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Posted on Fri, Oct 05, 2018

Immobilized enzymes

 Enzymes are the catalysts for biochemical reactions. As such, they speed up the transition from reactants to products without being consumed in the process. Multiple enzymes can be found in every cell, from bacteria up through to humans. We as humans have found ways to exploit enzymes to produce food products, fuel, pharmaceutical products, biotechnological tools, sensors, and much more. The potential uses for enzymes are seemingly limitless. The creation of solid support structures with immobilized enzymes has improved our ability to reuse enzymes in a controlled manner for a variety of applications. Immobilized enzymes can be reused multiple times before their efficacy is lost. This allows them to be an affordable part of industrial processes.

Enzymes of interest

Some common enzymes used in industry include lipase, polyphenol oxidases, lignin peroxidase, horseradish peroxidase, amylase, nitrite reductase, and urease. Many of these enzymes are used for biosensors because of the fairly specific affinity between a substrate and its enzyme. Others, such as horseradish peroxidase, are used for chemical detection of biomarkers in tissue. Enzymes are used in the food, agricultural, and pharmaceutical industries to control and speed up reactions in order to quickly and accurately obtain a valuable final product. Enzymes are also increasingly being used in the production of biofuels. The enzymes can be harvested from microbial sources or can be made synthetically. No matter the source, the ability to immobilize the enzymes onto support systems makes it easier to retrieve and reuse the enzymes for multiple reactions. Additionally, immobilized enzymes are oriented to improve performance and binding efficiency. In the case of magnetic solid support systems, the enzymes are easily recovered for reuse.

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Immobilized enzymes on a solid supports

The support material for the immobilized enzymes is as varied as the application. The advent of new polymeric materials and the advancement of synthesis methods has created a wealth of possible support systems. Perhaps the most traditional solid support system is a resin or matrix to which the enzymes are attached. Newer methods include attaching immobilized enzymes to microbeads, nanoparticles, and electrospun fibers. Commonly used materials include natural polymers, synthetic polymers, and inorganic materials.

  • Natural polymers: alginate, chitosan, chitin, collagen, carrageenan, gelatin, cellulose, starch, pectin, sepharose
  • Synthetic polymers: DEAE cellulose, polyethylene glycol, polyvinyl chloride, polyurethane, polyaniline
  • Inorganic materials: zeolites (microporous crystalline solids), ceramics, celite, silica, glass, activated carbon, charcoal

 

Methods of creating immobilized enzymes on solid support systems

Sometimes enzymes are more stable on a support because sites of degradation are hidden. Chemical cross-linking agents are often used to help maintain the stability of the attachments. There are four main methods of attaching enzymes to a support material. Each method exploits the chemical properties of the amino acid side chains that make up the enzyme:

  1. Adsorption: hydrophobic interactions and salt linkages. This is the least stable conjugation method, and is the most susceptible to changes in pH or salt concentration.

  2. Covalent binding: amino acid functional groups and peptide-modified surfaces are used to  control protein orientation. This is the most stable method of protein immobilization: 
    • NHS/EDC chemistry to combine carboxyl and amine functional groups
    • Amine-reactive chemistry such as glutaraldehyde and silanes
    • Click chemistry 

  3. Affinity immobilization: an affinity ligand serves as a linkage between the support and the enzym
    • Biotin/streptavidin
    • Histidine tags and columns with metal chelators- recombinant enzyme proteins can be created with a poly-histidine tag. This tag has an affinity for metal chelators such as Ni-, Co-, Cu-, and Fe-NTC (Nitrilotriacetic acid) or IDA (iminodiacetic acid). The affinity between histidine and the metal chelators is strong enough to withstand harsh pH and high salt conditions. 
  4. Entrapment: caging of enzymes by covalent or non-covalent bonds within gels or fibers
    • A dried or lyophilized enzyme is mixed with an epoxy or polymer resin prior to polymerization.
    • It is critical that an optimal ratio of enzyme to polymer is defined in order to preserve the active site of the enzyme

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