An alpha-amylase mutant with improved enzymatic activity and applications thereof

A technology of amylase and mutants, which is applied in the field of genetic engineering and microbial engineering, can solve the problems of unfavorable human body, difficult and limited application of α-amylase

Active Publication Date: 2018-12-18
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0007] The expression level of high-temperature α-amylase in Escherichia coli is higher than that in Bacillus subtilis, but because Escherichia coli will produce harmful substances such as endotoxin in the process of fermentation and enzyme production, which is harmful to the human body and greatly limits its application ; and the cell wall of Bacillus subtilis does not contain endotoxin, is a kind of non-pathogenic soil microorganism, has been identified as food safety grade bacterial strain GRAS (Generally recognized as safe) by the U.S. Food and Drug Administration and relevant Chinese departments, but high temperature α -The expression level and activity of amylase heterologously expressed in Bacillus subtilis are very low
[0008] The above defects all make it difficult for the high-temperature α-amylase derived from bacteria to be widely used in industry

Method used

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  • An alpha-amylase mutant with improved enzymatic activity and applications thereof
  • An alpha-amylase mutant with improved enzymatic activity and applications thereof
  • An alpha-amylase mutant with improved enzymatic activity and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Preparation of α-amylase mutants

[0051] (1) Preparation of α-amylase single mutant

[0052] According to the gene sequence of α-amylase whose amino acid sequence is shown in SEQ ID NO.1, primers for introducing K82E and S405R mutations were designed and synthesized, and rapid PCR technology was used to carry the recombination of the gene encoding wild-type α-amylase The vector pET20b-amyS template was used to perform site-directed mutation on the α-amylase gene, and the DNA coding sequence was determined. It was identified that the 82nd Lys codon was changed into a Glu codon, and the 405th Ser codon was changed into an Arg codon. single mutant alpha-amylase;

[0053] Among them, the recombinant vector pET20b-amyS was preserved in the laboratory (Ref: L Z, D X, W J. Improving thethermostability and enhancing the Ca 2+ binding of the maltohexaose-forming α-amylase from Bacillus stearothermophilus. Journal of Biotechnology. 2016; 222:65–72.).

[0054] The s...

Embodiment 2

[0072] Embodiment 2: α-amylase mutant shakes flask fermentation to produce enzyme verification in Escherichia coli

[0073] (1) Shake flask fermentation of α-amylase mutants in Escherichia coli to produce enzymes

[0074] Pick recombinant bacteria E.coli BL21(DE3) / pET20b-amyS(K82E), E.coli BL21(DE3) / pET20b-amyS(S405), E.coli BL21(DE3) / pET20b-amyS(K82E / S405R ) at 37°C in LB liquid medium (containing 100 μg / mL ampicillin) for 8 to 10 hours, and the seed fermentation liquid was connected to the fermentation medium (containing 100 μg / mL ampicillin) according to the inoculum size of 5%, at 25 After culturing in a shaking table at ℃ for 48 hours, the fermentation broth was centrifuged at 4 ℃ and 8000 rpm for 10 minutes to remove bacteria, and the centrifuged supernatant was collected to obtain a crude enzyme solution.

[0075] (2) Determination of enzyme activity of α-amylase mutants in Escherichia coli shake flask fermentation

[0076] Determination of α-amylase mutants K82E, S40...

Embodiment 3

[0079] Example 3: Construction of recombinant vector pHY-SP amyE -amyS, pHY-SP amyE -amyS(K82E / S405R)

[0080] The specific steps are as follows (the construction process see figure 1 ):

[0081] (1) Design primers amyS-F and amyS-R containing homology arms, use recombinant vectors pET20b-amyS and pET20b-amyS (K82E / S405R) as templates, and PCR amplify amyS and amyS with homology arms ( K82E / S405R)

[0082] (2) Design primers pHY-F and pHY-R, and use the recombinant vector pHY300PLK-β-CGTase as a template to amplify the vector pHY-SP by PCR amyE , wherein the recombinant vector pHY300PLK-β-CGTase is preserved in the laboratory (Ref: Zhang, K., Duan, X., Wu, J. 2016. Multigene disruption in undomesticated Bacillus subtilis ATCC 6051 ausing the CRISPR / Cas9 system. Scientific Reports, 6. Bacillus subtilis).

[0083] The primer sequences are shown in Table 3:

[0084] Table 3 Primer Sequence

[0085]

[0086]

[0087] Note: The underlined part is the homology arm sequ...

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Abstract

An alpha-amylase mutant with improved enzymatic activity and applications thereof are disclosed and belong to the technical field of genetic engineering and microbial engineering. The mutant is obtained by mutating the amino acid at the number 82 site of alpha-amylase having an amino acid sequence of SEQ ID NO.1 from lysine into glutamic acid or mutating the amino acid at the number 405 site of the alpha-amylase having an amino acid sequence of SEQ ID NO.1 from serine into arginine or simultaneously mutating the amino acid at the number 82 site of the alpha-amylase having an amino acid sequence of SEQ ID NO.1 from lysine into glutamic acid and mutating the amino acid at the number 405 site from serine into arginine. The enzymatic activity of alpha-amylase in a fermentation solution can beincreased to 474.7 U/mL by fermenting a recombinant Bacillus subtilis engineering bacterium for 48 h, with the recombinant Bacillus subtilis engineering bacterium being constructed by adopting a genecoding the mutant as a target gene, adopting pHY300PLK as an expression vector, and adopting Bacillus subtilis WS5 as an expression host.

Description

technical field [0001] The invention relates to an α-amylase mutant with improved enzyme activity and application thereof, belonging to the technical fields of genetic engineering and microbial engineering. Background technique [0002] α-amylase (α-amylase, EC.3.2.1.1) is an important glycoside hydrolase, which has a wide range of substrate preferences and product specificity, and can cut off starch and related α-glucan molecules. α-1,4-glucosidic bond, and hydrolyze starch into soluble dextrin, oligosaccharides, maltose and glucose, and at the same time, it can retain the α-isomer conformation of the product. [0003] Therefore, α-amylase is widely used in industries such as food, washing, paper making, textile, alcohol and medicine. [0004] According to the different action temperature, α-amylase can also be divided into high temperature, medium and low temperature α-amylase. Among them, the high-temperature α-amylase has good thermal stability and has a wide range of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/28C12N15/56C12N15/63C12N1/21C12P19/14C12P19/04C12P19/02C12P19/12C12R1/125
CPCC12N9/2417C12P19/02C12P19/04C12P19/12C12P19/14C12Y302/01001
Inventor 吴敬宿玲恰姚动邦
Owner JIANGNAN UNIV
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