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A mutant α-amylase and its preparation method and application

A technology of amylase and PET30-PFAMY, which is applied in the field of bioengineering, can solve problems such as poor thermal stability and pH inadaptability, and achieve the effects of simplifying post-processing, improving enzyme activity, and reducing costs

Active Publication Date: 2019-06-28
河北华石生物科技有限公司
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Problems solved by technology

[0007] Because common α-amylases often have problems such as poor thermal stability and pH unsuitability for industrial production, researchers are currently focusing on the study of acid-resistant and high-temperature resistant α-amylases, which are more conducive to industrial production and are of great significance for saving energy and reducing costs.

Method used

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  • A mutant α-amylase and its preparation method and application
  • A mutant α-amylase and its preparation method and application
  • A mutant α-amylase and its preparation method and application

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Embodiment

[0034] 1 Gene design and synthesis

[0035] (1) Gene design: According to the protein sequence of the archaea Pyrococcus furiosus DSM 3638 in Genebank (SEQ ID NO: 1, Genebank: AAL80601.1), mutation sites were introduced into its W65, C204, and N366 sites, and the obtained sequence was as SEQ ID Amino acid sequence shown in NO: 2; use the online design tool Jcat (http: / / www.jcat.de / ) to reverse design the gene sequence. For the preferred codons required for host Escherichia coli expression and the G+C base content required for high-efficiency gene expression, the gene sequence designed above was optimized as SEQ ID NO: 3, which is similar to the wild-type gene sequence SEQ ID NO: 4 Ratio: The number of rare codons in Escherichia coli has been reduced from 13 to 0 in the wild type (SEQ ID NO:4), and the NdeI and SacI restriction sites have been removed during the design process, which is beneficial for later gene manipulation. The above-mentioned serial design is favorable for ...

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Abstract

The invention belongs to the field of biological engineering, and in particular relates to mutational alpha-amylase as well as a preparation method and application thereof. The mutational alpha-amylase provided by the invention has a high-temperature-resistant amino acid sequence, amino acids at the following one or more sites of the amino acid sequence are replaced with other amino acids on the basis of an amino acid sequence of wild type pyrococcus furiosus alpha-amylase (PFAMY), the replaced amino acid sites are W65, C204 and N366, the expression quantity of mutated PFAMY genes reaches 10000u / l in escherichia coli, and the enzyme activity of the mutational alpha-amylase is greatly improved compared with the wild type PFAMY; the heat stability of the mutated PFAMY is improved, and the optimum temperature is changed to 100 DEG C from the original 90 DEG C, so that the mutational alpha-amylase is higher in heat stability and more stable in enzyme activity; therefore, the mutational alpha-amylase has better industrial application prospect.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a mutant alpha-amylase and its preparation method and application. Background technique [0002] Starch is the second largest sugar reserve in nature after cellulose and is an important component of many food and non-food industries. Until the 19th century, starch was hydrolyzed using acid hydrolysis with dilute hydrochloric acid, but it is now almost completely replaced by enzymatic hydrolysis because of its low sugar content and the production of some unnecessary compounds. [0003] α-amylase, that is, α-1,4-glucan hydrolase, is widely distributed in animals, plants and microorganisms, and can randomly act on α-1 in starch, glycogen, oligosaccharide or polysaccharide molecules. 4-glycosidic bonds, hydrolyzing starch into a series of small molecular substances such as dextrin, maltose, oligosaccharides and glucose. [0004] The world enzyme market presents a phenomeno...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/28C12N15/56C12N15/70
CPCC12N9/2417C12N15/70C12N2800/22C12Y302/01001
Inventor 葛文广刘红厚蒋北华马彩霞
Owner 河北华石生物科技有限公司
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