October 1, 2008 (Vol. 28, No. 17)
Amberlite and Duolite Strive to Improve Catalysis Economics Through Reuse
Enzymes are proteins that catalyze biochemical reactions. In addition to performing biochemical reactions in living organisms, enzymes also function in vitro, which allows for the use of enzymes in industrial processes. Enzymes are used in a variety of large-scale industries including food, sugar, pharmaceuticals, and chemical processing. For industrial applications, biocatalytic processes based on enzymes have advantages over chemical processes.
Enzymes often occur under more mild (e.g., physiological) conditions and the number of processing steps using enzymes is often reduced while yielding faster reaction rates. Enzymes typically have highly specific stereo-, regio-, and chemo-selectivity, which leads to fewer by-products and higher purity. Lastly, processes using enzymes are often more environmentally friendly—with lower pressure, milder solvents, and reduced waste.
In addition, the range of reactions possible has been extended by extremophile bacteria that thrive in harsh environments such as hot springs.
While enzymes are not consumed during a biochemical reaction, they do have a finite lifetime. The ability of an enzyme to catalyze multiple cycles of a chemical reaction is important in the development of a cost-effective and efficient process. Assuming the enzyme can be separated from the reaction mixture, its reuse/lifetime potential is often limited by the loss of structural conformation. As the conformation of the enzyme degrades, its ability to catalyze reactions decreases, as it is primarily the 3-D structure that is critical to the specificity of the catalyzation.
A standard method for enabling the reuse of enzymes is to immobilize them onto solid supports that can easily be filtered from the reaction mixture. The immobilization of enzymes onto solid supports provides numerous advantages over their use in free solution, including: longer duration of activity (due to protection of structural features of the protein), multiple cycle reuse, and elimination of the need to remove the enzyme downstream. In addition, immobilizing enzymes on solid surfaces can work in either a stirred tank reactor or packed column system, allowing flexibility in scale-up.
Enzyme immobilization is certainly not a new technique. As early as 1916, it was shown that invertase could be immobilized on charcoal, and numerous techniques were demonstrated for immobilizing various enzymes on polymeric supports.
Immobilization techniques can be broadly classified as either noncovalent or covalent. Noncovalent attachment includes hydrophobic, ionic, and nonspecific binding. Covalent attachment is achieved by a chemical reaction between amino acids on the enzyme surface and the active functionality of the carrier. Rohm and Haas Amberlite™ and Duolite™ polymeric ion exchange and adsorbent resins have been used for the immobilization of a variety of different enzymes using a number of noncovalent and covalent techniques.
Enzymes can be immobilized onto solid media using nonspecific hydrophobic adsorption or ionic-bonding techniques. Noncovalent immobilization on Amberlite or Duolite resins is typically done using neutral, or near-neutral, pH conditions. The acrylic ester chemistry of the Amberlite XAD7HP resin and phenol-formaldehyde chemistries of Amberlite XAD761 and Duolite A7 resins provide gentle binding, which can lead to improved activity of the bound enzyme compared to other supports. Duolite A7 resin is particularly effective for the immobilization of b-glycosidases.
More hydrophobic chemistries such as the polystyrenic Amberlite XAD16 resin can be modified by grafting acrylate polymer on the adsorbent, creating a more hydrophilic surface for enzyme attachment. Nonmodified polystyrenic resins such as Amberlite XAD1180 can be used directly for enzyme adsorption in processes to produce enantiomerically pure compounds. With adsorption, enzyme leakage from the support is sometimes problematic. Gluteraldehyde, however, can be used for either preimmobilization activation of the resin or post-immobilization crosslinking of the immobilized enzyme improves binding stability.
Amberlite IRC50 resin (the precursor to Amberlite FPC3500 resin) is a cation exchange resin that has been used for ionic immobilization of enzymes such as fructozyme L, lipase, and phospholipase D. For b-galactosidase, Amberlite IRC50 is first reacted with polyethyleneimine and then reacted with gluteraldehyde. The gluteraldehyde acts as a crosslinker for the polyethyleneimine and provides a spacer arm for enzyme attachment.
An example of high-capacity binding of enzymes to nonmodified Amberlite IRC50 resin is shown in the Figure. Here, two different enzymes are bound to IRC50 using buffered conditions at a pH of 7.3 and contact times ranging from 4 to 12 hours. The data demonstrates that basic enzymes such as cytochrome C (pI of 9.6) and lysozyme (pI of 10.0) are well adsorbed on Amberlite IRC50 resin.
Another form of ionic attachment employs the use of anion exchange materials. Duolite A568 and Duolite A561 resins provide significant immobilization yields and protein loading, however, the immobilized enzyme specific activity can vary depending on the enzyme. Enzyme stability can improve with immobilization. For example, N-carbamyl-d-amino acid amidohydrolase demonstrates superior heat stability when immobilized on Duolite A568 resin.
Covalent enzyme attachment can be used to address some of the concerns about enzyme desorption in noncovalent systems. Modification of adsorbents such as Amberlite XAD7HP resin provides a method for covalent attachment. Aminoalkylation with 1,2-diaminoethane followed by gluteraldehyde activation of Amberlite XAD7HP resin allows covalent attachment of enzymes such as penicillin G acylase and glutaryl-7-ACA.
Activation of Amberlite XAD7HP resin using 2.5 M sodium hydroxide in 15% isopropanol followed by trichlorotriazine treatment provides a good support for immobilized pectinylase activity and stability. Another example of covalent immobilization is in the bioaffinity purification of biomolecules. The most common example is the purification of monoclonal antibodies using Protein A immobilized onto solid media.
With the increased emphasis on process economics and improved efficiency, the use of immobilized enzymes will continue to grow in the food, pharmaceutical, and chemical industries. It allows for the reduction of process steps, increases productivity, and decreases waste. The wide variety of immobilization supports provides suitable options for different enzymes, process systems, and cost requirements so that immobilization is an excellent process choice.
Jon R. Fisher (JonFisher@rohmhaas.com) is bioprocessing/healthcare technical service manager for the Americas at Rohm and Haas. Web: www.rohmhaas.com.