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...
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2022-11-01
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| Series: | International Journal of Molecular Sciences |
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| author | Andrey Valentinovich Zadorozhny Mikhail Evgenyevich Voskoboev Denis Vladimirovich Bochkov Alexei Sergeyevich Rozanov Elizaveta Dmitrievna Shedko Irina Anatolyevna Mescheryakova Alexander Gennadyevich Blinov Anton Vladimirovich Korzhuk Valeria Nikolayevna Shlyakhtun Natalia Vladimirovna Bogacheva Egor Vladimirovich Antonov Svetlana Valerevna Bannikova Tatiana Nikolayevna Goryachkovskaya Sergey Evgenyevich Peltek |
| author_facet | Andrey Valentinovich Zadorozhny Mikhail Evgenyevich Voskoboev Denis Vladimirovich Bochkov Alexei Sergeyevich Rozanov Elizaveta Dmitrievna Shedko Irina Anatolyevna Mescheryakova Alexander Gennadyevich Blinov Anton Vladimirovich Korzhuk Valeria Nikolayevna Shlyakhtun Natalia Vladimirovna Bogacheva Egor Vladimirovich Antonov Svetlana Valerevna Bannikova Tatiana Nikolayevna Goryachkovskaya Sergey Evgenyevich Peltek |
| author_sort | Andrey Valentinovich Zadorozhny |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-09T17:45:37Z |
| format | Article |
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| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T17:45:37Z |
| publishDate | 2022-11-01 |
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| spelling | doaj.art-8b58ab02bdcb4abb9ee455e7780fe29a2023-11-24T11:12:05ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-11-0123231503510.3390/ijms232315035Heterologous Expression of Extracellular Proteinase pAsPs of <i>Aspergillus pseudotamarii</i> in <i>Komagataella phaffii</i>Andrey Valentinovich Zadorozhny0Mikhail Evgenyevich Voskoboev1Denis Vladimirovich Bochkov2Alexei Sergeyevich Rozanov3Elizaveta Dmitrievna Shedko4Irina Anatolyevna Mescheryakova5Alexander Gennadyevich Blinov6Anton Vladimirovich Korzhuk7Valeria Nikolayevna Shlyakhtun8Natalia Vladimirovna Bogacheva9Egor Vladimirovich Antonov10Svetlana Valerevna Bannikova11Tatiana Nikolayevna Goryachkovskaya12Sergey Evgenyevich Peltek13Laboratory of Molecular Biotechnology, The Institute of Cytology and Genetics, SB RAS, 630090 Novosibirsk, RussiaLaboratory of Molecular Biotechnology, The Institute of Cytology and Genetics, SB RAS, 630090 Novosibirsk, RussiaKurchatov Genomic Center of the Institute of Cytology and Genetics, SB RAS, 630090 Novosibirsk, RussiaLaboratory of Molecular Biotechnology, The Institute of Cytology and Genetics, SB RAS, 630090 Novosibirsk, RussiaLaboratory of Molecular Biotechnology, The Institute of Cytology and Genetics, SB RAS, 630090 Novosibirsk, RussiaLaboratory of Molecular Biotechnology, The Institute of Cytology and Genetics, SB RAS, 630090 Novosibirsk, RussiaLaboratory of Molecular Biotechnology, The Institute of Cytology and Genetics, SB RAS, 630090 Novosibirsk, RussiaLaboratory of Molecular Biotechnology, The Institute of Cytology and Genetics, SB RAS, 630090 Novosibirsk, RussiaLaboratory of Molecular Biotechnology, The Institute of Cytology and Genetics, SB RAS, 630090 Novosibirsk, RussiaLaboratory of Molecular Biotechnology, The Institute of Cytology and Genetics, SB RAS, 630090 Novosibirsk, RussiaLaboratory of Molecular Biotechnology, The Institute of Cytology and Genetics, SB RAS, 630090 Novosibirsk, RussiaKurchatov Genomic Center of the Institute of Cytology and Genetics, SB RAS, 630090 Novosibirsk, RussiaLaboratory of Molecular Biotechnology, The Institute of Cytology and Genetics, SB RAS, 630090 Novosibirsk, RussiaLaboratory of Molecular Biotechnology, The Institute of Cytology and Genetics, SB RAS, 630090 Novosibirsk, RussiaNeutral 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.https://www.mdpi.com/1422-0067/23/23/15035proteinase<i>Komagataella phaffii</i>multicopyheterologous expression<i>Aspergillus pseudotamarii</i>industrial application |
| spellingShingle | Andrey Valentinovich Zadorozhny Mikhail Evgenyevich Voskoboev Denis Vladimirovich Bochkov Alexei Sergeyevich Rozanov Elizaveta Dmitrievna Shedko Irina Anatolyevna Mescheryakova Alexander Gennadyevich Blinov Anton Vladimirovich Korzhuk Valeria Nikolayevna Shlyakhtun Natalia Vladimirovna Bogacheva Egor Vladimirovich Antonov Svetlana Valerevna Bannikova Tatiana Nikolayevna Goryachkovskaya Sergey Evgenyevich Peltek Heterologous Expression of Extracellular Proteinase pAsPs of <i>Aspergillus pseudotamarii</i> in <i>Komagataella phaffii</i> International Journal of Molecular Sciences proteinase <i>Komagataella phaffii</i> multicopy heterologous expression <i>Aspergillus pseudotamarii</i> industrial application |
| title | Heterologous Expression of Extracellular Proteinase pAsPs of <i>Aspergillus pseudotamarii</i> in <i>Komagataella phaffii</i> |
| title_full | Heterologous Expression of Extracellular Proteinase pAsPs of <i>Aspergillus pseudotamarii</i> in <i>Komagataella phaffii</i> |
| title_fullStr | Heterologous Expression of Extracellular Proteinase pAsPs of <i>Aspergillus pseudotamarii</i> in <i>Komagataella phaffii</i> |
| title_full_unstemmed | Heterologous Expression of Extracellular Proteinase pAsPs of <i>Aspergillus pseudotamarii</i> in <i>Komagataella phaffii</i> |
| title_short | Heterologous Expression of Extracellular Proteinase pAsPs of <i>Aspergillus pseudotamarii</i> in <i>Komagataella phaffii</i> |
| title_sort | heterologous expression of extracellular proteinase pasps of i aspergillus pseudotamarii i in i komagataella phaffii i |
| topic | proteinase <i>Komagataella phaffii</i> multicopy heterologous expression <i>Aspergillus pseudotamarii</i> industrial application |
| url | https://www.mdpi.com/1422-0067/23/23/15035 |
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