Friday, November 13, 2015

Enzyme immobilization techniques

Different techniques used for immobilization


Enzyme adsorption results from hydrophobic interactions and salt linkages where either the support is bathed in enzyme for physical adsorption or the enzyme is dried on electrode surfaces. Adsorbed enzymes are shielded from aggregation, proteolysis and interaction with hydrophobic interfaces (Spahn and Minteer 2008). Researchers have used eco-friendly supports like coconut fibers having good water-holding capacity and high cation exchange property; microcrystalline cellulose with irreversible binding capacity; kaolin with high enzyme retainability by chemical acetylation; and micro/mesoporous materials having thiol functionalized, large surface area ideally suited for reduction and oxidation reactions (Dey et al. 2002; Hernández et al. 2007; Karagulyan et al. 2008; Brígida et al. 2010; Mitchell and Ramírez 2011; Huang et al. 2011). Silanized molecular sieves have also been successfully used as supports for enzyme adsorption owing to the presence of silanols on pore walls that facilitate enzyme immobilization by hydrogen bonding (Diaz and Balkus 1996). Various chemical modifications of the currently used supports would definitely help in better immobilization. Water activity profiles of lipase adsorbed using polypropylene-based hydrophobic granules/Accurel EP-100 has been reported (Persson et al. 2000). It would be important to note that Accurel with smaller particle sizes increases reaction rates and enantiomeric ratios during biocatalyzation (Sabbani et al. 2006).
For better process control and economic production, Yarrowia lipolytica lipase was immobilized on octyl-agarose and octadecyl-sepabeads supports by physical adsorption that resulted in higher yields and greater (tenfold) stability than that of free lipase. This was accounted by the hydrophobicity of octadecyl-sepabeads that enhances affinity between the enzyme and support (Cunha et al. 2008). Candida rugosa lipase adsorbed on biodegradable poly (3-hydroxybutyrate-co-hydroxyvalerate) showed 94 % residual activity after 4 h at 50 °C and reusability till 12 cycles (Cabrera-Padilla et al. 2011). These supports were preferred because they are less tough and crystalline than polyhydroxybutyrate. 1, 4-Butenediol diglycidyl ether-activated byssus threads have been suitable basement for urease that increased pH stability and retained 50 % enzyme activity under dried conditions (Mishra et al. 2011). Eco-friendly supports of biological origin not only prevent cropping up of ethical issues, but also cut down the production costs. Of late, biocompatible mesoporous silica nanoparticles (MSNs) supports have been used for biocatalysis in energy applications owing to their long-term durability and efficiency (Popat et al. 2011).


Covalent binding

Covalent association of enzymes to supports occurs owing to their side chain amino acids like arginine, aspartic acid, histidine and degree of reactivity based on different functional groups like imidazole, indolyl, phenolic hydroxyl, etc. (D’Souza 1998; Singh 2009). Peptide-modified surfaces when used for enzyme linkage results in higher specific activity and stability with controlled protein orientation (Fu et al. 2011). Cyanogen bromide (CNBr)-agarose and CNBr-activated-Sepharose containing carbohydrate moiety and glutaraldehyde as a spacer arm have imparted thermal stability to covalently bound enzymes (Hsieh et al. 2000; Cunha et al. 2008). Highly stable and hyperactive biocatalysts have been reported by covalent binding of enzymes to silica gel carriers modified by silanization with elimination of unreacted aldehyde groups and to SBA-15 supports containing cage-like pores lined by Si–F moieties (Lee et al. 2006; Szymańska et al. 2009). Increase in half-life and thermal stability of enzymes has been achieved by covalent coupling with different supports like mesoporous silica, chitosan, etc. (Hsieh et al. 2000; Ispas et al. 2009). Cross-linking of enzymes to electrospun nanofibers has shown greater residual activity due to increased surface area and porosity. Use of such nanodiametric supports have brought a turning point in the field of biocatalyst immobilization (Wu et al. 2005; Kim et al. 2006; Ren et al. 2006; Li et al. 2007; Huang et al. 2008; Sakai et al. 2010). Covalent binding of alcohol dehydrogenase on attapulgite nanofibers (hydrated magnesium silicate) has been opted owing to its thermal endurance and variable nano sizes (Zhao et al. 2010). Biocatalytic membranes have been useful in unraveling effective covalent interactions with silicon-coated enzymes (Hilal et al. 2006). Cross-linked enzyme aggregates produced by precipitation of enzyme from aqueous solution by addition of organic solvents or ionic polymers have been reported (Sheldon 2011). Different orientations of immobilized enzyme on magnetic nanoclusters obtained by covalent binding have found their applications in pharmaceutical industries owing to their enhanced longevity, operational stability and reusability (Yusdy et al. 2009). Maintaining the structural and functional property of enzymes during immobilization is one of the major roles played by a cross-linking agent. One such agent is glutaraldehyde, popularly used as bifunctional cross-linker, because they are soluble in aqueous solvents and can form stable inter- and intra-subunit covalent bonds.


Affinity immobilization

Affinity immobilization exploits specificity of enzyme to its support under different physiological conditions. It is achieved by two ways: either the matrix is precoupled to an affinity ligand for target enzyme or the enzyme is conjugated to an entity that develops affinity toward the matrix (Sardar et al. 2000). Affinity adsorbents have also been used for simultaneous purification of enzymes (Ho et al. 2004). Complex affinity supports like alkali stable chitosan-coated porous silica beads and agarose-linked multilayered concanavalin A harbor higher amounts of enzymes which lead to increased stability and efficiency (Shi et al. 2003; Sardar and Gupta 2005). Bioaffinity layering is an improvisation of this technique that exponentially increases enzyme-binding capacity and reusability due to the presence of non-covalent forces such as coulombic, hydrogen bonding, van der Waals forces, etc. (Sardar and Gupta 2005; Haider and Husain 2008).



Entrapment is caging of enzymes by covalent or non-covalent bonds within gels or fibers (Singh 2009). Efficient encapsulation has been achieved with alginate–gelatin–calcium hybrid carriers that prevented enzyme leakage and provided increased mechanical stability (Shen et al. 2011). Entrapment by nanostructured supports like electrospun nanofibers and pristine materials have revolutionalized the world of enzyme immobilization with their wide-ranging applications in the field of fine chemistry, biomedicine biosensors and biofuels (Dai and Xia 2006; Kim et al. 2006; Wang et al. 2009; Wen et al. 2011). Prevention of friability and leaching and augmentation of entrapment efficiency and enzyme activity by Candida rugosa lipase entrapped in chitosan have been reported. This support has also been reported to be non-toxic, biocompatible and amenable to chemical modification and highly affinitive to protein due to its hydrophilic nature (Betigeri and Neau 2002). Entrapment by mesoporous silica is attributed to its high surface area, uniform pore distribution, tunable pore size and high adsorption capacity (Ispas et al. 2009). Simultaneous entrapment of lipase and magnetite nanoparticles with biomimetic silica enhanced its activity in varying silane additives (Chen et al. 2011a). Sol–gel matrices with supramolecular calixarene polymers have been used for entrapment of C. rugosa lipase keeping in view their selective binding and carrying capacities (Erdemir and Yilmaz 2011). Lipases entrapped κ-carrageenan has been reported to be highly thermostable and organic solvent tolerant (Tümtürk et al. 2007; Jegannathan et al. 2010).


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