Overexpression and Purification of Gracilariopsis chorda Carbonic Anhydrase (GcCAα3) in Nicotiana benthamiana, and Its Immobilization and Use in CO2 Hydration Reactions

Abstract

Carbonic anhydrase (CA; EC 4.2.2.1) is a Zn-binding metalloenzyme that catalyzes the reversible hydration of CO 2. Recently, CAs have gained a great deal of attention as biocatalysts for capturing CO 2 from industrial flue gases owing to their extremely fast reaction rates and simple reaction mechanism. However, their general application for this purpose requires improvements to stability at high temperature and under in vitro conditions, and reductions in production and scale-up costs. In the present study, we developed a strategy for producing GcCAα3, a CA isoform from the red alga Gracilariopsis chorda, in Nicotiana benthamiana. To achieve high-level expression and facile purification of GcCAα3, we designed various constructs by incorporating various domains such as translation-enhancing M domain, SUMO domain and cellulosebinding domain CBM3. Of these constructs, MC-GcCAα3 that had the M and CBM3 domains was expressed at high levels in N. benthamiana via agroinfiltration with a yield of 1.0 g/kg fresh weight. The recombinant protein was targeted to the endoplasmic reticulum (ER) for high-level accumulation in plants. Specific and tight CBM3-mediated binding of recombinant GcCAα3 proteins to microcrystalline cellulose beads served as a means for both protein purification from total plant extracts and protein immobilization to a solid surface for increased stability, facilitating multiple rounds of use in CO 2 hydration reactions.

Production of Gloeophyllum trabeum Endoglucanase Cel12A in Nicotiana benthamiana for Cellulose Degradation

“…Next, we transformed the construct MSC-GtCel12A alone or together with BiP-bdSENP1-HA into the N. benthamiana leaves using agroinfiltration ( Figures 1B,C ) (Islam et al, 2019 ; Razzak et al, 2020 ). At 3, 5, and 7 days post infiltration (dpi), we isolated total protein extracts from the transformed leaves and analyzed them by western blotting using an anti-CBM3 antibody.…”

“…However, this tight binding can be considered beneficial for not only the efficient capture and recovery of target proteins but also the repeated utilization of target proteins. Previous studies have indicated that industrial enzymes such as carbonic anhydrases from different origins exhibit not only efficient enzyme activity but also high endurability, thereby allowing multiple reuses of produced enzymes (Kumari et al, 2020 ; Razzak et al, 2020 ).…”

“…In this study, the sequence corresponding to the N-terminal hydrophobic signal sequence (amino acids 1–20) was deleted from the full-length GtCel12A . To generate the BiP-M-bdSUMO-GtCel12A-CBM3-HDEL construct, we digested with XmaI and Acc65I the pCambia1300 plant expression vector (Komori et al, 2007 ; Razzak et al, 2020 ), containing the sequences encoding for the BiP signal sequence, M domain of the human receptor-type tyrosine-protein phosphatase C, SUMO domain, and CBM3-HDEL, and ligated the GtCel12A sequence into it that was digested with the same restriction endonucleases.…”

“…The constructs BiP-M-bdSUMO-GtCel12A-CBM3-HDEL or BiP:bdSENP1:HA (Islam et al, 2020 ) were transformed into Agrobacterium tumefaciens (EHA105). A. tumefaciens cells harboring binary vector constructs were introduced into N. benthamiana leaves via syringe infiltration as described previously (Islam et al, 2019 ; Razzak et al, 2020 ). In every agro-infiltration experiment, A. tumefaciens harboring p38, which is derived from Turnip crinkle virus and encodes a suppressor of host gene-silencing, was co-infiltrated at OD 600 value of 0.8 (Qu et al, 2003 ; Islam et al, 2019 ).…”

“…Further, the conditions for plant growth are much less affected by microorganisms that are detrimental to mammalian cell cultures (Buyel et al, 2017 ; Moon et al, 2019 ; Muthamilselvan et al, 2019 ; Knodler and Buyel, 2021 ; Schillberg and Finnern, 2021 ). In addition, previous studies indicated that various useful proteins of different origins, such as bacteria, bacteriophage, animals, and red algae, remained functional when produced in tobacco ( Nicotiana benthamiana ) (Garvey et al, 2013 ; Islam et al, 2019 , 2020 ; Kumari et al, 2020 ; Razzak et al, 2020 ). One of the most important considerations in protein production using plants is how target proteins can be expressed in large amounts because the cost-effectiveness of protein production is often highly dependent on the protein amounts yielded from a unit cultivation area.…”