Five Crucial Variables to Control in Your Enzymatic Reactor

You’ve finally cracked it at the benchtop. Moving from a successful milliliter scale assay to a scalable manufacturing reactor is a massive, highly anticipated milestone for any biotech team. But as you scale, new challenges arise. Variables that you optimized perfectly for your lab scale equipment are no longer appropriate in a large vessel.

Enzymatic reactors come in a wide range of sizes and complexities. Some reactors are simple and only require substrate to be pumped in and product collected as it flows out. Other reactors require multiple inputs and vigilant monitoring of conditions as the reaction progresses. If you are experiencing unexplained drops in yield, rapid catalyst degradation, or contaminated batches during scale-up, the culprit is often environmental.

For most enzyme reactors there are five variables that are important to control to maximize yield during scale up. Mastering these will protect your biocatalyst and maximize your return on investment.

1. Temperature: The Engine of Reaction Kinetics

Temperature is a double-edged sword in biocatalysis. Finding the exact thermal sweet spot for your engineered enzyme is critical for balancing speed and stability.

• Reducing mass transfer limitations: Increasing the temperature can increase the rate of diffusion limited enzymatic reactions. This is because molecules in hotter solutions move with higher energy and bump into enzyme active sites more often.
• Keeping your enzymes in shape: Sometimes increasing the temperature can increase enzymatic activity by changing the conformation(s) of the enzyme’s active site. If your enzyme evolved in an organism that lives at a certain temperature, that temperature is often optimal.
• Preventing thermal burnout: If your enzyme is losing activity too quickly, decreasing the temperature can reduce thermal denaturation of enzyme molecules leading to increased stability and yield over time.
• Maintaining thermal baselines: Whatever temperature is selected for your enzymatic reactor, it needs to be maintained. So temperature probes and appropriate heating elements are important. Mixing can also be important to disperse heated liquid from the heating element to the rest of the reactor.

2. pH: Mastering the Chemical Microenvironment

Even small deviations in pH can stall a reaction. The pH inside an enzymatic reactor affects enzyme activity by changing the charge on molecular surfaces. In all cases, pH probes and monitors are important tools for maintaining the efficiency of the reactor.

• Protecting the catalytic engine: Controlling the pH is important sometimes because a proton is used as part of the enzyme’s catalytic mechanism. Even when no proton is directly involved in catalysis, pH changes alter the surface charge of the enzyme resulting in changes in conformation or substrate “steering” around the active site.
• Active process control: Each enzyme has evolved to work best within a certain pH range, and it is best to keep the reactor at a pH the enzyme prefers. This can be done by including a buffer in the reactor, or by active titration of base or acid while the reaction is in progress.
• The solid-state advantage: Sometimes, the enzyme immobilization support also acts as a solid state buffer by exchanging protons with the bulk solution.

3. Mixing: Delivering the Goods

You can have the perfect temperature and pH, but adding heat, acid / base, or even substrate for the enzyme does not do any good without mixing it into the reactor.

• Engineering the flow: It is important to engineer the enzymatic reactor with appropriate pumps / mixers to move liquid where it needs to go.
• Packed Bed: In packed bed reactors, mixing of the soluble components such as substrates, cofactors, and buffers is accomplished in a separate tank. Then the mixture is pumped through a bed of immobilized enzymes in a second tank that does not require further mixing. This is ideal if no adjustments to the reaction conditions are needed during the enzymatic transformation.
• Stirred Tank: If additional chemical ingredients need to be added during the course of the reaction, or if heat or oxygen needs to be dispersed then a stirred tank reactor is preferred.

4. Solvent: Balancing Solubility and Enzyme Integrity

Bridging the gap between organic chemistry and biological catalysts requires careful solvent selection.

• The solubility dilemma: Most enzymes operate best with water as a solvent. However, this may not be ideal for the manufacturing process. Sometimes substrates / products of the enzymatic reaction require organic solvents to aid in their solubility. In this case, a balance has to be struck between maintaining the structural integrity of enzymes, and dissolving the small molecule ingredients of the reaction.
• The role of immobilization: Immobilizing the enzymes on a solid surface can help because it creates a different localized environment around the enzyme. For example, an enzyme immobilized in a hydrophilic polymer can remain in a relatively aqueous environment while the bulk solution is organic solvent.

5. Microbial Growth Prevention: Protecting Your Batch

The harsh irony of bioprocessing is that the conditions that support enzymatic activity also support microbial life. The same warm temperatures, neutral pH, and aqueous environment preferred by most enzymes is also preferred by microbes. And enzymatic reactors have plenty of protein to feed microbes as well, so it is only a matter of time before these opportunists begin to colonize the process equipment.

• Thermal sterilization: Temperature can be used to prevent microbial growth if it is above 60°C. This high temperature can denature many enzymes, so it is helpful to use enzymes that have natural, or engineered, thermal stability.
• pH Shock: A pH below 4.6 can be used to prevent microbial growth. If this is unobtainable, even a pH below 5.5 can slow the growth of many microorganisms.
• Chemical barriers: Organic solvents, such as 20% ethanol, can be used to prevent microbial growth. Antibiotics can also be used to prevent microbial growth, however, these are often too expensive or disallowed by regulatory agencies in manufacturing processes.

Summary: Reactor Optimization at a Glance

Let Solidzymes Scale Your Biocatalyst

In summary, the five crucial variables for an enzymatic reactor are temperature, pH, mixing, solvent, and microbial growth prevention. Optimizing and controlling these will allow optimal usage of the enzymes and reagents in the biocatalytic process.

You don’t have to tackle the challenges of scale-up alone. Solidzymes offers enzyme reactor optimization services for companies interested in improving the efficiency of their enzymatic reactors before, during, or after scale-up.

References

1. Dimopoulos, A. Enzyme Bioreactors: Scaling Up for Industrial Biotechnology. Journal of Bioprocess Engineering, 2024, 10 (1).
2. Coloma, J., Guiavarc’h, Y., Hagedoorn, P.-L., & Hanefeld, U. (2021). Immobilisation and flow chemistry: tools for implementing biocatalysis. Chemical Communications, 57, 11416–11428.
3. Stepankova, V., Bidmanova, S., Koudelakova, T., Prokop, Z., Chaloupkova, R., & Damborsky, J. (2013). Strategies for Stabilization of Enzymes in Organic Solvents. ACS Catalysis, 3, 2823–2836.