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Preparation method and application of carboxypeptidase A with function of degrading ochratoxin A

A technology of ochratoxin and carboxypeptidase, which is applied in the field of preparation of mature peptide of carboxypeptidase A, can solve the problems of complex preparation methods, reduced catalytic activity, and harsh catalytic conditions, and achieve high-efficiency and stable degradation, good application potential, and The effect of good environmental adaptability

Inactive Publication Date: 2019-04-23
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the bovine pancreas CPA obtained by expressing CPA zymogen and then treated with trypsin has problems such as complex preparation methods, harsh catalytic conditions, and activity easily affected by environmental conditions (such as reduced catalytic activity under acidic conditions). Therefore, it is urgently needed Develop a simple and efficient preparation method of CPA enzyme, in order to obtain CPA with better environmental tolerance and higher catalytic activity under different environmental conditions

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  • Preparation method and application of carboxypeptidase A with function of degrading ochratoxin A
  • Preparation method and application of carboxypeptidase A with function of degrading ochratoxin A
  • Preparation method and application of carboxypeptidase A with function of degrading ochratoxin A

Examples

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Effect test

Embodiment 1

[0048] Example 1 Codon Optimization of Carboxypeptidase A Mature Peptide Encoding Gene

[0049] First, the secondary and tertiary structures of bovine pancreatic carboxypeptidase A were analyzed using bioinformatics software. After a large number of screenings of different protein truncated expression modes, the carboxypeptidase containing 307 amino acids was determined to remove the C-terminal signal peptide and leader peptide. The truncated expression of peptidase A mature peptide (M-CPA), the sequence structure of the pro-CPA enzyme is shown in figure 1 shown. The amino acid sequence of the mature peptide of bovine pancreatic carboxypeptidase A (M-CPA) is shown in SEQ ID NO.1, and the nucleotide sequence of the coding gene is shown in SEQ ID NO.3. According to the codon preference of Pichia pastoris, combined with codon optimization software and manual screening, the coding gene of the codon-optimized CPA mature peptide shown in SEQ ID NO.2 was obtained, and the coding gen...

Embodiment 2

[0050] Embodiment 2 expresses the carrier of carboxypeptidase A mature peptide and the construction of recombinant yeast

[0051] The gene encoding the mature CPA peptide obtained in Example 1 was connected between the EcoR I and Not I restriction sites of the pPIC9K plasmid to construct the Pichia pastoris expression vector pPIC9K / M-CPA. The expression vector was transformed into Pichia pastoris GS115 (Pichia pastoris GS115), and the positive recombinant strain was obtained through screening, and the recombinant Pichia strain in which the pPIC9K / M-CPA vector was stably integrated into the chromosome was obtained through passage.

Embodiment 3

[0052] The cultivation of embodiment 3 recombinant Pichia pastoris and the preparation of carboxypeptidase A

[0053] This example provides a method for culturing and inducing expression of recombinant Pichia pastoris for preparing a mature peptide of carboxypeptidase A, specifically as follows:

[0054] (1) Activation culture on a solid plate: the recombinant Pichia pastoris strain constructed in Example 2 was inoculated on a fresh YPD plate, and cultured upside down in a yeast incubator at 30° C. for 2 days.

[0055] (2) Seed culture: Pick single colonies of the recombinant strains, inoculate them in 25mL BMGY medium (containing 4% glycerol), and cultivate them in a 250mL Erlenmeyer flask until the cell concentration reaches OD 600 =2~3. The culture conditions are: 28°C, 225r / min shaker culture.

[0056] (3) Induced expression: collect the above culture, centrifuge at room temperature at 4000rpm for 5min, pour off the supernatant, and resuspend the obtained bacterial cells...

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Abstract

The invention relates to the technical field of biology, in particular to a preparation method and application of carboxypeptidase A with a function of degrading ochratoxin A. The invention disclosesthe encoding gene of the mature peptide of the beef pancreas gland carboxypeptidase A optimized by specific codons, an expression vector and a host of the encoding gene and the preparation method of the carboxypeptidase A. The amino acid sequence of the mature peptide of the carboxypeptidase A is shown in SEQ ID NO.1; the nucleotide sequence of the encoding gene of the mature peptide of the beef pancreas gland carboxypeptidase A optimized by the codons is shown in SEQ ID NO.2. Abundant expression of the carboxypeptidase A is achieved by using a pastoris picha yeast expression system, the carboxypeptidase A prepared by using the method has biological catalytic activity without treatment and can effectively and stably degrade the ochratoxin A, moreover, the activity of degrading the ochratoxin A can be stably given play to in different environments, and the carboxypeptidase A can be used for treatment of products contaminated by the ochratoxin A in practice.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method and application of a carboxypeptidase A mature peptide capable of degrading ochratoxin A. Background technique [0002] In practical production, the phenomenon of agricultural products being polluted by Ochratoxin A (OTA) is widespread, and products exceeding the national standard limit can only be discarded, and the resulting economic losses are immeasurable. However, a considerable part of the products can be put on the market after degrading OTA into non-toxic products through biochemical methods, among which enzymatic degradation method is currently the safest and most effective detoxification method. Although the removal of mycotoxins in the market currently mainly uses adsorbents, some toxins cannot be adsorbed by them, and the adsorption effect will be desorbed due to the influence of temperature and pH. With the change of the global environment, the poll...

Claims

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Application Information

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IPC IPC(8): C12N9/48C12N15/57C12N15/70C12N15/81A23L5/20
CPCA23L5/25C12N9/485C12N15/70C12N15/81C12Y304/17001
Inventor 梁志宏熊露赵萌胡海宁
Owner CHINA AGRI UNIV
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