Characterization and application of natural and recombinant butelase-1 to improve industrial enzymes by end-to-end circularization
| Autor: | James P. Tam, Chuan-Fa Liu, Aida Serra, Xiaohong Zhang, Siu Kwan Sze, Giang K. T. Nguyen, Xinya Hemu, Janet To, Shining Loo |
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| Přispěvatelé: | School of Biological Sciences |
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Glycosylation General Chemical Engineering Peptide Legumain 01 natural sciences law.invention 03 medical and health sciences chemistry.chemical_compound law Ligase activity Lipase Cyclic-Peptides chemistry.chemical_classification DNA ligase biology 010405 organic chemistry Biological sciences [Science] General Chemistry Unprotected Peptide Segments 0104 chemical sciences 030104 developmental biology Enzyme chemistry Biochemistry biology.protein Recombinant DNA |
| Zdroj: | RSC advances. 11(37) |
| ISSN: | 2046-2069 |
| Popis: | 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). |
| Databáze: | OpenAIRE |
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