Preparation of aminopeptidase and application of aminopeptidase in protein debitterizing

A technology of aminopeptidase and protein, applied in the field of enzyme engineering, can solve problems affecting food quality and restricting applications, and achieve the effects of improving flavor and quality, reducing bitterness, and mild debittering conditions

Pending Publication Date: 2021-11-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are a large number of peptides with hydrophobic amino acid residues at the N-terminal (molecular weight between 500-1000Da) in the protein. These hydrophobi

Method used

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  • Preparation of aminopeptidase and application of aminopeptidase in protein debitterizing
  • Preparation of aminopeptidase and application of aminopeptidase in protein debitterizing
  • Preparation of aminopeptidase and application of aminopeptidase in protein debitterizing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1: Construction of recombinant bacteria expressing aminopeptidase

[0052] Aminopeptidase derived from Aspergillus oryzae, Aspergillus flavus, Aspergillus sojae, Aspergillus minisclerotigenes, Aspergillus transmontanensis, Aspergillus novoparasiticus, Aspergillus arachidicola (the accession number or gene number on the NCBI corresponding to the aminopeptidase is respectively NCBI accession number: XP_001825745.1 , GenBank: RAQ51025.1, NCBI accession number: Q8J2N2, GenBank: KAB8272165.1, GenBank: KAE8313457.1, GenBank: KAB8217617.1, GenBank: KAE8342201.1), construct recombinant bacteria respectively, and the specific implementation steps are as follows:

[0053] (1) Construction of recombinant plasmids

[0054] Using chemical methods to synthesize the gene encoding aminopeptidase AoAPase whose nucleotide sequence is shown in SEQ ID NO.2 on the vector pPIC9K, directly obtain the recombinant plasmid pPIC9K-AoAPase, and transform the recombinant plasmid into Esc...

Embodiment 2

[0059] Embodiment 2: Recombinant bacterial expression aminopeptidase

[0060] (1) Using the recombinant bacteria constructed in Example 1, shake the flask to ferment to produce enzyme.

[0061] Pick large single colonies from the MD plate into labeled small tubes, add 4mL of BMGY medium to each small tube, and culture for 2-3 days. When there is obvious bacterial precipitation at the bottom of the small tube, centrifuge at 5000rpm for 5min, pour off the supernatant in an ultra-clean bench, add 2mL of BMMY medium, add 1% methanol after resuspension, and incubate at 30°C for 2-3 days. 24h added 1% volume of methanol. Measure the enzyme activity and protein content of the bacterial solution, select 5-10 transformants with enzyme activity, and then carry out shake flask screening.

[0062] Add 5-10 transformants with high enzyme activity to 50 mL of BMGY medium and culture for 2-3 days. When the bottom of the shake flask has obvious bacterial precipitation, centrifuge at 5000rp...

Embodiment 3

[0067] Example 3: Application of different aminopeptidases in protein debittering

[0068] Embodiment 2 obtains the aminopeptidase of different sources and carries out debittering to albumen:

[0069] Weigh soybean protein, fish protein, and shrimp protein to prepare 100mL protein solution with a concentration of 20g / L, add aminopeptidase 400U / g substrate to the protein solution, and use a constant temperature water bath shaker to maintain the hydrolysis temperature at 50°C and 150rpm enzyme Decompose for 5 hours, take a boiling water bath for 10 minutes, cool for a period of time, centrifuge the enzymolysis solution at 8000 rpm for 10 minutes, and take the supernatant to test the bitterness.

[0070] The results are shown in Table 2: the aminopeptidases from different sources obtained in Example 2 have significant differences in debittering effects on different types of proteins. Among them, aminopeptidase AoAPase has a more obvious debittering effect on soybean protein, fis...

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Abstract

The invention discloses preparation of aminopeptidase and application of aminopeptidase in protein debitterizing, and belongs to the technical field of enzyme engineering. The invention provides aminopeptidase with an amino acid sequence as shown in SEQ ID No.1. The aminopeptidase is expressed in saccharomycetes, and the enzyme activity of the aminopeptidase reaches 545 U/mL. The aminopeptidase has strong debitterizing capability on protein, the bitterness of soybean protein, fish protein and shrimp protein can be eliminated by adding the aminopeptidase according to the adding amount of 700U/g protein, the reaction condition is mild, and other characteristics of peptide are not changed. Therefore, the aminopeptidase disclosed by the invention has an extremely high application prospect in protein debitterizing.

Description

technical field [0001] The invention relates to the preparation of aminopeptidase and its application in protein debittering, belonging to the technical field of enzyme engineering. Background technique [0002] Protein is a polypeptide mixture obtained under the action of acid, alkali, heat treatment or enzymatic hydrolysis, and has good biological activity. Some peptides in the protein are easily absorbed by the human body due to their small molecular weight, and some of these low-molecular-weight peptides can also play unique physiological functions. Soy protein has the effect of lowering blood pressure, blood fat and cholesterol, can regulate blood sugar, promote blood circulation, improve human absorption function, enhance immunity, and resist oxidation. Fish protein has the functions of nourishing the skin, relieving fatigue, improving immunity and whitening. Antarctic krill protein has a complete range of amino acids and high nutrition. It can provide many trace ele...

Claims

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Application Information

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IPC IPC(8): C12N9/48A23L5/20
CPCC12N9/485A23L5/25
Inventor 颜正飞袁帅宿玲恰吴敬
Owner JIANGNAN UNIV
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