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A kind of alkaline protease and its gene and application

A protease, alkaline technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc.

Active Publication Date: 2020-10-23
北京科为博生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Compared with foreign countries, although my country's enzyme preparation industry has developed rapidly, there is still a certain gap
At present, the market demand for alkaline protease is relatively large, but the production level of existing alkaline protease production strains needs to be improved, and it is urgent to develop high-efficiency expression strains of alkaline protease

Method used

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  • A kind of alkaline protease and its gene and application
  • A kind of alkaline protease and its gene and application
  • A kind of alkaline protease and its gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The acquisition of embodiment 1 alkaline protease gene

[0040] Bacillus licheniformis CRVAB300 was isolated from soil samples collected near the reservoir. Specifically, the soil sample is dissolved in sterile water, after enrichment culture, a suitable dilution gradient is applied to a flat plate containing 1% skimmed milk, and it is obtained by screening according to the size of the transparent circle on the flat plate.

[0041] Extraction of Bacillus licheniformis CRVAB300 (Bacillus licheniformis) genomic DNA:

[0042] (1) Take 0.5-2mL of cultured bacteria solution, centrifuge at 10000rpm for 30s, absorb the supernatant as much as possible, and collect the bacteria;

[0043] (2) Add 200 μL buffer RB to the EP tube to resuspend, centrifuge at 10,000 rpm for 30 s, and discard the supernatant;

[0044] (3) For Gram-positive bacteria: add 120 μL lysozyme, mix by inverting, and bathe in 37°C water bath for 30-60 minutes;

[0045] (4) Centrifuge at 12000rpm for 2min, d...

Embodiment 2

[0059] The construction of embodiment 2 alkaline protease expression vector pwb980-apr

[0060] Subtilis expression plasmid pwb980 was used as a template to amplify the vector fragment by PCR. Amplification primers are: upstream primer: 5'-GCTAGCTTCAGCACAATTCCAAGAAAAAC-3'; downstream primer: 5'-GGCAAAAGCTTGAGTTGCGCCTCCTGCCAG-3'. The carrier DNA fragment of about 3738bp size was amplified by PCR, and after the fragment was recovered, polymer fusion PCR was carried out with the above-mentioned alkaline protease gene fragment (You C, Zhang XZ, Zhang YH (2012) Simple cloning via direct transformation of PCR product (DNA Multimer)toEscherichia coli and Bacillus subtilis.Appl Environ Microbiol78:1593–1595), the fusion product was transformed into Bacillus subtilis B.subtilis 168 competent cells, spread on the LB plate containing kanamycin (25μg / mL), and selected Positive transformants were extracted for plasmid sequencing verification, and it was determined that the recombinant pla...

Embodiment 3

[0061] Embodiment 3 Expression vector pwb980-apr transforms Bacillus amyloliquefaciens K1

[0062] The preparation method of Bacillus amyloliquefaciens K1 competent cells is as follows:

[0063] (1) Inoculate Bacillus amyloliquefaciens K1 in 5 mL LB medium and culture overnight.

[0064] (2) Take 2.5mL of the overnight culture and put it into 40mL (LB+0.5M sorbitol), culture at 37°C and shake at 200rpm until OD 600 It is between 0.6 and 0.8.

[0065] (3) Bath the bacterial solution in ice water for 10 minutes, then centrifuge at 5000g at 4°C for 5 minutes to collect the bacterial cells.

[0066] (4) Resuspend the cells with 50 mL pre-cooled electroporation medium (0.5M sorbitol, 0.5M mannitol, 10% glucose), centrifuge at 5000g for 5min at 4°C, remove the supernatant, and rinse 4 times in this way.

[0067] (5) Resuspend the washed bacteria in 1 mL of electroporation medium, aliquot into EP tubes, and aliquot 60 μL in each tube.

[0068] The conversion conditions are as fol...

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Abstract

The invention belongs to the field of bioengineering and particularly relates to an alkaline protease as well as gene and application thereof. The amino acid sequence of the alkaline protease gene is expressed as SEQ ID NO.1. The base sequence of the alkaline protease gene is expressed as SEQ ID NO.2. The invention also provides a recombinant vector containing the alkaline protease gene and a recombination strain containing the alkaline protease gene. Recombinant plasmids containing the alkaline protease gene are built by using a gene engineering technique; efficient expression of the alkaline protease in bacillus amyloliquefaciens is achieved; the built alkaline protease recombination strain has a higher application value and has extremely high capability of producing alkaline protease; compared with the wild bacteria under the same fermentation conditions, the enzyme activity of the built alkaline protease recombination strain is increased by about 55%.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to an alkaline protease and its gene and application. Background technique [0002] Alkaline protease, also known as serine protease, belongs to the protease class. Its optimum pH is 9-11, and it is relatively stable at pH 5-10. Alkaline protease is mainly used in the washing industry, and is also widely used in food, medical, brewing, silk, leather and other fields. [0003] Alkaline protease was first discovered in the pancreas of pigs. In 1913, Rohm first used trypsin in a washing soak. In 1945, Dr. Jaag in Switzerland discovered alkaline protease from microbial sources, which made it possible for protease to be widely used in the detergent industry. At present, protease is the enzyme with the largest proportion among industrial enzymes, accounting for about 60% of the total annual sales in the world, of which alkaline protease accounts for about 25%. Therefore, alkaline proteas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/54C12N15/57C12N15/75C12N1/21
CPCC12N9/54
Inventor 吴培均李富伟张士彬罗建杰
Owner 北京科为博生物科技有限公司
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