Solid-Phase Assembly of Multienzyme Systems into Artificial Cellulosomes

Abstract

We herein describe a bioinspired solid-phase assembly of a multienzyme system scaffolded on an artificial cellulosome. An alcohol dehydrogenase and an ω-transaminase were fused to cohesin and dockerin domains to drive their sequential and ordered coimmobilization on agarose porous microbeads. The resulting immobilized scaffolded enzymatic cellulosome was characterized through quartz crystal microbalance with dissipation and confocal laser scanning microscopy to demonstrate that both enzymes interact with each other and physically colocalize within the microbeads. Finally, the assembled multifunctional heterogeneous biocatalyst was tested for the one-pot conversion of alcohols into amines. By using the physically colocalized enzymatic system confined into porous microbeads, the yield of the corresponding amine was 1.3 and 10 times higher than the spatially segregated immobilized system and the free enzymes, respectively. This work establishes the basis of a new concept to organize multienzyme systems at the nanoscale within solid and porous immobilization carriers.

Is enzyme immobilization a mature discipline? Some critical considerations to capitalize on the benefits of immobilization

“…In this way, at least we can know the final distribution of the different enzymes in the biocatalyst. 332,[404][405][406][407] Although the optimization may be complex, finally the control of the enzyme order may be possible and confirmed, provided the researcher understands the phenomena occurring in the co-immobilization.…”

Engineering Enzyme Properties for Improved Biocatalytic Processes in Batch and Continuous Flow

“…An artificial cellulosome was designed by Zeballos et al to accommodate a two-enzyme cascade with a dimeric transaminase from Aspergillus terreus and a tetrameric alcohol dehydrogenase from Bacillus (Geobacillus) stearothermophilus. 91 The authors discussed the benefits of this highly organized system to the efficiency of the cascade toward 3-methyl-1butylamine from the corresponding alcohol. Natural enzyme scaffolding systems have been investigated for their potential to organize non-natural cascades, often in the context of improved metabolic engineering, and may provide opportunities for cell-free systems too.…”

“…Enzyme scaffolding also occurs in nature, in structures such as the cellulosome, , which uses cohesin to scaffold dockerin-tagged enzymes involved in cellulose decomposition. An artificial cellulosome was designed by Zeballos et al to accommodate a two-enzyme cascade with a dimeric transaminase from Aspergillus terreus and a tetrameric alcohol dehydrogenase from Bacillus (Geobacillus) stearothermophilus . The authors discussed the benefits of this highly organized system to the efficiency of the cascade toward 3-methyl-1-butylamine from the corresponding alcohol.…”

Protein Mediated Enzyme Immobilization

“…[ 33 ] Cellulosome building blocks are also being used to direct enzyme immobilization for in vitro biocatalysis. [ 34 ] For example, alcohol dehydrogenase and ω‐transaminase were immobilized on the surface of porous agarose beads functionalized with Co 2+ by mixing the beads with His tagged cohesin domains and enzymes fused to dockerin domains. The coimmobilization of both enzymes improves yield of acetophenone from 7% for soluble enzyme to 69% for coimmobilized enzymes.…”