Method for preparing N-glycosylated proline aminopeptidase and application

Abstract

The invention discloses a method for preparing N-glycosylated proline aminopeptidase and application and belongs to the technical field of enzyme engineering. Proline aminopeptidase N-end expressed by pichia pastoris comprises histidine tag, a metal-chelating chromatography is utilized to further purify expressed proteins, the recovery rate reaches 86.5%, and the purification fold is 13.4 times. It is proved that N-galactosylated modification occurs in purified aminopeptidase, and a galactosylated modification effect enables stability of an enzyme to be greatly improved. According to a glutenin hydrolysis method, the pure enzyme and alkaline protease are subjected to a synergistic effect, and the free amino acid content is improved from 16.2 mg / L before hydrolysis to 2770.1 mg / L.

Description

technical field [0001] The invention relates to a method and application for preparing N-glycosylated proline aminopeptidase, belonging to the technical field of enzyme engineering. Background technique [0002] Proline aminopeptidase (PAP) is a highly specific exoprotease, which has important applications in many fields such as food, medicine and chemical industry. However, the production of PAP mainly faces the following problems: (1) the yield of wild bacteria is poor, and the purification of proline aminopeptidase reported at home and abroad needs to pass through 4 chromatographic columns, and the recovery rate is only 0.5%-10% (as shown in the table 1). (2) The optimum temperature range for common proline aminopeptidase is 30-45°C, and it will be rapidly inactivated if it exceeds this temperature, and there is no research on the thermal stability of this enzyme. [0003] Purification results and properties of proline aminopeptidase from different sources in table 1 ...

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Problems solved by technology

But the production of PAP mainly faces the following problems: (1) the yield of wild bacteria is poor, and the purification of proline aminopeptidase reported both at home and abroad needs to pass through 4 chromatographic columns, and the recovery rate is only 0.5%-10% (as shown in the table 1)

(2) The optimum temperature range for common proline aminopeptidase is 30-45°C, and it will be rapidly inactivated if it exceeds this temperature, and there is no research on the thermal stability of this enzyme

[0005] At present, research on heterologous expression of proline aminopeptidase focuses on prokaryotic hosts such as Escherichia coli and Bacillus subtilis. Although the expression level of recombinant protein is relatively high, it has the following disadvantages: 1) It is not easy to secrete and express, and it is easy to form inclusion bodies ; 2) Aminopeptidase has poor stability, and the residual enzyme activity is only 10% after being incubated at the optimum reaction temperature of 50° C. for 1 h; 3) The purification efficiency is poor, and the yield is only 1.7% after ion exchange chromatography and gel filtration

Matysiak-Budnik T and others use proline endopeptidase to degrade gluten to prevent the disease, but due to the poor decomposition ability of endopeptidase, a high concentration of endopeptidase is required to degrade the target polypeptide (length About 30-40 amino acids) complete cleavage

In addition, the latest research shows that none of the five commercially available enzyme supplements (the active ingredients are acid protease, dipeptidase and leucine aminopeptidase) that claim to have the function of degrading gluten immunogen can not effectively degrade and cause immune stress response peptide

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