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A kind of method utilizing recombinant subtilis proline aminopeptidase to degrade casein

A technology for proline aminopeptidase and leucine aminopeptidase, which is applied in the field of casein degradation, can solve problems such as inability to cut polypeptides, and achieves the effects of improving hydrolysis depth, good application prospect and simplifying extraction process

Active Publication Date: 2019-05-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For non-specific aminopeptidases, they can hydrolyze most amino acid residues from the end of the polypeptide, but they cannot cleave polypeptides with a proline at the N-terminus

Method used

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  • A kind of method utilizing recombinant subtilis proline aminopeptidase to degrade casein
  • A kind of method utilizing recombinant subtilis proline aminopeptidase to degrade casein
  • A kind of method utilizing recombinant subtilis proline aminopeptidase to degrade casein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: Preparation of recombinant subtilis proline aminopeptidase

[0036] Take 40mL of recombinant Bacillus subtilis WB600 fermentation broth cultured for 24h, centrifuge at 10,000rpm and 4°C for 10min, take the supernatant, discard the precipitate, and obtain the crude enzyme solution, which is then concentrated by ultrafiltration, using ultrafiltration with a molecular weight cut-off of 30kDa Centrifuge tube, the ultrafiltration condition is 5000rpm, centrifuge at 4°C for 20min, the concentration factor is controlled at 3-4 times, the ultrafiltration concentrated solution is dialyzed in 1L buffer A at 4°C overnight, and 5mL of the dialyzate is used for nickel affinity chromatography Column, the flow rate is maintained at 1mL / min, first eluted with buffer A of 5 column volumes, and then eluted with buffer B of 5 column volumes, according to the changes in the readings of the UV detector, the eluate is collected in batches, and the obtained The eluate was dialyz...

Embodiment 2

[0037] Embodiment 2: Recombinant subtilis proline aminopeptidase small molecule substrate selectivity

[0038] Prepare 1mM synthetic dipeptides and peptides with 0.05M PBS pH 7.5 buffer: Pro-Leu, Pro-Lys, Pro-Pro, Leu-Pro, Pro-Phe-Gly-Lys, Pro-Leu-Ser-Arg- Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys.

[0039] Take 0.9 mL of dipeptide and polypeptide solutions respectively, and then add 0.1 mL of enzyme solution diluted with 0.05 M PBS pH 7.5 buffer solution (the enzyme solution is obtained by the method described in Example 1, and the enzyme activity is 15.4 U / mL).

[0040]After reacting at 50°C for 10 minutes, immediately add 2.5mL acetic acid and 2.5mL ninhydrin solution, bathe in boiling water for 30min, measure the absorbance at 480nm, the blank control is 0.1mL 0.05M PBS pH 7.5 buffer solution instead of the diluted enzyme solution, to The highest hydrolysis ability is 100%. The results showed that the relative activity of hydrolyzing Pro-Leu was 69.5%, the relative activity of Pro...

Embodiment 3

[0040]After reacting at 50°C for 10 minutes, immediately add 2.5mL acetic acid and 2.5mL ninhydrin solution, bathe in boiling water for 30min, measure the absorbance at 480nm, the blank control is 0.1mL 0.05M PBS pH 7.5 buffer solution instead of the diluted enzyme solution, to The highest hydrolysis ability is 100%. The results showed that the relative activity of hydrolyzing Pro-Leu was 69.5%, the relative activity of Pro-Lys was 74.4%, the relative activity of Pro-Pro was 44.1%, the relative activity of Leu-Pro was 0, and the relative activity of Pro-Phe- The relative activity to Gly-Lys was 100%, and the relative activity to Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys was 17.4%. The ability of recombinant proline aminopeptidase to hydrolyze different small molecular substrates is different, and it is selective for the hydrolysis of small molecular substrates. Embodiment 3: the effect of recombinant subtilis proline aminopeptidase in casein degradation

[0041] Prepar...

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Abstract

The invention discloses a method for degrading casein by utilizing recombinant bacillus subtilis proline aminopeptidase, and belongs to the fields of enzyme engineering and food additives. The method for degrading the casein by utilizing the recombinant bacillus subtilis proline aminopeptidase hydrolyzes the casein by utilizing proline aminopeptidase and through cooperation with alkaline protease and leucine aminopeptidase; the content of free amino acids is 3.968mg / mL, and is 63 times of the content of free amino acids before hydrolysis; polypeptide above 2,000Da does not exist basically; the content of small peptide below 180Da reaches 36.66%; exposed N-terminal proline residues are sufficiently hydrolyzed; the content of free proline is 0.044mg / mL, and is 3.67 times of the content of free proline which is not hydrolyzed; a hydrolysis extent of the proline is improved. The method for degrading the casein by utilizing the recombinant bacillus subtilis proline aminopeptidase has quite good application prospects in fields of food, beverages, and processing and utilization of food protein resources.

Description

technical field [0001] The invention relates to a method for degrading casein by using recombinant subtilis proline aminopeptidase, belonging to the fields of enzyme preparations and food additives. Background technique [0002] Enzyme preparations refer to substances with enzymatic properties extracted from organisms, which have been used in various fields such as medicine, chemical industry, food, brewing, etc., with a very wide range of applications. The food processing industry is closely related to people's lives. Enzymes are used more and more in the food processing industry, and their role is becoming more and more important. It is greatly reflected in meat processing, deep hydrolysis of protein and as food additives. [0003] With the development of society, the application of enzyme preparations is becoming more and more extensive, and aminopeptidase is also constantly developing its application fields. Non-specific aminopeptidase can broadly hydrolyze amino acid r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/06C12N9/48C12R1/125
CPCC12N9/485C12P21/06C12Y304/14012
Inventor 田亚平汪克红王开道周楠迪
Owner JIANGNAN UNIV
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