Biocatalysis in Polymer Science

Biocatalysis, which involves the use of enzymes, microbes, and higher organisms to carry out chemical reactions, is well established in the production of pharmaceuticals, food, agrochemicals, and fine chemicals. Its application in polymer science and technology, however, is a more recent development, according to Richard A. Gross, professor of chemical and biological sciences and engineering at Polytechnic University, Brooklyn, N.Y. Only during the past seven years or so has research interest in this area escalated, he says.

Biocatalysis in polymer science is a highly interdisciplinary area, says H. N. Cheng, senior research fellow at Hercules Inc. in Wilmington, Del. Researchers involved come from organic chemistry, polymer chemistry, chemical engineering, biochemistry, molecular biology, protein chemistry, enzymology, and industrial microbiology.

Much of the research in this area has focused on the use of enzymes for the synthesis of novel monomers and polymers, for the catalysis of polymer modification reactions, and for polymer degradation.

For example, Cheng, Richard J. Riehle, and colleagues at Hercules have used biocatalysis to modify and improve the properties of polymeric materials. One such material is Kymene G3-X, a water-soluble polymer manufactured by Hercules that is used to make paper products stronger when wet.

"The polymer is a polyaminopolyamide-epichlorohydrin (PAE)," Riehle notes. "PAE resins are the predominant commercial products used to manufacture wet-strength paper."

Polyaminopolyamide has an amide backbone with a secondary amine functionality that reacts with epichlorohydrin. After polymerization, Hercules uses a combined microbial/enzymatic treatment to remove undesirable chlorine-containing by-products, such as 1,3-dichloropropanol and 3-chloropropanediol, from the resin.

The microbial reaction is essentially a fermentation process where specific microorganisms are used to carry out the dehalogenation reactions. The by-products are converted to CO₂ and NaCl.

IN THE ENZYMATIC reaction, a protease enzyme is used to remove chlorine-containing end groups on the polymer. This reaction ensures that these end groups do not contribute to the formation of more chlorine-containing by-products.

"The use of this combined microbial/enzymatic process produces a material that contains less than 10 ppm of undesirable by-products," Riehle explains. "As such, it satisfies European regulatory requirements for its use in food-contact paper, such as tea bags, coffee filters, and milk and juice cartons."

Cheng's group at Hercules also is using enzymes to modify and improve the properties of polysaccharides such as pectins. Pectins occur in cell walls of plants and cement the cells together. They are soluble in water but insoluble in alcohol and other organic solvents. The compounds consist mainly of galacturonic acid sequences interrupted by rhamnopyranose residues. Many of the galacturonic acid residues are methoxylated.

" Pectin is commercially available and is used extensively as gelling agents, thickeners, stabilizers, and emulsifiers," Cheng notes. "The degree and sequence distribution of methoxylation, the distribution of rhamnopyranoses, and the molecular weight influence its gelation properties. For pectin with a high methoxy content, gelation can occur through the addition of an aqueous solution of sucrose and citric acid."

In 2000, the Hercules team patented an enzymatic process for modifying high-methoxy pectin with l-amino acids to produce pectins with improved gelling properties.

Although pectic enzymes are known in nature, the enzymes used for the Hercules process are nonpectic, according to Cheng. "We used papain obtained from papaya to work on pectin, which is not a natural substrate of the enzyme," he tells C&EN. "Papain is a protease and is normally used to hydrolyze proteins--for example, to tenderize meat. Papain was not previously known to react with pectin. We brought an unnatural pair together and used them in synthesis--for the coupling reaction between pectin and amino acids."

The researchers determined the strength, in terms of rheological properties, of pectins modified by a variety of amino acids. The strongest gel was formed by high-methoxy pectin modified with L-arginine.

The group suggests that the amino acid-modified pectin derivatives may potentially be useful in industry as gelling or thickening agents and also in cosmetic formulations and drug delivery systems.

Gregory F. Payne, professor of chemical engineering, and coworkers at the Center for Biosystems Research at the University of Maryland Biotechnology Institute are looking into the use of enzymes to create functionally useful biopolymer-based materials that would be difficult to obtain by alternative synthetic routes.

"Our specific target has been to generate protein-polysaccharide conjugates," he tells C&EN. "In nature, protein-polysaccharide conjugates carry out important chemical, mechanical, and biological functions. Examples are the mucins that perform protective and lubricating functions for organs, and the proteoglycans, which are among the largest and most complex structures in mammalian cells and perform various mechanical and biological functions."

IN RECENT WORK, the group used tyrosinase to prepare conjugates of chitosan (a linear aminopolysaccharide) and green fluorescent protein (GFP) [Langmuir, 19, 9382 (2003)]. Tyrosinase is a copper-containing oxidative enzyme found in plant and animal tissues that catalyzes the production of melanin and other pigments.

"We mix the protein with chitosan and tyrosinase, and the conjugation occurs over a few hours," Payne says. "We do not need protection and deprotection steps or reactive reagents, so the method can be performed in 'one pot' without the need for special precautions or expensive reagents. We expect this method could be generalized to many proteins."

The method requires that the protein have accessible tyrosine residues for oxidation by the tyrosinase. Oxidation yields activated quinone residues that react with nucleophilic substituents of the chitosan.

"If a native protein lacks accessible tyrosine residues, we exploit molecular biological methods to 'genetically fuse' a tyrosine-rich peptide sequence at one end of the protein," Payne continues. "Our results indicate that a tyrosine-rich fusion tail enhances tyrosinase-initiated protein-chitosan conjugation."

The group members showed that their GFP-chitosan conjugates can be deposited onto micropatterned gold electrodes in response to an applied voltage. "The deposition occurs with high spatial resolution, and GFP's fluorescence--and therefore structure--remains intact," Payne says. "The incorporation of proteins into microfabricated devices offers considerable potential for biosensors and therapeutic devices. We are currently investigating this assembly procedure for applications in biosensors, microarrays, and microelectromechanical systems."

Gross, graduate student Soma Chakraborty, and coworkers at Polytechnic University are carrying out research on starch using an immobilized lipase known as Novozym 435 to esterify starch nanoparticles. The enzyme is a lipase from Candida antarctica immobilized on an acrylic macroporous resin. It has been shown to exhibit exceptional activity for a variety of polymerizations [Macromolecules, 36, 8219 (2003)].

Starch, Chakraborty points out, is an abundant, inexpensive, naturally occurring polysaccharide that is biocompatible, biodegradable, and nontoxic. Modified forms of starch are potentially useful as drug carriers.

Chakraborty's research has focused on regioselective acylation at the C-6 position of the starch's glycopyranose repeat units. To make the nanoparticles accessible for reaction, she incorporated them into reverse micelles stabilized by a commercially available compound known as Aerosol-OT (AOT, bis[2-ethylhexyl]sodium sulfosuccinate). The nanodimensions of AOT-coated starch particles and their solubility in nonpolar media, such as toluene, allow their diffusion through the pores of the macroporous Novozym 435 resin.

"The esterification occurs throughout the starch nanoparticles instead of being limited to the surface of large particles or films as was the case in previous attempts to acylate starch using enzymes," Chakraborty explains. "We used vinyl stearate, -caprolactone, and maleic anhydride as the acyl donors for the esterification reactions.

"We have shown that enzyme catalysis can be used to prepare a new family of structurally and dimensionally well-defined nanoparticles from an abundant, biocompatible, and natural building material," she says.

In related work, postdoc Bishwabhusan Sahoo and Gross have been using Novozym 435 to catalyze the conjugation of natural polyols, such as glycerol, with poly(acrylic acid). "The esterification occurs with extraordinary selectivity under mild conditions without the need for activation of pendant acid groups," Sahoo notes. "We conduct the reactions in bulk by forming monophasic poly(acrylic acid)/glycerol liquids. The high regioselectivity of this biotransformation results in water-soluble poly(acrylic acid-co-glycerolacrylate) instead of cross-linked gels that would otherwise be formed by using traditional chemical catalysts."

SYNTHESIS OF SUCH polymers without enzymes requires multiple steps. Gross's group is now extending the enzymatic technique to other polyacids and polyols.

In a collaborative project in the Netherlands, researchers Andreas Heise at DSM in Geleen, Anja R. A. Palmans at Eindhoven University of Technology, and coworkers are combining enzymatic polymerization with "chemical" polymerization to synthesize block copolymers. "DSM is committed to innovative R&D work on sustainable routes employing technologies such as biocatalysis and fermentation," Heise says. "DSM is also convinced that biotechnology routes are set to become increasingly important as they offer economic advantages as well as being eco-efficient through the use of renewable resources. The aim of the collaborative project is to combine the best attributes of both enzymatic and chemical methods to obtain new material designs."

In 2002, the team showed that ring-opening polymerization (ROP) of -caprolactone catalyzed by Novozym 435 can be combined with atomic transfer radical polymerization (ATRP) of styrene using a CuBr catalyst to synthesize block copolymers [Macromolecules, 35, 2873 (2002)]. ATRP is a controlled, or living, radical polymerization process that typically employs an alkyl halide as an initiator and a transition-metal complex as a catalyst to create a polymer radical (C&EN, Sept. 9, 2002, page 36).

The researchers used a bifunctional initiator containing an activated bromide to initiate ATRP and a single primary alcohol group to initiate the enzymatic process. The block copolymers were synthesized in 90–95% yield in two consecutive steps starting with the enzymatic ROP followed by ATRP of styrene.

"More recently, we successfully combined enzymatic ROP of e-caprolactone with nitroxide-mediated, living free-radical polymerization of styrene from a bifunctional initiator to obtain block copolymers," Heise tells C&EN. "We achieved this using a cascade approach without an intermediate transformation or workup step.

"This is the first example of a chemoenzymatic polymer synthesis process in one pot," he continues. "This approach is a step toward the polymerization equivalent of academically and industrially important combined catalytic reactions in organic chemistry, such as the dynamic kinetic resolution of racemic mixtures in a chemoenzymatic procedure.

"Now that the proof of principle has been established, we will try to use this approach to obtain new functional polymers and develop a process for kinetic resolution polymerization," Heise adds.

At Johannes Gutenberg University in Mainz, Germany, professor of organic and polymer chemistry Holger Frey and coworkers are investigating the use of Novozym 435 as a catalyst for the synthesis of hyperbranched aliphatic polyesters. The group showed that e-caprolactone, or d-valerolactone, and 2,2-bis(hydroxymethyl)butyric acid can be copolymerized using the catalyst to synthesize dendritic copolyesters [Macromol. Rapid Commun., 23, 292 (2002)]. The copolymerizations can be carried out in solution and under bulk conditions.

Frey and coworkers used the method, which combines ROP of the lactone with polycondensation of the carboxylic acid, to prepare a series of hyperbranched copolyesters with different degrees of branching. "To the best of our knowledge, this is the first enzyme-catalyzed synthesis of hyperbranched polymers," Frey tells C&EN. "The strategy is broadly applicable to various other substrates. The synthetic conditions are mild, which means that it is possible to incorporate sensitive functional units."

Jan C. M. van Hest, professor of bioorganic chemistry at Nijmegen University, in the Netherlands, and coworkers are developing techniques for the "smart" assembly of hybrid biopolymers. The work has potential applications for protein purification and, in the biosensor field, for protein recognition.

"We have been working in the past year on creating stimulus-responsive enzymes and scaffolds," he tells C&EN. "We hope to be able to mimic cascade processes in nature, in which the positioning of the enzyme active site results in a high level of control over selectivity and efficiency within multistep reaction processes."

The work is being carried out within the European Research Training Network project Smart Assembly of Hybrid Biopolymers, which was set up to design and synthesize biologically active polymer materials with well-defined positional control over enzyme activity. The project, which is coordinated by van Hest, is funded by the European Union through its Fifth Framework Improving Human Potential Programme.

THE PROJECT has three lines of research. First, fusion proteins are constructed that consist of a component that functions as an enzyme and a structural component. The latter enables the protein to be assembled into larger biocatalytic aggregates in a controlled manner on a scaffold, or carrier material.

Second, mechanisms, such as leucine zipper interactions for assembling the proteins on the scaffold, are being tested. A leucine zipper is a motif found in some DNA-binding proteins in which every seventh amino acid in a region of around 35 amino acids is leucine. Zipper interactions enable two surfaces to be linked together.

The third line of research focuses on modified carrier materials that act as the scaffolds for the fusion proteins. Examples of such carriers are a porous polymer material known as polyHIPE (high internal phase emulsion) and virus coat proteins.

Van Hest and collaborators use C. antarctica Lipase B (CAL-B) as a model enzyme system. They have cloned the gene to produce the fusion protein and demonstrated its catalytic activity. They also have shown that anchoring units in the fusion protein, based on elastin polypeptides and leucine zipper fragments, do not have a negative effect on enzymatic activity.

"Our multidisciplinary approach is quite unique in that it combines techniques from polymer chemistry, peptide synthesis, and molecular biology," van Hest says. "We have shown it to be possible to introduce functional handles into polyHIPE that can be used for specific modification by oligopeptide moieties, such as small elastin fragments and leucine zipper molecules.

"Preliminary results also indicate successful grafting of enzymes onto a second scaffold material, namely virus particles," he continues. "Our current research is directed at assembling fusion proteins onto our functionalized scaffolds and at investigating if and to what extent we can position enzymes accurately onto our carrier materials."

At the University of Massachusetts, Lowell, Ashok L. Cholli and coworkers--including Muthiah Thiyagarajan, who recently moved to the University of North Carolina, Charlotte--are using biocatalysts to create novel electrically conducting and optically active polymers that have potential applications in optoelectronics. A focus of the group's research is the enzymatic synthesis of polyaniline nanocomposites that are not only conducting and optically active but also chiral and water soluble.

In recent work, the group synthesized a chiral conducting nanocomposite of poly(acrylic acid), polyaniline, and camphorsulfonic acid (CSA) using the enzyme horseradish peroxidase [J. Am. Chem. Soc., 125, 11502 (2003)] CSA acts as a chiral dopant.

"We induce chirality in the achiral aniline monomer by complexing it with chiral molecules, such as camphorsulfonic acid," Cholli explains. "We create water-soluble nanocomposites by using the enzyme to polymerize the aniline-CSA monomers in the presence of a polyelectrolyte, such as poly(acrylic acid)."

The polyaniline chains in the nanocomposites have a helical conformation. Cholli and coworkers discovered that the enzyme plays a dual role in the synthesis: It not only acts as a catalyst but also determines the conformation of the polymer chain.

"The handedness of the helices is dictated by the enzyme," Cholli says. "Circular dichroism shows that the specificity of the helical handedness is the same regardless of whether the monomer entering the active site of the enzyme is (+) or (–).

"We are now interested to know whether there are other naturally occurring enzymes that dictate the conformation in biocatalytically synthesized polymers," Cholli adds. "We are also focusing on the role of size and shape of dopants in the helical chain formation."

MEANWHILE, Kurt F. Brandstadt, a bioscience researcher at Dow Corning in Midland, Mich., and coworkers are working at the interface between silicon chemistry and biotechnology to design bioprocesses for the production of polymeric silicon-based materials, biosensors, delivery systems, and solid-phase separation products for the fermentation industry.

"We want to make what nature makes," Brandstadt tells C&EN. "But unfortunately, diatoms (small single-celled plants) still have the edge in producing ordered, long-range silica structures.

"Diatoms bioprocess multigigatons of silicic acid each year," he says. "We want to know how silicic acid is transported across biological membranes, concentrated, and stabilized for the creation of beautiful and delicate structures with significant long-range order in a reproducible process."

The tetrafunctional nature of the silicon atom and the possibility that four reactions can occur at each silicon site in silicic acid and its esters may yield clues to the mechanisms of these bioprocesses. The role of proteins or polypeptides in constructing the silicic acid matrices is complicated, however.

"We have been collaborating with colleagues at Open University, Milton Keynes, England, and Genencor International, Palo Alto, Calif., to investigate whether proteins act as enzymes or just as templates in the formation of silica structures," Brandstadt notes. "We decided to take several steps back and focus on a simple model compound for our investigation."

Last year, the team reported model studies on the ability of enzymes to catalyze the formation of molecules with a single siloxane bond during the in vitro hydrolysis and condensation of alkoxysilanes [J. Inorg. Biochem., 96, 401 (2003)].

"We have clearly demonstrated that the active site of trypsin, a proteolytic enzyme, is specifically involved in the formation of a siloxane bond by the condensation of silanols," Brandstadt says. "We also showed that trypsin, as well as several other proteins and polypeptides, catalyzes the hydrolysis of alkoxysilanes in a nonspecific manner.

"We are now continuing to evaluate the catalytic role of trypsin, but in more complicated model silicon-based systems," he concludes. "The information should help us better understand how Nature does her job. The work is only one step on our journey into silicon biotechnology."

According to Cheng and Gross, the use of biocatalysis in polymer science is now receiving welcome attention from industry, with several companies looking at the technology. The researchers suggest that promising areas for industrial development include biotransformations that require chemo-, regio-, or enantioselectivities, or monomers and polymers that are difficult to synthesize chemically. Biotransformation may also minimize the problems of waste generation, color formation, and high-temperature operation often encountered in chemical processes.

"There is no shortage of opportunities either for fundamental research or for product development," Cheng and Gross conclude.