What is Enzyme Immobilization?
Fundamentally, enzyme immobilization is a way to improve the economics of biocatalysis. The ability to recycle an enzyme, especially an expensively produced orlowabundance enzyme, results in a more efficient process while simultaneously reducing the need to perform extra postreaction enzymeremoval steps.
In chemical terms, enzyme immobilization is the attachment of an enzyme to a solid support. This effectively converts the biocatalyst from a homogeneous catalyst to a heterogeneous catalyst
There are a lot of different ways of accomplishing this. Enzymes can be immobilized on solid supports ranging from gels to plastics and ceramics. They can be immobilized using virtually any intermolecular interaction. Hydrophobic polymers have been used to stick hydrophobic proteins in place and ionic materials have beenused to hold charged proteins in place
Advantages of Enzyme Immobilization
ImmobilizationImmobilizing an enzyme can have interesting effects on its behaviour. It can lead to increased selectivity, stability or activity. Associating an enzyme with a solid support surrounds it with a new microenvironment which can restrict its movements and offer protection against harsh conditions.
Immobilization also eliminates the enzyme’s ability to aggregate, increasing the amount of enzyme which can be loaded into a reaction. For this reason, the potentialproductivity of biocatalytic processes that use immobilized enzymes is often higher than those that use enzymes free in solution.
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When enzymes are immobilized, a continuousflow format can be used which enables substrate to be continuously added to the system while the product is simultaneously removed. This allows processes to run more efficiently, using lower reaction volumes and generating less waste.
By anchoring the enzyme to a solid support, the enzyme can be reused rather than discarded during product isolation. Reusing an enzyme improves the efficiency of a biocatalytic process, saving production time and money.
The immobilization of the enzyme to the carrier also means that the product will be enzymefree, which simplifies product purification. However, depending on the method by which the enzyme has been immobilized, it is possible for the enzyme to leach into the product over time.
Lastly, enzymes can be coimmobilized meaning that different types of enzymes can be immobilized closely together on the same support. Their proximity improves the efficiency of multistep reactions. This lends an artificial structure to the enzyme arrangement, mimicking enzyme cascades commonly found in living systems and facilitating more complex chemical transformations in a smaller space.
Disadvantages of Traditional Enzyme Immobilization Methods
From an industry perspective, there are perceived limitations associated with the cost of preparing an enzyme for immobilization. Traditionally, the enzyme of interest must be isolated before immobilization because contaminants will compete for the surface of the solid support. The recovery of an enzyme from cell debris and the separation of the active enzyme from other proteins is a costly process, requiring time, equipment, reagents, and laboratory space. Using EziG technology, purificationand immobilization happen simultaneously, so that you can immobilize your enzyme directly out of cellfree extract or culture supernatant.
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Attaching an enzyme to many solid supports can also reduce its catalytic efficiency or completely eliminate its activity altogether. The attachment site between the enzyme and the solid support can hinder access to the catalytic pocket of the enzyme. It can also physically restrain the enzyme and reduce its ability to accommodate changes in its structure that occur during catalysis, reducing its efficiency. EziG avoids this problem by using a flexible and welldefined site of attachment between the enzyme and the solid support surface.
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Attachment to most solid supports also introduces mass transfer limitations. When the enzyme is immobilized on the surface of the carrier, the diffusion of the substratefrom the bulk phase into the microenvironment surrounding the enzyme and the diffusion of the product out of this microenvironment limit reaction rates. Porous solidsupports can slow this process down further because the substrate and product molecules both need to diffuse into and out of the pores of the solid support. The high mass transfer efficiency of EziG leads to high process efficiency.
Lastly, the use of a solid support necessitates the use of a clean reaction mixture. The presence of high molecular weight materials or other solid matter in the reaction can foul the solid support, reducing access of the enzyme to its substrate and possibly causing other process failures. This means that starting materials always need to be filtered. However, should anything get through, the EziG platform makes it easy to recover your enzyme. The robust nature of the EziG solid support allows it to be rigorously cleaned so that it can be reused again and again.
Methods of Enzyme Immobilization
Until the development of EziG (see below), there was no general method of immobilization that works for all enzymes. Each immobilization method that has beenestablished is an attempt to find a reliable and affordable technique that immobilizes the enzyme while retaining its catalytic activity. Immobilization methods differ from one another based on the properties of the material used for the solid support, the characteristics of the enzyme, and the conditions under which the immobilization process is conducted.
The method used to immobilize an enzyme will have some impact on the activity of the enzyme and given that each enzyme has unique characteristics, finding the right method for a particular process can be challenging. There are several general methods by which enzymes are immobilized:
- Adsorption
- Covalent Bonding
- Entrapment/encapsulation
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Ever since the first investigations of invertase activity on charcoal and alumina by Nelson and Griffin over a century ago, adsorption has been a goto technique for immobilizing enzymes. Adsorption takes advantage of the unique surface chemistry of an enzyme to immobilize it. For example, enzymes can have hydrophobic patcheson their surface. When a hydrophobic material like charcoal or acrylic is mixed into a solution containing the enzyme, a significant amount of the enzyme will bind to its surface. The effect of this interaction can be hard to predict. Some enzymes are stabilised, others are destabilised. Many enzymes lose activity and some gain activity. The relative ease of adsorption and the low cost of adsorbents makes this method attractive, but it suffers from many drawbacks including enzyme leaching, activity loss, and low enzyme loading.
To prevent enzyme leaching and increase enzyme loading, many biochemists have turned to covalent bonding. Protein surfaces are chemically complex, displaying lots of different functional groups. One, the primary amine of lysine, has been used to linkenzymes to solid supports more than any other thanks to its robust and versatile chemistry. A wide variety of chemical reactions taking advantage of surface lysine residues have been employed in enzyme immobilization, including the wellknown NHS ester coupling, carbodiimide coupling, reductive amination, and epoxide ring opening. Unfortunately, the inherent reactivities of the solid supports needed to prepare these formulations often make them a challenge to work with and the added cost of synthesizing the chemicallyactivated solid support is not trivial.
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Precipitation, a wellknown method of enzyme purification. Crosslinking reagents, such as glutaraldehyde, can be added to an enzyme solution to create porous beadsmade entirely out of the enzyme. This creates a stable and highly concentrated form of the enzyme, but often significantly reduces enzyme activity because randomly crosslinking enzymes can limit substrate diffusion and damage or block their catalytic sites.
Preventing activity loss is a big challenge in enzyme immobilization. Reduced enzyme stability and activity is often attributed to unfavourable interactions with the solid support. Entrapment or encapsulation is generally regarded as a gentler immobilization technique since the enzyme is simply trapped in a tiny pocket, not adhered to a surface. Trapped enzymes are prepared by synthesizing a porous matrix in a solution containing an enzyme. The matrix is then washed to remove excess enzyme, leaving behind an immobilized catalyst. This method works well witha wide range of enzymes but the small pores that prevent enzyme escape limit the diffusion of substrate and product to and from the enzyme, slowing the enzymedriven process down.
Overall, it is hard to predict how an enzyme will respond to different immobilization methods. Often the outcome is highly materialdependent. This has led biotechnologists to develop a wide range of materials for enzyme immobilization.
