widely used in industry are about 6.5 and 4.5, so acid and alkali need to be added to adjust the pH during the liquefaction and saccharification process. Adding a large amount of acid and alkali in the liquefaction and saccharification process not only complicates the processing technology, but also increases the production cost. If an alpha-amylase...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/28C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/2417C12N15/815C12N2800/102C12Y302/01001
Inventor 李阳源王建荣黄江聂金梅陈丽芝何小梅杨玲黄佳乐
Owner GUANGDONG VTR BIO TECH
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