Thermostability enhancement of the Pseudomonas fluorescens esterase I by in vivo folding selection in Thermus thermophilus
Prolonged stability is a desired property for the biotechnological application of enzymes since it allows its reutilization, contributing to making biocatalytic processes more economically competitive with respect to chemical synthesis. In this study, we have applied selection by folding interferenc...
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| Veröffentlicht in: | Biotechnology and bioengineering 2020-01, Vol.117 (1), p.30-38 |
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| creator | Mate, Diana M. Rivera, Noé R. Sanchez‐Freire, Esther Ayala, Juan A. Berenguer, José Hidalgo, Aurelio |
| description | Prolonged stability is a desired property for the biotechnological application of enzymes since it allows its reutilization, contributing to making biocatalytic processes more economically competitive with respect to chemical synthesis. In this study, we have applied selection by folding interference at high temperature in Thermus thermophilus to obtain thermostable variants of the esterase I from Pseudomonas fluorescens (PFEI). The most thermostable variant (Q11L/A191S) showed a melting temperature (Tm) of 77.3 ± 0.1°C (4.6°C higher than the wild‐type) and a half‐life of over 13 hr at 65°C (7.9‐fold better than the wild‐type), with unchanged kinetic parameters. Stabilizing mutations Q11L and A191S were incorporated into PFEI variant L30P, previously described to be enantioselective in the hydrolysis of the (−)‐enantiomer of the Vince lactam. The final variant Q11L/L30P/A191S showed a significant improvement in thermal stability (Tm of 80.8 ± 0.1°C and a half‐life of 65 min at 75°C), while retaining enantioselectivity (E > 100). Structural studies revealed that A191S establishes a hydrogen bond network between a V‐shaped hairpin and the α/β hydrolase domain that leads to higher rigidity and thus would contribute to explaining the increase in stability.
Prolonged stability is a desired property for the biotechnological application of enzymes since it allows its reutilization, contributing to making biocatalytic processes more economically competitive with respect to chemical synthesis. In this study, we have applied selection by folding interference at high temperature in Thermus thermophilus to obtain thermostable variants of the esterase I from Pseudomonas fluorescens (PFEI). The most thermostable variant (Q11L/A191S) showed a melting temperature (Tm) of 77.3 ± 0.1°C (4.6°C higher than the wild‐type) and a half‐life of over 13 hr at 65°C (7.9‐fold better than the wild‐type), with unchanged kinetic parameters. |
| doi_str_mv | 10.1002/bit.27170 |
| format | Article |
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Prolonged stability is a desired property for the biotechnological application of enzymes since it allows its reutilization, contributing to making biocatalytic processes more economically competitive with respect to chemical synthesis. In this study, we have applied selection by folding interference at high temperature in Thermus thermophilus to obtain thermostable variants of the esterase I from Pseudomonas fluorescens (PFEI). The most thermostable variant (Q11L/A191S) showed a melting temperature (Tm) of 77.3 ± 0.1°C (4.6°C higher than the wild‐type) and a half‐life of over 13 hr at 65°C (7.9‐fold better than the wild‐type), with unchanged kinetic parameters.</description><identifier>ISSN: 0006-3592</identifier><identifier>EISSN: 1097-0290</identifier><identifier>DOI: 10.1002/bit.27170</identifier><identifier>PMID: 31529702</identifier><language>eng</language><publisher>United States: Wiley Subscription Services, Inc</publisher><subject>Amides ; Bacterial Proteins - chemistry ; Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Chemical synthesis ; directed evolution ; Directed Molecular Evolution ; Enantiomers ; Enzyme Stability ; Esterase ; Esterases - chemistry ; Esterases - genetics ; Esterases - metabolism ; Folding ; High temperature ; Hot Temperature ; Hydrogen bonds ; Hydrolase ; in vivo selection ; Melt temperature ; Models, Molecular ; Mutation ; Organic chemistry ; Protein Engineering ; Protein Folding ; Pseudomonas fluorescens ; Pseudomonas fluorescens - enzymology ; Pseudomonas fluorescens - genetics ; Rigidity ; Thermal stability ; thermostability ; Thermus thermophilus ; Thermus thermophilus - genetics ; Thermus thermophilus - metabolism ; Yeast</subject><ispartof>Biotechnology and bioengineering, 2020-01, Vol.117 (1), p.30-38</ispartof><rights>2019 Wiley Periodicals, Inc.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c3900-20f2d2cfef40570b75a282efd79003ebda6ff062c59077efb9de9be81e3ecc453</citedby><cites>FETCH-LOGICAL-c3900-20f2d2cfef40570b75a282efd79003ebda6ff062c59077efb9de9be81e3ecc453</cites><orcidid>0000-0001-5740-5584 ; 0000-0002-9689-6272</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Fbit.27170$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fbit.27170$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,776,780,1411,27901,27902,45550,45551</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/31529702$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Mate, Diana M.</creatorcontrib><creatorcontrib>Rivera, Noé R.</creatorcontrib><creatorcontrib>Sanchez‐Freire, Esther</creatorcontrib><creatorcontrib>Ayala, Juan A.</creatorcontrib><creatorcontrib>Berenguer, José</creatorcontrib><creatorcontrib>Hidalgo, Aurelio</creatorcontrib><title>Thermostability enhancement of the Pseudomonas fluorescens esterase I by in vivo folding selection in Thermus thermophilus</title><title>Biotechnology and bioengineering</title><addtitle>Biotechnol Bioeng</addtitle><description>Prolonged stability is a desired property for the biotechnological application of enzymes since it allows its reutilization, contributing to making biocatalytic processes more economically competitive with respect to chemical synthesis. In this study, we have applied selection by folding interference at high temperature in Thermus thermophilus to obtain thermostable variants of the esterase I from Pseudomonas fluorescens (PFEI). The most thermostable variant (Q11L/A191S) showed a melting temperature (Tm) of 77.3 ± 0.1°C (4.6°C higher than the wild‐type) and a half‐life of over 13 hr at 65°C (7.9‐fold better than the wild‐type), with unchanged kinetic parameters. Stabilizing mutations Q11L and A191S were incorporated into PFEI variant L30P, previously described to be enantioselective in the hydrolysis of the (−)‐enantiomer of the Vince lactam. The final variant Q11L/L30P/A191S showed a significant improvement in thermal stability (Tm of 80.8 ± 0.1°C and a half‐life of 65 min at 75°C), while retaining enantioselectivity (E > 100). Structural studies revealed that A191S establishes a hydrogen bond network between a V‐shaped hairpin and the α/β hydrolase domain that leads to higher rigidity and thus would contribute to explaining the increase in stability.
Prolonged stability is a desired property for the biotechnological application of enzymes since it allows its reutilization, contributing to making biocatalytic processes more economically competitive with respect to chemical synthesis. In this study, we have applied selection by folding interference at high temperature in Thermus thermophilus to obtain thermostable variants of the esterase I from Pseudomonas fluorescens (PFEI). The most thermostable variant (Q11L/A191S) showed a melting temperature (Tm) of 77.3 ± 0.1°C (4.6°C higher than the wild‐type) and a half‐life of over 13 hr at 65°C (7.9‐fold better than the wild‐type), with unchanged kinetic parameters.</description><subject>Amides</subject><subject>Bacterial Proteins - chemistry</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Chemical synthesis</subject><subject>directed evolution</subject><subject>Directed Molecular Evolution</subject><subject>Enantiomers</subject><subject>Enzyme Stability</subject><subject>Esterase</subject><subject>Esterases - chemistry</subject><subject>Esterases - genetics</subject><subject>Esterases - metabolism</subject><subject>Folding</subject><subject>High temperature</subject><subject>Hot Temperature</subject><subject>Hydrogen bonds</subject><subject>Hydrolase</subject><subject>in vivo selection</subject><subject>Melt temperature</subject><subject>Models, Molecular</subject><subject>Mutation</subject><subject>Organic chemistry</subject><subject>Protein Engineering</subject><subject>Protein Folding</subject><subject>Pseudomonas fluorescens</subject><subject>Pseudomonas fluorescens - enzymology</subject><subject>Pseudomonas fluorescens - genetics</subject><subject>Rigidity</subject><subject>Thermal stability</subject><subject>thermostability</subject><subject>Thermus thermophilus</subject><subject>Thermus thermophilus - genetics</subject><subject>Thermus thermophilus - metabolism</subject><subject>Yeast</subject><issn>0006-3592</issn><issn>1097-0290</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kcFO3DAQhq0KVBbaQ1-gssQFDoGJTeL1sSCgKyHBYXu2HGfcNXLsrZ2Atk9fL0s5IKE5WKP59M1YPyHfajirAdh558YzJmoBn8isBikqYBL2yAwA2oo3kh2Qw5wfSyvmbfuZHPC6YVIAm5G_yxWmIeZRd867cUMxrHQwOGAYabR0XCF9yDj1cYhBZ2r9FBNmgyFTzCMmnZEuaLehLtAn9xSpjb534TfN6NGMLobt5GXLlLe6sm29cn7KX8i-1T7j19f3iPy6uV5e_azu7m8XVz_uKsMlQMXAsp4Zi_YCGgGdaDSbM7S9KFOOXa9ba6FlppEgBNpO9ig7nNfI0ZiLhh-Rk513neKfqRytBlc-4L0OGKesGJMcoOHACnr8Dn2MUwrlOsV4Kdnydkud7iiTYs4JrVonN-i0UTWobSCqBKJeAins91fj1A3Yv5H_EyjA-Q54dh43H5vU5WK5U_4DTL6XIw</recordid><startdate>202001</startdate><enddate>202001</enddate><creator>Mate, Diana M.</creator><creator>Rivera, Noé R.</creator><creator>Sanchez‐Freire, Esther</creator><creator>Ayala, Juan A.</creator><creator>Berenguer, José</creator><creator>Hidalgo, Aurelio</creator><general>Wiley Subscription Services, Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QF</scope><scope>7QO</scope><scope>7QQ</scope><scope>7SC</scope><scope>7SE</scope><scope>7SP</scope><scope>7SR</scope><scope>7T7</scope><scope>7TA</scope><scope>7TB</scope><scope>7U5</scope><scope>8BQ</scope><scope>8FD</scope><scope>C1K</scope><scope>F28</scope><scope>FR3</scope><scope>H8D</scope><scope>H8G</scope><scope>JG9</scope><scope>JQ2</scope><scope>KR7</scope><scope>L7M</scope><scope>L~C</scope><scope>L~D</scope><scope>P64</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0001-5740-5584</orcidid><orcidid>https://orcid.org/0000-0002-9689-6272</orcidid></search><sort><creationdate>202001</creationdate><title>Thermostability enhancement of the Pseudomonas fluorescens esterase I by in vivo folding selection in Thermus thermophilus</title><author>Mate, Diana M. ; 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In this study, we have applied selection by folding interference at high temperature in Thermus thermophilus to obtain thermostable variants of the esterase I from Pseudomonas fluorescens (PFEI). The most thermostable variant (Q11L/A191S) showed a melting temperature (Tm) of 77.3 ± 0.1°C (4.6°C higher than the wild‐type) and a half‐life of over 13 hr at 65°C (7.9‐fold better than the wild‐type), with unchanged kinetic parameters. Stabilizing mutations Q11L and A191S were incorporated into PFEI variant L30P, previously described to be enantioselective in the hydrolysis of the (−)‐enantiomer of the Vince lactam. The final variant Q11L/L30P/A191S showed a significant improvement in thermal stability (Tm of 80.8 ± 0.1°C and a half‐life of 65 min at 75°C), while retaining enantioselectivity (E > 100). Structural studies revealed that A191S establishes a hydrogen bond network between a V‐shaped hairpin and the α/β hydrolase domain that leads to higher rigidity and thus would contribute to explaining the increase in stability.
Prolonged stability is a desired property for the biotechnological application of enzymes since it allows its reutilization, contributing to making biocatalytic processes more economically competitive with respect to chemical synthesis. In this study, we have applied selection by folding interference at high temperature in Thermus thermophilus to obtain thermostable variants of the esterase I from Pseudomonas fluorescens (PFEI). The most thermostable variant (Q11L/A191S) showed a melting temperature (Tm) of 77.3 ± 0.1°C (4.6°C higher than the wild‐type) and a half‐life of over 13 hr at 65°C (7.9‐fold better than the wild‐type), with unchanged kinetic parameters.</abstract><cop>United States</cop><pub>Wiley Subscription Services, Inc</pub><pmid>31529702</pmid><doi>10.1002/bit.27170</doi><tpages>9</tpages><orcidid>https://orcid.org/0000-0001-5740-5584</orcidid><orcidid>https://orcid.org/0000-0002-9689-6272</orcidid></addata></record> |
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| subjects | Amides Bacterial Proteins - chemistry Bacterial Proteins - genetics Bacterial Proteins - metabolism Chemical synthesis directed evolution Directed Molecular Evolution Enantiomers Enzyme Stability Esterase Esterases - chemistry Esterases - genetics Esterases - metabolism Folding High temperature Hot Temperature Hydrogen bonds Hydrolase in vivo selection Melt temperature Models, Molecular Mutation Organic chemistry Protein Engineering Protein Folding Pseudomonas fluorescens Pseudomonas fluorescens - enzymology Pseudomonas fluorescens - genetics Rigidity Thermal stability thermostability Thermus thermophilus Thermus thermophilus - genetics Thermus thermophilus - metabolism Yeast |
| title | Thermostability enhancement of the Pseudomonas fluorescens esterase I by in vivo folding selection in Thermus thermophilus |
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