Specifically engineered, biobased additives possess many advantages when compared to conventional ingredients, including higher performance and a more environmentally friendly profile. In addition, they add novel functionality to coatings not possible with traditionally available additives, creating new opportunities for high performance formulations. The possibilities for creating value through the use of bioengineered ingredients is limited only by the imagination. Think of the functionality that you would like your coating to bring to a surface and chances are nature has already perfected a solution.

Biologically-derived additives offer a novel approach for functionalizing coatings and surfaces (see, "Formulating with Bioengineered Additives: Enhancing the Performance and Functionality of Paints and Coatings," Coatings World, March 2010). The blending of molecules isolated from nature with the synthetic environment of a coating represents a novel route for functionalizing coatings with unique properties. Biological catalysts (enzymes) have a variety of desirable characteristics, including catalytic specificity and efficiency, manufacturing sustainability through utilization of renewable resources and environmental compatibility through natural degradative processes for disposal. As such, these novel additives present an immediate opportunity for coatings companies to diversify, distinguish and expand markets for their product lines by providing coatings that not only protect and beautify but also functionalize the surfaces they coat (see, "Functional Additives: A Platform for Revitalizing the Paint and Coatings Industry," Coatings World, February 2010).

Recent biotechnical advances have provided important tools for the efficient development of enzymes with improved properties for both established and new areas of application. Case studies published by the Organization for Economic Cooperation and Development (OECD) document how biotechnology has been implemented and assessed, in terms of cost and sustainability, across a variety of industries (Table 1). The conclusions were noteworthy for those industries that have not yet embraced this technology: 1) the approaches for incorporation of biotechnology were rarely systematic, i.e. each company took a different approach; 2) adoption of the technology was attempted even though biotech skills had to be acquired, usually through industrial or academic partners; 3) lead times for expanding development throughout processes or products improved with succeeding developments; and 4) cost was the primary motivating factor with environmental improvements a secondary concern.1 Although biotechnology is currently a major contributor to many clean industrial products and processes as illustrated in Table 1, its potential in the paint and coatings industry is far from realized.

Reactive Surfaces has established the incredible potential that bioactive compounds present for creating new modes of functionality and thus increasing the value proposition for paints and coatings to both consumers and industrial users. The company possesses the ability to evaluate these natural materials with regard to their potential viability in a coatings environment and thus is able to build a robust portfolio of targeted, highly functional bioengineered additives. Access to and understanding of state-of-the-art genetic engineering technologies is a key component of the company's product development program. Knowledge of various coating formulations and their desired characteristics as well as production processes and application techniques is also a fundamental aspect of the company's R&D efforts.

Since its inception, Reactive Surfaces has organized its operations to ensure this combination of capabilities. Our scientific team is comprised of internationally recognized experts in molecular biology as well as biochemistry, polymer science and coatings formulation. An advisory committee comprised of business and industry leaders provides further guidance on commercialization of novel technologies. An extensive network of external experts in biotechnology (Texas A&M University), fermentation engineering (University of Georgia, Athens) and polymer chemistry and coatings science (University of Southern Mississippi) provide additional evaluation support.

In this third article on bioengineered additives, we outline the strategy followed at Reactive Surfaces to identify and develop effective biobased additives that provide functionality that will enable paint and coating formulations to meet unmet market needs. By way of example as to how we would approach a new project to deliver a formulation-ready, biobased additive to your company for functionalizing your coatings, we outline the sequence of steps that has led to the successful introduction of our DeGreez additive for self-cleaning coatings. We have shown that careful selection of the biocatalyst can lead to bioadditives that retain activity after incorporation in commercial coatings, thus providing coatings that exhibit oil-resistance not through physical properties, but through its biocatalytic properties.2 We have also shown that the physical and chemical properties of the polymer are considered critical and important for optimal efficacy and to meet the activity demands of the application.3 This example of an enzyme-based additive clearly demonstrates the comprehensive approach necessary to ensure that the resulting additives are cost effective, high performing and sustainable, and that they provide unique opportunities for novel formulation development. It is the intent of this product development approach to assure formulators that they can make multiples of present margins on any given product line in their present markets and facilitate the expansion of their product lines into presently untapped markets.

The development of bio-functionalized coatings requires a series of progressive steps, starting with identification of opportunities for adding functionality to paints and coatings (see Figure 1). Once a specific market need has been identified, the judicious selection of an appropriate biocatalyst from nature's palette of functionalities follows. An assessment of polymer compatibility and functional limits in the solid state must then be conducted. Finally, formulation of biocatalytic coatings relative to industry accepted cast/cure coating standards is required. Let's discuss how this worked for Reactive Surfaces' DeGreez additives.

Step One: Identify the Market Need

In recent years, a major effort has been made by coating manufacturers to develop oil-resistant, self-cleaning coatings for applications such as optics, electronic displays, kitchen surfaces and textiles. The use of these coatings would eliminate or reduce the need for corrosive/caustic soaps, detergents and solvents; prevent oil-degradation of coatings and underlying substrates; and improve odor control and hygiene. For low oil-load applications, the industry has offered novel coatings with good roll-off properties and very high water and oil contact angles to reduce the visual impact of oil contamination. For high oil-load applications, such as kitchen surfaces, more traditional protective coatings have been used that are resistant to penetration/uptake. However, these surfaces remain difficult to clean without the use of excessive surfactants or solvents.

An alternative and novel approach is to functionalize coatings with lipid hydrolyzing enzymes from nature, enabling the auto-decontamination and removal of oil from surfaces. Lipases (triacylglycerol acylhydrolase, E.C. 3.1.1.3) have emerged as key enzymes in the biotechnology sector. The natural substrates of lipases are long-chain triacylglycerols, which are hydrolyzed by the addition of water across the carboxyl ester bonds to liberate fatty acids and glycerol (Figure 2). They are robust and versatile with respect to the range of substrates they can act upon, while at the same time being highly specific for the reactions they catalyze. In addition to their hydrolytic activity on triglycerides, lipases catalyze esterification, transesterification, acidolysis, alcoholysis and aminolysis (Table 3). They also exhibit good chemoselectivity, regioselectivity and enantioselectivity, and as hydrolases they do not require cofactors. Finally, lipases possess broad substrate specificity and exhibit optimum activities over a wide range of temperatures. This versatility makes lipases important industrial biocatalysts.4-11

Reactive Surfaces identified a number of different markets being served by formulators whose products could be differentiated with a degreasing function. One such market was in floor sealants used in commercial facilities subject to oil and grease contamination. The market is highly fractured with hundreds of suppliers. The margins, while respectable, were constantly dwindling as the companies battled each other for market share. The end users interviewed were clearly capable of and willing to pay for products that would significantly reduce their labor-intensive cleaning requirements and the environmental liability of large scale use of industrial cleaners.

Step Two: Identify Potential Bioactive Additives

At Reactive Surfaces, we develop natural additives such that the biocompounds and reactants are dispersed, embedded and maintained in an active state within a continuous polymer phase, either as solid films or aqueous dispersions. To create a single macroscopic phase without affecting biomolecule activity, the selection and/or modification of the biocatalyst is the first step forward in harnessing biomaterials in functional coatings (Figure 1). Selection of the most appropriate bioactive (enzyme or peptide) from the diverse options that nature has to offer is critical.

Fortunately, there are classification systems that help narrow down the choices once a desired functionality has been identified. The Enzyme Classification (EC) system places enzymes into different categories (EC 1-6) based on the general type of chemical reaction they mediate (Table 2). Enzymes are classified differently depending upon whether they catalyze oxidations, cause the transfer of a functional group from one molecule to another, add or remove water from a molecule, break or create carbon-carbon bonds or change the arrangement of bond connections within a molecule. Sublevels within each of these categories provide increasingly specific information. Lipases, for example, are a subclass in the group of enzymes known as hydrolases (category EC 3), and each different lipase has been assigned a specific classification number along with a description of its specific characteristics (selected examples of which are shown in Table 3). In the case of the self-degreasing additive DeGreez, the lipases were our first choice, although whether these enzymes could withstand the rigors of existence in a dry-film environment and exhibit a degreasing function was entirely unknown. Selecting from the vast number of lipases (over 100 have been identified) presented another significant challenge.

This information is helpful in narrowing the selection of appropriate enzyme candidates for use in a specific biobased additive. There are, in fact, some defining characteristics that should guide the selection of an appropriate biocatalyst. In addition to identifying an enzyme that meets the application parameters such as temperature, pH and type of substrate (the term "substrates" is used here in the biochemical sense as the molecule that the enzyme acts upon), characteristics such as stability, catalytic rate and substrate specificity must also be considered. In searching for appropriate lipase enzymes for our DeGreez additive, for example, we were looking for lipases that had broad specificity for different substitution patterns and chain lengths within the triglycerides in order to develop the most effective product possible. A clear understanding of both the characteristics of the different lipase enzymes and the desired performance profile of the final additive were required before this step could be completed. It cannot be stressed enough that proper assessment of application requirements and a detailed understanding of the functionalities that are available within a set of related bioactives (enzymes, peptides, etc.) is an essential step in enabling the development of a reactive coating.

Step Three: Establish Assay Method

Once the biocompounds of interest have been selected, it is necessary to establish a method or methods for detection and measurement of bioactivity under various conditions during the development process. In some cases, there will be widely known techniques that can be adopted for use with the laboratory experiments employed in analyzing bioadditive performance. In other cases, no obvious or universal assay will be available and thus a method must be designed to match the specific requirements of the specific test conditions. This is especially the case where a dry, cast film must be tested for its enzymatic activity against the target molecule of interest (e.g., in this case, grease, cooking oils, natural fats, etc.) In either case, it is very important to consider the sensitivity and availability of substrates.

For our study of lipolytic enzymes, a visual method was selected based on previous literature reports of its effectiveness in quickly assessing the specificity of lipase enzymes. Our colleagues at the University of Southern Mississippi developed a rapid colorimetric method to detect degreasing of a surface using pH indicators embedded in a gel matrix containing the oil or grease of interest. When the indicator material was applied to surfaces coated with leading floor sealants (five mil thickness) containing DeGreez, a color change was noted within 10 minutes, with the assay completed in 30 minutes (Figure 3). The indicator material is initially dark green and changes to yellow in the presence of active DeGreez additive in the coating being tested. No color change is observed in control surfaces that do not contain any DeGreez. This method enables in situ visual evaluation and real-time observation of the catalytic activity of the biobased additive immobilized in the applied coating.

Step 4: Screen for Compatibility with Polymer Systems

After confirming that catalytic activity of the selected biocompound candidates can be correctly assayed in the coating as a cast film, the next step in the development process is to determine if the candidates are compatible with the wide range of resin systems commonly used in commercial paints and coatings. Several questions must be answered at this stage: a) is the bioactive stable in the polymer matrix? b) does the biocatalyst function properly when blended with the polymer? and c) is the activity level affected by the presence of the resin system? An understanding of the role that the physical and chemical properties of the polymer and the interplay between actives and polymer and the chemicals being reacted each control the efficiency of the reactive coating. To optimize the activity of embedded biocatalysts, selection criteria of the solid phase polymer type are as important as those of the biocatalyst. Although molecular interaction with polymeric materials may alter some of the enzymatic characteristics through activation or enhanced specificities, the fundamental properties must remain unchanged in a successful functional coating. If chemical interactions take place between the resin system and the biological molecule, the activity of the bioadditive can be reduced or eliminated.

To screen for compatibility with polymer systems, the bioadditive is typically blended with solution phase polymers and then assessed for impact on the desired bioactivity. For the DeGreez additive, three different lipases with attractive reactivity profiles were evaluated with several different resin systems. They performed well in most resins with the exception of the isocyanates (Figure 4), which reacted with the hydroxyl and amine functional groups in the enzymes, reducing their activity. While this limitation can be overcome by altering the blending protocols, an acrylic resin system was thus selected for further initial evaluations of DeGreez.

Step 5: Assess Reactivity in Polymer Systems

Polymer matrices provide a very different environment for a biocompound when compared with traditional solution-based systems. Therefore, it is necessary to determine if the biomolecule will exhibit the desired reactivity when blended with a resin. More specifically, when hydrolysis in solution is performed, the enzyme is solvated at the molecular level resulting in homogeneous biocatalysis. In a polymer system or coating, however, the biocompound is not dissolved but rather suspended in the polymer matrix, creating a heterogeneous system, and there is no way to easily predict if the desired functionality will be achieved. In this case, diffusional constraints derived from substrate accessibility to the bioactive become crucial. As a result, reactivity can be a function of total surface area or bulk volume, and it is necessary to understand which mechanism is operating (see, "Formulating with Bioengineered Additives: Enhancing the Performance and Functionality of Paints and Coatings," Coatings World, March 2010).

We have observed each mechanism operating for different products developed at Reactive Surfaces. Lipases investigated for our DeGreez additive have been shown to exhibit an increasing catalytic rate for hydrolysis with increasing surface area when blended with Avanse MV-100 emulsion and applied to polypropylene sheets. Activity was independent of bulk volume, however. In comparison, coatings containing our OPDtox enzyme additive for decontamination of organophosphorous compounds including chemical warfare agents and pesticides have been shown to exhibit catalytic activity that is directly related to bulk volume. Clearly, the property and performance optimization of the selected polymer type must match the sorption characteristics of the reactants.

Step 6: Analysis of Reactive Coatings

The final step in the development of reactive coatings must include assessment of activity by application specific property testing. This phase of the development process includes evaluation of both the performance of the bioactive and the conventional properties of the coating. It is imperative that the biobased additive exhibit long-term stability and activity without having any impact on desirable coating characteristics such as gloss, hardness, adhesion and impact resistance. In addition to employing specialized testing methods for determining the activity profile of the new bioadditive, typical chemical resistance, hydrolysis, thermal, scrub testing and other tests must be completed on representative coating formulations.
br /> In the case of our Degreez lipase additive, the addition of lipase had no discernable effect on coating performance at an enzyme addition level of three percent. All properties of the enzyme modified coating were equal to that of the un-modified standard. At an enzyme level of 14.3%, film softening and blistering were observed in the coatings, possibly as a result of the carrier solvent. The enzyme formulation is currently being investigated to minimize these impacts on coating performance.

With respect to bioadditive performance, the biocatalytic clearing of a heavy oil incident was demonstrated by contaminating prepared surfaces with a thick layer of vegetable oil (2 μL per cm). The panels were monitored over 72 hours, and during that time the lipase-blended coating cleared its surface of all oil (see Figure 2 in "Formulating with Bioengineered Additives: Enhancing the Performance and Functionality of Paints and Coatings," Coatings World, March 2010).

Positive results have also been obtained for scrub tests using three different types of coatings provided by Hillyard, Inc., St. Joseph, MO, containing the DeGreez additive (zinc crosslinked acrylic styrene copolymer, institutional grade tile sealer based on a modified acrylic polymer emulsion and a water dispersed silicone sealer used for tile, sandstone and marble). Degreasing activity was measured as the rate of hydrolysis of p-nitrophenyl acetate using a UV/Vis spectrophotometer. The activity of the enzyme after contact with a detergent solution and without scrubbing served as a control for the effects of the detergent on the enzyme's degreasing ability. In all cases, degreasing activity and gloss levels were retained after samples were scrubbed up to 200 times in a scrub machine, and performance was better for coatings containing DeGreez than for coatings cleaned with a detergent. The coatings other performance criteria were unaffected by the presence of the DeGreez additives. Similar results were obtained when a silicone topcoat containing DeGreez was applied over a modified acrylic sealant undercoat (Figure 5a&b).

Step 7: Determine Regulatory Status

Once performance testing has confirmed that the new bioadditive functions effectively in coatings formulations without impacting the key aesthetic and protective nature of the coating, it is time to take the necessary steps for bringing the product to the marketplace. Before any new chemical or biochemical-based product can be commercialized, regulatory bodies in the U.S., Europe and around the world require notification about the substance. Therefore, it is necessary to determine whether or not the product contains any new substances or substances that require registration due to their mode of action or intended application.

The active ingredients in DeGreez are enzymes. In the U.S. such compounds are regulated under the Toxic Substances Control Act (TSCA) and are handled by the Chemical Division of the Environmental Protection Agency (EPA). The specific enzyme in DeGreez, Lipase, Triacylglycerol, is already listed on the TSCA Chemical Inventory and thus can be offered commercially without the need for any TSCA notification. In addition, no special record keeping is required, and Lipase, Triacylglycerol is not regulated by any of the 50 states. As long as Lipase, Triacylglycerol is not genetically or chemically modified in any way during its isolation or use, it is not regulated in the U.S. In Europe, Lipase, Triacylglycerol is listed on the EINECS inventory and is preregistered under the REACH regulation. No chemical notification in Europe is required, but importers of Lipase, Triacylglycerol must conform to the REACH registration process that is triggered by certain volume thresholds.

Step 8: Address Commercialization Issues

Commercial production of bioengineered additives requires an understanding of large scale biological production processes and access to appropriate production equipment. For pilot scale manufacturing, Reactive Surfaces partners with the Bioprocessing Fermentation Facility at the University of Georgia, Athens and Boston-based 21st Century Biochemicals. We also have a relationship with biotechnology industry leader Lonza, which currently serves as the commercial toll producer of both enzyme and peptide products. In the case of DeGreez, there were a number of avenues for large scale, cost effective commercial production.

And there you have it. From the initial identification of need at the end-user and formulator level, to the selection from the palette of nature of a collection of suitable candidate bioadditives, to the proof-of-concept testing, to the selection of the coating systems, to the completion of standard coatings tests, and finally to the pre-commercialization steps of regulatory compliance and commercial-scale production. The complete development stages of a new bioengineered additive was all accomplished in the course of less than three years.

Building a Robust Pipeline of Bioadditives

In addition to its three products at or near the commercialization stage-Protecoat, OPDTox and DeGreez-Reactive Surfaces has several other development programs in place. Potential new applications for the existing products are being investigated, as are new bioadditives with unique functionalities. Protecoat antimicrobial peptides, for example, have been shown in preliminary tests to exhibit both antiviral and antialgal properties. Early studies of the DeGreez additive in polymethylmethacrylate films indicate that it may be possible to create self-healing coatings with this product. We are also investigating the use of cell-based particulate material from various different organisms as biodegradable fillers, opacifiers, UV stabilizers, colorants and other important coating ingredients. Other programs are investigating the use of bioengineered additives for antifouling surfaces, rechargeable/reprogrammable coatings, anti-corrosion surfaces, deodorizing coatings and catalytic column coatings for liquid and gaseous waste-stream decontamination, among others.

For each of these programs, we follow the various steps described above in order to ensure development of products that will provide added value to the paint and coatings industry. In many cases, each step can be, and often is, iterative. Ultimately, it is the integration of these various influences and development of the methodology to monitor and assess the critical variables that makes development of functional coating systems possible and enables the movement of bioadditive candidates through development into commercial products.

It is important to note, too, that while an immense diversity of function can be found in nature, we are not limited to what is readily provided in the natural world. At Reactive Surfaces, we have the ability, through the tools of genetic engineering, to create unique functionalities for specific purposes. Genetic engineering induces cell alterations in organisms based on the artificial manipulation and transfer of genetic material. Enzyme enhancement can be achieved via several different methodologies. Directed evolution using mutation and recombinant technologies, for example, ultimately leads to creation of microorganisms designed to express enzymes that can achieve specific chemical reactions. Metabolic engineering tools enable researchers to improve the microorganisms that are used to produce the enzymes and aim to affect metabolic pathways to achieve higher yields while reducing energy consumption and waste generation. When combined, these technologies make it possible to design commercially viable biocatalysts with specific properties targeted for application as additives in paints and coatings.

Are You Ready to Functionalize and Improve Your Present Formulations?

As the paint and coatings industry recovers from the recession, analysts predict that significant consolidation will ensue. Companies lacking differentiating technologies and notable value-added formulations may not survive the fierce competition. Bioengineered additives can enable coatings suppliers to develop sustainable coating solutions with unique functionalities. You don't have to break the bank to achieve differentiation, you can just functionalize your formulations.

What functionalities do you want to bring to your future product lines?

With its present range of available products and broad portfolio of other additives under development, Reactive Surfaces has successfully demonstrated the viability of biobased additives as a means of introducing novel activity to surfaces. We have the unique combination of expertise and capabilities in biotechnology and coatings formulation to identify further candidates and functionalities you need. Forward-thinking manufacturers will seriously investigate the advantages of integrating such bioadditives into their coating products, including functionalities highlighted in these articles as well as those not yet discussed. We are eager to work with such market leaders to explore all of the possible opportunities presented by nature, and in doing so to help you reinvigorate the coatings industry.