Advances in Biopolymers - American Chemical Society

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Chapter 18

Functionalizing Chitosan Using Tyrosinase: From the Construction of Bio-Based Products to the Assembly of Stimuli-Responsive Materials for Biofabrication 1,*

1,2

GregoryF.Payne and Tianhong Chen 1

Center for Biosystems Research, University of Maryland Biotechnology Institute, 5115 Plant Sciences Building, College Park, MD 20742-4450 Current address: TA Instruments, 109 Lukens Drive, New Castle,DE19720 *Corresponding author: [email protected] 2

Tyrosinase is a versatile enzyme that oxidizes a broad range of substrates that include low molecular weight phenols, peptides that contain tyrosine, and proteins with accessible tyrosine residues. The products of tyrosinase-catalyzed reactions are reactive o-quinones (or o-quinone residues) that can undergo uncatalyzed reactions with various nucleophiles. We are studying the use tyrosinase to generate quinones to undergo grafting reactions with the aminopolysaccharide chitosan. Initially, our goal was to demonstrate that tyrosinase could initiate the grafting of various plant phenols onto chitosan to create functional polymers from bio-based renewable resources. More recently, we are examining how tyrosinase can be enlisted to generate protein-chitosan conjugates with stimuli-responsive properties. Here we review our group's efforts over the last decade.

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Chitosan is an aminopolysaccharide derived from chitin. The distinctive feature of chitosan is that it has primary amines at nearly every repeating residue, and these primary amines confer two important properties. At low pH, these amines are protonated making chitosan a water-soluble cationic polyelectrolyte. Raising the pH above about 6.3 leads to deprotonation of these amines, a reduction in chitosan's charge and ultimately to chitosan becoming insoluble. Thus, the first important property conferred by chitosan's primary amines is the pH-responsive solubility. Secondly, these amines are nucleophilic allowing chitosan to be derivatized using a range of electrophilic reagents.

CH2OH

CH2OH

+ ntf

In our studies, we are derivatizing chitosan using the enzyme tyrosinase to in Mta-generate the electrophile that subsequently reacts with chitosan. Tyrosinases, and related phenol oxidases, are ubiquitous in nature. These enzymes oxidize a diverse range of phenolic reactants using molecular oxygen as the oxidant (i.e. complex cofactors are not required for this oxidation). The product of this oxidation is an oquinone that is reactive - it diffuses from the enzyme's active site and can undergo a cascade of uncatalyzed reactions. These reactions are familiar for their role in the enzymatic browning of foods, but these reactions are also integral to various natural processes - the hardening of the insect integument (i.e. quinone tanning) and the setting of the mussel's waterresistant adhesive. For the last decade, our group has been examining how tyrosinase can be enlisted to initiate reactions that lead to the grafting of fimctionalizing moieties onto chitosan's backbone.(i) Our hope when we started was that tyrosinase would provide an environmentally-friendly means to confer useful functional properties to chitosan - that is, to create useful biobased products from renewable natural resources.

Quinone

Phenol Enzyme (Tyrosinase)

Polysaccharide ~ Chitosan

emlted Chitosan

R'

Activated'


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Tyrosinase-catalyzed modification with low molecular weight natural phenols The suggestion that tyrosinase could provide a generic means to derivatize chitosan is supported by the facts that; (i) a diverse array of phenolic compounds are abundantly available from nature (i.e. plant phenols), and (ii) tyrosinases have a broad substrate range for phenols. To demonstrate the broad potential, we have performed reactions with various low molecular weight natural phenols and observed a variety of functional properties can be conferred to chitosan. In one of our early studies, we grafted chlorogenic acid onto chitosan. The name, chlorogenic acid, is a misnomer as this phenol has no chlorines in its structure. Rather, chlorogenic acid is a natural product that is abundant in coffee. The tyrosinase-catalyzed grafting of chlorogenic acid to chitosan yielded a derivative that was soluble under acid conditions but insoluble under neutral conditions (characteristic of chitosan). Unlike chitosan, however, the chlorogenic acidmodified chitosan became soluble under mildly basic conditions - presumably due to the hydrophilic and acidic functionality of the quinic acid moiety of chlorogenic acid.(2) Arbutin is a natural phenol found in pears. When this phenol was reacted with tyrosinase in the presence of low concentrations of chitosan (0.5 %), the solution was observed to undergo gel formation^.?) Similarly, reactions between tyrosinase, dopamine and chitosan led to gel formation, and when this reaction was performed between two surfaces, the surfaces were bonded together even when "curing" occurred under wet conditions.(4)

H

Chlorogenic Acid

OH

Arbutin

Gallates are abundant in tannins and various gallate esters are used as food antioxidants. We observed that gallates could be oxidized by tyrosinase although our results suggest that the gallate ester concentration has a significant effect on the reactions in this system (either the enzymatic and/or non-enzymatic reactions).(5) To react the hydrophobic octylgallate with tyrosinase we used a 30 % ethanol-water solution and performed the reaction in the presence of a chitosan film (i.e. chitosan modification was performed heterogeneously). After


265 reaction, the films were washed extensively, dried and then the contact angle was measured. These measurements indicate that the surface of the octylgallate modified-chitosan films was more hydrophobic than the surface of the unmodified chitosanfilm.(6) COOR H NCH CH Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 27, 2017 | http://pubs.acs.org Publication Date: August 28, 2006 | doi: 10.1021/bk-2006-0935.ch018

2

2

:

OH

Dihydroxyphenethylamine (Dopamine)

Gallate Ester

In a final example, we examined the tyrosinase-catalyzed modification of chitosan with catechin. After reaction, the polymer was purified by a series of precipitation, washing and resolubilization steps. Rheological measurements on the re-dissolved polymer indicated that the catechin-modified chitosan offers associative thickening properties.(7) OH

Catechin

The above results indicate that a broad range of low molecular weight phenols can be enzymatically-grafted onto chitosan to yield modified chitosans with various functional properties. Thus, tyrosinase allows access to the diversity of natural phenols available from plants and provides a facile means for creating functional chitosan derivatives. While these studies did not focus on the covalent linkage between the phenol and chitosan, chemical evidence based on mass spectrometry is consistent with the formation of either (or both) Michael-type adducts or Schiff-bases.(7)


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Extending tyrosinase-catalyzed modification to peptides and proteins Tyrosinase's activity is not confined to low molecular weight phenols rather tyrosinase can oxidize the phenolic moieties (i.e. the tyrosine residues) of peptides and proteins. Thus, tyrosinase can access an even broader palate of compounds for grafting onto chitosan. Recent studies demonstrated that a model di-petide (Tyr-Ala) could be enzymatically-grafted to chitosan. Further, peptides from casein hydrolyzate could be enzymatically grafted onto chitosan without the need for purification of the peptides from this complex mixture. Thus, the molecular recognition capabilities of tyrosinase can be exploited to selectively oxidize only peptides that have tyrosine residues - potentially this molecular recognition capability might eliminate the need for fractionation/purification of the peptide raw materials. Rheological studies with this peptide-modified chitosan indicated that it offered associative thickening properties.(S)

Tyrosyl residue

Peptide/Protein

weWue

"Activated"

Polysaccharide Chitosan

Chitosan

Grafted Peptide/Protein

In addition to recognizing and reacting with tyrosine residues of peptides, the enzyme can oxidize accessible tyrosine residues on proteins. Once these residues are "activated", by conversion into o-quinone residues, they can be grafted onto chitosan to yield protein-chitosan conjugates. This was first demonstrated using the open-chain protein gelatin that has a small fraction of tyrosine residues localized to its telopeptide region. The addition of tyrosinase to a gelatin-chitosan blend was observed to result in a sol-gel transition to yield a relatively weak gel.(9,70) Interestingly, the tyrosinase-catalyzed gelatinchitosan gels could be reversibly strengthened by cooling below the transition temperature that gelatin solutions normally undergo gel formation. This result suggests that tyrosinase-catalyzed reactions do not destroy gelatin's ability to undergo coil-to-triple helix transitions. Also interesting is that the gelatinchitosan gel is transient and spontaneously breaks over time in processes that appears to follow percolation-type physical models.(Ji)


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The ability of tyrosinase to initiate the grafting of peptides and proteins allows a broad range of bio-based products to be grafted onto chitosan. Importantly, the results with gelatin demonstrate that protein grafting is achieved with retention of structural characteristics important for functionality (i.e. gelatin's ability to undergo coil-to-helix transition). Also important is that the macromolecular conjugate offers characteristics unique from those of the starting materials (i.e. the tyrosinase-catalyzed gelatin-chitosan gel forms a transient network). To further extend the tyrosinase-initiated grafting of proteins to chitosan, we collaborated with Dr. Bentley's group who engineered a green fluorescent protein (GFP) to have a C-terminal penta-tyrosine tail. It was reasoned that this tail would provide additional tyrosine residues in an unstructured region that would be readily accessible for tyrosinase-catalyzed activation. Results demonstrated that this GFP construct could be grafted onto the chitosan backbone and the GFP-chitosan conjugate retained the fluorescent properties characteristic of the protein. In addition, the conjugate also possesses the pHresponsive solubility that is characteristic of chitosan.(i2) Thus, tyrosinase permits the facile conjugation of proteins to chitosan to yield a conjugate with a combination of properties contributed by each of the biomacromolecules.

Exploiting chitosan's stimuli-responsive properties to direct the assembly of protein-chitosan conjugates Through a series of collaborations, we are exploring the concept of biofabrication - the use of biological materials for micro/nano-scale fabrication.(ii) A key motivation for biofabrication is the recognition that many biological materials can self-assemble - potentially offering a "bottom-up" approach to constructing precise structures at the nanoscale. However, biological materials offer more than self-assembly capabilities. The ability of biological materials to be acted upon by precise biocatalysts (i.e. enzymes) allows macromolecular structures to be built through enzymatic-assembly. Tyrosinase-mediated conjugation of proteins to chitosan provides an example of such an enzymatic-assembly operation. Another feature of biological polymers is that they are generally charged and therefore can respond to applied electrical signals. We are exploiting chitosan's pH-dependent electrostatic properties to direct its assembly in response to locally-applied electrical signals. Scheme 1 shows the mechanism for chitosan's directed-assembly. At a cathode surface, protons can be electrochemically reduced generating a localized pH-gradient. Chitosan chains that experience a localized pH that exceeds its pKa (-6.3) will become insoluble


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and will deposit at the cathode surface. After electrodeposition, the deposited chitosan film can be rinsed and neutralized after which it will remain deposited as a thin film (thesefilmscan be re-dissolved by rinsing with mild acid).(74,75) There are two important features of chitosan's directed-assembly (i.e electrodeposition). First, electrodeposition does not require macroscopic electrodes but has been performed on microscopic cathode surfaces that have been patterned onto silicon wafers(76) or onto the surfaces of microfluidic channels.(77) With these microfabricated devices, the applied electrical signals can be controlled spatially (by the initial patterning of the electrodes) and temporally (based on when the voltage is applied), allowing the directedassembly of chitosan to be well-controlled in space and time. The second important feature of this electrodeposition, is that protein-chitosan conjugates that retain chitosan's pH-responsive solubility can also be directed to assemble in response to localized electrical signals. In fact, recent studies have demonstrated that separate proteins can be guided to sequentially-assemble at different addresses (i.e. different electrode surfaces).(7S)

TU

Proton consumption Low bulk pH (soluble chitosan)

pH gradient pH = pK

a

Scheme 7. Mechanism for the electrodeposition (Le. directed-assembly) of chitosan in response to a localized applied voltage at the cathode. While our studies are on-going, chitosan appears to provide unique properties for biofabrication. Its nucleophilic amines permit the enzymaticassembly of proteins onto its backbone (via tyrosinase-mediated reactions). And, its pH-responsive solubility allows the directed-assembly of proteinchitosan conjugates at specific addresses (in response to localized electrical stimuli).


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Conclusions Tyrosinase's broad substrate range enables a diverse array of substrates (phenols, peptides and proteins) to be grafted onto chitosan to create bio-based polymeric products with a variety of macromolecular architectures and functional properties. In many cases, the substrates could be obtained as byproducts from food processing or agricultural operations - potentially providing value-added opportunities for these renewable resources. Further, many of these substrates are obtained from edible materials such that all components in some systems (i.e. chitosan, tyrosinase, and the substrate) could be food-grade while products derived from these components would be expected to offer safety and environmental benefits. Finally, tyrosinase is simple to use with reactions occurring under mild conditions without the need for reactive reagents (the reactive 0-quinone is in wYw-generated by the enzyme). Thus, tyrosinase provides interesting means to graft substrates to chitosan to create functional bio-based products. Tyrosinase's amino-acid-residue-specificity provides an interesting opportunity to enzymatically-assemble protein-chitosan conjugates with defined macromolecular architectures. Importantly, tyrosinase-mediated conjugation is performed under sufficiently mild conditions that the protein retains structural features critical to functional properties. Further, molecular biological methods can be enlisted to add accessible tyrosine residues to proteins (i.e. through tyrosine-rich fusion tags) suggesting that tyrosinase-mediated conjugation could provide a generic construction method. Potentially, such fusion tags could also allow control of the protein's orientation with respect to chitosan backbone. In addition to offering properties characteristic of the protein, the protein-chitosan conjugates may offer the pH-responsive properties characteristic of chitosan. These pH-responsive properties have been shown to allow conjugates to be directed to assemble in response to localized electrical signals. Thus, our hope is that tyrosinase mediated coupling to chitosan will emerge as an enabling technique for biofabrication.

Acknowledgements Financial support was provided by the United States Department of Agriculture (2001-35504-10667) and the National Science Foundation (grant BES-0114790).


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