Carnegie Mellon U. Develops Microgel To Recover Enzymes For Manufacturing, Research Assays

"By enabling efficient enzyme recovery, this microgel system overcomes significant obstacles in using natural enzymes in laboratory and industrial settings," said Bruce Armitage, associate professor of chemistry at Carnegie Mellon, who developed the recoverable enzymatic microgel in collaboration with chemistry professor Gary Patterson, and graduate students Rong Cao and Zhenyu Gu.

Enzymes are routinely used in manufacturing to catalyze important reactions, such as the breakdown of sugars to create lactose-free milk or cheese products for the dairy industry. In many cases, industrial enzymes are embedded in a solid, synthetic matrix to easily separate them from their chemical product after a reaction takes place. But embedded enzymes may eventually leach from a matrix. The chemical cross-linking reaction used to attach the enzymes to the matrix also can inadvertently inactivate the enzymes, rendering them useless. What's more, large chemicals cannot enter a dense matrix to react with embedded enzymes.

The microgel developed by Armitage and Patterson bypasses these limitations. The enzyme is tightly connected to the microgel matrix, but remains fully functional. Molecules can diffuse into the porous microgel and undergo chemical reaction when they encounter an enzyme. The product can then diffuse out of the microgel. Separation of the product from the enzyme takes advantage of the fact that the microgel particles precipitate from solution at a low temperature. After the product has been removed, the microgel particles can be re-suspended by adding fresh water and heating the solution.

"The complete recoverability of the enzymatic activity is encouraging, and we are excited about extending this concept to other enzymes," noted Armitage. In addition to the food and dairy industries, enzymes are used in a wide variety of applications, including DNA testing in forensic labs, clinical tests for diagnosing diseases and synthesis of new pharmaceutical agents.

Armitage and Patterson relied on biomolecular recognition to create their microgel particles. For each particle, single DNA strands were used to create a three-way junction (TWJ) that looks like spokes of a wheel. To the end of each spoke, they tethered a strand of peptide nucleic acid (PNA), a synthetic material that recognizes and binds to DNA. Finally, enzyme complexes were then attached irreversibly to the tips of up to four PNA strands in a way that led to cross-linking of different TWJs, resulting in the microgel. At room temperature, individual microgel particles are suspended in solution, but lowering the temperature causes the microgel particles to cluster together and precipitate from solution.

To test these enzymatic microgel particles, the Carnegie Mellon chemists used an analogue of lactose that changes color when broken down by the microgel enzyme. When the investigators added this substrate to the microgel, a yellow color instantly appeared, indicating the reaction's success. Once the reaction was complete, the temperature was lowered, causing the microgel particles to precipitate. The product remained in solution and was separated simply by pouring the liquid into a separate container. The microgel particles could be subjected to several cycles of reaction, precipitation and reconstitution with no loss of activity, meaning that the enzyme was not leaching from the microgel, nor was it becoming inactivated.

The project demonstrates how effectively chemistry can bridge the biological and materials sciences, according to Armitage. "This is becoming a dominant theme in interdisciplinary research, particularly relating to nanotechnology. Our microgel is just one more example of Carnegie Mellon's leadership in this important area."

This research was funded by Carnegie Mellon University, 3M Corporation and the National Science Foundation. It was reported in the January 28 issue of the Journal of the American Chemical Society.