Amylase mutants with improved thermal stability as well as coding gene and application of amylase mutants

A technology encoding gene and amylase, which is applied in application, genetic engineering, plant gene improvement, etc., can solve the problems of poor thermal stability and achieve excellent thermal stability

Active Publication Date: 2015-11-18
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Abstract
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  • Claims
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Problems solved by technology

[0004] The present invention aims at the deficiency of poor thermostability of α-amylase AMY121 in the prior art, and provides a kind of amylase mutant with improved thermostability, its coding gene and application

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  • Amylase mutants with improved thermal stability as well as coding gene and application of amylase mutants
  • Amylase mutants with improved thermal stability as well as coding gene and application of amylase mutants
  • Amylase mutants with improved thermal stability as well as coding gene and application of amylase mutants

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Embodiment 1

[0025] After inserting two amino acids between the 208th and 209th amino acid residues of the α-amylase AMY121 in the thermostable bacterium Bacillussp.SCSIO15121 from deep sea vents, its thermal stability decreased significantly. Through homology modeling, it was found that the insertion of two amino acids significantly changed the conformation of Lys209. The amino acid sequence of the α-amylase AMY121 is shown in SEQ ID NO.1, and the nucleotide sequence of its coding gene—α-amylase gene amy121 has been deposited in Genbank, and its accession number is KJ577547.

[0026] 1 Identification of amino acid residue Lys209 related to thermostability

[0027] 1.1 Site-directed mutation of Lys at position 209 of α-amylase AMY121 mediated by inverse PCR

[0028] In order to further study the amino acid residue Lys209 related to the thermostability of α-amylase AMY121, starting from the α-amylase amy121 gene (KJ577547) of deep-sea hydrothermal vent bacteria Bacillussp. The primers wer...

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Abstract

The invention discloses amylase mutants with improved thermal stability as well as a coding gene and application of the amylase mutants. Starting from hydrothermal vent derived bacterium alpha-amylase AMY121, the amylase mutants with improved thermal stability are obtained by virtue of site-saturation mutagenesis technology; the hydrothermal vent bacterium alpha-amylase AMY121 has an amino acid residue sequence as shown in SEQ ID NO.1; and the amino acid sequences of screened amylase mutants Y187E, K205L and Y187E / K205L are respectively as shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4. At corresponding optimum action temperature, on the basis of an evaluation standard of T5015 and half-life period t1 / 2 at 75 DEG C, the thermal stability of the amylase mutant is improved compared with wild amylase; and the application scope of the hydrothermal vent bacterium alpha-amylase AMY121 is extended.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kind of amylase mutant with improved thermostability, its coding gene and application. Background technique [0002] α-amylase (EC3.2.1.1) can hydrolyze high-molecular-weight starch into low-molecular-weight oligosaccharides, which has wide application value in bioenergy, feed, food and other industries. The first step in the enzymatic utilization of native starch is often to gelatinize the starch at high temperature, and add α-amylase during this process, so the market has a strong demand for high-temperature-resistant α-amylase. However, the wild-type amylase screened from the natural environment often cannot fully adapt to the harsh external environment in industrial applications. The inventor cloned a new α-amylase gene amy121 (KJ577547) from the heat-resistant bacterium Bacillussp.SCSIO15121 in deep-sea hot springs. The optimum temperature for the recombinant enzy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/28C12N15/56
CPCC12N9/2417C12Y302/01001
Inventor 龙丽娟杨键李丽珍李洁肖运柱齐振雄尹团张偲
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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