A kind of high temperature resistant α-amylase and its preparation method and application

A technology of high temperature resistance and amylase, applied in the field of genetic engineering, can solve problems such as reducing economic benefits and increasing process complexity, and achieve the effects of reducing energy consumption, obvious economic and social benefits, and improving stability

Active Publication Date: 2017-03-15
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004] At present, the optimum temperature and pH of high-temperature-resistant α-amylases used in the starch liquefaction process are generally around 95°C and 6.5. In order to maintain activity, a certain concentration of Ca is generally required. 2+ , in order to ensure that the high temperature resistant α-amylase exerts a higher activity in the process of starch liquefaction, the original pH of the natural slurry needs to be adjusted from (3.2-4.5) to (5.8-6.2), and in the next step of saccharification The pH of the saccharification solution is adjusted to 4.2-4.5. The two-step pH adjustment process not only increases the complexity of the process but also reduces the economic benefits. Therefore, in the ideal process step, it is necessary to develop a stable and active product under high temperature and acidic environment. thermostable alpha-amylase

Method used

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  • A kind of high temperature resistant α-amylase and its preparation method and application
  • A kind of high temperature resistant α-amylase and its preparation method and application
  • A kind of high temperature resistant α-amylase and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1: Obtaining of wild-type high temperature resistant α-amylase gene

[0030] (1) Extraction of Bacillus licheniformis genomic DNA:

[0031] ①Cultivate the bacteria at 37°C, 200 r / min for 18-24 hours, and collect the bacteria.

[0032] ② Add 500 μL ddH 2 O, resuspend the cells, wash the cells, centrifuge at 5000 r / min for 5 min, and discard the supernatant.

[0033] ③ Add 500 μL ddH 2 O, resuspend the bacteria, add lysozyme (15 μg / mL), and digest in a water bath at 37°C for 1 h.

[0034] ④ Add 50 μL of 10% SDS and 30 μL of proteinase K, and digest in a water bath at 60°C for 2 h.

[0035] ⑤ Add 10% volume of 5 mol / L NaCl, transfer the supernatant tube to add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), invert the centrifuge tube several times, and centrifuge at 12000 r / min for 10 min.

[0036] ⑥ Take the supernatant, add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), invert the centrifuge tube several times, and centri...

Embodiment 2

[0046] Example 2: Site-directed mutation of the wild-type high temperature resistant α-amylase gene

[0047] (1) The wild-type thermostable α-amylase gene was ligated into the vector pUC19.

[0048] The target gene obtained by PCR amplification was purified with Bam HI and Hin Digested with dIII, the digested product was purified and detected by agarose gel electrophoresis. At the same time, the plasmid pUC19 was also used Bam HI and Hin dIII was subjected to double digestion, purification, and finally T 4 DNA ligase was ligated at 12°C for 8 hours to construct the recombinant plasmid pUC19- amy . The recombinant plasmid was transformed into Escherichia coli JM109 by electroporation method, and the results of double enzyme digestion and PCR verification showed that amy The gene has been successfully cloned into the vector pUC19.

[0049] Its sequencing shows that it is amplified to the wild-type high temperature resistant α-amylase gene sequence such as SEQ ID N...

Embodiment 3

[0064] Example 3: Construction of a novel high temperature resistant α-amylase expression vector

[0065] pBAPR is based on the Escherichia coli-Bacillus subtilis shuttle cloning vector pBE2 as the backbone, cloned into a strong promoter of Bacillus alkalophilus alkaline protease P APR and a fructan sucrase signal sequence that enables direct secretion of recombinant proteins into the culture medium sacB And get. It contains both the replicon of the Bacillus subtilis plasmid pUB110 and the replicon of the Escherichia coli plasmid pGEM3, and can autonomously replicate in Escherichia coli, Bacillus subtilis and Bacillus licheniformis cells. it comes with amp r and Km r Gene, ampicillin resistance can be used as a selection marker in Escherichia coli, and kanamycin resistance can be used as a selection marker in Bacillus subtilis and Bacillus licheniformis.

[0066] A new type of high temperature resistant α-amylase gene obtained by overlapping PCR construction amyM with p...

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Abstract

The invention relates to novel high-temperature resistant alpha-amylase, a preparing method of the novel high-temperature resistant alpha-amylase and an application of the novel high-temperature resistant alpha-amylase. As PCR site-specific mutagenesis is carried out on genes of wild-type high-temperature resistant alpha-amylase, and the genes are efficiently expressed in bacillus subtilis. The novel high-temperature resistant alpha-amylase is high in stability in the high-temperature environment and the acid environment, and can better adapt to industrial production to achieve the effects of saving energy, reducing consumption and improving efficiency. By means of the technical scheme, the genes of the wild-type high-temperature resistant alpha-amylase are separated from bacillus licheniformis, mutagenesis is carried out on His316 amino acid residues of the genes, efficient expression is carried out in the bacillus subtilis, and under the 90-DEG C condition and the pH-4.5 condition, the stability of the novel high-temperature resistant alpha-amylase (His316 to Arg) is improved compared with the wild-type high-temperature resistant alpha-amylase.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to site-directed mutation and recombination technology of genes, in particular to a novel high-temperature-resistant alpha-amylase and its preparation method and application. Background technique [0002] At present, most of the strains used in the production of high-temperature-resistant α-amylase at home and abroad are Bacillus stearothermophilus ( Bacillus stearothermophilus ) and Bacillus licheniformis ( Bacillus licheninformis ), compared with the α-amylase produced by other bacteria, it has better heat resistance, and Bacillus licheniformis has higher growth temperature, good product synthesis efficiency, excellent protein synthesis and secretion ability, so it is widely used Used in industrial production. [0003] B. licheninformis The high temperature resistant α-amylase (BLA) produced is widely used in starch hydrolysis process due to its good heat resistanc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/28C12N15/56C12N15/75C12N1/21C12R1/125
CPCC12N9/2414C12N15/75C12Y302/01001
Inventor 刘逸寒王正祥路福平王春霞王建玲
Owner TIANJIN UNIV OF SCI & TECH
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