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A kind of α-amylase gene and its application

A technology of amylase and gene, which is applied in genetically engineered bacteria and its application fields, can solve the problems of poor enzyme activity, instability, and insufficient purity of α-amylase, and achieve the effects of short cycle, easy purification, and large-scale expression

Active Publication Date: 2021-05-25
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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  • A kind of α-amylase gene and its application
  • A kind of α-amylase gene and its application
  • A kind of α-amylase gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Nucleotide sequence of amylase gene isolated from Bacillus subtilis 48-1.

[0029] Genomic DNA of Bacillus subtilis was extracted by the 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'-GGAATTCCATATGGTGAGAAGCAAAAAAA-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] Table 1 The reaction parameters of PCR

[0034] 10×Taq Buffer 5μl dNTP (2.5mmol / L) 4μl Template (genomic DNA) 2μl Primer F 2μl Primer R 2μl Taq DNA polymerase 0.5μl wxya 2 o

[0035] Amplification conditions: pre-denatur...

Embodiment 2

[0036] Embodiment 2: Construction of recombinant expression vector

[0037] The fragment amplified in Example 1, because the 5' end of the primer F contains the Nde I restriction site (the 8th base to the 13th base) and the 5' end of the primer R contains the BamH I respectively Restriction site (from the 3rd base to the 8th base), so use these two enzymes to double-enzyme digest the fragment, and pET28a is double-enzyme-digested with the same enzyme at the same time, and the large fragment is recovered by electrophoresis. And use T4 ligase to ligate 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 )...

Embodiment 3

[0038] Embodiment 3: the preparation of amylase

[0039] The recombinant engineering bacterium obtained in Example 2 is connected to 100ml LB liquid medium (containing 1‰50mg / ml kanamycin) by 1% inoculum, 37 DEG C, 150rpm is cultivated to OD600=0.6, adds 1‰ of 1mM IPTG (isopropyl-β-D-thiogalactopyranoside), transferred to 16 ° C, 80rpm after induction for 10h, centrifuged to collect the bacteria, the bacteria were washed with 5ml 50mmol / l imidazole buffer (pH8.2 ) suspension, ultrasonic crushing. Centrifuge to take supernatant and precipitate respectively, then suspend the precipitate with 5ml 50mmol / l imidazole buffer (pH8.2), take 50μl respectively for SDS polyacrylamide gel electrophoresis (SDS-PAGE), bands appear in the precipitate, indicating The resulting recombinant protein is an inclusion body, such as Figure 4 Lane 3 shows. Example 4: Denaturation and renaturation of inclusion bodies and determination of enzyme activity

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Abstract

The invention discloses an α-amylase gene, which is obtained from Bacillus Bacillus It is isolated from sp.48‑1, and contains its constructed recombinant vector, genetically engineered bacteria and its produced recombinant amylase preparation. The amylase amino acid sequence with endo-α-1,4 glycosidic bond activity is shown in SEQ ID NO.1, and the coding sequence of its gene is the nucleotide sequence shown in SEQ ID NO.2. By constructing a recombinant vector and expressing it in Escherichia coli, the expression product has the function of endocutting α-1,4 glycosidic bonds, and the present invention describes that amylase plays an important role in promoting alcoholization of tobacco leaves.

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 Patents(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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