Spatially confined enzymatic tandem system with GOx and HRP compartmentalized in ultrafiltration membrane

Sara Barricella; Jordi M. Fuertes; Kevin H. Putera; Aubrey E. Quigley; Victoria Haritos; Benny D. Freeman; Gil Garnier

doi:10.1016/j.memsci.2023.122214

Spatially confined enzymatic tandem system with GOx and HRP compartmentalized in ultrafiltration membrane

Sara Barricella, Jordi M. Fuertes, Kevin H. Putera, Aubrey E. Quigley, Victoria Haritos, Benny D. Freeman, Gil Garnier

Research output: Contribution to journal › Article › Research › peer-review

3 Citations (Scopus)

Abstract

The precise control of timely and spatially defined enzymatic reactions is of considerable interest in biology, biomedical engineering, and bio-catalysis. In this study, we report a robust multi-enzymatic cascade system with glucose oxidase (GOx) and horseradish peroxidase (HRP) spatially compartmentalized in a polyether sulfone ultrafiltration (UF) membrane by co-deposition of polydopamine (PDA) and polyethyleneimine (PEI). The PDA/PEI nanolayers reduce enzyme leakage, retaining ∼98 and ∼70 % of GOx and HRP, respectively, and facilitate substrates and intermediates passage and enrichment near the enzyme's active sites. The membrane serves as a porous scaffold facilitating enzyme immobilization and recyclability, improving stability and catalytic efficiency. The spatially defined cascade system exhibits superior catalytic activity, leading to a 1.48-fold and 3-fold increase compared to free GOx and HRP, respectively. This improvement is likely due to substrate channeling, substrate diffusion and product convection. Furthermore, the dynamics of the GOx and HRP tandem catalysis was investigated in a closed system which demonstrated the crucial roles of dissolved oxygen in both the oxidoreductase and peroxidase reactions. The concept of tandem enzyme reactions in UF membranes can be generalized to create efficient and stable enzyme systems for applications in biosensing and industrial processes.

Original language	English
Article number	122214
Number of pages	11
Journal	Journal of Membrane Science
Volume	690
DOIs	https://doi.org/10.1016/j.memsci.2023.122214
Publication status	Published - 15 Jan 2024

Keywords

Cascade catalysis
Compartmentalization
Glucose oxidase (GOx)
Horseradish peroxidase (HRP)
Ultrafiltration membrane

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10.1016/j.memsci.2023.122214

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@article{20a6a0e7f49043b69e55f7bbd7356fa0,

title = "Spatially confined enzymatic tandem system with GOx and HRP compartmentalized in ultrafiltration membrane",

abstract = "The precise control of timely and spatially defined enzymatic reactions is of considerable interest in biology, biomedical engineering, and bio-catalysis. In this study, we report a robust multi-enzymatic cascade system with glucose oxidase (GOx) and horseradish peroxidase (HRP) spatially compartmentalized in a polyether sulfone ultrafiltration (UF) membrane by co-deposition of polydopamine (PDA) and polyethyleneimine (PEI). The PDA/PEI nanolayers reduce enzyme leakage, retaining ∼98 and ∼70 % of GOx and HRP, respectively, and facilitate substrates and intermediates passage and enrichment near the enzyme's active sites. The membrane serves as a porous scaffold facilitating enzyme immobilization and recyclability, improving stability and catalytic efficiency. The spatially defined cascade system exhibits superior catalytic activity, leading to a 1.48-fold and 3-fold increase compared to free GOx and HRP, respectively. This improvement is likely due to substrate channeling, substrate diffusion and product convection. Furthermore, the dynamics of the GOx and HRP tandem catalysis was investigated in a closed system which demonstrated the crucial roles of dissolved oxygen in both the oxidoreductase and peroxidase reactions. The concept of tandem enzyme reactions in UF membranes can be generalized to create efficient and stable enzyme systems for applications in biosensing and industrial processes.",

keywords = "Cascade catalysis, Compartmentalization, Glucose oxidase (GOx), Horseradish peroxidase (HRP), Ultrafiltration membrane",

author = "Sara Barricella and Fuertes, {Jordi M.} and Putera, {Kevin H.} and Quigley, {Aubrey E.} and Victoria Haritos and Freeman, {Benny D.} and Gil Garnier",

note = "Funding Information: This project was supported by the Australian Research Council (ARC) through the ARC Research Hub for Energy-efficient Separation ( IH170100009 ). The authors acknowledge the facilities and technical assistance of the Monash Centre for Electron Microscopy and Monash Micro Imaging, a Node of Microscopy Australia, and Monash X-ray Platform. The authors also acknowledge use of the facilities, equipment and technical assistance of Monash Histology Platform, Department of Anatomy and Development Biology, Monash University. Funding Information: The distribution of embedded enzymes in the GR80PP membrane was analyzed by confocal laser scanning microscopy (CLSM). Excitation at 488 nm yielded the green fluorescence of the FITC-labelled GOx, while excitation at 640 nm produced the red fluorescence of the Alexa Fluor 647-labelled HRP (Fig. 2a). The merged image confirms the successful compartmentalization of GOx and HRP in the membrane, with GOx located beneath the skin filtration layer and HRP deposited in the support layer (Fig. 2a). The results are consistent with IR spectra of the membrane cross-section collected using O-PTIR (Fig. 2 c-f). The IR spectra of the pristine membrane (Fig. S1), covering the range 2700–3588 cm−1, clearly showed the expected peaks associated with PES (3096 cm−1, typically attributed to the C–H stretching of alkene aromatics), and PP (2948 and 2913 cm−1, characteristic of C–H stretching in alkane), while also confirming the lack of interference of the embedding medium in the area of interest. Single wavenumber images were taken using O-PTIR at 3388 cm−1 (O–H stretching) in the embedded membrane (Fig. 2e) to identify the PDA coating, and then IR spectra were acquired at selected locations (Fig. 2f). The hydroxyl spectral image was chosen because it is clearly distinct from the C–H stretch of the PES, thus allowing the identification of the PDA coating. A layered distribution of PDA is observed in Fig. 2e, with the coating localized at ca. 30 μm from the PES skin surface. The water contact angles on the pristine and biocatalytic membranes (Fig. S2b) confirm that the hydrophilicity of the skin layer did not change after enzyme immobilization and PDA/PEI coating, suggesting the successful retention of PDA between the skin and support layer. FT-IR spectra of the pristine and biocatalytic membranes (Fig. S2a) confirm that GOx, HRP, and PDA/PEI are retained between the skin filtration layer and the non-woven support. The surface charge measured for the pristine and enzymatic membranes as a function of pH is presented in Supplementary Material (Fig. S3). Both membranes have similar zeta potential curves, exhibiting a slight positive charge at acidic pH that transit to a negative charge at alkaline pH. Due to the amine group, the isoelectric point of the enzymatic membrane shifted from 4.2 to 4.5 [53]. However, at the pH investigated (pH = 5), both the pristine and enzymatic membrane are essentially neutral.This project was supported by the Australian Research Council (ARC) through the ARC Research Hub for Energy-efficient Separation (IH170100009). The authors acknowledge the facilities and technical assistance of the Monash Centre for Electron Microscopy and Monash Micro Imaging, a Node of Microscopy Australia, and Monash X-ray Platform. The authors also acknowledge use of the facilities, equipment and technical assistance of Monash Histology Platform, Department of Anatomy and Development Biology, Monash University. Publisher Copyright: {\textcopyright} 2023 The Authors",

year = "2024",

month = jan,

day = "15",

doi = "10.1016/j.memsci.2023.122214",

language = "English",

volume = "690",

journal = "Journal of Membrane Science",

issn = "0376-7388",

publisher = "Elsevier BV",

}

TY - JOUR

T1 - Spatially confined enzymatic tandem system with GOx and HRP compartmentalized in ultrafiltration membrane

AU - Barricella, Sara

AU - Fuertes, Jordi M.

AU - Putera, Kevin H.

AU - Quigley, Aubrey E.

AU - Haritos, Victoria

AU - Freeman, Benny D.

AU - Garnier, Gil

N1 - Funding Information: This project was supported by the Australian Research Council (ARC) through the ARC Research Hub for Energy-efficient Separation ( IH170100009 ). The authors acknowledge the facilities and technical assistance of the Monash Centre for Electron Microscopy and Monash Micro Imaging, a Node of Microscopy Australia, and Monash X-ray Platform. The authors also acknowledge use of the facilities, equipment and technical assistance of Monash Histology Platform, Department of Anatomy and Development Biology, Monash University. Funding Information: The distribution of embedded enzymes in the GR80PP membrane was analyzed by confocal laser scanning microscopy (CLSM). Excitation at 488 nm yielded the green fluorescence of the FITC-labelled GOx, while excitation at 640 nm produced the red fluorescence of the Alexa Fluor 647-labelled HRP (Fig. 2a). The merged image confirms the successful compartmentalization of GOx and HRP in the membrane, with GOx located beneath the skin filtration layer and HRP deposited in the support layer (Fig. 2a). The results are consistent with IR spectra of the membrane cross-section collected using O-PTIR (Fig. 2 c-f). The IR spectra of the pristine membrane (Fig. S1), covering the range 2700–3588 cm−1, clearly showed the expected peaks associated with PES (3096 cm−1, typically attributed to the C–H stretching of alkene aromatics), and PP (2948 and 2913 cm−1, characteristic of C–H stretching in alkane), while also confirming the lack of interference of the embedding medium in the area of interest. Single wavenumber images were taken using O-PTIR at 3388 cm−1 (O–H stretching) in the embedded membrane (Fig. 2e) to identify the PDA coating, and then IR spectra were acquired at selected locations (Fig. 2f). The hydroxyl spectral image was chosen because it is clearly distinct from the C–H stretch of the PES, thus allowing the identification of the PDA coating. A layered distribution of PDA is observed in Fig. 2e, with the coating localized at ca. 30 μm from the PES skin surface. The water contact angles on the pristine and biocatalytic membranes (Fig. S2b) confirm that the hydrophilicity of the skin layer did not change after enzyme immobilization and PDA/PEI coating, suggesting the successful retention of PDA between the skin and support layer. FT-IR spectra of the pristine and biocatalytic membranes (Fig. S2a) confirm that GOx, HRP, and PDA/PEI are retained between the skin filtration layer and the non-woven support. The surface charge measured for the pristine and enzymatic membranes as a function of pH is presented in Supplementary Material (Fig. S3). Both membranes have similar zeta potential curves, exhibiting a slight positive charge at acidic pH that transit to a negative charge at alkaline pH. Due to the amine group, the isoelectric point of the enzymatic membrane shifted from 4.2 to 4.5 [53]. However, at the pH investigated (pH = 5), both the pristine and enzymatic membrane are essentially neutral.This project was supported by the Australian Research Council (ARC) through the ARC Research Hub for Energy-efficient Separation (IH170100009). The authors acknowledge the facilities and technical assistance of the Monash Centre for Electron Microscopy and Monash Micro Imaging, a Node of Microscopy Australia, and Monash X-ray Platform. The authors also acknowledge use of the facilities, equipment and technical assistance of Monash Histology Platform, Department of Anatomy and Development Biology, Monash University. Publisher Copyright: © 2023 The Authors

PY - 2024/1/15

Y1 - 2024/1/15

N2 - The precise control of timely and spatially defined enzymatic reactions is of considerable interest in biology, biomedical engineering, and bio-catalysis. In this study, we report a robust multi-enzymatic cascade system with glucose oxidase (GOx) and horseradish peroxidase (HRP) spatially compartmentalized in a polyether sulfone ultrafiltration (UF) membrane by co-deposition of polydopamine (PDA) and polyethyleneimine (PEI). The PDA/PEI nanolayers reduce enzyme leakage, retaining ∼98 and ∼70 % of GOx and HRP, respectively, and facilitate substrates and intermediates passage and enrichment near the enzyme's active sites. The membrane serves as a porous scaffold facilitating enzyme immobilization and recyclability, improving stability and catalytic efficiency. The spatially defined cascade system exhibits superior catalytic activity, leading to a 1.48-fold and 3-fold increase compared to free GOx and HRP, respectively. This improvement is likely due to substrate channeling, substrate diffusion and product convection. Furthermore, the dynamics of the GOx and HRP tandem catalysis was investigated in a closed system which demonstrated the crucial roles of dissolved oxygen in both the oxidoreductase and peroxidase reactions. The concept of tandem enzyme reactions in UF membranes can be generalized to create efficient and stable enzyme systems for applications in biosensing and industrial processes.

AB - The precise control of timely and spatially defined enzymatic reactions is of considerable interest in biology, biomedical engineering, and bio-catalysis. In this study, we report a robust multi-enzymatic cascade system with glucose oxidase (GOx) and horseradish peroxidase (HRP) spatially compartmentalized in a polyether sulfone ultrafiltration (UF) membrane by co-deposition of polydopamine (PDA) and polyethyleneimine (PEI). The PDA/PEI nanolayers reduce enzyme leakage, retaining ∼98 and ∼70 % of GOx and HRP, respectively, and facilitate substrates and intermediates passage and enrichment near the enzyme's active sites. The membrane serves as a porous scaffold facilitating enzyme immobilization and recyclability, improving stability and catalytic efficiency. The spatially defined cascade system exhibits superior catalytic activity, leading to a 1.48-fold and 3-fold increase compared to free GOx and HRP, respectively. This improvement is likely due to substrate channeling, substrate diffusion and product convection. Furthermore, the dynamics of the GOx and HRP tandem catalysis was investigated in a closed system which demonstrated the crucial roles of dissolved oxygen in both the oxidoreductase and peroxidase reactions. The concept of tandem enzyme reactions in UF membranes can be generalized to create efficient and stable enzyme systems for applications in biosensing and industrial processes.

KW - Cascade catalysis

KW - Compartmentalization

KW - Glucose oxidase (GOx)

KW - Horseradish peroxidase (HRP)

KW - Ultrafiltration membrane

UR - http://www.scopus.com/inward/record.url?scp=85175658879&partnerID=8YFLogxK

U2 - 10.1016/j.memsci.2023.122214

DO - 10.1016/j.memsci.2023.122214

M3 - Article

AN - SCOPUS:85175658879

SN - 0376-7388

VL - 690

JO - Journal of Membrane Science

JF - Journal of Membrane Science

M1 - 122214

ER -

Spatially confined enzymatic tandem system with GOx and HRP compartmentalized in ultrafiltration membrane

Abstract

Keywords

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ARC Research Hub for Energy-efficient Separation

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