Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium Sp. Strain Usm2
The synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly acti...
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| Format: | Article |
| Language: | en_US |
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American Society for Microbiology
2011
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| Online Access: | http://hdl.handle.net/1721.1/66933 https://orcid.org/0000-0002-1015-1270 https://orcid.org/0000-0002-6671-5987 |
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| author | Bhubalan, Kesaven Chuah, Jo-Ann Shozui, Fumi Taguchi, Seiichi Sudesh, Kumar Brigham, Christopher J. Sinskey, Anthony J Rha, Chokyun |
| author2 | Harvard University--MIT Division of Health Sciences and Technology |
| author_facet | Harvard University--MIT Division of Health Sciences and Technology Bhubalan, Kesaven Chuah, Jo-Ann Shozui, Fumi Taguchi, Seiichi Sudesh, Kumar Brigham, Christopher J. Sinskey, Anthony J Rha, Chokyun |
| author_sort | Bhubalan, Kesaven |
| collection | MIT |
| description | The synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolated Chromobacterium sp. USM2 (PhaC[subscript Cs]). PhaC[subscript Cs] showed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. An in vitro assay of recombinant PhaC[subscript Cs] expressed in Escherichia coli showed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strain C. necator (307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaC[subscript Cs] was 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC from C. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation in Escherichia coli expressing PhaC[subscript Cs] of up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaC[subscript Cs] is a naturally occurring, highly active PHA synthase with superior polymerizing ability. |
| first_indexed | 2024-09-23T17:08:03Z |
| format | Article |
| id | mit-1721.1/66933 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T17:08:03Z |
| publishDate | 2011 |
| publisher | American Society for Microbiology |
| record_format | dspace |
| spelling | mit-1721.1/669332022-10-03T10:38:55Z Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium Sp. Strain Usm2 Bhubalan, Kesaven Chuah, Jo-Ann Shozui, Fumi Taguchi, Seiichi Sudesh, Kumar Brigham, Christopher J. Sinskey, Anthony J Rha, Chokyun Harvard University--MIT Division of Health Sciences and Technology Massachusetts Institute of Technology. Biomaterials Science and Engineering Laboratory Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Engineering Systems Division Sinskey, Anthony J. Sinskey, Anthony J. Brigham, Christopher J. Sinskey, Anthony J. Rha, ChoKyun The synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolated Chromobacterium sp. USM2 (PhaC[subscript Cs]). PhaC[subscript Cs] showed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. An in vitro assay of recombinant PhaC[subscript Cs] expressed in Escherichia coli showed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strain C. necator (307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaC[subscript Cs] was 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC from C. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation in Escherichia coli expressing PhaC[subscript Cs] of up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaC[subscript Cs] is a naturally occurring, highly active PHA synthase with superior polymerizing ability. Ministry of Science, Technology and Innovation, Malaysia 2011-11-04T16:33:13Z 2011-11-04T16:33:13Z 2011-03 2010-08 Article http://purl.org/eprint/type/JournalArticle 1098-5336 0099-2240 http://hdl.handle.net/1721.1/66933 Bhubalan, K. et al. “Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium sp. Strain USM2.” Applied and Environmental Microbiology 77 (2011): 2926-2933. Web. 4 Nov. 2011. © 2011 American Society for Microbiology https://orcid.org/0000-0002-1015-1270 https://orcid.org/0000-0002-6671-5987 en_US http://dx.doi.org/10.1128/AEM.01997-10 Applied and Environmental Microbiology Creative Commons Attribution-Noncommercial-Share Alike 3.0 http://creativecommons.org/licenses/by-nc-sa/3.0/ application/pdf American Society for Microbiology Sinskey |
| spellingShingle | Bhubalan, Kesaven Chuah, Jo-Ann Shozui, Fumi Taguchi, Seiichi Sudesh, Kumar Brigham, Christopher J. Sinskey, Anthony J Rha, Chokyun Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium Sp. Strain Usm2 |
| title | Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium Sp. Strain Usm2 |
| title_full | Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium Sp. Strain Usm2 |
| title_fullStr | Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium Sp. Strain Usm2 |
| title_full_unstemmed | Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium Sp. Strain Usm2 |
| title_short | Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium Sp. Strain Usm2 |
| title_sort | characterization of the highly active polyhydroxyalkanoate synthase of chromobacterium sp strain usm2 |
| url | http://hdl.handle.net/1721.1/66933 https://orcid.org/0000-0002-1015-1270 https://orcid.org/0000-0002-6671-5987 |
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