Journal ArticleDOI
Immobilization of unspecific peroxygenase expressed in Pichia pastoris by metal affinity binding
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TLDR
This work investigates the immobilization of an UPO-variant originally from Agrocybe aegerita by fusion of a C-terminal hexa-histidine tag and subsequent metal affinity binding on porous glass carriers (EnginZyme EziG), which has the potential to greatly simplify immobilization as no purification steps are necessary.About:
This article is published in Molecular Catalysis.The article was published on 2020-08-01. It has received 10 citations till now. The article focuses on the topics: Unspecific peroxygenase & Pichia pastoris.read more
Citations
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The rise of continuous flow biocatalysis – fundamentals, very recent developments and future perspectives
TL;DR: This review describes the fundamentals of continuously operated reactors with a special focus on enzyme-catalyzed reactions and devoted special attention on future perspectives in this key emerging technological area ranging from process analytical technologies to digitalization.
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Hemoprotein Catalyzed Oxygenations: P450s, UPOs, and Progress toward Scalable Reactions.
TL;DR: Recent developments in the engineering of proteins, cells, and processes for oxygenations by hemoproteins and unspecific peroxygenases are surveyed and their potential and relative merits for scalable reactions are evaluated.
Journal ArticleDOI
Identification and Expression of New Unspecific Peroxygenases - Recent Advances, Challenges and Opportunities.
TL;DR: A review of the state of the art in identification, expression, and screening approaches of fungal peroxygenases can be found in this article, along with challenges associated with current protein production and screening strategies as well as potential solutions and opportunities.
Journal ArticleDOI
Immobilization of the peroxygenase from agrocybe aegerita. The effect of the immobilization ph on the features of an ionically exchanged dimeric peroxygenase
Diego Carballares,Roberto Morellon-Sterling,Xiaomin Xu,Frank Hollmann,Roberto Fernandez-Lafuente +4 more
TL;DR: The immobilization of the recombinant dimeric unspecific peroxygenase from Agrocybe aegerita (rAaeUPO) is outlined, showing how the immobilization pH greatly affects the final biocatalyst performance.
Journal ArticleDOI
A holistic carrier-bound immobilization approach for unspecific peroxygenase
TL;DR: Three different immobilization procedures suitable for UPOs are presented and the immobilized enzyme, indeed, shows longer stability and increased robustness to reaction conditions, and no enzyme deactivation occurred when incubated in organic media for 120 h.
References
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Journal ArticleDOI
Enzymatic assembly of DNA molecules up to several hundred kilobases
Daniel G. Gibson,Lei Young,Ray-Yuan Chuang,J. Craig Venter,Clyde A. Hutchison,Hamilton O. Smith +5 more
TL;DR: An isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5′ exonuclease, a DNA polymerase and a DNA ligase is described.
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Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris
Joan Lin-Cereghino,William W. Wong,See Xiong,William Giang,Linda T. Luong,Jane Vu,Sabrina D. Johnson,Geoff P. Lin-Cereghino +7 more
TL;DR: A condensed protocol for cell preparation and transformation that works reliably with either auxotrophic markers or antibiotic selection is described.
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Parameters necessary to define an immobilized enzyme preparation
TL;DR: In this review, the reasons for loss of activity during immobilization are outlined and suitable diagnostic tests are highlighted to elucidate the precise cause and thereby methods to restore activity.
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Directed evolution of unspecific peroxygenase from Agrocybe aegerita.
Patricia Molina-Espeja,Eva Garcia-Ruiz,David Gonzalez-Perez,René Ullrich,Martin Hofrichter,Miguel Alcalde +5 more
TL;DR: The directed-evolution platform described is a valuable point of departure for the development of customized UPOs with improved features and for the study of structure-function relationships.
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Reliable high‐throughput screening with Pichia pastoris by limiting yeast cell death phenomena
TL;DR: The protocol for protein expression by P. pastoris is simplified and the change from one host for protein engineering to another host for enzyme production becomes dispensable, and this accelerates the protein breeding cycles and makes predictions for large-scale production more accurate.