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Alkaline protease gene, alkaline protease as well as preparation method and application of alkaline protease

A protease and alkaline technology, applied in the field of genetic engineering, can solve the problem of high production cost of alkaline protease, achieve good stability and codon adaptability, improve codon adaptability, and reduce production costs

Pending Publication Date: 2020-11-06
深圳润康生态环境股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The object of the present invention is to provide alkaline protease gene aprbp and corresponding recombinant expression vector, recombinant expression bacterial strain, and alkaline protease AprBp and its preparation method and application, aiming at solving the production cost of existing bacillus patagoniensis alkaline protease high technical issues

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  • Alkaline protease gene, alkaline protease as well as preparation method and application of alkaline protease
  • Alkaline protease gene, alkaline protease as well as preparation method and application of alkaline protease
  • Alkaline protease gene, alkaline protease as well as preparation method and application of alkaline protease

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preparation example Construction

[0044] Correspondingly, the embodiment of the present invention also provides a kind of preparation method of alkaline protease AprBp, it comprises the steps:

[0045] S1. Provide alkaline protease gene aprbp, expression vector and expression strain;

[0046] S2. Amplify the alkaline protease gene aprbp, and connect the obtained amplified product to an expression vector to obtain a recombinant expression vector;

[0047] S3. Transforming the recombinant expression vector into an expression strain to obtain a recombinant expression strain;

[0048] S4. Cultivate the recombinant expression strain to obtain alkaline protease AprBp;

[0049] Wherein, the nucleotide sequence of the alkaline protease gene aprbp is shown in SEQ ID NO:1.

[0050] In the preparation method of alkaline protease AprBp provided by the embodiment of the present invention, the corresponding recombinant expression vector and recombinant expression strain are obtained by amplifying the alkaline protease gen...

Embodiment 1

[0073] This embodiment provides alkaline protease gene sequence analysis and codon optimization process, and the construction of corresponding expression vector, including the following steps:

[0074] Bacillus patagoniensis peptidase was entered in the NCBI protein database, and through comparative analysis, it was found that the protease with the accession number WP_078392427.1 was similar to the alkaline protease from Bacillus alkalophilus, Bacillus subtilis and Bacillus circulans (current industry The most widely used alkaline protease) has the highest similarity. Analysis from the angle of amino acid similarity shows that this protease is the most suitable for industrial application in the searched protein sequence, so this protease is used as the research object for experiments.

[0075] In the present invention, the protease is intended to be recombinantly expressed in Bacillus subtilis. Since the gene codes of Bacillus subtilis and Bacillus patagoniensis have certain d...

Embodiment 2

[0081] This embodiment provides a construction process of a recombinant Bacillus subtilis strain containing the recombinant expression vector obtained in Example 1, specifically as follows:

[0082] The first step in the construction process of recombinant Bacillus subtilis is to prepare competent cells. The preparation process of Bacillus subtilis WB800N competent cells is roughly as follows:

[0083] (4) Pick a single colony (2-3mm in diameter) from a plate cultured at 37°C for 16-20 hours, transfer it to a 50mL centrifuge tube containing 5ml LB medium, and shake vigorously at 37°C overnight;

[0084] (5) Inoculate 1% of the inoculum into 50ml GM (LB+0.5M sorbitol), measure the OD in the shaker tube, and control the inoculum so that the OD of the medium after inoculation is between 0.19-0.2. Cultivate at 37°C, 200rpm until OD600=0.8-1.0 (about 3-4 hours);

[0085] (6) Take all the bacterial liquid in an ice-water bath for 10 minutes, then centrifuge at 5000 rpm for 8 minute...

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Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to an alkaline protease gene, an alkaline protease as well as a preparation method and application of thealkaline protease. The nucleotide sequence of the alkaline protease gene aprbp is shown as SEQ ID NO:1, and the alkaline protease gene aprbp is obtained by comprehensively optimizing a sequence (GenBank: WP_078392427.1) of the alkaline protease derived from Bacillus patagoninesis. The amino acid sequence of the alkaline protease AprBP is shown in SEQ ID NO:2, and the alkaline protease not only enriches the types and sources of alkaline proteases, but also has higher enzymatic activity, and the highest enzymatic activity in a 5 L fermentation tank can reach 18035 U / mL. At the same time, the alkaline protease shows good stability in a wide temperature range and under an alkaline condition, and has good application prospects and industrial value.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to alkaline protease gene aprbp, a corresponding recombinant expression vector, a recombinant expression strain, alkaline protease AprBp, a preparation method and application thereof. Background technique [0002] Protease is a kind of enzyme that catalyzes the hydrolysis of protein. As an important industrial enzyme preparation, protease is widely used in feed processing, brewing, washing, food processing and other fields. Proteases are widely distributed. Currently, the proteases on the market are mainly derived from microorganisms. Compared with animals and plants, microbial proteases have a wider pH range and lower production costs. According to the optimal reaction pH environment of microbial protease, protease can be divided into acid protease, neutral protease and alkaline protease. Alkaline protease is a class of enzymes that hydrolyze proteins under ...

Claims

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Application Information

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IPC IPC(8): C12N15/57C12N9/54C12N15/75C12N1/21C12P21/06C12R1/125
CPCC12N9/54C12N15/75C12P21/06C12N2800/22
Inventor 王建荣祝木金余思王平孟建军刘小慧曹革
Owner 深圳润康生态环境股份有限公司
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