期刊
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
卷 23, 期 23, 页码 -出版社
MDPI
DOI: 10.3390/ijms232315035
关键词
proteinase; Komagataella phaffii; multicopy; heterologous expression; Aspergillus pseudotamarii; industrial application
资金
- Ministry of Science and Higher Education of the Russian Federation [075-11-2020-036]
- Ministry of Science and Higher Education [075-15-2019-1662]
The neutral protease pAsPs gene from Aspergillus pseudotamarii was successfully obtained and integrated into the genome of yeast strain Komagataella phaffii T07. The recombinant enzyme showed high specific activity towards casein and was inhibited by Cu2+. Mass-spectrometric analysis confirmed the consistency of the obtained pAsPs preparation with the predicted amino acid sequence.
Neutral protease pAsPs gene was obtained by sequence optimization of NpI protease from Aspergillus pseudotamarii. pAsPs was for the first time integrated in the genome of yeast strain Komagataella phaffii T07, and then produced in a 5 L bioreactor with an enzyme yield of 150,800 U/mL of culture liquid towards casein. The specific activity of the pAsPs was 7,657,000 U/mg toward casein, 2320 U/mg toward hemoglobin, and 25,344 U/mg toward azocasein per 1 mg of the protein. The enzyme was found to be inhibited by Cu2+. Optimal activity pH was shown in the range of pH 6.5-8.0, and optimal temperature-50-60 degrees C. The molecular mass of the recombinant protease pAsPs was shown to be 67.5 kDa. Mass-spectrometric analysis confirmed the identity of the amino acid sequence of the obtained pAsPs preparation with the predicted sequence, with 17% coverage and protein score 288. Thus, the novel neutral protease pAsPs is a promising candidate for large-scale use in manufacturing, including the food industry.
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