Enhancing Enzyme Activity and Immobilization in Nanostructured Inorganic–Enzyme Complexes

Abstract

Understanding the chemical and physical interactions at the interface of protein surfaces and inorganic crystals has important implications in the advancement of immobilized enzyme catalysis. Recently, enzyme-inorganic hybrid complexes have been demonstrated as effective materials for enzyme immobilization. The precipitation of phosphate nanocrystals in the presence of enzymes creates enzyme-Cu(PO)·3HO particles with high surface-to-volume ratios, enhanced activity, and increased stability. Here, we begin to develop a mechanistic understanding of enzyme loading in such complexes. Using a series of enzymes including horseradish peroxidase (HRP), a thermostable alcohol dehydrogenase (AdhD), diaphorase, catalase, glucose oxidase (GOx), and the protein bovine serum albumin (BSA), we identified a correlation between particle synthesis temperature, overall enzyme charge, and enzyme loading. The model enzyme HRP has a high predicted pI of ∼7.5 and maintains an overall positive charge under the synthesis conditions, phosphate buffer pH 7.4. HRP loading in HRP-Cu(PO) complexes was enhanced by 4.2-fold when synthesis was carried out at 37 °C in comparison with synthesis at 4 °C. HRP loading was further enhanced with synthesis at pH 8.0, correlating with a decrease in overall enzyme charge. Proteins with lower predicted pI values and negative overall charge (AdhD, pI of 5.6; diaphorase, pI of 6.8; GOx, pI of 5.2; catalase, pI of 6.9; and, BSA, pI of 5.7) exhibited higher enzyme loadings with 4 °C synthesis, 2.7-, 2.6-, 2.5-, 1.8-, and 1.7-fold protein loading enhancements, respectively. Using HRP as a model system, we also demonstrate that activity increased concomitantly with enzyme loading, and that particle nanostructure was minimally affected by synthesis temperature. Combined, the results presented here demonstrate the control of enzyme loading in enzyme-inorganic particles opening up new possibilities in enzyme and multienzyme catalysis.

Recent Advances in Enzyme-Nanostructure Biocatalysts with Enhanced Activity

“…carbon dots, carbon nanotubes) [40][41][42][43][44], and complex compounds (e.g. Cu3(PO4)2•3H2O, Ca3(PO4)2, Co3(PO4)2•8H2O, Mn3(PO4)2, Ca8H2(PO4)6, Cu4(OH)6)SO4, CaHPO4, Zn3(PO4)2, Mg-Al layered double hydroxide, CdSe/ZnS quantum dots) [17,[45][46][47][48][49][50][51][52][53][54][55][56][57][58][59]. These representative supports, which possess unique chemical and physical properties, such as controllable release of ion activator and synergic catalysts and response to external stimuli, are able to regulate the enzyme-support interaction and eventually lead to an unprecedented enhancement in immobilized enzyme activity [7,60,61].…”

“…The nanobiocatalysts highlighted here are expected to provide an insight into enzyme-nanostructure interaction, and provide a guideline for future design of high-efficiency nanobiocatalysts in both fundamental research and practical applications.Catalysts 2020, 10, 338 2 of 15 nanoscale supports, such as nanoparticles, nanowires, microspheres, metal-organic frameworks, and nanoflowers, has also drawn extensive attention in recent years [5,[16][17][18][19][20][21][22]. A great deal of effort has also been made to design nanostructured supports with a variety of components including noble metal (e.g., Au) [23,24], metal oxides (e.g., Cu 2 O, Fe 3 O 4 , SiO 2 , Ti 8 O 15 , alumina) [16,25-33], polymer (e.g., Cu 2+ /PAA/PPEGA matrix, aldehyde-derived Pluronic polymer, polycaprolactone) [34-36], metal-organic frameworks (e.g., zeolitic imidazolate framework) [37-39], carbon based (e.g., carbon dots, carbon nanotubes) [40-44], and complex compounds (e.g., Cu 3 (PO 4 ) 2 ·3H 2 O, Ca 3 (PO 4 ) 2 , Co 3 (PO 4 ) 2 ·8H 2 O, Mn 3 (PO 4 ) 2 , Ca 8 H 2 (PO 4 ) 6 , Cu 4 (OH) 6 )SO 4 , CaHPO 4 , Zn 3 (PO 4 ) 2 , Mg-Al layered double hydroxide, CdSe/ZnS quantum dots) [17,[45][46][47][48][49][50][51][52][53][54][55][56][57][58][59]. These representative supports, which possess unique chemical and physical properties, such as controllable release of ion activator and synergic catalysts and response to external stimuli, are able to regulate the enzyme-support interaction and eventually lead to an unprecedented enhancement in immobilized enzyme activity [7,60,61].Overall, recent years have witnessed great success in the interactions between artificial nanostructured supports and natural enzymes for enhanced activity.…”

Framework Nucleic Acid-Enabled Programming of Electrochemical Catalytic Properties of Artificial Enzymes

“…Molecular self-assembly is an elegant and powerful approach to pattern matter at the atomic scale, and there have been extensive studies on the development of self-assembling biomaterials. − DNA is a self-assembling biopolymer that is directed by canonical Watson–Crick base pairing to form predictable, double helical secondary structures, which enables it to be one of the most promising biomolecules for the construction of complex biomolecular networks. − The use of double helical DNA molecules for nanoscale engineering pursuit began with Seeman’s construction of artificial branched DNA tiles, where four rationally designed oligomeric nucleic acid strands self-assembled into an immobile four-way junction . Since then, structural DNA nanotechnology has enjoyed a rapid progress, and numerous complex nanostructures and different fabrication techniques have been introduced. − The nanoscale addressability of DNA nanostructures makes it an intriguing approach for developing artificial enzyme complexes. To date, numerous examples of organizing chemical reactions with programmability by taking advantages of DNA nanotechnology have been reported. − We have previously reported a rectangular DNA origami platform to anchor glucose oxidase (GOx) and horseradish peroxidase (HRP) .…”

Surface-Functionalized Mesoporous Nanoparticles as Heterogeneous Supports To Transfer Bifunctional Catalysts into Organic Solvents for Tandem Catalysis

“…1 In particular, the use of heterogeneous supports simplifies biocatalyst recycling and promotes continuous process development. [2][3] Today, any type of industrial enzymes can be immobilized onto a "solid" carrier whose properties can be further tuned by chemical or physical manipulation. In many examples, hydrogels [4][5] and synthetic resins [6][7][8] are used as enzyme hosts for improving catalyst stability and usability at ambient/elevated pressures and temperatures.…”

“…The fabrication of heterogeneous biocatalysts, through entrapping or immobilizing enzymes/whole cells onto organic and inorganic supports, offers the potential to revolutionize biocatalyst performance at the industrial process scale . In particular, the use of heterogeneous supports simplifies biocatalyst recycling and promotes continuous process development. , Today, any type of industrial enzymes can be immobilized onto a “solid” carrier whose properties can be further tuned by chemical or physical manipulation. In many examples, hydrogels , and synthetic resins − are used as enzyme hosts for improving catalyst stability and usability at ambient/elevated pressures and temperatures.…”