Introduction
Enzymes, as macromolecular biocatalysts, play a crucial role in accelerating biochemical reactions with high specificity and efficiency. Over recent years, enzymes have found extensive applications in various fields, including industrial bioprocesses, environmental remediation, biofuel production, pharmaceuticals, and diagnostics. However, the widespread use of free enzymes faces challenges such as poor stability, high cost, and difficulty in reuse. To overcome these limitations, various methods have been explored to enhance the activity and stability of enzymes for different applications.
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Immobilization is a key technique that has been employed to improve enzyme characteristics. The surfaces used in immobilization, whether macro or micro, play a significant role in enabling the use of enzymes in non-native applications, such as environmental remediation, biosensors, bioreactors, and other applied biotechnology fields. However, the interaction of enzymes with material surfaces can impact their activity and specificity. The immobilization process may lead to changes in enzyme properties, with some enzymes experiencing enhancement, while others may lose activity or undergo structural distortion.
Nanoparticles (NPs) have emerged as promising carriers for enzyme immobilization, aiming to address the challenges associated with traditional macro/micro carriers. Nanomaterials, with lengths scales below 100 nm, exhibit unique characteristics, including high surface area to volume ratios, making them suitable for various biotechnological applications.
Cholesterol Oxidase
Cholesterol oxidases (ChOxs) are a monomeric FAD-dependent oxido-reductase primarily found in bacteria. It catalyzes steroid substrates with a hydroxyl group at the 3-β position of the steroid ring. Cholesterol oxidase has commercial significance in determining cholesterol levels in various samples, acting as a catalyst in the bioconversion of sterol compounds and even exhibiting insecticidal properties.
Structure of Cholesterol Oxidase
Cholesterol oxidase can be broadly classified into two classes based on the binding of the cofactor FAD: Class-I with non-covalently bound FAD and Class-II with covalently bound FAD. The enzyme's general structure includes a substrate-binding domain and a FAD-binding domain, with the protein chain meandering between these regions. The substrate-binding domain typically consists of an eight-stranded mixed beta-pleated sheet and six alpha-helices. The active site contains a hydrophobic pocket sealed off from the outer environment by flexible loops.
Fig. 1 Structural views of different forms of ChOx (Vrielink A., Ghisla S. 2009).
Biological Functions of Cholesterol Oxidase
Cholesterol oxidase plays diverse roles in bacterial metabolism. In cholesterol-assimilating bacteria, it initiates the conversion of cholesterol to 4-cholesten-3-one. Some bacteria, like Mycobacterium and Rhodococcus equi, use cholesterol oxidase to damage host cell membranes, contributing to their pathogenicity. Additionally, cholesterol oxidase has applications in the production of pharmaceutical compounds and insecticidal proteins.
Applications of Cholesterol Oxidase
The enzyme's diagnostic applications include determining cholesterol levels in serum, HDL, LDL, and various clinical samples. Cholesterol oxidase is also employed in the bioconversion of cholesterol into bile acids and the production of pharmaceutically important products. Its insecticidal properties make it valuable in combating pests like the boll weevil. Moreover, cholesterol oxidase provides a potential target for bacterial infections, making it a crucial component in combating pathogenic bacteria.
Immobilization of Enzymes
Immobilization Methods
Immobilization methods can be categorized into physical and chemical methods. Physical methods involve weak interactions between the enzyme and the matrix, while chemical methods form covalent bonds between them. Common physical methods include adsorption, while chemical methods involve covalent coupling, entrapment, and cross-linking.
Adsorption relies on multipoint protein adsorption through ionic and hydrogen bonding, van der Waals forces, and hydrophobic interactions. Covalent coupling forms strong bonds between the surface amino acids of the enzyme and the matrix. Entrapment involves placing the enzyme within a polymer matrix, and cross-linking forms bonds between enzymes to create carrier-free macroparticles. Each method has its advantages and disadvantages, influencing factors such as enzyme recovery percentage, selectivity, operational stability, and reduction in inhibition.
Choice of Support Material for Immobilization
Selecting the right support material is crucial for enzyme immobilization. Support materials should possess characteristics such as large surface area, suitable shape and size, rigidity, hydrophilicity, permeability, insolubility, and stability. Organic and inorganic support materials can be classified based on morphology (porous and nonporous) and chemical nature. Nanomaterials, owing to their ideal size, have become popular matrices for enzyme immobilization. They offer advantages such as low cost, rapid reaction, high loading, and operational stability.
Immobilization of Cholesterol Oxidase
Cholesterol oxidase from bacterial sources has been immobilized using various carriers to enhance enzymatic properties, reusability, and operational stability.
Immobilization on Macro/Micro Carriers
Various macro/microcarriers have been employed to immobilize cholesterol oxidase, aiming to enhance its catalytic properties. For example, chitosan beads have been utilized to immobilize cholesterol oxidase from Rhodococcus sp. NCIM 2891, resulting in increased stability in organic solvents and reusability for multiple cycles. Co-immobilization of cholesterol oxidase with horseradish peroxidase and cholesterol esterase on Tetraethyl Orthosilicate (TEOS)-derived sol-gel films demonstrated high thermal stability and sensitivity for cholesterol detection.
N-ethyl-N'-3-dimethylaminopropyl carbodiimide-activated sepharose and conducting polymers like thiophene-3-boronic acid have also been used as matrices for cholesterol oxidase immobilization. Furthermore, cholesterol oxidase has been conjugated into various polymers, including electrochemically polymerized polyaniline and poly(acrylamide-co-acrylic acid)/polyethyleneimine supports.
Immobilization on Magnetic Nanoparticles
While reports on the immobilization of cholesterol oxidase on magnetic nanoparticles are limited, studies employing co-precipitation of magnetic Fe3O4 nanoparticles have shown promise. These nanoconjugates exhibited increased thermal and pH stability and were reusable for multiple cycles.
Immobilization on Metal Nanoparticles
Gold nanoparticles and other metal nanoparticles have been extensively used for cholesterol oxidase immobilization, particularly in biosensor applications. Gold electrodes modified with cholesterol oxidase nanoparticles have demonstrated enhanced sensitivity and detection limits. Moreover, novel biosensors incorporating molybdenum disulfide and gold nanoparticles have shown improved stability and sensitivity in cholesterol detection.
Immobilization on Other Nanoparticles
Cholesterol oxidase has been immobilized on various novel nanomaterials, including Mg2AlCO3 hydrotalcite layered double hydroxide nanomaterials and silica nanoparticles. These nanoconjugates exhibited improved stability and sensitivity and showed potential for biosensing applications.
Applications of Immobilized Cholesterol Oxidase
The immobilization of cholesterol oxidase has expanded its applications in both industrial and medical fields. Immobilized forms of cholesterol oxidase have been widely used in biosensors for detecting cholesterol in food samples, blood serum, and other clinical samples. The enhanced stability, reusability, and specificity of immobilized cholesterol oxidase make it superior to traditional detection methods.
Furthermore, the immobilized enzyme has been employed in the bioconversion of cholesterol into valuable products. Cholesterol oxidase immobilized on chitosan beads has demonstrated superior stability and reusability for catalyzing the bioconversion of cholesterol.
Conclusion
In conclusion, the immobilization of enzymes, particularly cholesterol oxidase, offers a versatile and effective strategy to enhance enzyme performance for diverse industrial and clinical purposes. Nanomaterials have emerged as promising matrices for enzyme immobilization, providing numerous advantages such as increased surface area, stability, and functionality. The applications of immobilized cholesterol oxidase range from biosensors for cholesterol detection to bioconversion processes. Continued research in this field is essential to address challenges and unlock the full potential of immobilized enzymes, contributing to advancements in biotechnology and healthcare.
References
- Vrielink A., Ghisla S. Cholesterol oxidase: biochemistry and structural features. The FEBS Journal. 2009, 276(23): 6826-6843.
- Ghosh S., et al. Immobilization of cholesterol oxidase: an overview. The Open Biotechnology Journal. 2018, 12(1).
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