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A novel α-amylase and its application

A technology of amylase and recombinant plasmid, applied in the direction of application, enzyme, introduction of foreign genetic material using vector, etc., to achieve the effect of wide temperature tolerance range

Active Publication Date: 2017-11-10
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few industrial application reports of low temperature and high activity α-amylase

Method used

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  • A novel α-amylase and its application
  • A novel α-amylase and its application
  • A novel α-amylase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Example 1: Acquisition of α-amylase gene laxh3357

[0013] Through the analysis of the whole genome of the new deep-sea bacteria XH031, three amylase genes and their gene sequences were obtained, and the upstream primer (5'-CGGAATTCATGCACCCTCGACCGGC-3') and the downstream primer (5'-CCCTCGAGACGCCGCCACATCCG -3') Using genomic DNA as a template, carry out PCR reaction, the PCR reaction composition is as follows (50 μ l reaction system): ddH 2 O 11.5 μl, upstream and downstream primers 0.5 μl each, DNA template 2 μl, 2×GCBuffer 25 μl, Taq 0.5 μl, dNTP Mixture 8 μl. The reaction conditions were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 65°C for 30 s, extension at 72°C for 3 min for 30 s, and final extension at 72°C for 10 min, a total of 30 cycles. After the reaction, the PCR product was recovered to obtain the α-amylase gene laxh3357. laxh3357 is one of the α-amylase genes, its nucleotide sequence is SEQ ID NO: 2, the encoded amino ...

Embodiment 2

[0014] Example 2: Construction of Escherichia coli cloning vector PUCm-T-laxh3357.

[0015] The α-amylase gene laxh3357 was connected to the carrier PUCm-T by using DNA Ligation Kit. The connection system (10 μl) was as follows: SolutionI 5 μl, DNA fragment 4 μl, PUCm-T carrier 1 μl. The connection solution obtained by ligation at 16°C for 16 hours can be used to obtain the E. coli cloning vector PUCm-T-laxh3357, which is used to transform E. coli JM109.

Embodiment 3

[0016] Example 3: Construction of Escherichia coli recombinant strain JM109-PUCm-T-laxh3357

[0017] Add 200 μl of thawed competent Ecoli.JM109 and 10 μl of the connection solution obtained in Example 2 to a 2 ml Eppendorf tube, ice-bath for 30 minutes, heat shock at 42°C for 90 seconds, ice-bath for 15 minutes, add 800 μl of LB medium, and culture with shaking at 37°C for 45 minutes. The bacterial solution was mixed with 4 μl IPTG and 40 μl X-gal, spread on the LB plate containing 100 μg / ml ampicillin, and incubated at 37°C for 12-14h. Pick white colonies for PCR and double enzyme digestion detection, the enzyme digestion system (20μl) is as follows: ddH 2 O 8 μl, PUCm-T-laxh3357 plasmid DNA 8 μl, EcoRI 1 μl, XholI 1 μl, 10×H Buffer 2 μl, those with 1428 bp specific band in agarose gel electrophoresis were positive transformation clones, that is, Escherichia coli JM109- containing PUCm-T-laxh3357 PUCm-T-laxh3357. Send 1ml of the positive clone bacteria solution for testing,...

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Abstract

The invention aims at providing novel alpha-amylase. The amino acid sequence of the novel alpha-amylase is SEQ ID NO: 1; the genes of the alpha-amylase are encoded, and one nucleotide sequence is SEQ ID NO: 2. The alpha-amylase has a wider temperature tolerance range, the specific activity of the amylase at 50 DEG C is up to 5776 U / mg, and the novel alpha-amylase still can keep about 38% activity at the low temperature of 10 DEG C.

Description

technical field [0001] The invention belongs to the technical field of functional gene screening, and in particular relates to a novel alpha-amylase and its application. Background technique [0002] α-amylase specifically catalyzes the hydrolysis of α-1,4 glycosidic bonds inside starch molecules, which can hydrolyze starch into smaller fragments. As the main hydrolase for degrading starch, α-amylase constitutes a very large family of glycosyl hydrolases. Most of the amylases found at present have the properties of acid resistance and high temperature resistance. Although many species can produce amylases, the α-amylases used commercially mainly come from the genus Bacillus (Bacillus licheniformis, Bacillus stearothermophilus and Bacillus amyloliquefaciens bacteria, etc.). The thermostable α-amylases widely put into commercial application are mainly from Bacillus subtilis, Bacillus stearothermophilus, Bacillus licheniformis and Bacillus amyloliquefaciens. However, there a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/28C12N15/70C12N15/56C12N1/21C12R1/19
CPCC12N9/2417
Inventor 王岩张晓华宋庆浩
Owner OCEAN UNIV OF CHINA
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