Journal of Polymers and the Environment, Vol. 10, No. 3, July 2002 (q 2002)

Renewable Resources and Enzymatic Processes to CreateFunctional Polymers: Adapting Materials and Reactionsfrom Food Processing

Christopher M. Aberg,1,2 Tianhong Chen,1,2 and Gregory F. Payne1,2,3

We are exploiting materials and concepts from food science to create functionalized, environmentallyfriendly derivatives of the biopolymer chitosan, a byproduct of seafood processing. Functionalgroups are grafted onto chitosan using tyrosinase, the enzyme responsible for food browning. Thefunctionalizing groups studied include low-molecular-weight phenols derived from natural sourcesand high-molecular-weight proteins. The approach of using low-molecular-weight phenols to func-tionalize chitosan is illustrated with arbutin, a natural phenol found in pears. Results demonstratethat tyrosinase initiates reactions that lead to the conversion of arbutin–chitosan solutions into gels.These gels can be rapidly broken by treatment with the chitosan-hydrolyzing enzyme chitosanase,demonstrating that the chitosan derivatives remain biodegradable. We briefly review other studiesin which low-molecular-weight natural phenols are enzymatically grafted onto chitosan to conferfunctional properties. The creation of co-polymers is illustrated by results in which tyrosinase isused to couple gelatin onto chitosan. Gelatin is a proteinaceous byproduct of meat production. Thetyrosinase-generated gelatin–chitosan conjugates have been observed to offer interesting rheologicaland thermal properties. These results demonstrate the potential for using renewable resources andenzymatic processing to create environmentally friendly polymers with useful functional properties.

KEY WORDS: Biodegradable; chitosan; enzymes; flavonoids; gelatin; natural phenol; renewable resources;sustainability; tyrosinase.

INTRODUCTION istries for synthesis and derivatization; and to developproducts that are biodegradable. To meet these goals aThree emerging goals for polymer manufacturingvariety of approaches have been advocated, includingare: to better utilize renewable resources as a source ofthe integration of biotechnology into polymer synthesisraw materials—either monomers (e.g., lactic acid and(e.g., polyhydroxyalkanoates). We believe there is a1,3-propanediol) or polymers (e.g., proteins and poly-substantial opportunity to employ materials and proc-saccharides); to employ environmentally friendly chem-essing concepts from the food industry to meet thesegoals. Specifically, the food industry has a long history1 Center for Biosystems Research, 5115 Plant Sciences Building, Uni-of exploiting renewable resources, manufacturing prod-versity of Maryland Biotechnology Institute, College Park, Mary-

land 20742. ucts using safe chemistries, and creating products that2 Department of Chemical and Biochemical Engineering, University of have no adverse environmental impacts. Here, we

Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, Mary-review our efforts to create functional polymers byland 21250.adapting materials and reactions common to the food3 To whom all correspondence should be addressed. E-mail: payne@

umbi.umd.edu industry.

771566-2543/02/0700-0077/0 q 2002 Plenum Publishing Corporation

78 Aberg, Chen, and Payne

RECOVERING MATERIALS FROM“UNDER-UTILIZED” RESOURCES

A common challenge to utilizing renewable re-sources is that the individual components may be presentat low levels. Additionally, these components are oftenincorporated within a complex network (e.g., the celluloseof plants exists within a complex lignocellulosic matrix).Recovery of the individual components from these matri-ces can be challenging. Nevertheless, the economic suc-cess of many natural resource-based industries relies onthe creation of value-added byproducts from the variousfractions present in the raw material. For instance, cornprocessors generate numerous commercial byproductsfrom the oil, carbohydrate, and protein fractions. Simi-larly, meat producers generate substantial quantities ofthe gelatin byproduct. Even lignin “wastes” generatedfrom the pulp and paper industry are used as a sourceof fuel.

It is obvious that value-added byproducts offeropportunities for increased revenues. We believe a lessobvious motivation for byproduct development is alsoemerging. Over time, economies of scale have led tohigh-intensity farming practices and large-scale food Scheme 1. Chitosan Production.processing operations. The scale of these operations leadsto the generation of nutrient-rich wastes at quantities thatpose environmental problems. In these cases, value-added demineralized, typically utilizing HCl, and deproteinated

with NaOH to yield the chitin intermediate [3–5]. Deace-byproducts may not only provide an opportunity for addi-tional income but may provide the best option for “waste” tylation of chitin to chitosan is typically achieved using

strong base (40–60% NaOH) and moderately high tem-management. This is illustrated by the following example.In the state of Maryland, the Chesapeake Bay is a peratures (100–1508C). As indicated by the dashed arrow

in Scheme 1, the difficulty of chemical deacetylation hasmajor aesthetic and economic asset. Over the last decadethere have been increasing concerns that excess nutrient stimulated interest in the discovery and development of

deacetylating enzymes [6].addition is jeopardizing the health of the bay. One result ofthese concerns is that a government–industry–university As noted, chitosan and its monomer glucosamine

are manufactured as dietary supplements for human con-partnership was established to explore alternatives to thelandfilling of “wastes” generated from the state’s crab- sumption. We believe an industrial grade of chitosan

could find broader applications not only because it ispacking industry. This partnership succeeded in establish-ing a manufacturing plant to convert the wastes into a inherently safe but because it offers unique physicochemi-

cal properties. Specifically, chitosan is a basic polysac-value-added biopolymer, chitosan [1]. Chitosan is derivedfrom chitin, and currently chitosan is sold as a dietary charide with primary amino groups in nearly every

repeating unit (because deacetylation is incomplete, chi-aid for individuals trying to lose weight. Glucosamine,the monomeric unit of chitosan, is also marketed (in tosan is formally a co-polymer of glucosamine and N-

acetylglucosamine). Chitosan’s primary amino groupsconjunction with chondroitin) as a dietary supplement forarthritis sufferers. have a pKa of about 6.3 [4, 7, 8]. Below the pKa, the

amino groups are protonated, making chitosan a cationicChitosan is derived from chitin that exists in theexoskeleton of crustaceans at levels of 10 to 25% (dry polyelectrolyte that is water soluble; water solubility is

uncommon for b-1,4-linked polysaccharides (e.g., cellu-basis) [2–4]. Chitin is also present in insects and fungalcell walls and is considered the second (or third) most lose and chitin). Above the pKa, chitosan’s amino groups

become deprotonated and the polymer is no longer waterabundant natural material after cellulose (and possiblylignin). Scheme 1 shows a typical operation for con- soluble. This pH-dependent solubility allows chitosan to

be formed into various shapes (e.g., beads, films, andverting crustacean wastes into chitosan. The wastes are

Renewable Resources and Enzymatic Processes to Create Functional Polymers 79

Scheme 2 shows that tyrosinase-catalyzed reactions arealso believed to be responsible for insect sclerotization(i.e., the hardening of insect “shells”) and the setting ofmussel “glue.”

The dashed line in Scheme 2 indicates that we areexamining tyrosinase-catalyzed reactions for the func-

Scheme 2. Processes Initiated by Tyrosinase. tionalization of chitosan. Initial studies showed that tyro-sinase-generated quinones reacted with chitosan and thatthe resulting chitosan derivatives had dramatically altered

membranes) using aqueous processing. This pH-depen- functional properties. Not surprisingly, the specific func-dent solubility also confers pH-responsive properties to tional properties of the chitosan derivatives dependedchitosan [9]. on the phenolic substrate selected for reaction. We have

explored a variety of phenolic substrates to examine therange of properties that can be conferred to chitosan. Ourfocus has been on natural phenols and, whenever possible,on phenolic compounds common in foods.

In addition to conferring basic properties to chitosan, TYROSINASE-CATALYZED REACTIONSthe amino groups also confer nucleophilic properties to WITH LOW-MOLECULAR-WEIGHTthis polymer. Specifically, the deprotonated amino groups PHENOLScan undergo reaction with a variety of reagents (e.g.,aldehydes, anhydrides, and acid chlorides) under rela-

Tyrosinases react with a broad range of phenolictively mild conditions. Because the amino groups are

substrates and a diverse array of natural phenols is presentconsiderably more reactive than chitosan’s hydroxyl

in foods [12]. Interestingly, the tyrosinase from mush-groups, modification can be regioselective in the sense

room is active over a pH range that also spans the regionthat reactions performed under mild conditions lead to

where chitosan can be either soluble or insoluble. Thus,modification exclusively at the amino group (i.e., the 2-

enzymatic reactions can be conducted under either homo-position of the repeating sugar residue) [10, 11]. In con-

geneous conditions (i.e., chitosan, the enzyme, and thetrast, cellulose must be reacted under harsh conditions

phenolic substrate are all soluble) or heterogeneous condi-and, under such conditions, modification occurs nonselec-

tions (i.e., the enzyme and phenolic substrate are in solu-tively on all three of the hydroxyl groups (i.e., at the

tion while chitosan is present as an insoluble film) as a2-, 3-, and 6-positions). In our studies, we are using an

result of chitosan’s pH-dependent solubility.enzymatic reaction to generate an electrophile for subse-

The ability of tyrosinase-catalyzed reactions to dra-quent reaction with chitosan’s amino groups.

matically alter the properties of chitosan is illustrated byresults with arbutin. Arbutin is a natural phenol commonin pears [13, 14]. In our studies, we performed homoge-EXPLOITING AN ENZYMATIC REACTION

COMMON IN FOODS neous reactions by mixing arbutin and tyrosinase with achitosan solution at a pH of 5.8 to 6. This pH is justsufficient to maintain chitosan in solution. During theTo generate electrophiles capable of reacting with

chitosan’s amino group, we are utilizing the oxidative course of the reaction, we observed the solution changefrom colorless to reddish and then to brown/black. Byenzyme tyrosinase (enzymes with similar activities are

also known as phenol oxidases or polyphenol oxidases). the time the solution had become black, we also visuallyobserved that it had formed a gel. Gel formation is illus-These enzymes are ubiquitous in nature and are responsi-

ble for the familiar browning of foods. As illustrated in trated by Fig. 1, which shows a dramatic increase inviscosity after about 7 hr of reaction.Scheme 2, enzymatic browning occurs when the plant

tissue is disrupted and the oxidative enzymes come in After reaction, the mechanical spectrum of the gelwas measured using rheometry. Figure 2 shows that forcontact with the plant’s phenolic substrates. Oxidation

leads to the generation of o-quinones that diffuse away the sample reacted with tyrosinase, the elastic modulus(G8) was greater than the viscous modulus (G9), bothfrom the enzyme’s active site. These quinones are reactive

and undergo a variety of reactions that lead to the forma- moduli were independent of frequency (v), and the com-plex viscosity (h*) decreased with increasing v. Thesetion of colored oligomeric phenols. As will be discussed,

80 Aberg, Chen, and Payne

Fig. 1. Tyrosinase-catalyzed gel formation. Homogeneous reactionswere performed in solutions of chitosan (0.5%, equivalent to 31 mMrepeating units), arbutin (5mM), and tyrosinase (60 U/ml) at roomtemperature. The “control” solution was incubated without tyrosinase.The viscosity was measured using a Brookfield viscometer at a rotationalspeed of 1 rpm and a spindle of S34 and S25.

observations are characteristic of a gel. In contrast, Fig.2 shows that a control in which chitosan and arbutin hadbeen incubated in the absence of tyrosinase behaved asa solution with G9 exceeding G8, both moduli increasingwith v, and h* being independent of v.

A final experiment was conducted in which chitosanand tyrosinase were incubated with varying concentra-tions of arbutin. When low levels of arbutin were used,the solutions were observed to change from colorless topink while only small increases in viscosity wereobserved (data not shown). Higher levels of arbutin led

Fig. 2. The mechanical spectrum of the conjugated arbutin–chitosanto the formation of dark black gels. Figure 3 shows thegel. Gels were prepared by reacting chitosan (0.5%), arbutin (5 mM),strength of these gels (i.e., the elastic modulus, G8)and tyrosinase (60 U/ml) for 24 hr at room temperature. The “sample”

increased monotonically with arbutin concentration. incubated with tyrosinase formed a gel, which was measured using aThese results indicate that the properties of the enzymati- controlled strain of 5 6 1%. The “control” incubated without tyrosinase

was a solution and it was measured at a controlled stress of 0.5 Pa.cally generated gels can be controlled based on the reac-The frequency range used to examine this solution was limited intion conditions.order to remain within the linear viscoelastic region. All samples wereThe observation that tyrosinase-catalyzed reactionsmeasured using a ThermoHakke RS1 with a parallel plate sensor

lead to gel formation may not be surprising in light of (PP60Ti) at a gap of 1 mm.the putative role of tyrosinase in insect sclerotization. Insclerotization, it is believed that tyrosinase converts low-molecular-weight phenolic compounds (typically deriva- crosslinked chitosan network, although chemical evi-

dence for covalent crosslinking has been difficult totives of tyrosine) into quinones [15–17]. The quinonesthen undergo reactions with proteins in the insect’s integu- attain.

As suggested above, the chemistry of the “quinonement, leading to hardening of the “shell.” These reactionsare often referred to as “quinone tanning,” and although tanning” reactions is complex and poorly understood.

Nevertheless, the facts that the starting materials (chitosanthe details of the reaction remain controversial, it appearsthat these reactions may lead to a crosslinked network. and arbutin) are natural and the reactions are mediated

by an enzyme common in nature suggest that the finalSimilarly, the reactions of arbutin may also lead to a

Renewable Resources and Enzymatic Processes to Create Functional Polymers 81

tion that chitosan derivatives remain biodegradable isnoteworthy in light of observations that highly substitutedcellulose derivatives are less biodegradable [19].

The ability to modify chitosan with tyrosinase is notlimited to the arbutin substrate. Table 1 lists several othernatural phenols that have been enzymatically grafted tochitosan. Chlorogenic acid is found in high concentrationsin coffee [20]; however its name is a misnomer, becausethere is no chlorine in chlorogenic acid. This phenol israpidly oxidized by tyrosinase and the product can begrafted onto chitosan. After precipitating and washingthe chlorogenic-acid-modified chitosan, we observed thatthis chitosan derivative is soluble under acidic and basicconditions but is insoluble under near-neutral pHs [21].Presumably, the carboxylate and hydroxyl groups of thequinic acid moiety of chlorogenic acid confer base solu-Fig. 3. The effect of arbutin concentration on the rheological propertiesbility to the chlorogenic-acid-modified chitosan.of arbutin–chitosan gels. Chitosan (0.5%), arbutin, and tyrosinase (60

U/ml) were incubated for 24 hr prior to measurement. The elastic Another phenolic substrate used for the modificationmodulus (G8) of each sample was measured with a ThermoHaake RS1 of chitosan is the flavonoid catechin (Table 1). Catechinrheometer using a parallel plate (PP60Ti) at a gap of 1 mm. Oscillatory can be oxidized by tyrosinase to its o-quinone, which cantests were performed with a controlled stress of 0.5 Pa at frequency of

then be grafted onto chitosan. After precipitation and0.1 Hz.washing, the catechin-modified chitosan was dissolvedat differing concentrations under acidic conditions. Theviscosity of these solutions was observed to increasematerials should be biodegradable. Biodegradability ismarkedly with concentration [18]—a behavior character-indicated in Fig. 4, which shows the arbutin-chitosanistic of associative thickeners. Associative thickeninggel can be readily broken by the chitosan-hydrolyzingresults when a water-soluble polymer has a small numberenzyme, chitosanase. Mass spectrometry analysis of anof moieties that can interact with each other to form aanalogous enzymatically modified chitosan showed thatphysically crosslinked network. Such networks are inter-low-molecular-weight sugar monomers and oligomersesting because at low shear they behave as gels but atwere formed by chitosanase treatment [18]. The observa-high shear the crosslinks are broken and the materialflows. After the shear is removed, the network can reform.The associative thickening properties of catechin-modi-fied chitosan suggest that the catechin moieties can asso-ciate with each other, although the molecular details ofsuch an association are currently unknown.

Using insect sclerotization as a model, we also exam-ined reactions with the tyrosine derivative 3,4-dihydroxy-phenethylamine (dopamine). As in the previouslydescribed studies, reactions were conducted under homo-geneous conditions with tyrosinase and dopamine beingincubated in chitosan solutions. During the course of thereaction, we observed browning of the solution and gelformation. As indicated in Table 1, these gels wereobserved to offer adhesive properties even in the presenceof water [22]. More recent studies suggest that althoughdopamine offers water-resistant adhesive properties, other

Fig. 4. Biodegradability of arbutin–chitosan gels. Gels were prepared phenolic substrates may offer superior performance.by incubating chitosan (0.5%), arbutin (5 mM), and tyrosinase (60 U/ Finally, Table 1 shows that heterogeneous reactionsml) for 24 hr. Then, chitosanase (1 U) was added the gel (15 ml)

can be used to modify the surface properties of a chitosanand incubated at room temperature. The viscosity was monitored as afilm. Specifically, we examined a series of gallic acid-function of time using a Brookfield viscometer at 1 rpm. Data are

shown for three separate experiments. derived esters. Gallic acids can be readily obtained from

82 Aberg, Chen, and Payne

Table I. Structures of Phenols and Functionalities of Chitosan Derivatives

Phenolic precursor Structure Functionality conferred

Arbutin Gel

Chlorogenic acid Base solubility

Catechin Associative thickening

3,4-Dihydroxyphenethylamine (dopamine) Water-resistant adhesion

Gallate esters (R 5 methyl, propyl, octyl) Surface hydrophobicity

plant tannins, and gallate esters can be created to offer a residues [24, 25]. For instance, Scheme 3 indicated thattyrosinase’s role in the setting of mussel “glue” is believedrange of functionalities depending on the ester substitu-

ent. Importantly, gallate esters such as propyl gallate are to be due to its ability to oxidize phenolic residues (e.g.,expected to be safe as they are currently used as foodantioxidants. To create a hydrophobically modified chito-san surface, we reacted octyl gallate with tyrosinase inan ethanol–water (30:70) mixture. The product(s) of theseenzymatic reactions (e.g., the o-quinones) were observedto react with and confer hydrophobic properties to thechitosan surface [23].

TYROSINASE-CATALYZED REACTIONSTO CREATE PROTEIN–CHITOSANCONJUGATES

As illustrated above, tyrosinases can react with abroad range of phenolic substrates. This substrate rangeis not limited to low-molecular-weight phenols because

Scheme 3. Tyrosinase-Catalyzed Oxidation of Proteins.tyrosinase is also able to oxidize polymers with phenolic

Renewable Resources and Enzymatic Processes to Create Functional Polymers 83

tyrosine or dihydroxyphenylalanine residues) of the mus-sel’s adhesive protein [26–28]. The oxidized quinoneresidues then undergo nonenzymatic, crosslinking reac-tions, yielding a three-dimensional gel network that con-fers cohesive strength.

For a protein to undergo tyrosinase-catalyzed oxida-tion, it must have tyrosine residues that are accessible.The mussel’s adhesive protein is reported to be rich inphenolic residues and to have an open chain structure[29]. In contrast, many proteins have a compact globularstructure, and we observed that globular proteins are notreadily oxidized by tyrosinase (at least not the handful ofglobular proteins we have studied). One readily availableprotein that does have an open chain structure is gelatin.Gelatin is well known for its repeating tripeptide sequence(Gly-X-Y), where Gly is a glycine, X is commonly pro- Fig. 5. Tyrosinase-catalyzed gel formation of the gelatin–chitosan mix-

ture. Experiments were performed at 358C using a Brookfield viscometerline, and Y is commonly hydroxyproline. This tripeptidewith a rotational speed of 1 rpm and an S34 spindle. All samplesrepeat enables gelatin to undergo a transition between acontained gelatin (3%) and chitosan (0.5%) and tyrosinase (60 U/ml)random coil and a triple helix. This coil-to-triple helixwere added as indicated in the figure.

transition is responsible for the reversible gel-formingabilities characteristic of gelatin. Interestingly, tyrosineresidues are not present in this repeat sequence, but a A second observation about tyrosinase-catalyzed

gelatin–chitosan gels is that they could be destroyed bysmall number of tyrosine residues are present in gelatin’stelopeptide region [30, 31]. the chitosan-hydrolyzing enzyme chitosanase. Specifi-

cally, we observed that when tyrosinase-catalyzed gelsScheme 3 shows that we examined whether tyrosi-nase could be used to create graft co-polymers of gelatin were incubated with chitosanase at temperatures above

gelatin’s gel point, then the gels were rapidly convertedand chitosan. For this, we dissolved gelatin above its gelpoint temperature, blended it with chitosan, and then into a solution. This observation demonstrates the impor-

tance of chitosan to the gel network. [32].incubated this solution with tyrosinase. During the reac-tion, we observed that the solution changed from colorless The third observation about the tyrosinase-catalyzed

gelatin–chitosan gels is that they are transient. Specifi-to pink, and the solution was converted to a gel [32].This is illustrated in Fig. 5 by the dramatic increase in cally, we observed that these gels break over the course

of hours, days, or weeks depending on the conditionsviscosity observed after 20 min of incubation. Interest-ingly, a control in which gelatin (but not chitosan) was (e.g., concentrations and temperature). We do not yet

understand the macromolecular architecture of the tyrosi-incubated with tyrosinase was observed to change color(indicative of tyrosinase-catalyzed oxidation), although nase-catalyzed gelatin–chitosan gels, so the mechanism

for gel breaking is unknown and currently under investi-the increase in viscosity was small.From more extensive rheological studies, we gation. Nevertheless, it is possible to speculate that if the

gel-breaking process can be controlled, then these gelsobserved three interesting features of the tyrosinase-cata-lyzed gelatin–chitosan gels. First, the strength of these may be suitable for time-release applications.

At a broader level, we believe the ability to enzymat-gels (i.e., G8) increased as the temperature was loweredbelow gelatin’s gel point and then G8 decreased as the ically couple proteins to the polysaccharide chitosan may

be important for a couple of reasons. First, proteins aretemperature was raised above gelatin’s melting tempera-ture. This suggests that tyrosinase-catalyzed reactions do readily available byproducts from agriculture and food

processing, and protein–chitosan conjugates may providenot destroy gelatin’s ability to undergo the coil-to-triplehelix transition. Additionally, the mild heating that is a range of environmentally friendly materials (e.g., biode-

gradable packaging materials). Second, protein–poly-capable of melting gelatin’s triple helix did not convertthe tyrosinase-catalyzed gels into a solution (i.e., although saccharide conjugates commonly confer important visco-

elastic properties to natural materials. Examples includeheating decreased G8, this modulus did not decreasebelow G9) [32]. This suggests that tyrosinase-catalyzed peptidoglycans of bacterial cell walls, proteoglycans of

connective tissue, and mucins of mucous membranes. Thereactions lead to a network that is different from thecharacteristic triple helix network of gelatin. ability to employ tyrosinase to create protein–chitosan

84 Aberg, Chen, and Payne

ference, National Academy of Sciences, Washington, DC, pp.conjugates suggests the potential to create water-soluble115–117.

polymers with useful rheological properties. One limita- 2. C. J. Brine and P. R. Austin (1981) Comp. Biochem. Physiol.tion to the development of protein–chitosan-based prod- 69B, 283–286.

3. N. K. Mathur and C. K. Narang (1990) J. Chem. Edu. 67, 938–942.ucts is the difficulty in predicting how the properties4. S. M. Hudson and C. Smith (1998) in D. L. Kaplan (Ed.), Biopoly-will vary with composition and processing conditions.

mers from Renewable Resources, Springer Press, Berlin, pp. 96–To overcome this limitation, we are also developing com- 118.

5. H. K. No and S. P. Meyers (1995) J. Aquat. Food Prod. Technol.binatorial methods to efficiently screen for mechanical4, 27–50.[33] and biological [34] properties.

6. N. N. Win and W. F. Stevens (2001) Appl. Microbiol. Biotechnol.57, 334–341.

7. A. Domard, (1987) Int. J. Biol. Macromol. 9, 98–103.CONCLUSIONS8. M. Rinaudo, G. Pavlov, and J. Desbrieres (1999) Polymer 40, 7029–

7032.Food chemistry and food processing provide a rich9. R. Vazquez-Duhalt, R. Tinoco, P. D’Antonio, L. D. T. Topoleski,

source of ideas of how to exploit renewable resources, and G. F. Payne. (2001) Bioconjugate Chemistry 12, 301–306.10. J. Xu, S. P. McCarthy, R. A. Gross, and D. L. Kaplan (1996)employ “green” chemistries, and create environmentally

Macromolecules 29, 3436–3440.friendly products. Here we have reviewed our efforts11. J. V. Gruber, V. Rutar, J. Bandekar, and P. N. Konish (1995) Macro-

to use the enzyme responsible for food browning (i.e., molecules 28, 8865–8867.tyrosinase) to create functional derivatives of the biopoly- 12. A. J. Parr and G. P. Bolwell (2000) J. Sci. Food Agricul. 80, 985–

1012.mer chitosan. Tyrosinases convert a broad range of pheno-13. M. N. Clifford (2000) J. Sci. Food Agric. 80, 1126–1137.lic substrates into reactive o-quinones that can be 14. A. Schieber, P. Keller, and R. Carle (2001) J. Chrom. A 910, 265–

subsequently grafted onto chitosan. To create chitosan 273.15. M. Sugumaran (1988) Adv. Insect. Physiol. 21, 179–231.derivatives with useful functional properties, we selected16. M. G. Peter (1989) Angew. Chem. Int. Ed. Engl. 28, 555–570.from the diverse array of low-molecular-weight phenols17. S. O. Andersen, M. G. Peter, and P. Roepstorff (1996) Comp.

present in nature—particularly phenols common in foods. Biochem. Physiol. 113B, 689–705.18. L.-Q. Wu, H. D. Embree, B. M. Balgley, P. J. Smith, and G. F.The potential of this enzymatic approach is illustrated in

Payne (2002) Environ. Sci. Technol. 36, 3446–3454.Table 1, which shows that chitosan derivatives with vari-19. W. G. Glasser, B. K. McCartney, and G. Samaranayake (1994)ous functional properties have been generated. Biotechnol. Progr. 10, 214–219.

The ability of tyrosinase to react with tyrosine resi- 20. M. N. Clifford (2000) J. Sci. Food Agric. 80, 1033–1043.21. G. Kumar, P. J. Smith, and G. F. Payne (1999) Biotechnol. Bioeng.dues of proteins provides the opportunity to create pro-

63, 154–165.tein–polysaccharide conjugates. Such conjugates may be22. K. Yamada, T. Chen, G. Kumar, O. Vesnovsky, L. D. T. Topoleski,

useful for numerous applications, ranging from edible and G. F. Payne (2000) Biomacromolecules 1, 252–258.23. C. J. Govar, T. Chen, N.-C. Liu, M. T. Harris, and G. F. Paynepackaging to medical materials. Although much needs to

(2001) in H. N. Chen and R. A. Gross (Eds.), Biocatalysis: Frontiersbe done to characterize the chemistry and physics of thesein Polymer Reactions, in press.

conjugates, the observed properties and the expected 24. K. Marumo and J. H. Waite (1986) Biochim. Biophys. Acta. 872, 98–safety of these materials make them exciting candidates 103.

25. L. Shao, G. Kumar, J. L. Lenhart, P. J. Smith, and G. F. Paynefor further work.(1999) Enzyme Microb. Technol. 25, 660–668.

26. J. H. Waite, (1990) Comp. Biochem. Physiol. 97B, 19–29.27. M. Yu, J. Hwang, and T. J. Deming (1999) J. Am. Chem. Soc.ACKNOWLEDGMENTS

121, 5825–5826.28. L. A. Burzio and J. H. Waite (2000) Biochemistry 39, 11147–11153.Financial support for this research was provided by29. M. P. Deacon, S. S. Davis, J. H. Waite, and S. E. Harding (1998)

the United States Department of Agriculture (2001- Biochemistry 37, 14108–14112.35504-10667) and the National Science Foundation 30. K. H. Mayo (1996) Biopolymers 40, 359–370.

31. G. King, E. M. Brown, and J. M. Chen (1996) Protein Eng. 9, 43–49.(grant BES-0114790).32. T. Chen, H. D. Embree, L.-Q. Wu, and G. F. Payne (2002) Biopoly-

mers, 64, 292–302.33. H. D. Embree, T. Chen, R. Vazquez-Duhalt, L. D. T. Topoleski,REFERENCES and G. F. Payne (2002), submitted for publication.34. T. Chen, R. Vazquez-Duhalt, C.-F. Wu, W. E. B. Bentley, and G.1. C. P. Condon, J. Cowan, D. Healey, and G. F. Payne. (2001)

Proceedings of 4th Annual Green Chemistry and Engineering Con- F. Payne (2001). Biomacromolecules 2, 456–462.