Alpha-amylase gene and application thereof

A technology of amylase and gene, applied in genetically engineered bacteria and its application fields, can solve the problems of poor enzyme activity, instability, and insufficient purity of α-amylase, etc., and achieve simple culture medium and culture conditions, short cycle, and enzyme The effect of live stability

Active Publication Date: 2019-01-11
CHINA TOBACCO YUNNAN IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are few reports on the use of α-amylase to promote tobacco alcoholization. Although there are also reports that the amylase gene is cloned from Bacillus, based on the selection of bacterial strains, the selection of expression plasmids, and the methods for constructing plasmids, etc., The obtained α-amylase has insufficient purity, poor enzymatic activity, and instability

Method used

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  • Alpha-amylase gene and application thereof
  • Alpha-amylase gene and application thereof
  • Alpha-amylase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: from bacillus subtilis ( Bacillus subtilis ) The nucleotide sequence of the amylase gene isolated in 48-1.

[0029] Genomic DNA of Bacillus subtilis was extracted by CTAB method, and 2 μl was used as a template for polymerase chain reaction (PCR). According to the conserved region of the published amylase homologous sequence, primers (primer F and primer R) were designed in combination with the vector used. , the primers, components and amplification conditions used in this PCR reaction process are as follows:

[0030]F: 5'-GGAATTCCATATGGTGAGAAGCAAAAAA-3' (SEQ ID NO.3)

[0031] R: 5'-CGGGATCCTTATTGTGCAGCTGCTTGTA-3' (SEQ ID NO.4)

[0032] The composition of the PCR amplification system is shown in Table 1.

[0033] 10×Taq Buffer

[0034] Amplification conditions: pre-denaturation at 94°C for 4min, denaturation at 94°C for 45S, annealing at 57°C for 45S, extension at 72°C for 90S, end filling at 72°C for 10min, three steps of denaturation, anne...

Embodiment 2

[0035] Embodiment 2: Construction of recombinant expression vector

[0036] The fragment amplified in embodiment 1, because the 5 ' end of its primer F contains respectively Nde The I restriction site (from the 8th base to the 13th base) and the 5' end of the primer R contain Bam H I restriction site (3rd base to 8th base), so use these two enzymes to double-digest the fragment, and pET28a is double-digested with the same enzyme, and the large fragment is recovered by electrophoresis , and ligated with T4 ligase at 16°C for 6h, the schematic diagram of the successfully constructed vector is as follows figure 1 shown. The ligation product was transformed into Escherichia coli BL21(DE3) by chemical transformation method, cultured on LB solid plate containing kanamycin (100 μg / ml), and the white colonies growing on the plate were picked and analyzed by colony PCR ( figure 2 ) and extracted plasmid single and double digestion ( image 3 ) positive clones were screened and ...

Embodiment 3

[0037] Embodiment 3: the preparation of amylase

[0038] The recombinant engineered bacteria obtained in Example 2 was connected to 100ml LB liquid medium (containing 1‰ 50mg / ml kanamycin) according to 1% inoculation amount, and cultivated to OD at 37°C and 150rpm 600 =0.6, add 1‰ of 1mM IPTG (isopropyl-β-D-thiogalactopyranoside), turn to 16°C, induce 10h at 80rpm, collect the bacteria by centrifugation, and use 5ml 50mmol / l Suspended in imidazole buffer (pH 8.2) and sonicated. Take the supernatant by centrifugation, then suspend it with 5ml 50mmol / l imidazole buffer (pH8.2), take 50μl respectively for SDS polyacrylamide gel electrophoresis (SDS-PAGE), the band appears in the supernatant after centrifugation, Indicates that the obtained recombinant protein is not an inclusion body, such as Figure 4 Shown in lane 1.

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Abstract

The invention discloses an alpha-amylase gene. The alpha-amylase gene is prepared from Bacillus sp. 48-1, containing the constructed recombinant vector, the genetically engineered bacterium and the recombinant amylase preparation produced by the genetically engineered bacterium. Having an endotangent. The amino acid sequence of the amylase with alpha-1, 4 glycosidic bond activity is shown in SEQ ID NO: 1, and the coding sequence of the gene is the nucleotide sequence shown in SEQ ID NO: 2. The recombinant vector is constructed and expressed in E. coli, and the product of expression has Alpha-1, 4 endoglycosidic bond function. The present invention describe that amylase plays an important role in promote tobacco leaf alcoholization and the like.

Description

technical field [0001] The invention relates to an amylase, especially an α-amylase, a gene encoding the enzyme, a recombinant vector containing the encoding gene and its construction method, a genetically engineered bacterium and its application, belonging to the field of biotechnology and enzyme engineering . Background technique [0002] Starch is formed by the polymerization of glucose molecules. It can be divided into two types: amylose and amylopectin. The general formula is (C6H10O5)n. Amylose is an unbranched helical structure, and amylopectin is composed of 24 to 30 glucose residues connected end to end by α-1,4-glycosidic bonds, and α-1,6-glycosidic bonds at the branched chains. Starch is a nutrient stored in plants, stored in seeds and tubers, and the content of starch in all kinds of plants is relatively high. In addition to being edible, starch can also be used as a raw material for making dextrin, maltose, glucose, and alcohol. It can also be used to prepare ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/28C12N15/56C12N15/70C12N1/21C12R1/19
CPCC12N9/2417C12N15/70C12Y302/01001
Inventor 吴丽君白晓莉王毅朱杰杨德中段如敏孙万万魏云林季秀玲
Owner CHINA TOBACCO YUNNAN IND
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