Heterologous Expression of Extracellular Proteinase pAsPs of <i>Aspergillus pseudotamarii</i> in <i>Komagataella phaffii</i>
Neutral protease pAsPs gene was obtained by sequence optimization of NpI protease from <i>Aspergillus pseudotamarii.</i> pAsPs was for the first time integrated in the genome of yeast strain <i>Komagataella phaffii</i> T07, and then produced in a 5 L bioreactor with an enzyme...
| Main Authors: | , , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-11-01
|
| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/23/23/15035 |
| Summary: | Neutral protease pAsPs gene was obtained by sequence optimization of NpI protease from <i>Aspergillus pseudotamarii.</i> pAsPs was for the first time integrated in the genome of yeast strain <i>Komagataella phaffii</i> 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 Cu<sup>2+</sup>. Optimal activity pH was shown in the range of pH 6.5–8.0, and optimal temperature—50–60 °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. |
|---|---|
| ISSN: | 1661-6596 1422-0067 |