Protein engineering of cellobiose dehydrogenase from Phanerochaete chrysosporium in yeast Saccharomyces cerevisiae InvSc1 for increased activity and stability
Аутори
Blažić, MarijaBalaž, Ana Marija
Tadić, Vojin
Draganić, Bojana
Ostafe, Raluca
Fischer, Rainer
Prodanović, Radivoje
Чланак у часопису (Рецензирана верзија)
Метаподаци
Приказ свих података о документуАпстракт
Cellobiose dehydrogenase (CDH) can be used in industry for lactobionic acid production, as a part of biosensors for disaccharides and in wound healing. In fungi it is involved in lignocellulose degradation. CDH gene from Phanerochaete chrysosporium has been cloned in pYES2 plasmid for extracellular expression and protein engineering in yeast Saccharomyces cerevisiae InvSC1 for the first time. A CDH gene library was generated using error-prone PCR and screened by spectrophotometric enzymatic assay
based on 2,6-dichloroindophenol reduction detection in microtiter plates. Several mutants with increased activity and specificity towards lactose and cellobiose were found, purified and characterized in detail. Recombinant CDH enzymes showed a broad molecular weight between 120 and 150 KDa due to hyperglycosylation and the best S137N mutant showed 2.2 times increased kcat and 1.5 and 2 times increased specificity constant for lactose and cellobiose compared to the wild type enzyme. pH optimum... of mutants was not changed while thermostability of selected mutants improved and S137N mutant retained 30% of it’s original activity after 15 minutes at 70oC compared to 10% of activity that the wild type enzyme retained. Mutants M65S and S137N showed also 1.6 and 1.5 times increased productivity of hydrogen peroxide in the presence of 30mM lactose compared to the wild type.
Кључне речи:
cellobiose dehydrogenase / directed evolution / lactose / Saccharomyces cerevisiaeИзвор:
Biochemical Engineering Journal, 2019, 146, 179-185Издавач:
- Elsevier
Финансирање / пројекти:
- Алергени, антитела, ензими и мали физиолошки значајни молекули: дизајн, структура, функција и значај (RS-172049)
- Испитивања односа структура-функција у ћелијском зиду биљака и измене структуре зида ензимским инжењерингом (RS-173017)
Напомена:
- This is the peer-reviewed version of the article: https://doi.org/10.1016/j.bej.2019.03.025
- http://cer.ihtm.bg.ac.rs/handle/123456789/2656
DOI: 10.1016/j.bej.2019.03.025
ISSN: 1369-703X
WoS: 000466999900020
Scopus: 2-s2.0-85063648705
Институција/група
IHTMTY - JOUR AU - Blažić, Marija AU - Balaž, Ana Marija AU - Tadić, Vojin AU - Draganić, Bojana AU - Ostafe, Raluca AU - Fischer, Rainer AU - Prodanović, Radivoje PY - 2019 UR - https://cer.ihtm.bg.ac.rs/handle/123456789/2655 AB - Cellobiose dehydrogenase (CDH) can be used in industry for lactobionic acid production, as a part of biosensors for disaccharides and in wound healing. In fungi it is involved in lignocellulose degradation. CDH gene from Phanerochaete chrysosporium has been cloned in pYES2 plasmid for extracellular expression and protein engineering in yeast Saccharomyces cerevisiae InvSC1 for the first time. A CDH gene library was generated using error-prone PCR and screened by spectrophotometric enzymatic assay based on 2,6-dichloroindophenol reduction detection in microtiter plates. Several mutants with increased activity and specificity towards lactose and cellobiose were found, purified and characterized in detail. Recombinant CDH enzymes showed a broad molecular weight between 120 and 150 KDa due to hyperglycosylation and the best S137N mutant showed 2.2 times increased kcat and 1.5 and 2 times increased specificity constant for lactose and cellobiose compared to the wild type enzyme. pH optimum of mutants was not changed while thermostability of selected mutants improved and S137N mutant retained 30% of it’s original activity after 15 minutes at 70oC compared to 10% of activity that the wild type enzyme retained. Mutants M65S and S137N showed also 1.6 and 1.5 times increased productivity of hydrogen peroxide in the presence of 30mM lactose compared to the wild type. PB - Elsevier T2 - Biochemical Engineering Journal T1 - Protein engineering of cellobiose dehydrogenase from Phanerochaete chrysosporium in yeast Saccharomyces cerevisiae InvSc1 for increased activity and stability VL - 146 SP - 179 EP - 185 DO - 10.1016/j.bej.2019.03.025 ER -
@article{ author = "Blažić, Marija and Balaž, Ana Marija and Tadić, Vojin and Draganić, Bojana and Ostafe, Raluca and Fischer, Rainer and Prodanović, Radivoje", year = "2019", abstract = "Cellobiose dehydrogenase (CDH) can be used in industry for lactobionic acid production, as a part of biosensors for disaccharides and in wound healing. In fungi it is involved in lignocellulose degradation. CDH gene from Phanerochaete chrysosporium has been cloned in pYES2 plasmid for extracellular expression and protein engineering in yeast Saccharomyces cerevisiae InvSC1 for the first time. A CDH gene library was generated using error-prone PCR and screened by spectrophotometric enzymatic assay based on 2,6-dichloroindophenol reduction detection in microtiter plates. Several mutants with increased activity and specificity towards lactose and cellobiose were found, purified and characterized in detail. Recombinant CDH enzymes showed a broad molecular weight between 120 and 150 KDa due to hyperglycosylation and the best S137N mutant showed 2.2 times increased kcat and 1.5 and 2 times increased specificity constant for lactose and cellobiose compared to the wild type enzyme. pH optimum of mutants was not changed while thermostability of selected mutants improved and S137N mutant retained 30% of it’s original activity after 15 minutes at 70oC compared to 10% of activity that the wild type enzyme retained. Mutants M65S and S137N showed also 1.6 and 1.5 times increased productivity of hydrogen peroxide in the presence of 30mM lactose compared to the wild type.", publisher = "Elsevier", journal = "Biochemical Engineering Journal", title = "Protein engineering of cellobiose dehydrogenase from Phanerochaete chrysosporium in yeast Saccharomyces cerevisiae InvSc1 for increased activity and stability", volume = "146", pages = "179-185", doi = "10.1016/j.bej.2019.03.025" }
Blažić, M., Balaž, A. M., Tadić, V., Draganić, B., Ostafe, R., Fischer, R.,& Prodanović, R.. (2019). Protein engineering of cellobiose dehydrogenase from Phanerochaete chrysosporium in yeast Saccharomyces cerevisiae InvSc1 for increased activity and stability. in Biochemical Engineering Journal Elsevier., 146, 179-185. https://doi.org/10.1016/j.bej.2019.03.025
Blažić M, Balaž AM, Tadić V, Draganić B, Ostafe R, Fischer R, Prodanović R. Protein engineering of cellobiose dehydrogenase from Phanerochaete chrysosporium in yeast Saccharomyces cerevisiae InvSc1 for increased activity and stability. in Biochemical Engineering Journal. 2019;146:179-185. doi:10.1016/j.bej.2019.03.025 .
Blažić, Marija, Balaž, Ana Marija, Tadić, Vojin, Draganić, Bojana, Ostafe, Raluca, Fischer, Rainer, Prodanović, Radivoje, "Protein engineering of cellobiose dehydrogenase from Phanerochaete chrysosporium in yeast Saccharomyces cerevisiae InvSc1 for increased activity and stability" in Biochemical Engineering Journal, 146 (2019):179-185, https://doi.org/10.1016/j.bej.2019.03.025 . .