Chaperone assisted recombinant expression of a mycobacterial aminoacylase in Vibrio natriegens and Escherichia coli capable of N-lauroyl-L-amino acid synthesis
Abstract Background Aminoacylases are highly promising enzymes for the green synthesis of acyl-amino acids, potentially replacing the environmentally harmful Schotten-Baumann reaction. Long-chain acyl-amino acids can serve as strong surfactants and emulsifiers, with application in cosmetic industrie...
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2023-04-01
|
| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12934-023-02079-1 |
| _version_ | 1797840760344674304 |
|---|---|
| author | Gerrit Haeger Jessika Wirges Nicole Tanzmann Sven Oyen Tristan Jolmes Karl-Erich Jaeger Ulrich Schörken Johannes Bongaerts Petra Siegert |
| author_facet | Gerrit Haeger Jessika Wirges Nicole Tanzmann Sven Oyen Tristan Jolmes Karl-Erich Jaeger Ulrich Schörken Johannes Bongaerts Petra Siegert |
| author_sort | Gerrit Haeger |
| collection | DOAJ |
| description | Abstract Background Aminoacylases are highly promising enzymes for the green synthesis of acyl-amino acids, potentially replacing the environmentally harmful Schotten-Baumann reaction. Long-chain acyl-amino acids can serve as strong surfactants and emulsifiers, with application in cosmetic industries. Heterologous expression of these enzymes, however, is often hampered, limiting their use in industrial processes. Results We identified a novel mycobacterial aminoacylase gene from Mycolicibacterium smegmatis MKD 8, cloned and expressed it in Escherichia coli and Vibrio natriegens using the T7 overexpression system. The recombinant enzyme was prone to aggregate as inclusion bodies, and while V. natriegens Vmax™ could produce soluble aminoacylase upon induction with isopropyl β-d-1-thiogalactopyranoside (IPTG), E. coli BL21 (DE3) needed autoinduction with lactose to produce soluble recombinant protein. We successfully conducted a chaperone co-expression study in both organisms to further enhance aminoacylase production and found that overexpression of chaperones GroEL/S enhanced aminoacylase activity in the cell-free extract 1.8-fold in V. natriegens and E. coli. Eventually, E. coli ArcticExpress™ (DE3), which co-expresses cold-adapted chaperonins Cpn60/10 from Oleispira antarctica, cultivated at 12 °C, rendered the most suitable expression system for this aminoacylase and exhibited twice the aminoacylase activity in the cell-free extract compared to E. coli BL21 (DE3) with GroEL/S co-expression at 20 °C. The purified aminoacylase was characterized based on hydrolytic activities, being most stable and active at pH 7.0, with a maximum activity at 70 °C, and stability at 40 °C and pH 7.0 for 5 days. The aminoacylase strongly prefers short-chain acyl-amino acids with smaller, hydrophobic amino acid residues. Several long-chain amino acids were fairly accepted in hydrolysis as well, especially N-lauroyl-L-methionine. To initially evaluate the relevance of this aminoacylase for the synthesis of N-acyl-amino acids, we demonstrated that lauroyl-methionine can be synthesized from lauric acid and methionine in an aqueous system. Conclusion Our results suggest that the recombinant enzyme is well suited for synthesis reactions and will thus be further investigated. |
| first_indexed | 2024-04-09T16:19:56Z |
| format | Article |
| id | doaj.art-f601b43b5c2f478885297c9b9a20b33b |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-04-09T16:19:56Z |
| publishDate | 2023-04-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-f601b43b5c2f478885297c9b9a20b33b2023-04-23T11:32:17ZengBMCMicrobial Cell Factories1475-28592023-04-0122111510.1186/s12934-023-02079-1Chaperone assisted recombinant expression of a mycobacterial aminoacylase in Vibrio natriegens and Escherichia coli capable of N-lauroyl-L-amino acid synthesisGerrit Haeger0Jessika Wirges1Nicole Tanzmann2Sven Oyen3Tristan Jolmes4Karl-Erich Jaeger5Ulrich Schörken6Johannes Bongaerts7Petra Siegert8Institute of Nano- and Biotechnologies, Aachen University of Applied SciencesInstitute of Nano- and Biotechnologies, Aachen University of Applied SciencesInstitute of Nano- and Biotechnologies, Aachen University of Applied SciencesInstitute of Nano- and Biotechnologies, Aachen University of Applied SciencesTH Köln - Campus LeverkusenInstitute of Molecular Enzyme Technology, Heinrich Heine University DüsseldorfTH Köln - Campus LeverkusenInstitute of Nano- and Biotechnologies, Aachen University of Applied SciencesInstitute of Nano- and Biotechnologies, Aachen University of Applied SciencesAbstract Background Aminoacylases are highly promising enzymes for the green synthesis of acyl-amino acids, potentially replacing the environmentally harmful Schotten-Baumann reaction. Long-chain acyl-amino acids can serve as strong surfactants and emulsifiers, with application in cosmetic industries. Heterologous expression of these enzymes, however, is often hampered, limiting their use in industrial processes. Results We identified a novel mycobacterial aminoacylase gene from Mycolicibacterium smegmatis MKD 8, cloned and expressed it in Escherichia coli and Vibrio natriegens using the T7 overexpression system. The recombinant enzyme was prone to aggregate as inclusion bodies, and while V. natriegens Vmax™ could produce soluble aminoacylase upon induction with isopropyl β-d-1-thiogalactopyranoside (IPTG), E. coli BL21 (DE3) needed autoinduction with lactose to produce soluble recombinant protein. We successfully conducted a chaperone co-expression study in both organisms to further enhance aminoacylase production and found that overexpression of chaperones GroEL/S enhanced aminoacylase activity in the cell-free extract 1.8-fold in V. natriegens and E. coli. Eventually, E. coli ArcticExpress™ (DE3), which co-expresses cold-adapted chaperonins Cpn60/10 from Oleispira antarctica, cultivated at 12 °C, rendered the most suitable expression system for this aminoacylase and exhibited twice the aminoacylase activity in the cell-free extract compared to E. coli BL21 (DE3) with GroEL/S co-expression at 20 °C. The purified aminoacylase was characterized based on hydrolytic activities, being most stable and active at pH 7.0, with a maximum activity at 70 °C, and stability at 40 °C and pH 7.0 for 5 days. The aminoacylase strongly prefers short-chain acyl-amino acids with smaller, hydrophobic amino acid residues. Several long-chain amino acids were fairly accepted in hydrolysis as well, especially N-lauroyl-L-methionine. To initially evaluate the relevance of this aminoacylase for the synthesis of N-acyl-amino acids, we demonstrated that lauroyl-methionine can be synthesized from lauric acid and methionine in an aqueous system. Conclusion Our results suggest that the recombinant enzyme is well suited for synthesis reactions and will thus be further investigated.https://doi.org/10.1186/s12934-023-02079-1AminoacylaseVibrio natriegensChaperone co-expressionInclusion bodiesAcyl-amino acids |
| spellingShingle | Gerrit Haeger Jessika Wirges Nicole Tanzmann Sven Oyen Tristan Jolmes Karl-Erich Jaeger Ulrich Schörken Johannes Bongaerts Petra Siegert Chaperone assisted recombinant expression of a mycobacterial aminoacylase in Vibrio natriegens and Escherichia coli capable of N-lauroyl-L-amino acid synthesis Microbial Cell Factories Aminoacylase Vibrio natriegens Chaperone co-expression Inclusion bodies Acyl-amino acids |
| title | Chaperone assisted recombinant expression of a mycobacterial aminoacylase in Vibrio natriegens and Escherichia coli capable of N-lauroyl-L-amino acid synthesis |
| title_full | Chaperone assisted recombinant expression of a mycobacterial aminoacylase in Vibrio natriegens and Escherichia coli capable of N-lauroyl-L-amino acid synthesis |
| title_fullStr | Chaperone assisted recombinant expression of a mycobacterial aminoacylase in Vibrio natriegens and Escherichia coli capable of N-lauroyl-L-amino acid synthesis |
| title_full_unstemmed | Chaperone assisted recombinant expression of a mycobacterial aminoacylase in Vibrio natriegens and Escherichia coli capable of N-lauroyl-L-amino acid synthesis |
| title_short | Chaperone assisted recombinant expression of a mycobacterial aminoacylase in Vibrio natriegens and Escherichia coli capable of N-lauroyl-L-amino acid synthesis |
| title_sort | chaperone assisted recombinant expression of a mycobacterial aminoacylase in vibrio natriegens and escherichia coli capable of n lauroyl l amino acid synthesis |
| topic | Aminoacylase Vibrio natriegens Chaperone co-expression Inclusion bodies Acyl-amino acids |
| url | https://doi.org/10.1186/s12934-023-02079-1 |
| work_keys_str_mv | AT gerrithaeger chaperoneassistedrecombinantexpressionofamycobacterialaminoacylaseinvibrionatriegensandescherichiacolicapableofnlauroyllaminoacidsynthesis AT jessikawirges chaperoneassistedrecombinantexpressionofamycobacterialaminoacylaseinvibrionatriegensandescherichiacolicapableofnlauroyllaminoacidsynthesis AT nicoletanzmann chaperoneassistedrecombinantexpressionofamycobacterialaminoacylaseinvibrionatriegensandescherichiacolicapableofnlauroyllaminoacidsynthesis AT svenoyen chaperoneassistedrecombinantexpressionofamycobacterialaminoacylaseinvibrionatriegensandescherichiacolicapableofnlauroyllaminoacidsynthesis AT tristanjolmes chaperoneassistedrecombinantexpressionofamycobacterialaminoacylaseinvibrionatriegensandescherichiacolicapableofnlauroyllaminoacidsynthesis AT karlerichjaeger chaperoneassistedrecombinantexpressionofamycobacterialaminoacylaseinvibrionatriegensandescherichiacolicapableofnlauroyllaminoacidsynthesis AT ulrichschorken chaperoneassistedrecombinantexpressionofamycobacterialaminoacylaseinvibrionatriegensandescherichiacolicapableofnlauroyllaminoacidsynthesis AT johannesbongaerts chaperoneassistedrecombinantexpressionofamycobacterialaminoacylaseinvibrionatriegensandescherichiacolicapableofnlauroyllaminoacidsynthesis AT petrasiegert chaperoneassistedrecombinantexpressionofamycobacterialaminoacylaseinvibrionatriegensandescherichiacolicapableofnlauroyllaminoacidsynthesis |