Enhancing RGI lyase thermostability by targeted single point mutations
Rhamnogalacturonan I lyase (RGI lyase) (EC 4.2.2.-) catalyzes the cleavage of rhamnogalacturonan I in pectins by β-elimination. In this study the thermal stability of a RGI lyase (PL 11) originating from Bacillus licheniformis DSM 13/ATCC14580 was increased by a targeted protein engineering approach...
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| Veröffentlicht in: | Applied microbiology and biotechnology 2013-11, Vol.97 (22), p.9727-9735 |
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| creator | Silva, Inês R Larsen, Dorte M Jers, Carsten Derkx, Patrick Meyer, Anne S Mikkelsen, Jørn D |
| description | Rhamnogalacturonan I lyase (RGI lyase) (EC 4.2.2.-) catalyzes the cleavage of rhamnogalacturonan I in pectins by β-elimination. In this study the thermal stability of a RGI lyase (PL 11) originating from Bacillus licheniformis DSM 13/ATCC14580 was increased by a targeted protein engineering approach involving single amino acid substitution. Nine individual amino acids were selected as targets for site-saturated mutagenesis by the use of a predictive consensus approach in combination with prediction of protein mutant stability changes and B-factor iteration testing. After extensive experimental verification of the thermal stability of the designed mutants versus the original wild-type RGI lyase, several promising single point mutations were obtained, particularly in position Glu434 on the surface of the enzyme protein. The best mutant, Glu434Leu, produced a half-life of 31 min at 60 °C, corresponding to a 1.6-fold improvement of the thermal stability compared to the original RGI lyase. Gly55Val was the second best mutation with a thermostability half-life increase of 27 min at 60 °C, and the best mutations following were Glu434Trp, Glu434Phe, and Glu434Tyr, respectively. The data verify the applicability of a combinatorial predictive approach for designing a small site saturation library for improving enzyme thermostability. In addition, new thermostable RGI lyases suitable for enzymatic upgrading of pectinaceous plant biomass materials at elevated temperatures were produced. |
| doi_str_mv | 10.1007/s00253-013-5184-3 |
| format | Article |
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In this study the thermal stability of a RGI lyase (PL 11) originating from Bacillus licheniformis DSM 13/ATCC14580 was increased by a targeted protein engineering approach involving single amino acid substitution. Nine individual amino acids were selected as targets for site-saturated mutagenesis by the use of a predictive consensus approach in combination with prediction of protein mutant stability changes and B-factor iteration testing. After extensive experimental verification of the thermal stability of the designed mutants versus the original wild-type RGI lyase, several promising single point mutations were obtained, particularly in position Glu434 on the surface of the enzyme protein. The best mutant, Glu434Leu, produced a half-life of 31 min at 60 °C, corresponding to a 1.6-fold improvement of the thermal stability compared to the original RGI lyase. Gly55Val was the second best mutation with a thermostability half-life increase of 27 min at 60 °C, and the best mutations following were Glu434Trp, Glu434Phe, and Glu434Tyr, respectively. The data verify the applicability of a combinatorial predictive approach for designing a small site saturation library for improving enzyme thermostability. In addition, new thermostable RGI lyases suitable for enzymatic upgrading of pectinaceous plant biomass materials at elevated temperatures were produced.</description><identifier>ISSN: 0175-7598</identifier><identifier>EISSN: 1432-0614</identifier><identifier>DOI: 10.1007/s00253-013-5184-3</identifier><identifier>PMID: 23995225</identifier><language>eng</language><publisher>Berlin/Heidelberg: Springer-Verlag</publisher><subject>amino acid substitution ; Amino acids ; Analysis ; Applied Genetics and Molecular Biotechnology ; Bacillus - enzymology ; Bacillus - genetics ; Bacillus licheniformis ; Bacteria ; Biomass ; Biomedical and Life Sciences ; Biotechnology ; Cellular biology ; DNA Mutational Analysis ; DNA, Bacterial - chemistry ; DNA, Bacterial - genetics ; Engineering ; Enzyme Stability ; Enzymes ; Evolution ; Genetic aspects ; half life ; High temperature ; Libraries ; Life Sciences ; Lyases ; Microbial Genetics and Genomics ; Microbiology ; Molecular Sequence Data ; Mutagenesis ; Mutagenesis, Site-Directed ; Mutant Proteins - chemistry ; Mutant Proteins - genetics ; Mutant Proteins - metabolism ; mutants ; Mutation ; Optimization ; pectins ; Pectins - metabolism ; Plant biomass ; Point Mutation ; Polysaccharide-Lyases - chemistry ; Polysaccharide-Lyases - genetics ; Polysaccharide-Lyases - metabolism ; Polysaccharides ; prediction ; protein engineering ; Protein Stability ; Proteins ; Sequence Analysis, DNA ; Studies ; Temperature ; Thermal properties ; thermal stability</subject><ispartof>Applied microbiology and biotechnology, 2013-11, Vol.97 (22), p.9727-9735</ispartof><rights>Springer-Verlag Berlin Heidelberg 2013</rights><rights>COPYRIGHT 2013 Springer</rights><rights>Springer-Verlag 2013</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c591t-f8ce04670ca61c0d6951da343ab7874cd6985f83181c1ec5995808cc5191af93</citedby><cites>FETCH-LOGICAL-c591t-f8ce04670ca61c0d6951da343ab7874cd6985f83181c1ec5995808cc5191af93</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s00253-013-5184-3$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00253-013-5184-3$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,780,784,27924,27925,41488,42557,51319</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/23995225$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Silva, Inês R</creatorcontrib><creatorcontrib>Larsen, Dorte M</creatorcontrib><creatorcontrib>Jers, Carsten</creatorcontrib><creatorcontrib>Derkx, Patrick</creatorcontrib><creatorcontrib>Meyer, Anne S</creatorcontrib><creatorcontrib>Mikkelsen, Jørn D</creatorcontrib><title>Enhancing RGI lyase thermostability by targeted single point mutations</title><title>Applied microbiology and biotechnology</title><addtitle>Appl Microbiol Biotechnol</addtitle><addtitle>Appl Microbiol Biotechnol</addtitle><description>Rhamnogalacturonan I lyase (RGI lyase) (EC 4.2.2.-) catalyzes the cleavage of rhamnogalacturonan I in pectins by β-elimination. In this study the thermal stability of a RGI lyase (PL 11) originating from Bacillus licheniformis DSM 13/ATCC14580 was increased by a targeted protein engineering approach involving single amino acid substitution. Nine individual amino acids were selected as targets for site-saturated mutagenesis by the use of a predictive consensus approach in combination with prediction of protein mutant stability changes and B-factor iteration testing. After extensive experimental verification of the thermal stability of the designed mutants versus the original wild-type RGI lyase, several promising single point mutations were obtained, particularly in position Glu434 on the surface of the enzyme protein. The best mutant, Glu434Leu, produced a half-life of 31 min at 60 °C, corresponding to a 1.6-fold improvement of the thermal stability compared to the original RGI lyase. Gly55Val was the second best mutation with a thermostability half-life increase of 27 min at 60 °C, and the best mutations following were Glu434Trp, Glu434Phe, and Glu434Tyr, respectively. The data verify the applicability of a combinatorial predictive approach for designing a small site saturation library for improving enzyme thermostability. In addition, new thermostable RGI lyases suitable for enzymatic upgrading of pectinaceous plant biomass materials at elevated temperatures were produced.</description><subject>amino acid substitution</subject><subject>Amino acids</subject><subject>Analysis</subject><subject>Applied Genetics and Molecular Biotechnology</subject><subject>Bacillus - enzymology</subject><subject>Bacillus - genetics</subject><subject>Bacillus licheniformis</subject><subject>Bacteria</subject><subject>Biomass</subject><subject>Biomedical and Life Sciences</subject><subject>Biotechnology</subject><subject>Cellular biology</subject><subject>DNA Mutational Analysis</subject><subject>DNA, Bacterial - chemistry</subject><subject>DNA, Bacterial - genetics</subject><subject>Engineering</subject><subject>Enzyme Stability</subject><subject>Enzymes</subject><subject>Evolution</subject><subject>Genetic aspects</subject><subject>half life</subject><subject>High temperature</subject><subject>Libraries</subject><subject>Life Sciences</subject><subject>Lyases</subject><subject>Microbial Genetics and Genomics</subject><subject>Microbiology</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis</subject><subject>Mutagenesis, Site-Directed</subject><subject>Mutant Proteins - chemistry</subject><subject>Mutant Proteins - genetics</subject><subject>Mutant Proteins - metabolism</subject><subject>mutants</subject><subject>Mutation</subject><subject>Optimization</subject><subject>pectins</subject><subject>Pectins - metabolism</subject><subject>Plant biomass</subject><subject>Point Mutation</subject><subject>Polysaccharide-Lyases - chemistry</subject><subject>Polysaccharide-Lyases - genetics</subject><subject>Polysaccharide-Lyases - metabolism</subject><subject>Polysaccharides</subject><subject>prediction</subject><subject>protein engineering</subject><subject>Protein Stability</subject><subject>Proteins</subject><subject>Sequence Analysis, DNA</subject><subject>Studies</subject><subject>Temperature</subject><subject>Thermal properties</subject><subject>thermal 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genetics</topic><topic>Engineering</topic><topic>Enzyme Stability</topic><topic>Enzymes</topic><topic>Evolution</topic><topic>Genetic aspects</topic><topic>half life</topic><topic>High temperature</topic><topic>Libraries</topic><topic>Life Sciences</topic><topic>Lyases</topic><topic>Microbial Genetics and Genomics</topic><topic>Microbiology</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis</topic><topic>Mutagenesis, Site-Directed</topic><topic>Mutant Proteins - chemistry</topic><topic>Mutant Proteins - genetics</topic><topic>Mutant Proteins - metabolism</topic><topic>mutants</topic><topic>Mutation</topic><topic>Optimization</topic><topic>pectins</topic><topic>Pectins - metabolism</topic><topic>Plant biomass</topic><topic>Point Mutation</topic><topic>Polysaccharide-Lyases - chemistry</topic><topic>Polysaccharide-Lyases - genetics</topic><topic>Polysaccharide-Lyases - metabolism</topic><topic>Polysaccharides</topic><topic>prediction</topic><topic>protein 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β-elimination. In this study the thermal stability of a RGI lyase (PL 11) originating from Bacillus licheniformis DSM 13/ATCC14580 was increased by a targeted protein engineering approach involving single amino acid substitution. Nine individual amino acids were selected as targets for site-saturated mutagenesis by the use of a predictive consensus approach in combination with prediction of protein mutant stability changes and B-factor iteration testing. After extensive experimental verification of the thermal stability of the designed mutants versus the original wild-type RGI lyase, several promising single point mutations were obtained, particularly in position Glu434 on the surface of the enzyme protein. The best mutant, Glu434Leu, produced a half-life of 31 min at 60 °C, corresponding to a 1.6-fold improvement of the thermal stability compared to the original RGI lyase. Gly55Val was the second best mutation with a thermostability half-life increase of 27 min at 60 °C, and the best mutations following were Glu434Trp, Glu434Phe, and Glu434Tyr, respectively. The data verify the applicability of a combinatorial predictive approach for designing a small site saturation library for improving enzyme thermostability. In addition, new thermostable RGI lyases suitable for enzymatic upgrading of pectinaceous plant biomass materials at elevated temperatures were produced.</abstract><cop>Berlin/Heidelberg</cop><pub>Springer-Verlag</pub><pmid>23995225</pmid><doi>10.1007/s00253-013-5184-3</doi><tpages>9</tpages></addata></record> |
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| subjects | amino acid substitution Amino acids Analysis Applied Genetics and Molecular Biotechnology Bacillus - enzymology Bacillus - genetics Bacillus licheniformis Bacteria Biomass Biomedical and Life Sciences Biotechnology Cellular biology DNA Mutational Analysis DNA, Bacterial - chemistry DNA, Bacterial - genetics Engineering Enzyme Stability Enzymes Evolution Genetic aspects half life High temperature Libraries Life Sciences Lyases Microbial Genetics and Genomics Microbiology Molecular Sequence Data Mutagenesis Mutagenesis, Site-Directed Mutant Proteins - chemistry Mutant Proteins - genetics Mutant Proteins - metabolism mutants Mutation Optimization pectins Pectins - metabolism Plant biomass Point Mutation Polysaccharide-Lyases - chemistry Polysaccharide-Lyases - genetics Polysaccharide-Lyases - metabolism Polysaccharides prediction protein engineering Protein Stability Proteins Sequence Analysis, DNA Studies Temperature Thermal properties thermal stability |
| title | Enhancing RGI lyase thermostability by targeted single point mutations |
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