Biocatalytic Applications Research Articles

PH- and Redox-Responsive Pickering Emulsions Based on Cellulose Nanocrystal Surfactants.

Nanoparticle surfactants (NPSs), formed by using dynamic interactions between nanoparticles and complementary ligands at the liquid-liquid interface, have emerged as "smart emulsifiers" with attributes of high emulsification efficiency, long-term stability, and on-demand emulsification/demulsification capabilities. However, only pH-responsiveness can be adopted for the assembly of reported NPSs formed by electrostatic interactions. Here, we propose an alternative design strategy, by taking advantage of the ferrocenium (Fc+ ) sulfate ion pair, to develop a new type of cellulose nanocrystal (CNC) surfactant. The Fc+ groups are sensitive to pH, redox reagents and voltage, imparting the CNC surfactants and derived Pickering emulsions with multi-stimuli-responsiveness, and showing promising applications in controllable delivery, release, and biphasic biocatalysis.

PH‐ and Redox‐Responsive Pickering Emulsions Based on Cellulose Nanocrystal Surfactants

AbstractNanoparticle surfactants (NPSs), formed by using dynamic interactions between nanoparticles and complementary ligands at the liquid‐liquid interface, have emerged as “smart emulsifiers” with attributes of high emulsification efficiency, long‐term stability, and on‐demand emulsification/demulsification capabilities. However, only pH‐responsiveness can be adopted for the assembly of reported NPSs formed by electrostatic interactions. Here, we propose an alternative design strategy, by taking advantage of the ferrocenium (Fc+) sulfate ion pair, to develop a new type of cellulose nanocrystal (CNC) surfactant. The Fc+ groups are sensitive to pH, redox reagents and voltage, imparting the CNC surfactants and derived Pickering emulsions with multi‐stimuli‐responsiveness, and showing promising applications in controllable delivery, release, and biphasic biocatalysis.

Self-sufficient biocatalysts constructed using chitin-based microspheres

The concomitant recycling of enzymes and co-factors is a big challenge in biocatalysis. In this study, co-immobilization of the enzyme and the co-factor pyridoxal 5′-phosphate was achieved using chitin-based microspheres as the support. Chitin/chitosan composite microspheres were prepared and then modified with polyethyleneimine. Three pyridoxal 5′-phosphate-independent enzymes were used to demonstrate that the self-sufficient biocatalysts based on chitin/chitosan composite microspheres and polyethyleneimine modified microspheres displayed good generalizability for high recycling efficiency of both enzyme and pyridoxal 5′-phosphate. Furthermore, the self-sufficient biocatalysts were applied in the continuous flow production of cadaverine and maintained good continuous catalysis without the exogenous addition of pyridoxal 5′-phosphate, suggesting that this co-immobilization platform shows promise for application in industrial biocatalysis.

MOF-Immobilized Two-in-One Engineered Enzymes Enhancing Activity of Biocatalytic Cascade for Tumor Therapy.

Biocatalytic systems based on enzyme cascade reactions have attracted growing interest in the field of biocatalytic medicine. However, it is a major challenge to reasonably construct enzyme cascade reactions with high stability, selectivity, and catalytic efficiency for the in vivo biocatalytic application. Herein, two-in-one engineered glucose oxidase (GOx-Fe0 ) isfabricated by a biomineralization strategy, through which a nanozyme (Fe0 NP) is anchored within the inner cavity of GOx. Then, GOx-Fe0 is immobilized in a pH-sensitive metal-organic framework (MOF) zeolitic imidazolate framework-8 (ZIF-8) to establish a stable and effective MOF-immobilized two-in-one engineered enzyme, GOx-Fe0 @ZIF-8. In vitro studies show that GOx-Fe0 @ZIF-8 exhibits excellent stability and high pH/glucose selectivity, and the shorter spacing between cascade enzymes can increase the cascade throughput and effectively improve the reaction efficiency of the enzyme cascade. In vivo experiments exhibit that GOx-Fe0 @ZIF-8 solves the instability and systemic toxicity of free enzymes, and achieves deep tumor penetration and significant chemodynamic therapeutic efficacy through a pH/glucose-selective enzyme cascade reaction in tumor site. Taken together, such an orchestrated enzyme engineering strategy can effectively improve enzyme stability, selectivity, and enzyme cascade reaction efficiency via chemical transformations, and also provide a promising strategy for the application of biocatalytic cascade reactions in vivo.

Enzymatic Conversion of CO2: From Natural to Artificial Utilization.

Enzymatic carbon dioxide fixation is one of the most important metabolic reactions as it allows the capture of inorganic carbon from the atmosphere and its conversion into organic biomass. However, due to the often unfavorable thermodynamics and the difficulties associated with the utilization of CO2, a gaseous substrate that is found in comparatively low concentrations in the atmosphere, such reactions remain challenging for biotechnological applications. Nature has tackled these problems by evolution of dedicated CO2-fixing enzymes, i.e., carboxylases, and embedding them in complex metabolic pathways. Biotechnology employs such carboxylating and decarboxylating enzymes for the carboxylation of aromatic and aliphatic substrates either by embedding them into more complex reaction cascades or by shifting the reaction equilibrium via reaction engineering. This review aims to provide an overview of natural CO2-fixing enzymes and their mechanistic similarities. We also discuss biocatalytic applications of carboxylases and decarboxylases for the synthesis of valuable products and provide a separate summary of strategies to improve the efficiency of such processes. We briefly summarize natural CO2 fixation pathways, provide a roadmap for the design and implementation of artificial carbon fixation pathways, and highlight examples of biocatalytic cascades involving carboxylases. Additionally, we suggest that biochemical utilization of reduced CO2 derivates, such as formate or methanol, represents a suitable alternative to direct use of CO2 and provide several examples. Our discussion closes with a techno-economic perspective on enzymatic CO2 fixation and its potential to reduce CO2 emissions.

Template-Directed Incorporation of Functional Molecules into DNA.

We report a versatile method for the incorporation of functional molecules into oligonucleotides carrying reactive groups by using a template-directed postsynthetic approach in the solution phase. For this purpose, we prepared oligonucleotides carrying an amino group on the backbone by using an acylic threoninol scaffold. The resulting oligonucleotides could be used to introduce almost any molecule carrying aldehyde, which can be, among other things, a metal-binding ligand or a fluorophore. In our study, we incorporated aldehyde-bearing phenanthroline, a metal-binding ligand, into oligonucleotides by template-directed reversible conjugation. We observed that the use of an abasic sugar site instead of a natural nucleobase in the template strand increased the yield of conjugation and induced selective incorporation of the phenanthroline. This method could lead progress in the development of probes for the recognition of abasic regions in duplex DNA. Moreover, template-directed formation of metal ligand-oligonucleotide conjugates might have potential applications in hybrid biocatalysis for enantioselective transformations.

Moving towards the Application of Biocatalysis in Food Waste Biorefinery

Waste valorization is an important strategy to reduce environmental pollution and dependency on petroleum-based fuels. In this regard, utilization of food waste as a versatile and low-cost resource is important. Several advanced catalytic methods for the valorization of food waste have been widely investigated for the production of liquid biofuels. Along this line, chemical catalysts have been explored for the synthesis of liquid biofuels. Chemo-catalysis is mainly metal based, which requires harsh process conditions. Alternatively, biocatalysts are currently being investigated as a result of several advantages such as mild reaction conditions, recyclability, selectivity and biodegradability. In this work, recent biocatalytic technologies for the preparation of liquid biofuels through food waste valorization are discussed thoroughly. Lipases are employed for the synthesis of biodiesel and the upgradation of bio-oil, whereas methane mono-oxygenases could be explored for the production of methanol via the oxidation of methane generated from food wastes. Industrial production of ethanol from food waste using bioconversion technologies is a success story. To date, there has been no specific report on the use of food waste for propanol preparation using enzymes. The ABE process (Acetone–Butanol–Ethanol) (using suitable microorganisms) is used for butanol preparation, where the vacuum stripping system is integrated to remove butanol from the broth and circumvent inhibition. The synthesis of hydrocarbon fuels from fatty acids and triglycerides can be carried out using enzymes, such as carboxylic acid reductase and fatty acid photodecarboxylase (an algal photoenzyme). Both carboxylic acid reductase and fatty acid photodecarboxylase have not yet been applied in the direct valorization of food wastes. Furthermore, limitations of the reported methods, societal and economic aspects and a fresh perspective on the subject, along with important examples, are described.

Design, Production, and Characterization of Catalytically Active Inclusion Bodies.

Catalytically active inclusion bodies (CatIBs) are promising biologically produced enzyme/protein immobilizates for application in biocatalysis, synthetic chemistry, and biomedicine. CatIB formation is commonly induced by fusion of suitable aggregation-inducing tags to a given target protein. Heterologous production of the fusion protein in turn yields CatIBs. This chapter presents the methodology needed to design, produce, and characterize CatIBs.

Hydrophilic metal-chelated membrane for biocatalytic membrane reactor application

Hydrophilic metal-chelated membrane for biocatalytic membrane reactor application

Synthesis of Enzyme-based Organic-Inorganic Hybrid Nanoflower Particles

Enzyme-incorporated hybrid nanostructures are the immobilization of enzymes and inorganic components that exhibits promising characteristics in various industries. The immobilization of enzymes onto nanomaterial is naturally based to accommodate the enzymatic activity, stability, recyclability as well as their catalytic functions. The designing of these conjugates can improve the overall enzymatic performance by imparting their novel properties onto the system in comparison to conventional free enzymes which experience drawbacks in terms of deactivation or denaturing. A facile and ultrafast method is described in this paper to synthesize a novel enzyme-incorporated lipase/Cu 3 (PO 4 ) 2 hybrid nanoflower (NF). The physical properties of the hybrid NF allow easier retrieval which indicates its higher reusability and recyclability value. The enzyme loading capacity was found to be 95.1% whereas, the catalytic performance of lipase/Cu 3 (PO 4 ) 2 hybrid NF at the optimal conditions resulted in a specific enzyme activity of 1752 U/g corresponding to an increment of 90.5% to that of free lipase. This indicates that the well-designed lipase/Cu 3 (PO 4 ) 2 hybrid NF to be highly efficient in industrial biocatalytic applications. Meanwhile, in future work, we aim to study its operational stability and reusability to enzymatically degrade biopolymers through hydrolysis process.

IrO2 clusters loaded on dendritic mesoporous silica nanospheres with superior peroxidase-like activity for sensitive detection of acetylcholinesterase and its inhibitors

Nanomaterials-based enzyme mimics (nanozymes), by simulating enzyme catalysis, have shown potential in numerous biocatalytic applications, but nanozymes face significant challenges of catalytic activity and reusability that may restrict their practical uses. Herein, we report facile fabrication of surface-clean IrO2 clusters supported on dendritic mesoporous silica nanospheres (DMSNs), which exhibit superior peroxidase-like activity, high thermal/long-term stability, and good recyclability. The IrO2 clusters (1.4 ± 0.2 nm in size) are obtained by the laser ablation without any ligands and possess negative surface charge, which are efficiently loaded on the amino-functionalized DMSNs by electrostatic adsorption. Owing to morphological and structural advantages, the resulted DMSN/IrO2 heterostructure displays outstanding peroxidase-like catalytic performance. Compared with horseradish peroxidase, it shows comparable affinities but higher reaction rate (2.95 × 10−7 M·s−1) towards H2O2, resulting from rapid electron transfer during the catalysis. This value is also larger than those of mesoporous silicas supported metal or metal oxides nanoparticles/clusters in the previous studies. Benefitting from excellent peroxidase-catalysis of the DMSN/IrO2, the colorimetric assays are further successfully established for the detection of acetylcholine esterase and its inhibitor, showing high sensitivity and selectivity. The work provides novel design of supported nanozymes for biosensing.

Biochemical characterization, substrate and stereoselectivity of an outer surface putative α/β hydrolase from the pathogenic Leptospira

The genome of pathogenic leptospira encodes a plethora of outer surface and secretory proteins. The outer surface or secreted α/β hydrolases in a few pathogenic organisms are crucial virulent factors. They hydrolyze host immune factors and pathogen's immune-activating ligands, which help pathogens to evade the host's innate immunity. In this study, we report biochemical characterizations, substrate and stereoselectivity of one of the leptospiral outer surface putative α/β hydrolases, IQB77_09235 (LABH). Purified LABH displayed better kinetic parameters towards small water-soluble esters such as p-nitrophenyl acetate and p-nitrophenyl butyrate. The LABH exhibited moderate thermostability and displayed a pH optimum of 8.5. Remarkably, a phylogenetic study suggested that LABH does not cluster with other characterized bacterial esterases or lipases. Protein structural modeling revealed that some structural features are closely associated with Staphylococcus hycus lipase (SAH), a triacylglycerol hydrolase. The hydrolytic activity of the protein was found to be inhibited by a lipase inhibitor, orlistat. Biocatalytic application of the protein in the kinetic resolution of racemic 1-phenylethyl acetate reveals excellent enantioselectivity (E > 500) in the production of (R)-1-phenylethanol, a valuable chiral synthon in several industries. To our knowledge, this is the first detailed characterization of outer surface α/β hydrolases from leptospiral spp.

Solid Migration to Assemble a Flower-like Nanozyme with Highly Dense Single Copper Sites for Specific Phenol Oxidation

Nanozymes with high catalytic stability and sustainability have emerged as powerful competitors to natural enzymes for diverse biocatalytic applications. However, constructing a nanozyme with high specificity is one of their biggest challenges. Herein, we develop a facile solid migration strategy to access a flower-like single copper site nanozyme (Cu SSN) via direct transformation of copper foam activated by 2-methylimidazole. With highly clustered CuN3 sites whose local structure is similar to that of natural polyphenol oxidase, the Cu SSN exhibits excellent activity and specificity to oxidize phenols without peroxidase-like activity. Furthermore, the Cu SSN shows high sensitivity in the colorimetric detection of epinephrine with a low detection limit of 0.10 μg mL-1, exceeding that of most previously reported enzyme-mimicking catalysts. This work not only provides a simple method for the large-scale preparation of high-performance nanozymes but also offers an inspiration for the design of highly specific nanozymes by mimicking the synergy among sites in natural enzymes.

Flavofun: Exploration of fungal flavoproteomes

Fungi produce a plethora of natural products exhibiting a fascinating diversity of chemical structures with an enormous potential for medical applications. Despite the importance of understanding the scope of natural products and their biosynthetic pathways, a systematic analysis of the involved enzymes has not been undertaken. In our previous studies, we examined the flavoprotein encoding gene pool in archaea, eubacteria, the yeast Saccharomyces cerevisiae, Arabidopsis thaliana, and Homo sapiens. In the present survey, we have selected the model fungus Neurospora crassa as a starting point to investigate the flavoproteomes in the fungal kingdom. Our analysis showed that N. crassa harbors 201 flavoprotein-encoding genes amounting to 2% of the total protein-encoding genome. The majority of these flavoproteins (133) could be assigned to primary metabolism, termed the “core flavoproteome”, with the remainder of flavoproteins (68) serving in, as yet unidentified, reactions. The latter group of “accessory flavoproteins” is dominated by monooxygenases, berberine bridge enzyme-like enzymes, and glucose-methanol-choline-oxidoreductases. Although the exact biochemical role of most of these enzymes remains undetermined, we propose that they are involved in activities closely associated with fungi, such as the degradation of lignocellulose, the biosynthesis of natural products, and the detoxification of harmful compounds in the environment. Based on this assumption, we have analyzed the accessory flavoproteomes in the fungal kingdom using the MycoCosm database. This revealed large differences among fungal divisions, with Ascomycota, Basidiomycota, and Mucoromycota featuring the highest average number of genes encoding accessory flavoproteins. Moreover, a more detailed analysis showed a massive accumulation of accessory flavoproteins in Sordariomycetes, Agaricomycetes, and Glomeromycotina. In our view, this indicates that these fungal classes are proliferative producers of natural products and also interesting sources for flavoproteins with potentially useful catalytic properties in biocatalytic applications.

A Double Hemin Bonded G-Quadruplex Embedded in Metal-Organic Frameworks for Biomimetic Cascade Reaction.

Biocatalytic transformations in living cells, such as enzymatic cascades, function effectively in spatially confined microenvironments. However, mimicking enzyme catalytic cascade processes is challenging. Herein, we report a new dual-Hemin-G-quadruplex (dHemin-G4) DNAzyme with high catalytic activity over noncovalent G4/Hemin and monocovalent counterparts (G4-Hemin and Hemin-G4) by covalently linking hemin to both ends of an intramolecular G4. We use MAF-7, a hydrophilic metal-organic framework (MOF), as the protecting scaffold to integrate a biocatalytic cascade consisting of dHemin-G4 DNAzyme and glucose oxidase (GOx), by a simple and mild method with a single-step encapsulation of both enzymes. Such a MAF-7-confined cascade system shows superior activity over not only traditional G4/Hemin but also other MOFs (ZIF-8 and ZIF-90), which was mainly attributed to high-payload enzyme packaging. Notably, the introduction of hydrophilic G4 allows to avoid the accumulation of hydrophobic hemin on the surface of MAF-7, which decreases cascade biocatalytic activity. Furthermore, MAF-7 as protective coatings endowed the enzyme with excellent recyclability and good operational stability in harsh environments, including elevated temperature, urea, protease, and organic solvents, extending its practical application in biocatalysis. In addition, the incorporated enzymes can be replaced on demand to broaden the scope of catalytic substrates. Taking advantages of these features, the feasibility of dHemin-G4/GOx@MAF-7 systems for biosensing was demonstrated. This study is conducive to devise efficient and stable enzyme catalytic cascades to facilitate applications in biosensing and industrial processes.

The structure of His-tagged Geobacillus stearothermophilus purine nucleoside phosphorylase reveals a `spanner in the works'.

The 1.72 Å resolution structure of purine nucleoside phosphorylase from Geobacillus stearothermophilus, a thermostable protein of potential interest for the biocatalytic synthesis of antiviral nucleoside compounds, is reported. The structure of the N-terminally His-tagged enzyme is a hexamer, as is typical of bacterial homologues, with a trimer-of-dimers arrangement. Unexpectedly, several residues of the recombinant tobacco etch virus protease (rTEV) cleavage site from the N-terminal tag are located in the active site of the neighbouring subunit in the dimer. Key to this interaction is a tyrosine residue, which sits where the nucleoside ring of the substrate would normally be located. Tag binding appears to be driven by a combination of enthalpic, entropic and proximity effects, which convey a particularly high affinity in the crystallized form. Attempts to cleave the tag in solution yielded only a small fraction of untagged protein, suggesting that the enzyme predominantly exists in the tag-bound form in solution, preventing rTEV from accessing the cleavage site. However, the tagged protein retained some activity in solution, suggesting that the tag does not completely block the active site, but may act as a competitive inhibitor. This serves as a warning that it is prudent to establish how affinity tags may affect protein structure and function, especially for industrial biocatalytic applications that rely on the efficiency and convenience of one-pot purifications and in cases where tag removal is difficult.

Organizing Enzymes on Self‐Assembled Protein Cages for Cascade Reactions

AbstractCells use self‐assembled biomaterials such as lipid membranes or proteinaceous shells to coordinate thousands of reactions that simultaneously take place within crowded spaces. However, mimicking such spatial organization for synthetic applications in engineered systems remains a challenge, resulting in inferior catalytic efficiency. In this work, we show that protein cages as an ideal scaffold to organize enzymes to enhance cascade reactions both in vitro and in living cells. We demonstrate that not only enzyme‐enzyme distance but also the improved Km value contribute to the enhanced reaction rate of cascade reactions. Three sequential enzymes for lycopene biosynthesis have been co‐localized on the exterior of the engineered protein cages in Escherichia coli, leading to an 8.5‐fold increase of lycopene production by streamlining metabolic flux towards its biosynthesis. This versatile system offers a powerful tool to achieve enzyme spatial organization for broad applications in biocatalysis.

Organizing Enzymes on Self-Assembled Protein Cages for Cascade Reactions.

Cells use self-assembled biomaterials such as lipid membranes or proteinaceous shells to coordinate thousands of reactions that simultaneously take place within crowded spaces. However, mimicking such spatial organization for synthetic applications in engineered systems remains a challenge, resulting in inferior catalytic efficiency. In this work, we show that protein cages as an ideal scaffold to organize enzymes to enhance cascade reactions both in vitro and in living cells. We demonstrate that not only enzyme-enzyme distance but also the improved Km value contribute to the enhanced reaction rate of cascade reactions. Three sequential enzymes for lycopene biosynthesis have been co-localized on the exterior of the engineered protein cages in Escherichia coli, leading to an 8.5-fold increase of lycopene production by streamlining metabolic flux towards its biosynthesis. This versatile system offers a powerful tool to achieve enzyme spatial organization for broad applications in biocatalysis.

The Single-Component Flavin Reductase/Flavin-Dependent Halogenase AetF is a Versatile Catalyst for Selective Bromination and Iodination of Arenes and Olefins.

Flavin-dependent halogenases (FDHs) natively catalyze selective halogenation of electron rich aromatic and enolate groups. Nearly all FDHs reported to date require a separate flavin reductase to supply them with FADH2 , which complicates biocatalysis applications. In this study, we establish that the single component flavin reductase/flavin dependent halogenase AetF catalyzes halogenation of a diverse set of substrates using a commercially available glucose dehydrogenase to drive its halogenase activity. High site selectivity, activity on relatively unactivated substrates, and high enantioselectivity for atroposelective bromination and bromolactonization was demonstrated. Site-selective iodination and enantioselective cycloiodoetherification was also possible using AetF. The substrate and reaction scope of AetF suggest that it has the potential to greatly improve the utility of biocatalytic halogenation.

The Single‐Component Flavin Reductase/Flavin‐Dependent Halogenase AetF is a Versatile Catalyst for Selective Bromination and Iodination of Arenes and Olefins**

AbstractFlavin‐dependent halogenases (FDHs) natively catalyze selective halogenation of electron rich aromatic and enolate groups. Nearly all FDHs reported to date require a separate flavin reductase to supply them with FADH2, which complicates biocatalysis applications. In this study, we establish that the single component flavin reductase/flavin dependent halogenase AetF catalyzes halogenation of a diverse set of substrates using a commercially available glucose dehydrogenase to drive its halogenase activity. High site selectivity, activity on relatively unactivated substrates, and high enantioselectivity for atroposelective bromination and bromolactonization was demonstrated. Site‐selective iodination and enantioselective cycloiodoetherification was also possible using AetF. The substrate and reaction scope of AetF suggest that it has the potential to greatly improve the utility of biocatalytic halogenation.