Engineering pH-tolerant mutants of a cyanide dihydratase

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dc.contributor.authorWang, Lan
dc.contributor.authorWatermeyer, Jean M
dc.contributor.authorMulelu, Andani E
dc.contributor.authorSewell, Trevor B
dc.contributor.authorBenedik, Michael J
dc.date.accessioned2016-09-01T11:17:52Z
dc.date.available2016-09-01T11:17:52Z
dc.date.issued2012
dc.date.updated2016-09-01T11:13:20Z
dc.description.abstractCyanide dihydratase is an enzyme in the nitrilase family capable of transforming cyanide to formate and ammonia. This reaction has been exploited for the bioremediation of cyanide in wastewater streams, but extending the pH operating range of the enzyme would improve its utility. In this work, we describe mutants of Bacillus pumilus C1 cyanide dihydratase (CynDpum) with improved activity at higher pH. Error-prone PCR was used to construct a library of CynDpum mutants, and a high-throughput screening system was developed to screen the library for improved activity at pH 10. Two mutant alleles were identified that allowed cells to degrade cyanide in solutions at pH 10, whereas the wild-type was inactive above pH 9. The mutant alleles each encoded three different amino acid substitutions, but for one of those, a single change, E327G, accounted for the phenotype. The purified proteins containing multiple mutations were five times more active than the wild-type enzyme at pH 9, but all purified enzymes lost activity at pH 10. The mutation Q86R resulted in the formation of significantly longer fibers at low pH, and both E327G and Q86R contributed to the persistence of active oligomeric assemblies at pH 9. In addition, the mutant enzymes proved to be more thermostable than the wild type, suggesting improved physical stability rather than any change in chemistry accounts for their increased pH tolerance.en_ZA
dc.identifierhttp://dx.doi.org/10.1007/s00253-011-3620-9
dc.identifier.apacitationWang, L., Watermeyer, J. M., Mulelu, A. E., Sewell, T. B., & Benedik, M. J. (2012). Engineering pH-tolerant mutants of a cyanide dihydratase. <i>Applied Microbiology and Biotechnology</i>, http://hdl.handle.net/11427/21647en_ZA
dc.identifier.chicagocitationWang, Lan, Jean M Watermeyer, Andani E Mulelu, Trevor B Sewell, and Michael J Benedik "Engineering pH-tolerant mutants of a cyanide dihydratase." <i>Applied Microbiology and Biotechnology</i> (2012) http://hdl.handle.net/11427/21647en_ZA
dc.identifier.citationWang, L., Watermeyer, J. M., Mulelu, A. E., Sewell, B. T., & Benedik, M. J. (2012). Engineering pH-tolerant mutants of a cyanide dihydratase. Applied microbiology and biotechnology, 94(1), 131-140.en_ZA
dc.identifier.issn0175-7598en_ZA
dc.identifier.ris TY - Journal Article AU - Wang, Lan AU - Watermeyer, Jean M AU - Mulelu, Andani E AU - Sewell, Trevor B AU - Benedik, Michael J AB - Cyanide dihydratase is an enzyme in the nitrilase family capable of transforming cyanide to formate and ammonia. This reaction has been exploited for the bioremediation of cyanide in wastewater streams, but extending the pH operating range of the enzyme would improve its utility. In this work, we describe mutants of Bacillus pumilus C1 cyanide dihydratase (CynDpum) with improved activity at higher pH. Error-prone PCR was used to construct a library of CynDpum mutants, and a high-throughput screening system was developed to screen the library for improved activity at pH 10. Two mutant alleles were identified that allowed cells to degrade cyanide in solutions at pH 10, whereas the wild-type was inactive above pH 9. The mutant alleles each encoded three different amino acid substitutions, but for one of those, a single change, E327G, accounted for the phenotype. The purified proteins containing multiple mutations were five times more active than the wild-type enzyme at pH 9, but all purified enzymes lost activity at pH 10. The mutation Q86R resulted in the formation of significantly longer fibers at low pH, and both E327G and Q86R contributed to the persistence of active oligomeric assemblies at pH 9. In addition, the mutant enzymes proved to be more thermostable than the wild type, suggesting improved physical stability rather than any change in chemistry accounts for their increased pH tolerance. DA - 2012 DB - OpenUCT DP - University of Cape Town J1 - Applied Microbiology and Biotechnology LK - https://open.uct.ac.za PB - University of Cape Town PY - 2012 SM - 0175-7598 T1 - Engineering pH-tolerant mutants of a cyanide dihydratase TI - Engineering pH-tolerant mutants of a cyanide dihydratase UR - http://hdl.handle.net/11427/21647 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/21647
dc.identifier.urihttp://link.springer.com/article/10.1007/s00253-011-3620-9
dc.identifier.vancouvercitationWang L, Watermeyer JM, Mulelu AE, Sewell TB, Benedik MJ. Engineering pH-tolerant mutants of a cyanide dihydratase. Applied Microbiology and Biotechnology. 2012; http://hdl.handle.net/11427/21647.en_ZA
dc.languageengen_ZA
dc.publisherSpringer Verlagen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.sourceApplied Microbiology and Biotechnologyen_ZA
dc.source.urihttp://link.springer.com/journal/253
dc.subject.otherNitrilase
dc.subject.otherCyanide
dc.subject.otherBioremediation
dc.subject.otherCyanide dihydratase
dc.subject.otherpH tolerance
dc.subject.otherProtein stability
dc.titleEngineering pH-tolerant mutants of a cyanide dihydrataseen_ZA
dc.typeJournal Articleen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceArticleen_ZA
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