EziG: plug-and-play enzyme immobilization

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Get a quick overview of the EziG technology

Immobilization should be effortless

Historically speaking, biocatalysis has been difficult. Not only do you need to engineer an enzyme with high activity, but you also need to figure out how to purify that enzyme and use it in a cost-effective manner. This has limited the use of enzymes as catalysts.

Imagine that things were different: that a good enzyme was good enough; that all you needed to do was find an enzyme with reasonable activity and then immobilization was practically effortless; that the liquid handling aspects of purification were easy; that you could readily perform biocatalysis in flow without washing away your precious enzymes.

With EnginZyme’s universal immobilization matrix EziG, this is now reality.

With just a simple His-tag, EziG (pronounced “Easy G”) can bind nearly any enzyme with almost no loss in activity. You are no longer stuck with the undesirable choice of either not immobilizing at all or going through a long, tedious project to find a way to immobilize without losing significant activity.

EziG facilitates the rapid development of cost-effective enzymatic solutions by enabling immobilization without requiring extensive optimization. That means you save time and money on development costs. It means that binding will always be a simple, standardized, and robust procedure. EziG is universal. It just works. Not only does this always make immobilization an option – it makes it the default option.

General and reliable

Works with all enzyme types through the use of tags. The same material, any enzyme, all relevant conditions.

Easy to use

No tedious optimization required, any His-tagged enzyme will bind in active form through a simple and fast procedure.

High capacity

Large surface area and effective enzyme binding ensures high surface coverage — up to 30% active enzyme by weight.

High activity

Gentle and non-destructive binding through His-tags, on a porous carrier with minimal mass transfer limitations. Immobilized can be as active as dissolved!

Activity in organic media

EziG is the ideal support for transforming your biocatalysis process to a highly loaded efficient system without downstream processing issues.

Ideal for flow chemistry

A non-swelling and robust material with excellent fluid properties, filled with active enzyme. Get ready to pack your column.

"We have found EziG to be a very general and versatile platform for enzyme immobilisation and currently use it as the preferred option for all of our biocatalysts"

Prof. Nicholas J. Turner, Manchester Institute of Biotechnology

“The application of biocatalysts is our number 1 technology for chiral synthesis. EnginZyme’s technology has been a very powerful tool for this purpose”

Prof. Tom Moody, VP Technology Development & Commercialisation, Almac

“I have been really impressed with EnginZyme’s expertise”

Dr. Keith Mulholland, Associate Principal Scientist (Biocatalysis), AstraZeneca

Enzyme Immobilization In Depth

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 or low-abundance enzyme, results in a more efficient process while simultaneously reducing the need to perform extra post-reaction enzyme-removal 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 been used to hold charged proteins in place.

Advantages of
Enzyme Immobilization

Immobilizing 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 micro-environment 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 potential productivity of biocatalytic processes that use immobilized enzymes is often higher than those that use enzymes free in solution.

When enzymes are immobilized, a continuous-flow 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 enzyme-free, 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 co-immobilized meaning that different types of enzymes can be immobilized closely together on the same support. Their proximity improves the efficiency of multi-step 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, purification and immobilization happen simultaneously, so that you can immobilize your enzyme directly out of cell-free extract or culture supernatant.

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 well-defined site of attachment between the enzyme and the solid support surface. 

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 substrate from the bulk phase into the microenvironment surrounding the enzyme and the diffusion of the product out of this microenvironment limit reaction rates. Porous solid supports 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 been established 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:

  1. Adsorption
  2. Covalent Bonding
  3. Cross-linking
  4. Entrapment/encapsulation

Ever since the first investigations of invertase activity on charcoal and alumina by Nelson and Griffin over a century ago, adsorption has been a go-to technique for immobilizing enzymes. Adsorption takes advantage of the unique surface chemistry of an enzyme to immobilize it. For example, enzymes can have hydrophobic patches on 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 link enzymes 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 well-known 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 chemically-activated solid support is not trivial.

One innovative way of getting around this problem is to make the enzyme into its own solid support. Many enzymes can be aggregated into active gel beads under the right conditions, allowing their isolation. This is the basis of ammonium sulfate precipitation, a well-known method of enzyme purification. Cross-linking reagents, such as glutaraldehyde, can be added to an enzyme solution to create porous beads made entirely out of the enzyme. This creates a stable and highly concentrated form of the enzyme, but often significantly reduces enzyme activity because randomly cross-linking 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 with a 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 enzyme-driven process down.

Overall, it is hard to predict how an enzyme will respond to different immobilization methods. Often the outcome is highly material-dependent. This has led biotechnologists to develop a wide range of materials for enzyme immobilization.

Solid Supports for Enzyme Immobilization

Enzyme immobilization for industrial-scale biocatalysis requires a good support material. The support should have high enzyme binding capacity. It must be both mechanically and chemically stable to remain in use for long periods of time under challenging conditions. The support should also provide the highest level of enzyme activity per gram of enzyme immobilized. The search for the right support is often challenging, requiring experimentation with reaction conditions, enzyme loading, enzyme selectivity, preparation stability, process productivity, and overall cost.

Supports can be made from a bewildering array of materials ranging from biopolymers to inorganic materials, polymer resins, or any combination of these. Historically, materials have been developed to suit an enzyme or process of interest.

However, the industry needs to get away from this approach as it adds another time-consuming step to the development cycle of a new biocatalyst. A more general support is needed.

There are some general principles which can be applied to solid support design. As mass transfer and diffusion limitations are often issues for immobilized enzymes, the support should have a large surface area, with a well-defined particle size, and a well-connected network of pores. The support must be both physically and chemically stable. The support should bind the enzyme tightly in a well-defined way which does not interfere with activity. And the support should be made from cost-effective materials that are readily available. Is it possible to develop a generic support with these properties which suits most processes?

Applications of Enzyme Immobilization

The largest scale use of immobilized enzymes today is in the production of high-fructose corn syrup. Following the digestion of the thick starch slurry into glucose with inexpensive amylase enzyme, the high-cost glucose isomerase enzyme is used to convert the glucose into fructose, making the syrup sweeter. To make this process economical, the glucose isomerase is immobilized so that the reaction can be run in a more economical flow reactor and the isomerase can be reused between batches. Today, inexpensive glucose isomerase cross-linked with albumin is the catalyst of choice for the production of high-fructose corn syrup.

Lipases are heavily used industrial enzymes. They catalyze the breakdown of lipids or other ester-containing molecules. Lipases have been prepared as immobilized enzyme formulations commercially for many years and are now often used as a test-case for enzyme immobilization platforms.

One of the most widely used lipases is called Candida antarctica lipase B (CalB). It has been used as an immobilized enzyme in diverse industries, including in pharmaceutical production, the food industry, biodiesel fuel production, and in the synthesis of specialty esters used in cosmetics.  

The best-known lipase formulation is Novozym 435. Released in 1995, Novozym 435 is Candida antarctica lipase B (CAL-B) adsorbed onto a low-cost porous poly(methacrylic acid) support crosslinked with divinylbenzene. This formulation is particularly remarkable for the high activity of CAL-B on this support, for its low cost, and for its relatively high enzyme loading (8.5-20%). However, this support has only been used commercially for CAL-B and reliance on hydrophobic adsorption for enzyme binding means that it suffers from enzyme leaching, preventing long-term use and contaminating the product with trace enzymatic activity. The availability of a better performing support would better retain the enzyme, allowing longer reuse.

EziG: A Next-Generation Enzyme Immobilization Platform

The EziG platform was designed to be easy to use and broadly applicable while maintaining the maximum amount of enzyme activity. It harnesses the common poly-histidine tag engineered into countless enzymes. Its affinity-based adsorption method can specifically immobilize tagged enzymes from a crude mixture. Combining immobilization and purification steps saves time and reduces enzyme preparation costs. All of the materials used are non-toxic and the high affinity of the binding interaction prevents enzyme leaching.

EziG particles are made from coated controlled porosity glass particles specifically prepared to maximise surface area for high enzyme loading while allowing rapid mass transport to drive chemical processes as quickly as possible.

This leads to faster processing and higher yields with the same amount of enzyme in a smaller volume. EnginLipe, a CalB preparation on EziG, displays 10-fold higher activity per gram than Novozym 435.

The EziG platform brings reliability to the immobilization process, allowing it to be incorporated into the plan for a new biocatalytic process without having to worry about how the enzyme will tolerate immobilization. New, improved enzymes can be swapped into the process with a simple strip and reload step, allowing for more agile development of continuous biocatalytic processes.


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