Soluble expression and catalytic properties of codon-optimized recombinant bromelain from MD2 pineapple in Escherichia coli
Bromelain, a member of cysteine proteases, is found abundantly in pineapple (Ananas comosus), and it has a myriad of versatile applications. However, attempts to produce recombinant bromelain for commercialization purposes are challenging due to its expressibility and solubility. This study aims to...
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| Format: | Article |
| Language: | English English |
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Springer
2021
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| Online Access: | https://eprints.ums.edu.my/id/eprint/30896/1/Soluble%20expression%20and%20catalytic%20properties%20of%20codon-optimized%20recombinant%20bromelain%20from%20MD2%20pineapple%20in%20Escherichia%20coli_ABSTRACT.pdf https://eprints.ums.edu.my/id/eprint/30896/2/Soluble%20expression%20and%20catalytic%20properties%20of%20codon-optimized%20recombinant%20bromelain%20from%20MD2%20pineapple%20in%20Escherichia%20coli.pdf |
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| author | Rafida Razali Cahyo Budiman Khairul Azfar Kamaruzaman Vijay Kumar Subbiah |
| author_facet | Rafida Razali Cahyo Budiman Khairul Azfar Kamaruzaman Vijay Kumar Subbiah |
| author_sort | Rafida Razali |
| collection | UMS |
| description | Bromelain, a member of cysteine proteases, is found abundantly in pineapple (Ananas comosus), and it has a myriad of versatile applications. However, attempts to produce recombinant bromelain for commercialization purposes are challenging due to its expressibility and solubility. This study aims to express recombinant fruit bromelain from MD2 pineapple (MD2Bro; accession no: OAY85858.1) in soluble and active forms using Escherichia coli host cell. The gene encoding MD2Bro was codon-optimized, synthesized, and subsequently ligated into pET-32b(+) for further transformation into Escherichia coli BL21-CodonPlus(DE3). Under this strategy, the expressed MD2Bro was in a fusion form with thioredoxin (Trx) tag at its N-terminal (Trx-MD2Bro). The result showed that Trx-MD2Bro was successfully expressed in fully soluble form. The protein was successfully purified using single-step Ni2+-NTA chromatography and confirmed to be in proper folds based on the circular dichroism spectroscopy analysis. The purified Trx-MD2Bro was confirmed to be catalytically active against N-carbobenzoxyglycine p-nitrophenyl ester (N-CBZ-Gly-pNP) with a specific activity of 6.13 ± 0.01 U mg−1 and inhibited by a cysteine protease inhibitor, E-64 (IC50 of 74.38 ± 1.65 nM). Furthermore, the catalytic efficiency (kcat/KM) Trx-MD2Bro was calculated to be at 5.64 ± 0.02 × 10–2 µM−1 s−1 while the optimum temperature and pH were at 50 °C and pH 6.0, respectively. Furthermore, the catalytic activity of Trx-MD2Bro was also affected by ethylenediaminetetraacetic acid (EDTA) or metal ions. Altogether it is proposed that the combination of codon optimization and the use of an appropriate vector are important in the production of a soluble and actively stable recombinant bromelain. |
| first_indexed | 2024-03-06T03:11:36Z |
| format | Article |
| id | ums.eprints-30896 |
| institution | Universiti Malaysia Sabah |
| language | English English |
| last_indexed | 2024-03-06T03:11:36Z |
| publishDate | 2021 |
| publisher | Springer |
| record_format | dspace |
| spelling | ums.eprints-308962021-10-29T03:10:19Z https://eprints.ums.edu.my/id/eprint/30896/ Soluble expression and catalytic properties of codon-optimized recombinant bromelain from MD2 pineapple in Escherichia coli Rafida Razali Cahyo Budiman Khairul Azfar Kamaruzaman Vijay Kumar Subbiah QR1-502 Microbiology Bromelain, a member of cysteine proteases, is found abundantly in pineapple (Ananas comosus), and it has a myriad of versatile applications. However, attempts to produce recombinant bromelain for commercialization purposes are challenging due to its expressibility and solubility. This study aims to express recombinant fruit bromelain from MD2 pineapple (MD2Bro; accession no: OAY85858.1) in soluble and active forms using Escherichia coli host cell. The gene encoding MD2Bro was codon-optimized, synthesized, and subsequently ligated into pET-32b(+) for further transformation into Escherichia coli BL21-CodonPlus(DE3). Under this strategy, the expressed MD2Bro was in a fusion form with thioredoxin (Trx) tag at its N-terminal (Trx-MD2Bro). The result showed that Trx-MD2Bro was successfully expressed in fully soluble form. The protein was successfully purified using single-step Ni2+-NTA chromatography and confirmed to be in proper folds based on the circular dichroism spectroscopy analysis. The purified Trx-MD2Bro was confirmed to be catalytically active against N-carbobenzoxyglycine p-nitrophenyl ester (N-CBZ-Gly-pNP) with a specific activity of 6.13 ± 0.01 U mg−1 and inhibited by a cysteine protease inhibitor, E-64 (IC50 of 74.38 ± 1.65 nM). Furthermore, the catalytic efficiency (kcat/KM) Trx-MD2Bro was calculated to be at 5.64 ± 0.02 × 10–2 µM−1 s−1 while the optimum temperature and pH were at 50 °C and pH 6.0, respectively. Furthermore, the catalytic activity of Trx-MD2Bro was also affected by ethylenediaminetetraacetic acid (EDTA) or metal ions. Altogether it is proposed that the combination of codon optimization and the use of an appropriate vector are important in the production of a soluble and actively stable recombinant bromelain. Springer 2021 Article PeerReviewed text en https://eprints.ums.edu.my/id/eprint/30896/1/Soluble%20expression%20and%20catalytic%20properties%20of%20codon-optimized%20recombinant%20bromelain%20from%20MD2%20pineapple%20in%20Escherichia%20coli_ABSTRACT.pdf text en https://eprints.ums.edu.my/id/eprint/30896/2/Soluble%20expression%20and%20catalytic%20properties%20of%20codon-optimized%20recombinant%20bromelain%20from%20MD2%20pineapple%20in%20Escherichia%20coli.pdf Rafida Razali and Cahyo Budiman and Khairul Azfar Kamaruzaman and Vijay Kumar Subbiah (2021) Soluble expression and catalytic properties of codon-optimized recombinant bromelain from MD2 pineapple in Escherichia coli. Protein Journal, 40. pp. 406-418. ISSN 1572-3887 https://link.springer.com/content/pdf/10.1007/s10930-021-09974-9.pdf https://doi.org/10.1007/s10930-021-09974-9 https://doi.org/10.1007/s10930-021-09974-9 |
| spellingShingle | QR1-502 Microbiology Rafida Razali Cahyo Budiman Khairul Azfar Kamaruzaman Vijay Kumar Subbiah Soluble expression and catalytic properties of codon-optimized recombinant bromelain from MD2 pineapple in Escherichia coli |
| title | Soluble expression and catalytic properties of codon-optimized recombinant bromelain from MD2 pineapple in Escherichia coli |
| title_full | Soluble expression and catalytic properties of codon-optimized recombinant bromelain from MD2 pineapple in Escherichia coli |
| title_fullStr | Soluble expression and catalytic properties of codon-optimized recombinant bromelain from MD2 pineapple in Escherichia coli |
| title_full_unstemmed | Soluble expression and catalytic properties of codon-optimized recombinant bromelain from MD2 pineapple in Escherichia coli |
| title_short | Soluble expression and catalytic properties of codon-optimized recombinant bromelain from MD2 pineapple in Escherichia coli |
| title_sort | soluble expression and catalytic properties of codon optimized recombinant bromelain from md2 pineapple in escherichia coli |
| topic | QR1-502 Microbiology |
| url | https://eprints.ums.edu.my/id/eprint/30896/1/Soluble%20expression%20and%20catalytic%20properties%20of%20codon-optimized%20recombinant%20bromelain%20from%20MD2%20pineapple%20in%20Escherichia%20coli_ABSTRACT.pdf https://eprints.ums.edu.my/id/eprint/30896/2/Soluble%20expression%20and%20catalytic%20properties%20of%20codon-optimized%20recombinant%20bromelain%20from%20MD2%20pineapple%20in%20Escherichia%20coli.pdf |
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