High-level heterologous expression of active Chaetomium thermophilum FDH in Pichia pastoris
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Dosyalar
Tarih
2020
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Elsevier Inc.
Erişim Hakkı
info:eu-repo/semantics/closedAccess
Özet
Nowadays, the use of formate dehydrogenase (FDH, EC 1.17.1.9) is well established as a means of NADH regeneration from NAD+ via the coupled conversion of formate into carbon dioxide. Recent studies have been reported that specifically Chaetomium thermophilum FDH (CtFDH) is the most efficient FDH catalyzing this reaction in reverse (i.e. using CO2 as a substrate to produce formate, and thereby regenerating NAD+). However, to date the production of active CtFDH at high protein expression levels has received relatively little attention. In this study, we have tested the effect of batch and high cell density fermentation (HCDF) strategies in a small stirred fermenter, as well as the effect of supplementing the medium with casamino acids, on the expressed level of secreted CtFDH using P. pastoris. We have established that the amount of expressed CtFDH was indeed enhanced via a HCDF strategy and that extracellular protease activity was eliminated via the addition of casamino acids into the fermentation medium. On this basis, secreted CtFDH in an active form can be easily separated from the fermentation and can be used for subsequent biotechnological applications. © 2020 Elsevier Inc.
Açıklama
Anahtar Kelimeler
Casamino acid addition; CtFDH; HCDF strategy; P. pastoris expression system
Kaynak
Enzyme and Microbial Technology
WoS Q Değeri
Scopus Q Değeri
N/A
Cilt
137
