Advancements in technology have led to the development of novel methods for enzyme immobilization, including the use of inorganic-protein hybrid systems that are known as nanoflowers (NFs) because their morphology resembles that of flowers. In an established mechanism, NF synthesis occurs in phosphate-buffered saline through three steps: (i) nucleation via interaction of metal ions with a nitrogen group available in the protein, (ii) followed by aggregation, and (iii) anisotropic growth. Metals such as cobalt, copper, manganese, and zinc have been widely demonstrated for the immobilization of enzymes as NFs. These systems combine the unique properties of inorganic materials with the catalytic activity of enzymes, resulting in enhanced enzyme stability and effectiveness. Moreover, these inorganic-protein hybrids have been utilized for enzyme immobilization, which allows enzymes to be securely attached to a solid support. 

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1. Trametes versicolor Laccase-Based Magnetic Inorganic-Protein Hybrid Nanobiocatalyst for Efficient Decolorization of Dyes in the Presence of Inhibitors. Materials 2024, 17 (8), 1790.

2. Coriolus versicolor laccase-based inorganic protein hybrid synthesis for application in biomass saccharification to enhance biological production of hydrogen and ethanol. Enzyme and Microbial Technology 2024, 170, 110301.

3. Influence of metal ions on the immobilization of β-glucosidase through protein-inorganic hybrids. Indian Journal of Microbiology 2019, 59, 370-374.

The biological production of methanol from CH4 was found to be more effective than thermochemical processes due to its high efficiency and selectivity, as well as ambient reaction conditions. Methanotrophs are potential candidates for the use CH4 efficiently for the production of methanol by utilizing methane monooxygenase enzymes. Further, immobilized whole cells have been demonstrated to improve the properties of methanotrophs for oxidation of CH4.

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1. Encapsulation of Methanotrophs within a polymeric matrix containing copper-and Iron-based nanoparticles to enhance methanol production from a simulated Biogas. Polymer 2023, 15 (18) 3667.

2. Synthetic design of methanotroph co-cultures and their immobilization within polymers containing magnetic nanoparticles to enhance methanol production from wheat straw-based biogas. Bioresource Technology 2022, 364, 128032.

3. Integrating anaerobic digestion of potato peels to methanol production by methanotrophs immobilized on banana leaves. Bioresource Technology 2021, 323, 124550.

4. Conversion of biogas to methanol by methanotrophs immobilized on chemically modified chitosan. Bioresource Technology 2020, 315, 123791.

5. Biomethanol production from methane by immobilized co-cultures of methanotrophs. Indian Journal of Microbiology 2020, 60, 318-324.

6. Hierarchical macroporous particles for efficient whole-cell immobilization: application in bioconversion of greenhouse gases to methanol. ACS Applied Materials & Interfaces 2019, 11, 18968-18977.

Production of biofuels such as bioethanol and bio-hydrogen using lignocellulosic biomass has been hypothesized to be a very suitable alternative to fossil fuel while contributing to biowaste management. Generally, the lignocellulosic biomass produced from agriculture has been recognized as a cheap source of feed to produce bioethanol due to its high availability worldwide. In most bioprocesses, the feed accounts for more than 40% of the cost of production. The use of biowaste as a source of glucose to efficiently stimulate H2 production is an obvious alternative to reduce costs. Bacteria have been shown to completely degrade biowaste through multiple steps under anaerobic conditions. In the case of bioethanol, enzymatic saccharification of biomass is one of the most expensive steps in cellulosic bioethanol production because of the high cost of production of the enzyme. There are several methods to overcome this problem, such as enzyme immobilization and whole-cell immobilization.

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1. Coriolus versicolor laccase-based inorganic protein hybrid synthesis for application in biomass saccharification to enhance biological production of hydrogen and ethanol. Enzyme and Microbial Technology 2023, 170, 110301.

2. Continuous biohydrogen production from poplar biomass hydrolysate by a defined bacterial mixture immobilized on lignocellulosic materials under non-sterile conditions. Journal of Cleaner Production 2021, 287, 125037.

3. Enhanced saccharification and fermentation of rice straw by reducing the concentration of phenolic compounds using an immobilized enzyme cocktail. Biotechnology Journal 2019, 14(6), 1800468.

4. Bioprocessing of biodiesel industry effluent by immobilized bacteria to produce value-added products. Applied biochemistry and biotechnology 2018, 185, 179-190.

Immobilization strategies have been widely studied to improve enzyme loading, residual activity, and stability to overcome the significant limitations of stability. Various enzymatic immobilization procedures have been demonstrated, including (i) encapsulation, (ii) adsorption on supports, (iii) covalent immobilization, (iv) cross-linking using linkers such as glutaraldehyde, or combinations of these methods, such as covalent immobilization followed by cross-linking. Each method has its advantages and disadvantages for the immobilization of certain enzymes. The key benefits of enzyme immobilization methods include (i) easy biocatalyst separation; (ii) abatement of downstream processing; (iii) superior recycling of biocatalysts; (iv) improved stability, relative to higher temperatures, pH, or solvents; and (v) feasibility for use with other enzymes via co-immobilization. Significant disadvantages include (i) lower activity and reaction rates compared to free enzymes, (ii) surplus cost of immobilization supports, (iii) fouling, and (iv) disposal of exhausted immobilized biocatalysts through incineration or environmental issues due to material toxicity. 

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1. Novel cofactor regeneration-based magnetic metal-organic framework for cascade enzymatic conversion of biomass-derived bioethanol to acetoin. Bioresour. Technol. 2024, 408, 131175.

2. Sequential Co-Immobilization of Enzymes on Magnetic Nanoparticles for Efficient l-Xylulose Production. International Journal of Molecular Sciences 2024, 25 (5), 2746.

3. Recent strategies for the immobilization of therapeutic enzymes. Polymers 2022, 14 (7), 1409.

4. Rhus vernicifera Laccase Immobilization on Magnetic Nanoparticles to Improve Stability and its Potential Application in Bisphenol A Degradation. Indian Journal of Microbiology 2021, 61, 45-54.

5. Enhanced saccharification and fermentation of rice straw by reducing the concentration of phenolic compounds using an immobilized enzyme cocktail. Biotechnology Journal 2019, 14(6), 1800468.