Surface residues serine 69 and arginine 194 of metagenome-derived lipase influence catalytic activity

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dc.contributor.authorÖzgen, Fatma Feyza
dc.contributor.authorVardar-Yel, Nurcan
dc.contributor.authorRoth, Owen Scott
dc.contributor.authorShahbaz, Lersa Sayyad
dc.contributor.authorVardar-Schara, Gonul
dc.date.accessioned2021-05-15T11:33:52Z
dc.date.available2021-05-15T11:33:52Z
dc.date.issued2020
dc.departmentTıbbi Laboratuvar Teknikleri Anabilim Dalıen_US
dc.descriptionOzgen, Fatma Feyza/0000-0001-5114-8644
dc.description.abstractA lipase gene was identified and isolated from a fosmid metagenomics library constructed from forest topsoil samples. Error prone PCR and saturation mutagenesis were used to generate seven lipase variants: S69 P/R194Q, S69 G, S69 P, S69E, S69 P/T99S/N190S/R194Q, R194S, and R194A; by testing five different fatty acids of p-nitrophenyl (p-NP) ester, positions S69 and R194 were found to influence the catalytic activity. Specifically, variant R194S was identified that had 5.0, 4.5, 2.1, 2.2, and 1.4-fold higher catalytic efficiency (v(max)/K-m) towards p-NP acetate, p-NP butyrate, p-NP octanoate, p-NP dodecanoate, and p-NP palmitate, respectively, compared to the metagenome-derived native lipase. Among the S69 variants, variants S69 P/R194Q and S69 P hydrolyzed p-NP acetate 1.8- and 1.5-fold faster than wild-type in terms of apparent v(max)/K-m values, respectively. The activity is lowered by introducing mutations S69E, R194A, and S69 P/T99S/N190S/R194Q. Here, two additional residues, T99 and N190, were also identified that may have important roles in catalysis. Present work confirms the advantages of combining directed evolution and saturation mutagenesis approaches of protein engineering and expands the knowledge on the architecture of the industrially important lipase enzyme and its relation to its catalytic activity.en_US
dc.description.sponsorshipMarie Curie European grant; New Investigator grant of the California State University Program for Education & Research in Biotechnology; University Research, Scholarship, and Creative Activity Program of California State University Stanislausen_US
dc.description.sponsorshipThis work was supported by the Marie Curie European grant, New Investigator grant of the California State University Program for Education & Research in Biotechnology, and University Research, Scholarship, and Creative Activity Program of California State University Stanislaus.en_US
dc.identifier.doi10.1016/j.bej.2019.107442
dc.identifier.issn1369-703X
dc.identifier.issn1873-295X
dc.identifier.scopus2-s2.0-85075345331
dc.identifier.scopusqualityQ2
dc.identifier.urihttps://doi.org/10.1016/j.bej.2019.107442
dc.identifier.urihttps://hdl.handle.net/20.500.12939/241
dc.identifier.volume154en_US
dc.identifier.wosWOS:000518148400019
dc.identifier.wosqualityQ2
dc.indekslendigikaynakWeb of Science
dc.indekslendigikaynakScopus
dc.institutionauthorVardar-Yel, Nurcan
dc.language.isoen
dc.publisherElsevieren_US
dc.relation.ispartofBiochemical Engineering Journal
dc.relation.publicationcategoryMakale - Uluslararası Hakemli Dergi - Kurum Öğretim Elemanıen_US
dc.rightsinfo:eu-repo/semantics/closedAccessen_US
dc.subjectError-Prone PCRen_US
dc.subjectSaturation Mutagenesisen_US
dc.subjectMetagenomicsen_US
dc.subjectLipaseen_US
dc.subjectCatalytic Activityen_US
dc.titleSurface residues serine 69 and arginine 194 of metagenome-derived lipase influence catalytic activity
dc.typeArticle

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