Journal of Polymers and the Environment, Vol. 10, No.

3, July 2002 ( 2002)

Renewable Resources and Enzymatic Processes to Create

Functional Polymers: Adapting Materials and Reactions
from 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, environmentally
friendly derivatives of the biopolymer chitosan, a byproduct of seafood processing. Functional
groups are grafted onto chitosan using tyrosinase, the enzyme responsible for food browning. The
functionalizing groups studied include low-molecular-weight phenols derived from natural sources
and high-molecular-weight proteins. The approach of using low-molecular-weight phenols to functionalize chitosan is illustrated with arbutin, a natural phenol found in pears. Results demonstrate
that tyrosinase initiates reactions that lead to the conversion of arbutinchitosan 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 studies
in which low-molecular-weight natural phenols are enzymatically grafted onto chitosan to confer
functional properties. The creation of co-polymers is illustrated by results in which tyrosinase is
used to couple gelatin onto chitosan. Gelatin is a proteinaceous byproduct of meat production. The
tyrosinase-generated gelatinchitosan conjugates have been observed to offer interesting rheological
and thermal properties. These results demonstrate the potential for using renewable resources and
enzymatic 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 develop

products that are biodegradable. To meet these goals a
variety of approaches have been advocated, including
the integration of biotechnology into polymer synthesis
(e.g., polyhydroxyalkanoates). We believe there is a
substantial opportunity to employ materials and processing concepts from the food industry to meet these
goals. Specifically, the food industry has a long history
of exploiting renewable resources, manufacturing products using safe chemistries, and creating products that
have no adverse environmental impacts. Here, we
review our efforts to create functional polymers by
adapting materials and reactions common to the food
industry.

Three emerging goals for polymer manufacturing

are: to better utilize renewable resources as a source of
raw materialseither monomers (e.g., lactic acid and
1,3-propanediol) or polymers (e.g., proteins and polysaccharides); to employ environmentally friendly chem1

Center for Biosystems Research, 5115 Plant Sciences Building, University of Maryland Biotechnology Institute, College Park, Maryland 20742.
2
Department of Chemical and Biochemical Engineering, University of
Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250.
3
To whom all correspondence should be addressed. E-mail: payne@
umbi.umd.edu

77
1566-2543/02/0700-0077/0 2002 Plenum Publishing Corporation

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Aberg, Chen, and Payne

RECOVERING MATERIALS FROM

UNDER-UTILIZED RESOURCES
A common challenge to utilizing renewable resources is that the individual components may be present
at low levels. Additionally, these components are often
incorporated within a complex network (e.g., the cellulose
of plants exists within a complex lignocellulosic matrix).
Recovery of the individual components from these matrices can be challenging. Nevertheless, the economic success of many natural resource-based industries relies on
the creation of value-added byproducts from the various
fractions present in the raw material. For instance, corn
processors generate numerous commercial byproducts
from the oil, carbohydrate, and protein fractions. Similarly, meat producers generate substantial quantities of
the gelatin byproduct. Even lignin wastes generated
from the pulp and paper industry are used as a source
of fuel.
It is obvious that value-added byproducts offer
opportunities for increased revenues. We believe a less
obvious motivation for byproduct development is also
emerging. Over time, economies of scale have led to
high-intensity farming practices and large-scale food
processing operations. The scale of these operations leads
to the generation of nutrient-rich wastes at quantities that
pose environmental problems. In these cases, value-added
byproducts may not only provide an opportunity for additional income but may provide the best option for waste
management. This is illustrated by the following example.
In the state of Maryland, the Chesapeake Bay is a
major aesthetic and economic asset. Over the last decade
there have been increasing concerns that excess nutrient
addition is jeopardizing the health of the bay. One result of
these concerns is that a governmentindustryuniversity
partnership was established to explore alternatives to the
landfilling of wastes generated from the states crabpacking industry. This partnership succeeded in establishing a manufacturing plant to convert the wastes into a
value-added biopolymer, chitosan [1]. Chitosan is derived
from chitin, and currently chitosan is sold as a dietary
aid for individuals trying to lose weight. Glucosamine,
the monomeric unit of chitosan, is also marketed (in
conjunction with chondroitin) as a dietary supplement for
arthritis sufferers.
Chitosan is derived from chitin that exists in the
exoskeleton of crustaceans at levels of 10 to 25% (dry
basis) [24]. Chitin is also present in insects and fungal
cell walls and is considered the second (or third) most
abundant natural material after cellulose (and possibly
lignin). Scheme 1 shows a typical operation for converting crustacean wastes into chitosan. The wastes are

Scheme 1. Chitosan Production.

demineralized, typically utilizing HCl, and deproteinated

with NaOH to yield the chitin intermediate [35]. Deacetylation of chitin to chitosan is typically achieved using
strong base (4060% NaOH) and moderately high temperatures (100150C). As indicated by the dashed arrow
in Scheme 1, the difficulty of chemical deacetylation has
stimulated interest in the discovery and development of
deacetylating enzymes [6].
As noted, chitosan and its monomer glucosamine
are manufactured as dietary supplements for human consumption. We believe an industrial grade of chitosan
could find broader applications not only because it is
inherently safe but because it offers unique physicochemical properties. Specifically, chitosan is a basic polysaccharide with primary amino groups in nearly every
repeating unit (because deacetylation is incomplete, chitosan is formally a co-polymer of glucosamine and Nacetylglucosamine). Chitosans primary amino groups
have a pKa of about 6.3 [4, 7, 8]. Below the pKa, the
amino groups are protonated, making chitosan a cationic
polyelectrolyte that is water soluble; water solubility is
uncommon for -1,4-linked polysaccharides (e.g., cellulose and chitin). Above the pKa, chitosans amino groups
become deprotonated and the polymer is no longer water
soluble. This pH-dependent solubility allows chitosan to
be formed into various shapes (e.g., beads, films, and

Renewable Resources and Enzymatic Processes to Create Functional Polymers

Scheme 2. Processes Initiated by Tyrosinase.

membranes) using aqueous processing. This pH-dependent solubility also confers pH-responsive properties to
chitosan [9].

In addition to conferring basic properties to chitosan,

the amino groups also confer nucleophilic properties to
this polymer. Specifically, the deprotonated amino groups
can undergo reaction with a variety of reagents (e.g.,
aldehydes, anhydrides, and acid chlorides) under relatively mild conditions. Because the amino groups are
considerably more reactive than chitosans hydroxyl
groups, modification can be regioselective in the sense
that reactions performed under mild conditions lead to
modification exclusively at the amino group (i.e., the 2position of the repeating sugar residue) [10, 11]. In contrast, cellulose must be reacted under harsh conditions
and, under such conditions, modification occurs nonselectively on all three of the hydroxyl groups (i.e., at the
2-, 3-, and 6-positions). In our studies, we are using an
enzymatic reaction to generate an electrophile for subsequent reaction with chitosans amino groups.
EXPLOITING AN ENZYMATIC REACTION
COMMON IN FOODS
To generate electrophiles capable of reacting with
chitosans amino group, we are utilizing the oxidative
enzyme tyrosinase (enzymes with similar activities are
also known as phenol oxidases or polyphenol oxidases).
These enzymes are ubiquitous in nature and are responsible for the familiar browning of foods. As illustrated in
Scheme 2, enzymatic browning occurs when the plant
tissue is disrupted and the oxidative enzymes come in
contact with the plants phenolic substrates. Oxidation
leads to the generation of o-quinones that diffuse away
from the enzymes active site. These quinones are reactive
and undergo a variety of reactions that lead to the formation of colored oligomeric phenols. As will be discussed,

79

Scheme 2 shows that tyrosinase-catalyzed reactions are

also believed to be responsible for insect sclerotization
(i.e., the hardening of insect shells) and the setting of
mussel glue.
The dashed line in Scheme 2 indicates that we are
examining tyrosinase-catalyzed reactions for the functionalization of chitosan. Initial studies showed that tyrosinase-generated quinones reacted with chitosan and that
the resulting chitosan derivatives had dramatically altered
functional properties. Not surprisingly, the specific functional properties of the chitosan derivatives depended
on the phenolic substrate selected for reaction. We have
explored a variety of phenolic substrates to examine the
range of properties that can be conferred to chitosan. Our
focus has been on natural phenols and, whenever possible,
on phenolic compounds common in foods.
TYROSINASE-CATALYZED REACTIONS
WITH LOW-MOLECULAR-WEIGHT
PHENOLS
Tyrosinases react with a broad range of phenolic
substrates and a diverse array of natural phenols is present
in foods [12]. Interestingly, the tyrosinase from mushroom is active over a pH range that also spans the region
where chitosan can be either soluble or insoluble. Thus,
enzymatic reactions can be conducted under either homogeneous conditions (i.e., chitosan, the enzyme, and the
phenolic substrate are all soluble) or heterogeneous conditions (i.e., the enzyme and phenolic substrate are in solution while chitosan is present as an insoluble film) as a
result of chitosans pH-dependent solubility.
The ability of tyrosinase-catalyzed reactions to dramatically alter the properties of chitosan is illustrated by
results with arbutin. Arbutin is a natural phenol common
in pears [13, 14]. In our studies, we performed homogeneous reactions by mixing arbutin and tyrosinase with a
chitosan solution at a pH of 5.8 to 6. This pH is just
sufficient to maintain chitosan in solution. During the
course of the reaction, we observed the solution change
from colorless to reddish and then to brown/black. By
the time the solution had become black, we also visually
observed that it had formed a gel. Gel formation is illustrated by Fig. 1, which shows a dramatic increase in
viscosity after about 7 hr of reaction.
After reaction, the mechanical spectrum of the gel
was measured using rheometry. Figure 2 shows that for
the sample reacted with tyrosinase, the elastic modulus
(G) was greater than the viscous modulus (G), both
moduli were independent of frequency (), and the complex viscosity (*) decreased with increasing . These

80

Aberg, Chen, and Payne

Fig. 1. Tyrosinase-catalyzed gel formation. Homogeneous reactions

were performed in solutions of chitosan (0.5%, equivalent to 31 mM
repeating units), arbutin (5mM), and tyrosinase (60 U/ml) at room
temperature. The control solution was incubated without tyrosinase.
The viscosity was measured using a Brookfield viscometer at a rotational
speed 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 had
been incubated in the absence of tyrosinase behaved as
a solution with G exceeding G, both moduli increasing
with , and * being independent of .
A final experiment was conducted in which chitosan
and tyrosinase were incubated with varying concentrations of arbutin. When low levels of arbutin were used,
the solutions were observed to change from colorless to
pink while only small increases in viscosity were
observed (data not shown). Higher levels of arbutin led
to the formation of dark black gels. Figure 3 shows the
strength of these gels (i.e., the elastic modulus, G)
increased monotonically with arbutin concentration.
These results indicate that the properties of the enzymatically generated gels can be controlled based on the reaction conditions.
The observation that tyrosinase-catalyzed reactions
lead to gel formation may not be surprising in light of
the putative role of tyrosinase in insect sclerotization. In
sclerotization, it is believed that tyrosinase converts lowmolecular-weight phenolic compounds (typically derivatives of tyrosine) into quinones [1517]. The quinones
then undergo reactions with proteins in the insects integument, leading to hardening of the shell. These reactions
are often referred to as quinone tanning, and although
the details of the reaction remain controversial, it appears
that these reactions may lead to a crosslinked network.
Similarly, the reactions of arbutin may also lead to a

Fig. 2. The mechanical spectrum of the conjugated arbutinchitosan

gel. Gels were prepared by reacting chitosan (0.5%), arbutin (5 mM),
and tyrosinase (60 U/ml) for 24 hr at room temperature. The sample
incubated with tyrosinase formed a gel, which was measured using a
controlled strain of 5 1%. The control incubated without tyrosinase
was a solution and it was measured at a controlled stress of 0.5 Pa.
The frequency range used to examine this solution was limited in
order to remain within the linear viscoelastic region. All samples were
measured using a ThermoHakke RS1 with a parallel plate sensor
(PP60Ti) at a gap of 1 mm.

crosslinked chitosan network, although chemical evidence for covalent crosslinking has been difficult to
attain.
As suggested above, the chemistry of the quinone
tanning reactions is complex and poorly understood.
Nevertheless, the facts that the starting materials (chitosan
and arbutin) are natural and the reactions are mediated
by an enzyme common in nature suggest that the final

Renewable Resources and Enzymatic Processes to Create Functional Polymers

Fig. 3. The effect of arbutin concentration on the rheological properties

of arbutinchitosan gels. Chitosan (0.5%), arbutin, and tyrosinase (60
U/ml) were incubated for 24 hr prior to measurement. The elastic
modulus (G) of each sample was measured with a ThermoHaake RS1
rheometer using a parallel plate (PP60Ti) at a gap of 1 mm. Oscillatory
tests were performed with a controlled stress of 0.5 Pa at frequency of
0.1 Hz.

materials should be biodegradable. Biodegradability is

indicated in Fig. 4, which shows the arbutin-chitosan
gel can be readily broken by the chitosan-hydrolyzing
enzyme, chitosanase. Mass spectrometry analysis of an
analogous enzymatically modified chitosan showed that
low-molecular-weight sugar monomers and oligomers
were formed by chitosanase treatment [18]. The observa-

Fig. 4. Biodegradability of arbutinchitosan gels. Gels were prepared

by incubating chitosan (0.5%), arbutin (5 mM), and tyrosinase (60 U/
ml) for 24 hr. Then, chitosanase (1 U) was added the gel (15 ml)
and incubated at room temperature. The viscosity was monitored as a
function of time using a Brookfield viscometer at 1 rpm. Data are
shown for three separate experiments.

81

tion that chitosan derivatives remain biodegradable is

noteworthy in light of observations that highly substituted
cellulose derivatives are less biodegradable [19].
The ability to modify chitosan with tyrosinase is not
limited to the arbutin substrate. Table 1 lists several other
natural phenols that have been enzymatically grafted to
chitosan. Chlorogenic acid is found in high concentrations
in coffee [20]; however its name is a misnomer, because
there is no chlorine in chlorogenic acid. This phenol is
rapidly oxidized by tyrosinase and the product can be
grafted onto chitosan. After precipitating and washing
the chlorogenic-acid-modified chitosan, we observed that
this chitosan derivative is soluble under acidic and basic
conditions but is insoluble under near-neutral pHs [21].
Presumably, the carboxylate and hydroxyl groups of the
quinic acid moiety of chlorogenic acid confer base solubility to the chlorogenic-acid-modified chitosan.
Another phenolic substrate used for the modification
of chitosan is the flavonoid catechin (Table 1). Catechin
can be oxidized by tyrosinase to its o-quinone, which can
then be grafted onto chitosan. After precipitation and
washing, the catechin-modified chitosan was dissolved
at differing concentrations under acidic conditions. The
viscosity of these solutions was observed to increase
markedly with concentration [18]a behavior characteristic of associative thickeners. Associative thickening
results when a water-soluble polymer has a small number
of moieties that can interact with each other to form a
physically crosslinked network. Such networks are interesting because at low shear they behave as gels but at
high shear the crosslinks are broken and the material
flows. After the shear is removed, the network can reform.
The associative thickening properties of catechin-modified chitosan suggest that the catechin moieties can associate with each other, although the molecular details of
such an association are currently unknown.
Using insect sclerotization as a model, we also examined reactions with the tyrosine derivative 3,4-dihydroxyphenethylamine (dopamine). As in the previously
described studies, reactions were conducted under homogeneous conditions with tyrosinase and dopamine being
incubated in chitosan solutions. During the course of the
reaction, we observed browning of the solution and gel
formation. As indicated in Table 1, these gels were
observed to offer adhesive properties even in the presence
of water [22]. More recent studies suggest that although
dopamine offers water-resistant adhesive properties, other
phenolic substrates may offer superior performance.
Finally, Table 1 shows that heterogeneous reactions
can be used to modify the surface properties of a chitosan
film. Specifically, we examined a series of gallic acidderived esters. Gallic acids can be readily obtained from

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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 methyl, propyl, octyl)

Surface hydrophobicity

plant tannins, and gallate esters can be created to offer a

range of functionalities depending on the ester substituent. Importantly, gallate esters such as propyl gallate are
expected to be safe as they are currently used as food
antioxidants. To create a hydrophobically modified chitosan surface, we reacted octyl gallate with tyrosinase in
an ethanolwater (30:70) mixture. The product(s) of these
enzymatic reactions (e.g., the o-quinones) were observed
to react with and confer hydrophobic properties to the
chitosan surface [23].

residues [24, 25]. For instance, Scheme 3 indicated that

tyrosinases role in the setting of mussel glue is believed
to be due to its ability to oxidize phenolic residues (e.g.,

TYROSINASE-CATALYZED REACTIONS
TO CREATE PROTEINCHITOSAN
CONJUGATES
As illustrated above, tyrosinases can react with a
broad range of phenolic substrates. This substrate range
is not limited to low-molecular-weight phenols because
tyrosinase is also able to oxidize polymers with phenolic

Scheme 3. Tyrosinase-Catalyzed Oxidation of Proteins.

Renewable Resources and Enzymatic Processes to Create Functional Polymers

tyrosine or dihydroxyphenylalanine residues) of the mussels adhesive protein [2628]. The oxidized quinone
residues then undergo nonenzymatic, crosslinking reactions, yielding a three-dimensional gel network that confers cohesive strength.
For a protein to undergo tyrosinase-catalyzed oxidation, it must have tyrosine residues that are accessible.
The mussels adhesive protein is reported to be rich in
phenolic residues and to have an open chain structure
[29]. In contrast, many proteins have a compact globular
structure, and we observed that globular proteins are not
readily oxidized by tyrosinase (at least not the handful of
globular proteins we have studied). One readily available
protein 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 proline, and Y is commonly hydroxyproline. This tripeptide
repeat enables gelatin to undergo a transition between a
random coil and a triple helix. This coil-to-triple helix
transition is responsible for the reversible gel-forming
abilities characteristic of gelatin. Interestingly, tyrosine
residues are not present in this repeat sequence, but a
small number of tyrosine residues are present in gelatins
telopeptide region [30, 31].
Scheme 3 shows that we examined whether tyrosinase could be used to create graft co-polymers of gelatin
and chitosan. For this, we dissolved gelatin above its gel
point temperature, blended it with chitosan, and then
incubated this solution with tyrosinase. During the reaction, we observed that the solution changed from colorless
to pink, and the solution was converted to a gel [32].
This is illustrated in Fig. 5 by the dramatic increase in
viscosity observed after 20 min of incubation. Interestingly, a control in which gelatin (but not chitosan) was
incubated with tyrosinase was observed to change color
(indicative of tyrosinase-catalyzed oxidation), although
the increase in viscosity was small.
From more extensive rheological studies, we
observed three interesting features of the tyrosinase-catalyzed gelatinchitosan gels. First, the strength of these
gels (i.e., G) increased as the temperature was lowered
below gelatins gel point and then G decreased as the
temperature was raised above gelatins melting temperature. This suggests that tyrosinase-catalyzed reactions do
not destroy gelatins ability to undergo the coil-to-triple
helix transition. Additionally, the mild heating that is
capable of melting gelatins triple helix did not convert
the tyrosinase-catalyzed gels into a solution (i.e., although
heating decreased G, this modulus did not decrease
below G) [32]. This suggests that tyrosinase-catalyzed
reactions lead to a network that is different from the
characteristic triple helix network of gelatin.

83

Fig. 5. Tyrosinase-catalyzed gel formation of the gelatinchitosan mixture. Experiments were performed at 35C using a Brookfield viscometer
with a rotational speed of 1 rpm and an S34 spindle. All samples
contained gelatin (3%) and chitosan (0.5%) and tyrosinase (60 U/ml)
were added as indicated in the figure.

A second observation about tyrosinase-catalyzed

gelatinchitosan gels is that they could be destroyed by
the chitosan-hydrolyzing enzyme chitosanase. Specifically, we observed that when tyrosinase-catalyzed gels
were incubated with chitosanase at temperatures above
gelatins gel point, then the gels were rapidly converted
into a solution. This observation demonstrates the importance of chitosan to the gel network. [32].
The third observation about the tyrosinase-catalyzed
gelatinchitosan gels is that they are transient. Specifically, we observed that these gels break over the course
of hours, days, or weeks depending on the conditions
(e.g., concentrations and temperature). We do not yet
understand the macromolecular architecture of the tyrosinase-catalyzed gelatinchitosan gels, so the mechanism
for gel breaking is unknown and currently under investigation. Nevertheless, it is possible to speculate that if the
gel-breaking process can be controlled, then these gels
may be suitable for time-release applications.
At a broader level, we believe the ability to enzymatically couple proteins to the polysaccharide chitosan may
be important for a couple of reasons. First, proteins are
readily available byproducts from agriculture and food
processing, and proteinchitosan conjugates may provide
a range of environmentally friendly materials (e.g., biodegradable packaging materials). Second, proteinpolysaccharide conjugates commonly confer important viscoelastic properties to natural materials. Examples include
peptidoglycans of bacterial cell walls, proteoglycans of
connective tissue, and mucins of mucous membranes. The
ability to employ tyrosinase to create proteinchitosan

84
conjugates suggests the potential to create water-soluble
polymers with useful rheological properties. One limitation to the development of proteinchitosan-based products is the difficulty in predicting how the properties
will vary with composition and processing conditions.
To overcome this limitation, we are also developing combinatorial methods to efficiently screen for mechanical
[33] and biological [34] properties.
CONCLUSIONS
Food chemistry and food processing provide a rich
source of ideas of how to exploit renewable resources,
employ green chemistries, and create environmentally
friendly products. Here we have reviewed our efforts
to use the enzyme responsible for food browning (i.e.,
tyrosinase) to create functional derivatives of the biopolymer chitosan. Tyrosinases convert a broad range of phenolic substrates into reactive o-quinones that can be
subsequently grafted onto chitosan. To create chitosan
derivatives with useful functional properties, we selected
from the diverse array of low-molecular-weight phenols
present in natureparticularly phenols common in foods.
The potential of this enzymatic approach is illustrated in
Table 1, which shows that chitosan derivatives with various functional properties have been generated.
The ability of tyrosinase to react with tyrosine residues of proteins provides the opportunity to create proteinpolysaccharide conjugates. Such conjugates may be
useful for numerous applications, ranging from edible
packaging to medical materials. Although much needs to
be done to characterize the chemistry and physics of these
conjugates, the observed properties and the expected
safety of these materials make them exciting candidates
for further work.
ACKNOWLEDGMENTS
Financial support for this research was provided by
the United States Department of Agriculture (200135504-10667) and the National Science Foundation
(grant BES-0114790).
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