Coupling Peptide-Based Encapsulation of Enzymes with Bacteria for Paraoxon Bioremediation.

The catalytic efficiency of enzymes can be harnessed as an environmentally friendly solution for decontaminating various xenobiotics and toxins. However, for some xenobiotics, several enzymatic steps are needed to obtain nontoxic products. Another challenge is the low durability and stability of many native enzymes in their purified form. Herein, we coupled peptide-based encapsulation of bacterial phosphotriesterase with soil-originated bacteria, Arthrobacter sp. 4Hß as an efficient system capable of biodegradation of paraoxon, a neurotoxin pesticide. Specifically, recombinantly expressed and purified methyl parathion hydrolase (MPH), with high hydrolytic activity toward paraoxon, was encapsulated within peptide nanofibrils, resulting in increased shelf life and retaining ∼50% activity after 132 days since purification. Next, the addition of Arthrobacter sp. 4Hß, capable of degrading para-nitrophenol (PNP), the hydrolysis product of paraoxon, which is still toxic, resulted in nondetectable levels of PNP. These results present an efficient one-pot system that can be further developed as an environmentally friendly solution, coupling purified enzymes and native bacteria, for pesticide bioremediation. We further suggest that this system can be tailored for different xenobiotics by encapsulating the rate-limiting key enzymes followed by their combination with environmental bacteria that can use the enzymatic step products for full degradation without the need to engineer synthetic bacteria.

Trypsin Encapsulation in the Zeolitic Imidazolate Framework for Low-Molecular Weight Protein Analysis with High Selectivity and Efficiency.

Low-molecular weight proteins (LWPs) are important sources of biological information in biomarkers, signaling molecules, and pathology. However, the separation and analysis of LWPs in complex biological samples are challenging, mainly due to their low abundance and the complex sample pretreatment procedure. Herein, trypsin modified by poly(acrylic acid) (PAA) was encapsulated by a zeolitic imidazolate framework (ZIF-L). Mesopores were formed on the ZIF-L with the introduction of PAA. An alternative strategy for separation and pretreatment of LWPs was developed based on the prepared ZIF-L-encapsulated trypsin with adjustable pore size. The mesoporous structure of the prepared materials selectively excluded high-molecular weight proteins from the reaction system, allowing LWPs to enter the pores and react with the internal trypsin, resulting in an improved separation efficiency. The hydrophobicity of the ZIF-L simplified the digestion process by inducing significant structural changes in substrate proteins. In addition, the enzymatic activity was significantly enhanced by the developed encapsulation method that maintained the enzyme conformation, allowed low mass transfer resistance, and possessed a high enzyme-to-substrate ratio. As a result, the ZIF-L-encapsulated trypsin can achieve highly selective separation, valid denaturation, and efficient digestion of LWPs in a short time by simply mixing with substrate proteins, greatly simplifying the separation and pretreatment process of the traditional hydrolysis method. The prepared materials and the developed strategy demonstrated an excellent size-selective assay performance in model protein mixtures, showing great potential in the application of proteomics analysis.

Enzyme encapsulation in metal-organic frameworks using spray drying for enhanced stability and controlled release: A case study of phytase.

Encapsulating enzymes in metal-organic frameworks is a common practice to improve enzyme stability against harsh conditions. However, the synthesis of enzyme@MOFs has been primarily limited to small-scale laboratory settings, hampering their industrial applications. Spray drying is a scalable and cost-effective technology, which has been frequently used in industry for large-scale productions. Despite these advantages, its potential for encapsulating enzymes in MOFs remains largely unexplored, due to challenges such as nozzle clogging from MOF particle formation, utilization of toxic organic solvents, controlled release of encapsulated enzymes, and high temperatures that could compromise enzyme activity. Herein, we present a novel approach for preparing phytase@MIL-88 A using solvent-free spray drying. This involves atomizing two MOF precursor solutions separately using a three-fluid nozzle, with enzyme release controlled by manipulating defects within the MOFs. The physicochemical properties of the spray dried particles are characterized using X-ray diffraction, Fourier-transform infrared spectroscopy, and scanning electron microscopy. Leveraging the efficiency and scalability of spray drying in industrial production, this scalable encapsulation technique holds considerable promise for broad industrial applications.

Encapsulation of Enzymes into Hydrophilic and Biocompatible Metal Azolate Framework: Improved Functions of Biocatalyst in Cascade Reactions and its Sensing Applications.

Multiple enzyme-triggered cascade biocatalytic reactions are vital in vivo or vitro, considering the basic biofunction preservation in living organisms and signals transduction for biosensing platforms. Encapsulation of such enzymes into carrier endows a sheltering effect and can boost catalytic performance, although the selection and preparation of an appropriate carrier is still a concern. Herein, focusing on MAF-7, a category of metal azolate framework (MAF) with superiority against the topologically identical ZIF-8, this enzyme@MAF system can ameliorate the sustainability of encapsulating natural enzymes into carriers. The proposed biocatalyst composite AChE@ChOx@MAF-7/hemin is constructed via one-pot in situ coprecipitation method. Subsequently, MAF-7 is demonstrated to exhibit an excellent capacity of the carrier and protection against external factors in the counterpart of ZIF-8 through encapsulated and free enzymes. In addition, detections for specific substrates or inhibitors with favorable sensitivity are accomplished, indicating that the properties above expectation of different aspects of the established platform are successfully realized. This biofunctional composite based on MAF-7 can definitely provide a potential approach for optimization of cascade reaction and enzyme encapsulation.

Logic-gated tumor-microenvironment nanoamplifier enables targeted delivery of CRISPR/Cas9 for multimodal cancer therapy.

Recent innovations in nanomaterials inspire abundant novel tumor-targeting CRISPR-based gene therapies. However, the therapeutic efficiency of traditional targeted nanotherapeutic strategies is limited by that the biomarkers vary in a spatiotemporal-dependent manner with tumor progression. Here, we propose a self-amplifying logic-gated gene editing strategy for gene/H2O2-mediated/starvation multimodal cancer therapy. In this approach, a hypoxia-degradable covalent-organic framework (COF) is synthesized to coat a-ZIF-8 in which glucose oxidase (GOx) and CRISPR system are packaged. To intensify intracellular redox dyshomeostasis, DNAzymes which can cleave catalase mRNA are loaded as well. When the nanosystem gets into the tumor, the weakly acidic and hypoxic microenvironment degrades the ZIF-8@COF to activate GOx, which amplifies intracellular H+ and hypoxia, accelerating the nanocarrier degradation to guarantee available CRISPR plasmid and GOx release in target cells. These tandem reactions deplete glucose and oxygen, leading to logic-gated-triggered gene editing as well as synergistic gene/H2O2-mediated/starvation therapy. Overall, this approach highlights the biocomputing-based CRISPR delivery and underscores the great potential of precise cancer therapy.