Characterization and application of natural and recombinant butelase-1 to improve industrial enzymes by end-to-end circularization
Butelase-1, an asparaginyl endopeptidase or legumain, is the prototypical and fastest known Asn/Asp-specific peptide ligase. It is highly useful for engineering and macrocyclization of peptides and proteins. However, certain biochemical properties and applications of naturally occurring and recombin...
| Main Authors: | , , , , , , , , |
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| Other Authors: | |
| Format: | Journal Article |
| Language: | English |
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2022
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| Online Access: | https://hdl.handle.net/10356/160046 |
| _version_ | 1824453898584195072 |
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| author | Hemu, Xinya Zhang, Xiaohong Nguyen, Giang K. T. To, Janet Serra, Aida Loo, Shining Sze, Siu Kwan Liu, Chuan-Fa Tam, James P. |
| author2 | School of Biological Sciences |
| author_facet | School of Biological Sciences Hemu, Xinya Zhang, Xiaohong Nguyen, Giang K. T. To, Janet Serra, Aida Loo, Shining Sze, Siu Kwan Liu, Chuan-Fa Tam, James P. |
| author_sort | Hemu, Xinya |
| collection | NTU |
| description | Butelase-1, an asparaginyl endopeptidase or legumain, is the prototypical and fastest known Asn/Asp-specific peptide ligase. It is highly useful for engineering and macrocyclization of peptides and proteins. However, certain biochemical properties and applications of naturally occurring and recombinant butelase-1 remain unexplored. Here we report methods to increase the yield of natural and bacterial expressed recombinant butelase-1 and how they can be used to improve the stability and activity of two important industrial enzymes, lipase and phytase, by end-to-end circularization. First, the yield of natural butelase-1 was increased 3-fold to 15 mg kg(-1) by determining its highest distribution which is found in young tissues, such as shoots. The yield of recombinantly-produced soluble butelase-1 was improved by promoting cytoplasmic disulfide folding, codon changes, and truncation of the N-terminal pro-domain. Natural and recombinant butelase-1 displayed similar ligase activity, physical stability, and salt tolerance. Furthermore, the processing and glycosylation sites of natural and recombinant butelase-1 were determined by proteomic analysis. Storage conditions for both forms of butelase-1, frozen or lyophilized, were also optimized. Cyclization of lipase and phytase mediated by either soluble or immobilized butelase-1 was highly efficient and simple, and resulted in increased thermal stability and enhanced enzymatic activity. Overall, improved production of butelase-1 can be exploited to improve the biocatalytic efficacy of lipase and phytase by end-to-end cyclization. In turn, ligase-improved enzymes could be a general and environmentally friendly strategy for producing more stable and efficient industrial enzymes. |
| first_indexed | 2025-02-19T03:13:44Z |
| format | Journal Article |
| id | ntu-10356/160046 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2025-02-19T03:13:44Z |
| publishDate | 2022 |
| record_format | dspace |
| spelling | ntu-10356/1600462023-02-28T17:10:39Z Characterization and application of natural and recombinant butelase-1 to improve industrial enzymes by end-to-end circularization Hemu, Xinya Zhang, Xiaohong Nguyen, Giang K. T. To, Janet Serra, Aida Loo, Shining Sze, Siu Kwan Liu, Chuan-Fa Tam, James P. School of Biological Sciences Science::Biological sciences Unprotected Peptide Segments Cyclic-Peptides Butelase-1, an asparaginyl endopeptidase or legumain, is the prototypical and fastest known Asn/Asp-specific peptide ligase. It is highly useful for engineering and macrocyclization of peptides and proteins. However, certain biochemical properties and applications of naturally occurring and recombinant butelase-1 remain unexplored. Here we report methods to increase the yield of natural and bacterial expressed recombinant butelase-1 and how they can be used to improve the stability and activity of two important industrial enzymes, lipase and phytase, by end-to-end circularization. First, the yield of natural butelase-1 was increased 3-fold to 15 mg kg(-1) by determining its highest distribution which is found in young tissues, such as shoots. The yield of recombinantly-produced soluble butelase-1 was improved by promoting cytoplasmic disulfide folding, codon changes, and truncation of the N-terminal pro-domain. Natural and recombinant butelase-1 displayed similar ligase activity, physical stability, and salt tolerance. Furthermore, the processing and glycosylation sites of natural and recombinant butelase-1 were determined by proteomic analysis. Storage conditions for both forms of butelase-1, frozen or lyophilized, were also optimized. Cyclization of lipase and phytase mediated by either soluble or immobilized butelase-1 was highly efficient and simple, and resulted in increased thermal stability and enhanced enzymatic activity. Overall, improved production of butelase-1 can be exploited to improve the biocatalytic efficacy of lipase and phytase by end-to-end cyclization. In turn, ligase-improved enzymes could be a general and environmentally friendly strategy for producing more stable and efficient industrial enzymes. Ministry of Education (MOE) Nanyang Technological University Published version This work was supported by the Academic Research Grant Tier 3 (MOE2016-T3-1-003) from the Singapore Ministry of Education (MOE) and a Nanyang Technological University internal funding-Synzymes and Natural Products Center (SYNC). 2022-07-12T02:48:20Z 2022-07-12T02:48:20Z 2021 Journal Article Hemu, X., Zhang, X., Nguyen, G. K. T., To, J., Serra, A., Loo, S., Sze, S. K., Liu, C. & Tam, J. P. (2021). Characterization and application of natural and recombinant butelase-1 to improve industrial enzymes by end-to-end circularization. RSC Advances, 11(37), 23105-23112. https://dx.doi.org/10.1039/D1RA03763C 2046-2069 https://hdl.handle.net/10356/160046 10.1039/D1RA03763C 37 11 23105 23112 en MOE2016-T3-1-003 RSC Advances © 2021 The Author(s). Published by the Royal Society of Chemistry. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. application/pdf |
| spellingShingle | Science::Biological sciences Unprotected Peptide Segments Cyclic-Peptides Hemu, Xinya Zhang, Xiaohong Nguyen, Giang K. T. To, Janet Serra, Aida Loo, Shining Sze, Siu Kwan Liu, Chuan-Fa Tam, James P. Characterization and application of natural and recombinant butelase-1 to improve industrial enzymes by end-to-end circularization |
| title | Characterization and application of natural and recombinant butelase-1 to improve industrial enzymes by end-to-end circularization |
| title_full | Characterization and application of natural and recombinant butelase-1 to improve industrial enzymes by end-to-end circularization |
| title_fullStr | Characterization and application of natural and recombinant butelase-1 to improve industrial enzymes by end-to-end circularization |
| title_full_unstemmed | Characterization and application of natural and recombinant butelase-1 to improve industrial enzymes by end-to-end circularization |
| title_short | Characterization and application of natural and recombinant butelase-1 to improve industrial enzymes by end-to-end circularization |
| title_sort | characterization and application of natural and recombinant butelase 1 to improve industrial enzymes by end to end circularization |
| topic | Science::Biological sciences Unprotected Peptide Segments Cyclic-Peptides |
| url | https://hdl.handle.net/10356/160046 |
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