Novel high-temperature resistant alpha-amylase, preparing method of novel high-temperature resistant alpha-amylase and application of novel high-temperature resistant alpha-amylase

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

Active Publication Date: 2015-04-01
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Abstract
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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 starch liquefaction process, 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 the saccharification process, the pH must be adjusted to 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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  • Novel high-temperature resistant alpha-amylase, preparing method of novel high-temperature resistant alpha-amylase and application of novel high-temperature resistant alpha-amylase
  • Novel high-temperature resistant alpha-amylase, preparing method of novel high-temperature resistant alpha-amylase and application of novel high-temperature resistant alpha-amylase
  • Novel high-temperature resistant alpha-amylase, preparing method of novel high-temperature resistant alpha-amylase and application of novel high-temperature resistant alpha-amylase

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Experimental program
Comparison scheme
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, 200r / min for 18-24h, and collect the bacteria.

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

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

[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 hours.

[0035] ⑤Add 10% volume of 5mol / 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 12000r / min for 10min.

[0036] ⑥ Take the supernatant, add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), invert the centrifuge tube several times, and centrifuge at 1200r...

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 amplified by PCR was purified, digested with BamHI and HindIII, and the digested product was purified and detected by agarose gel electrophoresis. At the same time, plasmid pUC19 was double digested with BamHI and HindIII, purified, 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, and the results of double enzyme digestion and PCR verification showed that the amy gene had 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 NO:5.

[0050] (2) Site-directed mutation

[0051] A new type of high...

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 the fructan sucrase signal sequence sacB that can directly secrete the recombinant protein into the culture medium. 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] The novel high-temperature-resistant α-amylase gene amyM and pBAPR obtained by overlapping PCR construction were digested ...

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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 and Bacillus licheninformis, which have better heat resistance compared with α-amylase produced by other bacteria. Moreover, Bacillus licheniformis has high growth temperature, good product synthesis efficiency, and excellent protein synthesis and secretion ability, so it is widely used in industrial production. [0003] The high temperature-resistant α-amylase (BLA) produced by B.licheninformis is widely used in the starch hydrolysis process due to its good heat resistance. The fields of application are constantly expanding, su...

Claims

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Application Information

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Patent Type & Authority Applications(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 UNIVERSITY OF SCIENCE AND TECHNOLOGY
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