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dc.creatorMolloy, Susan
dc.creatorNikodinović-Runić, Jasmina
dc.creatorMartin, Leona B.
dc.creatorHartmann, Hermann
dc.creatorSolano, Francisco
dc.creatorDecker, Heinz
dc.creatorO'Connor, Kevin E.
dc.date.accessioned2018-11-22T00:23:16Z
dc.date.available2018-11-22T00:23:16Z
dc.date.issued2013
dc.identifier.issn0006-3592
dc.identifier.urihttp://cherry.chem.bg.ac.rs/handle/123456789/1359
dc.description.abstractThe tyrosinase gene from Ralstonia solanacearum (GenBank NP518458) was subjected to random mutagenesis resulting in tyrosinase variants (RVC10 and RV145) with up to 3.2-fold improvement in kcat, 5.2-fold lower Km and 16-fold improvement in catalytic efficiency for D-tyrosine. Based on RVC10 and RV145 mutated sequences, single mutation variants were generated with all variants showing increased kcat for D-tyrosine compared to the wild type (WT). All single mutation variants based on RV145 had a higher kcat and Km value compared to the RV145 and thus the combination of four mutations in RV145 was antagonistic for turnover, but synergistic for affinity of the enzyme for D-tyrosine. Single mutation variant 145_V153A exhibited the highest (6.9-fold) improvement in kcat and a 2.4-fold increase in Km compared to the WT. Two single mutation variants, C10_N322S and C10_T183I reduced the Km up to 2.6-fold for D-tyrosine but one variant 145_V153A increased the Km 2.4-fold compared to the WT. Homology based modeling of R. solanacearum tyrosinase showed that mutation V153A disrupts the van der Waals interactions with an -helix providing one of the conserved histidine residues of the active site. The kcat and Km values for L-tyrosine decreased for RV145 and RVC10 compared to the WT. RV145 exhibited a 2.1-fold high catalytic efficiency compared to the WT which is a 7.6-fold lower improvement compared to D-tyrosine. RV145 exhibited a threefold higher monophenolase:diphenolase activity ratio for D-tyrosine:D-DOPA and a 1.4-fold higher L-tyrosine:L-DOPA activity ratio compared to the WT. Biotechnol. Bioeng. 2013; 110: 1849-1857.en
dc.publisherWiley-Blackwell, Hoboken
dc.rightsrestrictedAccess
dc.sourceBiotechnology and Bioengineering
dc.subjecttyrosinaseen
dc.subjectD-tyrosineen
dc.subjectrandom mutagenesisen
dc.subjectsite specific mutagenesisen
dc.subjectenzyme catalysisen
dc.subjecthomology modelingen
dc.titleEngineering of a bacterial tyrosinase for improved catalytic efficiency towards D-tyrosine using random and site directed mutagenesis approachesen
dc.typearticle
dc.rights.licenseARR
dcterms.abstractХартманн, Херманн; Мартин, Леона Б.; Децкер, Хеинз; Никодиновић-Рунић, Јасмина; Солано, Францисцо; О'Цоннор, Кевин Е.; Моллоy, Сусан;
dc.citation.volume110
dc.citation.issue7
dc.citation.spage1849
dc.citation.epage1857
dc.identifier.wos000319525200006
dc.identifier.doi10.1002/bit.24859
dc.citation.other110(7): 1849-1857
dc.citation.rankM21
dc.identifier.pmid23381872
dc.description.otherPeer-reviewed manuscript: [http://cherry.chem.bg.ac.rs/handle/123456789/3491]
dc.description.otherSupplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3492]
dc.type.versionpublishedVersion
dc.identifier.scopus2-s2.0-84878415861
dc.identifier.rcubKon_2479


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