Recombinant Fungal Enzyme Purification

Recombinant CtPMO1 from C. thermophilum and HiPMO1 from S. thermophilum were expressed in Pichia pastoris and purified by nickel affinity chromatography as previously described [29 (link), 33 (link)]. Native TaAA9A was isolated by ion-exchange chromatography on DEAE-sepharose column (GE Healthcare) from a 7-day culture filtrate of T. aurantiacus grown at 50 °C in cellulose-containing medium [29 (link)]. The isolated TaAA9A was visualized on an SDS-PAGE gel for confirmation as an AA9 LPMO (TaAA9A) [35 (link)].

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Liu N., Yu W., Guo X., Chen J., Xia D., Yu J, & Li D. (2022). Oxidative cleavage of cellulose in the horse gut. Microbial Cell Factories, 21, 38.

Publication 2022

DEAE-sepharose column

Affinity chromatography Cellulose Deae Ion exchange chromatography Nickel Pichia pastoris Sds page Sepharose

Corresponding Organization :

Other organizations : Shandong Agricultural University

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Variable analysis

independent variables

Expression of recombinant CtPMO1 from C. thermophilum in Pichia pastoris
Expression of recombinant HiPMO1 from S. thermophilum in Pichia pastoris
Purification of CtPMO1 and HiPMO1 by nickel affinity chromatography
Isolation of native TaAA9A from T. aurantiacus culture filtrate by ion-exchange chromatography on DEAE-sepharose column

dependent variables

Purification and confirmation of CtPMO1, HiPMO1, and TaAA9A as AA9 LPMOs

control variables

Growth conditions for T. aurantiacus: 50 °C in cellulose-containing medium

positive controls

SDS-PAGE analysis to visualize and confirm TaAA9A as an AA9 LPMO

negative controls

No negative controls were explicitly mentioned.

Annotations

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