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Amylase, and encoding gene and application thereof

A technology of amylase and gene, applied in the field of amylase and its coding gene and its application, can solve the problems of limited application range, loss of activity, etc., and achieve strong enzyme activity and different sensitivity

Active Publication Date: 2014-12-31
SUN YAT SEN UNIV
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Problems solved by technology

[0003] Enzyme is a kind of biocatalyst, and the surrounding environment (temperature, pH, organic reagent, etc.) has a great influence on its activity. Many enzymes will lose their activity under some harsh reaction conditions (high temperature, low temperature or strong acid-base environment) , which limits its scope of application

Method used

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  • Amylase, and encoding gene and application thereof
  • Amylase, and encoding gene and application thereof
  • Amylase, and encoding gene and application thereof

Examples

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Embodiment Construction

[0034] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0035] 1: Screening of clones containing amylase sequence.

[0036] First compare the deep-sea hydrothermal vent metagenomic sequence with the NCBI non-redundant database to find information similar to the amylase gene; then search for the corresponding sequence information according to the annotation information, design specific primers, and screen the corresponding gene from the library. clones. In order to improve the screening efficiency, the 2880 clones participating in 454 sequencing were divided into 30 groups, that is, each 96-well plate was prepared into a mixed plasmid sample, and another plasmid sample mixed from 2880 clones was prepared as a positive control during screening In this way, the first round of screening is performed using the 30 mixed samples as PCR amplification templates, and the number of the mixed sample c...

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Abstract

The invention relates to two kinds of amylase protein and an encoding gene thereof, and application of the protein as starch hydrolase in industrial field. Fosmid library gene information fragment annotation information obtained by performing 454 sequencing on a sample coming from a deep-sea hydrothermal vent is taken as a research material, corresponding clones F_135 in a corresponding amylase gene cluster is screened out from a metagenome library, and the Fosmid clones is subjected to shotgun combined primer-walking sequencing and ORF predication, so that the gene sequence of the encoding amylase is obtained, and the DNA sequence of the encoding amylase is shown as a sequence table SEQ ID NO. 1. A target gene is inserted into pET-32a(+) expression vector, and through expression purification a polypeptide or a mature peptide encoded by the gene is obtained, and the amino acid sequence of the polypeptide or the mature peptide is shown as a sequence table SEQ ID NO. 2. The enzyme activity possessed by the amylase is subjected to preliminary research. The amylase is applicable to industrialized production correlated to starch hydrolysis because of the unique activity and the enzymological characteristics.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an amylase, its coding gene and its application. Background technique [0002] Amylases are a class of enzymes that can hydrolyze starch molecules into polymeric molecules composed of glucose, which are found widely distributed in animal, plant and microbial organisms. Amylase constitutes a large class of industrial enzymes, accounting for about 25% of the enzyme market. With the development of technology, the application range of amylase ranges from traditional starch degradation, textile, food, fermentation, papermaking, brewing and distillation industries field, extended to clinical, pharmaceutical and analytical chemistry and other fields. [0003] Enzyme is a kind of biocatalyst. The surrounding environment (temperature, pH, organic reagent, etc.) has a great influence on its activity. Many enzymes will lose their activity under some harsh reaction conditions (high temper...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/26C12N15/56C12N15/11C12N15/70C12N1/21C12R1/19
CPCC12N9/2417C12Y302/01001
Inventor 徐安龙
Owner SUN YAT SEN UNIV
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