Enzymatic Polymerization

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Introduction

Enzymes catalyze not only all in vivo biosynthetic reactions in living cells for main-
taining “life” but also many in vitro reactions of natural and unnatural substrates
under selected reaction conditions. Enzymatic catalysis for organic synthesis pos-
sesses advantages such as much acceleration of reaction rate, operation under
mild conditions, and high stereo-, regio-, and chemoselectivities of reactions in
comparison with those of chemical catalysts. Such characteristic properties have
brought about an extraordinarily rapid increase in interest in the area of biotrans-
formations (1–5).

All naturally occurring polymers are produced in vivo by enzymatic catalysis.

Recently, in vitro synthesis of polymers through enzymatic catalysis (“enzymatic
polymerization”) has been extensively studied (6–14); highly selective polymer-
izations catalyzed by enzymes have been developed to produce various functional
polymers in response to increasing demands of structural variation of synthetic
targets for polymers in material science.

This article deals with recent advances in enzymatic polymerizations. We

deﬁne enzymatic polymerization as “chemical polymer synthesis in vitro (in test
tubes) via nonbiosynthetic (nonmetabolic) pathways catalyzed by an isolated en-
zyme.” Enzymes are generally classiﬁed into six groups. Table 1 shows typical poly-
mers produced with catalysis by respective enzymes. The target macromolecules
for the enzymatic polymerization have been polysaccharides, poly(amino acid)s,
polyesters, polycarbonates, polyaromatics, vinyl polymers, etc. Here, enzymatic
polymerizations are described according to the polymer structure. In many cases,
enzymatic polymerization enables the synthesis of polymers, which otherwise are
difﬁcult to prepare. Enzymatic polymerization often provides an environmentally

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Table 1. Classiﬁcation of Enzymes and In Vitro Production of Typical
Polymers Catalyzed by Respective Enzymes

Enzymes

Typical polymers

Oxidoreductases

Polyphenols, polyanilines, vinyl polymers

Transferases

Polysaccharides, cyclic oligosaccharides, polyesters

Hydrolases

Polysaccharides, poly(amino acid)s, polyamides,

polyesters, polycarbonates

Lyases
Isomerases
Ligases

benign process, where starting materials and products are within the natural
material cycle; this is in the context of “green polymer chemistry” (13,14).

Polysaccharides

Polysaccharides are among the most important biopolymers as are proteins and
nucleic acids in nature. They are regarded as three important families of natu-
ral biomacromolecules. As to the enzymatic polymerization for polysaccharides,
hydrolases and transferases are reported to catalyze their synthetic reactions.

Hydrolases.

It is generally accepted that an enzymatic reaction is vir-

tually reversible, and hence, the equilibrium can be controlled by selecting the
reaction conditions. Based on this view, hydrolases, enzymes catalyzing a bond-
cleavage reaction by hydrolysis, have been developed as catalyst for the reverse
reaction of hydrolysis, leading to polymer production by a bond-forming reaction
(9,12).

It is believed that using a glycosidase for the glycosylation process is one of

the most promising methodologies for selective construction of a glycosidic link-
age under appropriate conditions, since chemical approach requires complicated
procedures including a regioselective blocking and deblocking of a hydroxy group
in the sugar moiety to achieve regioselectivity, and furthermore, complete stere-
ocontrol of the glycoside bond-formation has not often been achieved by chemical
catalysts.

Enzymatic formation of a glycosidic bond is realized by combined use of a

glycosyl donor and a glycosyl acceptor. The former is to be activated by an enzyme
to give a glycosyl-enzyme intermediate which can be attacked by a hydroxy group
of the acceptor, forming a glycosidic bond between the donor and the acceptor. The
repeated glycosylations are expected to produce polysaccharide molecules.

Glycosyl ﬂuorides, sugar derivatives whose anomeric hydroxy group is re-

placed by a ﬂuorine atom, are known to be recognized by glycosidases. Cellulose
is one of the most important biomacromolecules, which is the most abundant or-
ganic substance on the earth (12). Thus, in 1991, the ﬁrst in vitro synthesis of
cellulose via nonbiosynthetic pathway has been achieved by an enzymatic poly-
merization of

β-cellobiosyl ﬂuoride as substrate for Tricoderma viride cellulase,

an extracellular hydrolytic enzyme of cellulose (Fig. 1) (15–21). The polymeriza-
tion was performed in an aqueous organic solvent in order to make the desired

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ENZYMATIC POLYMERIZATION

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Fig. 1.

In vitro synthesis of artiﬁcial cellulose via cellulase catalysis.

polycondensation predominant in comparison with the competitive hydrolysis re-
action. A mixed solvent of acetonitrile/acetate buffer (pH 5) (5:1) gave the best
results in terms of the yield of water-insoluble “artiﬁcial cellulose.” The enzyme
promoted transglycosylation of the cellobiosyl moiety toward the 4

-hydroxy group

of another monomer eliminating hydrogen ﬂuoride. In this polymerization, regio-
and stereoselectivities were perfectly controlled.

The cellulase-catalyzed polycondensation of new cellobiosyl ﬂuoride deriva-

tives, 6-O-methyl and 6

-O-methyl-

β-cellobiosyl ﬂuorides, have been examined

(22,23). The 6-O-methylated monomer was polymerized using the puriﬁed en-
zyme in a regio- and stereoselective manner to give a novel cellulose derivative
having a methyl group alternatingly at the 6-position, which can never be realized
by the conventional modiﬁcation of natural cellulose, ie, methylation of cellulose.
On the other hand, the 6

-O-methylated monomer gave a mixture of low molecular

weight oligomers. The difference of the polymerization behavior can be explained
by the steric repulsion between the methyl group of the monomers and the active
site of the cellulase catalyst.

The process of the artiﬁcial cellulose was visually analyzed by using trans-

mission electron microscopy (24). Cellulose formation was detected as early as
30 s after the initial stage of the reaction in the aqueous acetonitrile. The elec-
tron diffraction pattern of the product showed the typical pattern of the crys-
tal structure of thermodynamically stable cellulose II with antiparallel orien-
tation between each glucan chains. When the puriﬁed cellulase (39 kDa) was
used, cellulose microﬁbrils with an electron diffraction pattern characteristic of
metastable cellulose I with parallel orientation, an allomorph of natural cellu-
lose, were ﬁrst observed in an artiﬁcial process (25). Based on these results,
a new concept of choroselectivity, selectivity concerning the relative ordering
of the polymer chain direction, in polymerization chemistry has been proposed
(26–28).

In some cases, the enzymatic polymerization afforded spherulites of arti-

ﬁcial cellulose II, composed of single crystals with the molecular axis orientated
perpendicular to the plane (29). Both positive- and negative-type spherulites were
observed by polarization optical microscopy. By changing the reaction parameters,

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331

the size, growth and formation/degradation rate, and number of spherulites could
be controlled.

α-Amylase catalyzed the polycondensation of α-

-glucosyl ﬂuoride in an

aqueous solution to produce maltooligosaccharides (mainly pentamer) (30).

α-

-Maltosyl ﬂuoride was also polymerized by

α-amylase catalyst in an aqueous

methanol, yielding a maltooligosaccharide with degree of polymerization (DP) up
to 7 (31). Enzymatic transglycosylation of

α-

-maltosyl ﬂuoride with a cyclodextrin

using pullulanase or isoamylase as a catalyst produced a branched cyclodextrin,
6-O-

α-maltosylcyclodextrin (32,33).

Synthetic xylan was synthesized by a cellulase-catalyzed polymerization us-

ing

β-xylobiosyl ﬂuoride as a substrate (34). The enzymatic polymerization pro-

ceeded in a perfect regio- and stereoselective manner to produce powdery arti-
ﬁcial xylan, which is insoluble in any organic solvent. Xylan, one of the most
important components of hemicellulose in plant cell walls, normally contains 4-
O-methylglucuronic acid or

-arabinose as a minor unit in the side chain. On

the other hand, the artiﬁcial xylan consists exclusively of a xylopyranose moiety
connected through a

β(1→4) glycosidic bond.

The ﬁrst synthesis of a cellulose–xylan hybrid polymer, a novel polysaccha-

ride having a glucose–xylose repeating unit, has been achieved by the xylanase-
catalyzed polymerization of

β-xylopyranosyl-glucopyranosyl ﬂuoride (Fig. 2) (35,

36). Identiﬁcation of the enzyme fraction promoting the polymerization showed
that endoxylanase was highly efﬁcient for production of the hybrid polymer.

Cellulase-catalyzed polycondensation of 4-thio-

β-cellobiosyl ﬂuoride pro-

duced hemithiocellodextrins having 4-thiocellobiosyl repeating units linked by
β(1→4) oxygen linkages (37). A water-soluble oligomer with DP up to 20 was
obtained in an aqueous acetonitrile.

Chitin is the most abundant organic macromolecules in the animal ﬁeld

found in invertebrates (12). The in vitro synthesis of this important biomacro-
molecule has been achieved for the ﬁrst time by enzymatic ring-opening polyad-
dition of a chitobiose oxazoline monomer (Fig. 3). Chitinase, a hydrolysis enzyme
of chitin, regio- and stereoselectively induced the polymerization of the monomer
in a basic buffer (38–41). It is postulated that the monomer is preferable as a

Fig. 2.

Enzymatic synthesis of cellulose–xylan hybrid polymer.

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ENZYMATIC POLYMERIZATION

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Fig. 3.

In vitro synthesis of artiﬁcial chitin via chitinase catalysis.

substrate because it can be recognized by the active site of chitinase readily due
to the oxazoline structure resembling that of the transition state of the chitin hy-
drolysis with chitinase as revealed by a later work (12,42). Thus, the monomer
is regarded as a “transition state analogue substrate” for chitinase. From x-ray
diffraction and nmr analysis, the product was found to show crystal structure of
α-chitin. A oxazoline monomer from N-acetyl glucosamine was also polymerized
at high substrate concentration to give chitooligosaccharides.

The visualization of high ordered structure formation during the enzymatic

synthesis of artiﬁcial chitin has been investigated (43). Plate-like single crystals of
α-chitin were ﬁrst formed and gradually shaped into ribbons by the rapid growth
along the a axis with the crystalline thickness being ca 10 nm. The

α-chitin rib-

bons then aggregated to form bundle-like or dendritic assemblies as the ribbon
concentration in solution increased. They grew up to spherulites by splaying and
branching. This artiﬁcial chitin spherulite, in which a number of

α-chitin rib-

bons radiated from a common center, is completely different from the helicoidal
textures composed of

α-chitin microﬁbrils known as a typical three-dimensional

organization of chitin (see C

HITIN

and C

HITOSAN

A cellulose–chitin hybrid polymer, a nonnatural polysaccharide having a

glucose unit and an N-acetyl glucosamine unit alternatingly in the main chain,
was synthesized by chitinase-catalyzed polyaddition of a disaccharide oxazoline
monomer in an aqueous solution (44).

Sugar-chain elongation from di-N-acetylchitobiose as initial substrate to

hexamer and heptamer of chitooligosaccharide was efﬁciently induced through
lysozyme catalysis in an acetate buffer containing 30% ammonium sulfate at 70

◦

The high concentration of ammonium sulfate resulted in a remarkable increase
of the hexamer and heptamer productions. In this reaction, a sugar-elongation
from the dimer to trimer was the rate-limiting step in the overall process of trans-
glycosylation (45).

Transferases.

Phosphorylase catalyzes polymerization of

α-

-glucose-1-

phosphate in the presence of primer, leading to in vitro synthesis of amylose
(Fig. 4) (46). By utilizing phosphorylase catalysis, various amylose derivatives
such as linear-, star-, and comb-shaped amylose polymers were synthesized (47).
The chain length could be controlled by a simultaneous start for all chains us-
ing a primer with a minimum length of four glucosyl residues. This method was

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333

Fig. 4.

Phosphorylase-catalyzed synthesis of amylose.

applied to production of styryl-type amylose macromonomer (48), amylose-graft-
poly(dimethylsiloxane) (49), amylose-graft-poly(

-glutamic acid) (50), amylose-

block-polystyrene (51), amylose-block-poly(ethylene oxide) (52), and amylose-
containing silica gel (53,54). Amylose-polytetrahydrofuran (PTHF) inclusion
complex was synthesized by the phosphorylse-catalyzed polymerization in the
presence of PTHF (55).

Cyclodextrin-

α(1→4)glucosyltransferase (CGTase) catalyzes formation of cy-

clodextrins from starch. By use of immobilized CGTase (silica gel support func-
tionalized with glutardialdehyde),

α-glucosyl ﬂuoride was transformed in high

yields, predominantly into cyclodextrin and maltooligomers as by-products (56).
α-Maltosyl ﬂuorides substituted at the 6- or 6

-position with H, F, Br, OCH

, and

OCOCH

have been tested as substrates for CGTase (57). Among these substrates,

only 6

-O-CH

and 6

-O-COCH

monomers were polymerized to give the cyclic

compounds, indicating that the afﬁnity of substrates toward the catalytic site of
CGTase (Bacillus marcerans) greatly affected the speciﬁcity of the cyclization.

Poly(amino acid)s

Biosynthesis of artiﬁcial polypeptides has been achieved by the expression of tar-
get proteins in living cells with a gene recombination technique; polypeptides
with precise control of the chain length, sequence, and stereochemistry have
been synthesized by genetic engineering. On the other hand, it is well known
that amino acid derivatives are subjected to protease-catalyzed coupling reac-
tion, yielding functional peptide compounds (58). In using amino acid esters as
monomer, poly(amino acid)s are obtained.

Papain catalyzed the polymerization of

-methionine methyl ester hydrochlo-

ride to give water-insoluble oligomer with DP

= 8–10 (59–61). The resulting

water-insoluble oligomer was converted to water-soluble sulfoxide and sulfone
derivatives by treatment of DMSO or hydrogen peroxide. Esters of phenylalanine,

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ENZYMATIC POLYMERIZATION

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threonine, and tyrosine were also subjected to the protease-catalyzed oligomeriza-
tion (62).

Polymerization of

-glutamic acid diethyl ester hydrochloride took place in

the presence of papain or

α-chymotrypsin as catalyst to give the correspond-

ing oligomer composed of 5–9 glutamic acid residues (63,64). An nmr analysis
showed that the product consisted exclusively of

α-peptide linkage (65). Diethyl

-aspartate was polymerized by alkanophilic protease from Streptomyces sp. to

give poly(ethyl

α,β-

-aspartate) with weight-average molecular weight (M

) up

to 3600 (66). The ratio of

α-linkage was about 88%, independent of the enzyme

concentration.

In order to enhance the molecular weight, protease was modiﬁed by mutation

technique to show high catalytic activity in an aqueous N,N-dimethylformamide
(DMF) solution. A subtilisin mutant (subtilisin 8350) derived from BPN

(sub-

tilisin from Bacillus amyloliquefaciens) via six site-speciﬁc mutation (Met 50
Phe, Gly 169 Ala, Asn 76 Asp, Gln 206 Cys, Tyr 217 Lys, and Asp 218 Ser) in-
duced the polymerization of

-methionine methyl ester in the aqueous DMF to

produce poly(methionine) with DP up to 50 (67). The increase of the molecular
weight is due to the improvement of the product solubility and minimization of
the enzymatic peptide cleavage under the high concentration of DMF. Another
mutant (subtilisin 8397) showing higher stability in DMF, the same as 8350 ex-
cept that there is no change for Tyr 217, has been applied as the catalyst for
the polymerization of single amino acid, dipeptide, and tripeptide methyl esters
(68).

A different type of peptide hydrolase, dipeptidyl transferase (dipeptidylpep-

tide hydrolase), catalyzed the polymerization of dipeptide amide in an aqueous
solution. In the case of glycyl-

-phenylalaninamide, trimer was formed in 78%

yield (69). The polymerization of glycyl-

-tyrosinamide produced the correspond-

ing oligomer with DP up to 8 (70).

Protease was used as catalyst for polymer modiﬁcation. Phenylalanine

residues at the side chain of methacrylamide polymers were coupled with ala-
nine t-butyl ester by

α-chymotrypsin catalyst in water–chloroform solvent (71).

Up to 35% peptide-bond formation was achieved for 7 days at room temperature.
Polyamide synthesis was performed by cellulase-assisted polycondensation of chi-
ral ﬂuorinated compound having carboxylic acid and amino groups (72).

Polyesters

Syntheses of aliphatic polyesters by fermentation and chemical processes have
been extensively studied in a viewpoint of biodegradable materials. Recently, an-
other approach of their production has been performed by using an isolated lipase
or esterase as catalyst via nonbiosynthetic pathways under mild reaction condi-
tions. Lipase and esterase are enzymes which catalyze hydrolysis of esters in an
aqueous environment in living systems. Some of them can act as a catalyst for the
reverse reactions, esteriﬁcations and transesteriﬁcations, in organic media (1–5).
These catalytic actions have been expanded to enzymatic synthesis of polyesters.
Figure 5 represents three major reaction types of lipase-catalyzed polymerization
leading to polyesters (6–14,73).

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335

Fig. 5.

Typical routes of polyester production using an isolated enzyme as catalyst.

Polymerization of Oxyacids and Their Esters.

Oxyacids.

1985,

lipase-catalyzed

polymerization

10-

hydroxydecanoic acid was reported. The monomer was polymerized in benzene
using poly(ethylene glycol) (PEG)-modiﬁed lipase soluble in the medium to give
an oligoester with DP more than 5 (74).

Ricinoleic acid, 12-hydroxyoctadecanoic acid, 16-hydroxyhexadecanoic acid,

and 12-hydroxydecanoic acid were polymerized by lipase from Candida cylin-
dracea (lipase CC) or Chromobacterium viscosum as a catalyst at 35

◦

C in wa-

ter, hydrocarbons, or benzene (75). The molecular weight of the polymers was ca
1

×10

. Oligomerization of ricinoleic acid proceeded in the presence of lipase CC

immobilized on ceramics (76).

In the polycondensation of 10-hydroxydecanoic and 11-hydroxyundecanoic

acids, a large amount of lipase CC catalyst (10 weight fold for the monomer)
afforded the corresponding polyesters with relatively high molecular weight (77,
78). From the latter monomer, the polymer with M

of 2.2

×10

was formed in the

presence of activated molecular sieves.

Porcine pancreas lipase (PPL) polymerized 3-hydroxybutyric and 12-

hydroxydodecanoic acids in anhydrous hydrophobic solvents (79). The molecular
weight of the polymer from the former was low (ca 500), whereas the polymeriza-
tion of the latter at 75

◦

C produced the polymer with molecular weight of 3

×10

A cellulase-assisted polymerization of chiral ﬂuorinated compound having car-
boxylic acid and phenolic groups produced the aromatic polyester (72).

Enzymatic synthesis of a methacrylamide-type polyester macromonomer

was reported (80,81). In the polymerization of 12-hydroxydodecanoic acid in the
presence of 11-methacryloylaminoundecanoic acid using lipase CC or Candida
antarctica lipase (lipase CA) as catalyst, the polymerizable group was quantita-
tively incorporated into terminal of the polymer chain.

By using characteristic catalysis of lipase, regio- and enantioselective poly-

merizations of oxyacids have been achieved. Lipase CA catalyzed regioselective

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ENZYMATIC POLYMERIZATION

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polymerization of cholic acid, in which the hydroxy group at 3-position was regios-
electively acylated to give the oligoester with molecular weight less than 1

×10

(82). An optically active oligoester was obtained by the enantioselective polymer-
ization of racemic 10-hydroxyundecanoic acid catalyzed by lipase CC. The result-
ing polymer was enriched in the (S) enantiomer with 60% enantiomeric excess (ee)
and the (R)-enriched unreacted monomer with 33% ee was recovered (83). In the
polymerization of racemic lactic acid catalyzed by lipase CA at 50

◦

C (84), nonamer

was detected in the product by MALDI-TOF mass measurement. A hplc analysis
showed that the

-enantiomer possessed higher enzymatic reactivity.

Oxyacid Esters.

The polymerization of ethyl glycolate using PEG-modiﬁed

esterase from hog liver and lipase from Aspergillus niger (lipase A) gave
oligo(glycolic acid) with DP up to 5 (85). PPL catalyzed the polymerization of
methyl esters of 5-hydroxypentanoic and 6-hydroxyhexanoic acids (86). In the
polymerization of the latter in hexane at 69

◦

C for more than 50 days, the polymer

with DP up to 100 was formed. Relationships between solvent type and polymer-
ization behaviors were systematically investigated; hydrophobic solvents such as
hydrocarbons and diisopropyl ether were suitable for the enzymatic production
of high molecular weight polymer. Polycondensation of various hydroxyesters,
ethyl esters of 3- and 4-hydroxybutyric acids, 5- and 6-hydroxyhexanoic acids,
5-hydroxydodecanoic acid, and 15-hydroxypentadecanoic acid, proceeded by Pseu-
domonas sp. lipase catalyst to give the corresponding polyesters with molecular
weight of several thousands (87).

A symmetrical hydroxy diester, dimethyl

β-hydroxyglutarate, was enantios-

electively polymerized by lipase catalyst to produce a chiral oligomer (dimer or
trimer) with 30–37% ee (88). The enantioselective polymerization of

ε-substituted-

ε-hydroxy esters took place in the presence of PPL catalyst, yielding optically
active oligomers (DP

< 6) (89). The enantioselectivity increased as a function of

bulkiness of the monomer substituent. Optically active polyesters with molecular
weight more than 1

×10

were obtained by the copolymerization of the racemic

oxyacid esters with methyl 6-hydroxyhexanoate.

Lipase-Catalyzed Polymerization of Dicarboxylic Acids or Their

Derivatives.

Enzymatic synthesis has been achieved via various combinations

of dicarboxylic acid derivatives and glycols. As to the diacid monomer, dicarboxylic
acids, activated and nonactivated esters, cyclic acid anhydrides, and polyanhy-
drides were enzymatically reacted with glycols under mild reaction conditions.

Dicarboxylic Acids.

Immobilized Mucor miehei lipase (lipase MM) cat-

alyzed polycondensation of adipic acid and 1,4-butanediol by using a horizontal
two-chamber reactor in the presence of molecular sieves as dehydrating agent
(90). A low dispersity polyester with DP

=20 was obtained by the two-stage poly-

merization.

The polymerization of dicarboxylic acids and glycols proceeded by using li-

pase CA catalyst in a solvent-free system, despite the initial heterogeneous mix-
ture of the substrates (91–94). The polymerization behaviors strongly depended
on the chain length of both monomers (93). The polymerization under reduced
pressure increased the molecular weight of polyesters. The detailed studies in the
combination of adipic acid (A) and 1,4-butanediol (B) showed that the propaga-
tion took place by the reaction of the preliminary adduct (AB) with a hydroxy-
terminated species (92).

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337

For the solvent-free polycondensation, a small amount of adjuvant was ef-

fective for the polymer production when both monomers were solid at the reaction
temperature (93). In the polymerization of adipic acid and 1,6-hexanediol, loss
of the enzymatic activity was small during the polymerization, whereas less than
half of the activity remained in using glycols with methylene chain length less than
4 (94). An attempted experiment allowed the polyester production from adipic acid
and 1,6-hexanediol in 200-kg scale. This solvent-free system has a good potential
as an environmentally friendly practical synthetic process of polymeric materials
owing to the mild reaction conditions without using organic solvents and toxic
catalysts.

A dehydration polymerization of dicarboxylic acids and glycols took place by

lipase catalyst even in water (95,96). This catalysis of lipase is quite speciﬁc since
a dehydration reaction in an aqueous solution is generally disfavored by water,
which is in equilibrium with starting materials because of the law of mass action.
Hydrophobic monomer combinations gave the polyesters in good yields.

Lipase CA catalyzed the polymerization of adipic acid and glycerol to give the

oligomeric products (97). The presence of molecular sieves improved the molecular
weight.

The molecular weight increase was achieved using vacuum system, which

removed the resulting water molecules during the polymerization (98,99). An
aliphatic polyester with M

of 4.2

×10

was obtained from sebacic acid and 1,4-

butandiol using lipase MM catalyst in diphenyl ether at 37

◦

C for 7 days under the

reduced pressure. The molecular weight was much larger than that obtained un-
der ambient pressure. The polymerization of isophthalic acid and 1,6-hexanediol
at 70

◦

C produced the corresponding aromatic polyesters with M

of 5.5

×10

(100).

Dicarboxylic Acid Diesters.

Since unactivated esters, typically alkyl esters,

show low reactivity toward lipase catalyst, the polycondensation with glycols was
often performed under vacuum to produce polyesters of high molecular weight.
Lipase MM-catalyzed polycondensation of diethyl sebacate and 1,4-butanediol un-
der vacuum produced the polymer with M

more than 2

×10

(98).

There is, of course, an equilibrium between the monomers and polymer in

the lipase-catalyzed polycondensation of dialkyl esters and glycols. In the lipase
CC- or MM-catalyzed polymerization of dimethyl succinate and 1,6-hexanediol in
toluene, adsorption of methanol by molecular sieves or elimination of methanol
by nitrogen bubbling shifted the thermodynamic equilibrium (101). When dicar-
boxylic acid dialkyl esters and

α,ω-alkylene glycols were used as monomers, cyclic

oligomers were formed from any monomer combinations examined (102). The yield
of the cyclics depended on the monomer structure, initial concentration of the
monomers, and reaction temperature. The ring-chain equilibrium was observed
and the molar distribution of the cyclic species obeyed the Jacobson–Stockmayer
equation.

Activated esters of halogenated alcohols, such as 2-chloroethanol, 2,2,2-

triﬂuoroethanol, and 2,2,2-trichloroethanol, have often been used as substrate
for enzymatic synthesis of esters (4), owing to the increase of the electrophilicity
(reactivity) of the acyl carbonyl and the avoidance of signiﬁcant alcoholysis of the
products by decreasing the nucleophilicity of the leaving alcohols.

Polymerization of bis(2,2,2-trichloroethyl) alkanediaoates with glycols pro-

ceeded by PPL catalyst in anhydrous solvents of low polarity to produce the

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ENZYMATIC POLYMERIZATION

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Fig. 6.

Lipase-catalyzed polycondensation of divinyl esters and glycols.

polyesters with molecular weight of several thousands (103,104). The oligomer
formation was observed in the polymerization of bis(2-chloroethyl) succinate
and 1,4-butanediol using Pseudomonas ﬂuorescens lipase (lipase PF) as catalyst
(105).

Vacuum method was applied to shift the equilibrium forward by removal of

the activated alcohol formed (98,99,106,107). In the polycondensation of bis(2,2,2-
triﬂuoroethyl) sebacate and aliphatic diols, lipases CC, MM, PPL, and Pseu-
domonas cepacia lipase (lipase PC) produced the polymer with M

more than

×10

. Among the enzymes examined, lipase MM showed the highest catalytic

activity (106). As to solvents, diphenyl ether and veratrole were suitable for the
production of the high molecular weight polyesters under vacuum. In the PPL-
catalyzed reaction of bis(2,2,2-triﬂuoroethyl) glutarate with 1,4-butanediol, the
increase of the molecular weight was attained by periodical vacuum using verat-
role or 1,3-dimethoxybenzene as less-volatile solvent (107).

In lipase-catalyzed transesteriﬁcations, enol esters have been used as acyl

agents (4), since the leaving unsaturated alcohol irreversibly tautomerizes to an
aldehyde or a ketone, leading to the desired product in high yields. Bis(enol ester)s
were reported to be much effective for the enzymatic synthesis of polyesters under
mild reaction conditions (Fig. 6) (108); the polymerization of divinyl adipate and
1,4-butanediol proceeded by lipase PF at 45

◦

C, and adipic acid and diethyl adipate

did not afford the polymeric materials under the similar reaction conditions.

Various lipases (lipases CA, MM, PC, and PF) catalyzed the polycondensation

of divinyl adipate or divinyl sebacate with

α,ω-glycols with different chain length

(109,110). A combination of divinyl adipate, 1,4-butanediol, and lipase PC afforded
the polymer with number-average molecular weight (M

) of more than 2

×10

The polymerization behaviors of the lipase-catalyzed polymerization of divinyl
adipate and 1,4-butanediol have been widely investigated (111–114). During the
polymerization, the hydrolysis of the terminal vinyl ester took place, resulting in
the signiﬁcant limitation of the formation of the polyester with high molecular
weight. A mathematical model describing the kinetics of this polymerization was
proposed, which effectively predicts the composition (terminal structure) of the
polyester.

Another irreversible approach was performed by using bis(2,3-butanedione

monoxime) alkanedioates as diester substrate (115). The polymerization with

α,ω-

alkylene glycols by lipase MM produced the polymer with M

up to 7.0

×10

An enantioselective polymerization of racemic substrates took place through

lipase catalysis, yielding optically active oligoesters and polyesters. The polymer-
ization of bis(2-chloroethyl) 2,5-dibromoadipate with excess of 1,6-hexanediol us-
ing lipase A catalyst produced optically active trimer and pentamer (116).

Vol. 2

ENZYMATIC POLYMERIZATION

339

Fig. 7.

Enantioselective polymerization of epoxy-containing diester with 1,4-butanediol.

PPL-catalyzed

polymerization

bis(2,2,2-trichloroethyl)

trans-3,4-

epoxyadipate with 1,4-butanediol enantioselectively proceeded in anhydrous
diethyl ether to give an optically active polyester with molecular weight of
5.3

×10

(Fig. 7) (117). The molar ratio of the diester to the diol was adjusted to

2:1 so as to produce the (

−) polymer with enantiomeric purity of >96%.

Polymerization of divinyl esters with triols regioselectively took place by

lipase CA catalyst to give the soluble polymers with M

of more than 1

×10

(118,119). MALDI-TOF MS analysis conﬁrmed the presence of a linear polyester
with hydroxy substituents. An nmr analysis of the product obtained from divinyl
sebacate and glycerol in bulk at 60

◦

C showed that 1,3-diglyceride was a main unit

and the branching unit (triglyceride) was contained in the resulting polymer. The
regioselectivity of the acylation between primary and secondary hydroxy groups
was 74:26. By choosing the reaction conditions, the polymer consisting exclusively
of 1,3-acylated unit of glycerol was formed.

Lipase CA catalyzed the regioselective polymerization of sugar alcohols such

as sorbitol and mannitol with divinyl sebacate to give polyesters containing sugar
group in the backbone (120). Some proteases show an esterase activity, especially
in their catalytic activity for regioselective acylation of sugars. By utilizing this
property, protease-catalyzed synthesis of sugar-containing polyesters was demon-
strated (121). Polycondensation of sucrose with bis(2,2,2-triﬂuoroethyl) adipate
using an alkaline protease from Bacillus sp. as catalyst proceeded to give the
polymer (M

=1.6×10

), which was claimed to have ester linkages at the C-6 and

C-1

positions on the sucrose (Fig. 8). In using divinyl adipate as diester monomer,

the molecular weight reached 1.1

×10

(122).

Another approach of enzymatic synthesis of sugar-containing polyesters was

demonstrated (123). Lipase CA-catalyzed reaction of sucrose or trehalose with an
excess of divinyl adipate produced 6,6

-diacylated product having vinyl esters at

both ends, which was employed as monomer in the enzymatic polycondensation
with various glycols, yielding linear polyesters with M

up to 2.2

×10

Unsaturated ester oligomers were synthesized by lipase-catalyzed polymer-

ization of diesters of fumaric acid and 1,4-butanediol (124). Mild reaction condi-
tions did not induce isomerization of the double bond to give all-trans oligomers
showing crystallinity, whereas the industrial unsaturated polyester having a mix-
ture of cis and trans double bonds is amorphous (125). The enzymatic polymeriza-
tion of bis(2-chloroethyl) fumarate with xylylene glycol produced the unsaturated
oligoester containing aromaticity in the backbone (126).

340

ENZYMATIC POLYMERIZATION

Vol. 2

Fig. 8.

Enzymatic synthesis of sucrose-containing polyester.

An unsaturated polyester possessing exclusively cis structure was syn-

thesized by lipase CA-catalyzed polymerization of dimethyl maleate and 1,6-
hexanediol in toluene (127). During the polymerization, formation of cyclic
oligomers was observed. The cycles were semicrystalline, whereas the linear poly-
mer was amorphous. In the lipase CA-catalyzed copolymerization of dimethyl
maleate and dimethyl fumarate with 1,6-hexanediol, the content of the cycliza-
tion was found to mainly depend on the conﬁguration and concentration of the
monomers (128).

Polyesters containing an aromatic moiety in the backbone were synthesized

by lipase CA-catalyzed polymerization of dicarboxylic acid divinyl esters and gly-
cols under mild reaction conditions. Divinyl esters of isophthalic acid, terephthalic
acid, and p-phenylene diacetic acid were enzymatically polymerized with

α,ω-

alkylene glycols to give the polymers with molecular weight of several thousands
(129). Aromatic polyesters were also synthesized from methyl esters of tereph-
thalic and isophthalic acids with 1,6-hexanediol in the presence of lipase CA (130).
In using methyl isophthalate as monomer, macrocyclic compounds were formed as
by-product. Protease from Bacillus licheniformis catalyzed the oligomerization of
esters of terephthalic acid with 1,4-butanediol (131). Lipase-catalyzed synthesis
of aromatic polyesters was achieved by the polymerization of divinyl esters with
xylylene glycols (129,132).

Enzymatic synthesis of ﬂuorinated polyesters was demonstrated (133). Fluo-

rinated diols such as 2,2,3,3-tetraﬂuoro-1,4-butanediol and 2,2,3,3,4,4-hexaﬂuoro-
1,5-pentanediol were used as glycol substrate and polymerized with divinyl adi-
pate using lipase CA catalyst. The enzymatic synthesis of polyester was also
achieved in supercritical ﬂuoroform solvent by the polymerization of bis(2,2,2-
trichloroethyl) adipate and 1,4-butanediol (134). The molecular weight increased
as a function of the pressure.

Anhydrides.

Ring-opening poly(addition-condensation) of cyclic acid anhy-

drides with glycols proceeded through lipase catalysis (135). The polymerization
of succinic anhydride with 1,8-octanediol proceeded using lipase PF catalyst at
room temperature to produce the polyester with M

of 3

×10

Vol. 2

ENZYMATIC POLYMERIZATION

341

Fig. 9.

Cyclic monomers polymerized by lipases.

Polyanhydrides were effective as diacid substrate for enzymatic synthesis of

polyesters (136). The reaction of poly(azelaic anhydride) and 1,8-octanediol took
place by lipase CA catalyst to give the corresponding polyester with molecular
weight of several thousands. In the reaction of poly(azelaic anhydride) and glyc-
erol, a highly branched polyester was obtained.

Oxiranes such as benzyl glycidate and glycidiyl phenyl ether were polymer-

ized with succinic anhydride in the presence of PPL at 60 or 80

◦

C (137,138).

The reaction of succinic anhydride with serine residue of the lipase catalyst pro-
duces a carboxylic acid moiety, which might act as acid catalyst for ring-opening of
oxirane.

Ring-Opening Polymerization of Cyclic Esters.

Polyester syntheses

have been achieved by enzymatic ring-opening polymerization of cyclic esters
with various ring-sizes. Figure 9 summarizes cyclic monomers so far polymerized
through lipase catalysis.

Lactones.

Small-size (four-membered) lactone derivatives have been re-

ported to be polymerized through lipase catalysis. The polymerization of

β-

propiolactone (

β-PL) proceeded by using Pseudomonas family lipases as catalyst

in bulk to give a mixture of linear and cyclic oligomers (139). By employing a very
small amount of lipase CC (0.5 versus for the monomer), high molecular weight
poly(

β-PL) was formed (140).

Ring-opening polymerization of racemic

α-methyl-β-propiolactone using li-

pase PC catalyst proceeded enantioselectively to produce an optically active (S)-
enriched polymer (141). The highest ee value of the polymer was 0.50. An nmr
analysis of the product showed that the stereoselectivity during the propagation
resulted from the catalyst enantiomorphic-site control.

β-Butyrolactone (β-BL) was enzymatically polymerized to give poly(β-

hydroxybutyrate) (PHB), which is a polyester produced in vivo by bacteria for
an energy-storage substance. PPL-catalyzed polymerization of

β-BL in bulk at

342

ENZYMATIC POLYMERIZATION

Vol. 2

room temperature produced PHB with molecular weight around 1

×10

(142). In

the polymerization at high temperature (80 or 100

◦

C), PHB with higher molecu-

lar weight was obtained by lipase CC, PF, or PPL catalyst (143,144). A signiﬁcant
amount of the cyclic PHB fraction was formed and the content of the cycles in-
creased with increasing the monomer conversion. Enantioselective polymerization
of

β-BL was achieved by using thermophilic lipase to give (R)-enriched PHB with

20–37% ee (145).

PHB depolymerase is an enzyme catalyzing hydrolysis of PHB and its cat-

alytic site is a serine residue, the same as lipase. The polymerization of

β-BL pro-

ceeded using two types of PHB depolymerase with or without substrate-binding
domains (SBD) as catalyst (146). The SBD-lacking PHB depolymerase showed
higher catalytic activity.

Chemoenzymatic synthesis of biodegradable poly(malic acid) was demon-

strated by lipase-catalyzed polymerization of benzyl

β-malolactone, followed by

the debenzylation (147). The molecular weight of poly(benzyl

β-malolactone) in-

creased by the copolymerization with a small amount of

β-PL (17 mol% for the

monomer) (148).

Five-membered unsubstituted lactone,

γ -butyrolactone, is not polymer-

ized by conventional chemical catalysts. However, oligomer formation from

γ -

butyrolactone was observed by using PPL or Pseudomonas sp. lipase as catalyst
(87,142).

Medium size lactones,

δ-valerolactone (δ-VL, six-membered) and ε-

caprolactone (

ε-CL, seven-membered), were subjected to lipase-catalyzed poly-

merizations. Lipases CC, PF, and PPL showed high catalytic activity for the poly-
merization of

δ-VL (149,150). The molecular weight of enzymatically obtained

poly(

δ-VL) was relatively low (less than 2×10

ε-CL was enzymatically polymerized by various lipases of different origin,

lipases CA, CC, PC, PF, and PPL (86,149–157). Among them, lipase CA was the
most active toward the

ε-CL polymerization; a very small amount of lipase CA (less

than 1 wt% for

ε-CL) was enough to induce the polymerization (151). Under appro-

priate reaction conditions, the molecular weight reached more than 4

×10

(157).

In the lipase CA-catalyzed polymerization in organic solvents, cyclic oligomers
were mainly formed, whereas the main product in the bulk polymerization was of
linear structure (155).

The detailed kinetics of the

ε-CL polymerization showed that termination

and chain transfer did not occur and the monomer consumption followed a ﬁrst-
order rate law under appropriate conditions, indicating that the system provided
controlled polymerizations where the molecular weight was a function of the
monomer to initiator stoichiometry (152,153,156).

Effect of reaction medium has been systematically investigated in the lipase

CA-catalyzed polymerization of

ε-CL (157). Solvents having log P values from −1.1

to 0.49 showed low propagation rates; on the other hand, solvents with log P values
from 1.9 to 4.5 efﬁciently induced the polymerization, leading to high molecular
weight polymer. The monomer-to-solvent ratio also affected the polymerization
behaviors.

Enzymatic hydrolytic degradation of poly(

ε-CL) in toluene also took place

using lipase CA catalyst to give oligomers with molecular weight of less than 500
(158). After the removal of the solvent from the reaction mixture, the residual

Vol. 2

ENZYMATIC POLYMERIZATION

343

oligomer was polymerized in the presence of the same catalyst of lipase. From
these data is proposed a basic concept that the degradation–polymerization could
be controlled by presence or absence of the solvent, providing a new methodology
of plastics recycling.

Substituted medium size lactones were polymerized by lipase catalyst.

Ring-opening polymerization of

α-methyl-substituted six- and seven-membered

lactones (

α-methyl-δ-valerolactone and α-methyl-ε-caprolactone, respectively)

proceeded using lipase CA catalyst in bulk (159). As to (R)- and (S)-3-methyl-4-oxa-
6-hexanolides (MOHELs), lipase PC induced the polymerization of both isomers.
The apparent initial rate of the S isomer was seven times larger than that of the R
isomer, suggesting that the enantioselective polymerization of MOHEL took place
through lipase catalysis (160).

Poly(1,4-dioxane-2-one) is a biocompatible polymer with good ﬂexibility and

tensile strength for medical applications. Metal-free poly(1,4-dioxane-2-one) with
M

up to 4.1

×10

was synthesized by lipase CA-catalyzed ring-opening polymer-

ization of 1,4-dioxan-2-one (161).

Lipase-catalyzed ring-opening polymerization of nine-membered lactone, 8-

octanolide (OL), has been reported (162). Lipases CA and PC showed the high
catalytic activity for the polymerization.

Four unsubstituted macrolides, 11-undecanolide (12-membered, UDL) (163,

164), 12-dodecanolide (13-membered, DDL) (164,165), 15-pentadecanolide (16-
membered, PDL) (163,164,166,167), and 16-hexadecanolide (17-membered) (168),
were subjected to the lipase-catalyzed polymerization. An nmr analysis showed
that the terminal structure of the polymer obtained in bulk was of carboxylic acid
at one end and of alcohol at the other terminal.

The bulk polymerization of PDL using lipase CA or MM as catalyst produced

the corresponding polyester with high molecular weight up to 3.4

×10

(167). The

polymerization behaviors (rate of the monomer consumption and molecular weight
of the polymer) depended on the water content in the reaction system. Enzymatic
ring-opening polymerization of macrolides (UDL, DDL, and PDL) proceeded even
in an aqueous medium (169).

The enzymatic polymerization of lactones is explained by considering the fol-

lowing reactions as the principal reaction course (Fig. 10) (160,163,170,171). The
key step is the reaction of the lactone with lipase involving the ring-opening of the
lactone to give the acyl-enzyme intermediate (enzyme-activated monomer, EM).
The initiation is a nucleophilic attack of water, which is probably contained in the
enzyme, onto the acyl carbon of the intermediate to produce

ω-hydroxycarboxylic

acid (n

= 1, the shortest propagating species). In the propagation stage, the in-

termediate is nucleophilically attacked by the terminal hydroxyl group of a prop-
agating polymer to produce a one-unit-more elongated polymer chain. This is a
monomer-activated mechanism in contrast to an active chain-end mechanism, the
widely known polymerization mechanism.

Macrolides have virtually no ring strain, and hence, show similar reactivi-

ties with acyclic fatty acid alkyl esters in the alkaline hydrolysis and lower an-
ionic ring-opening polymerizability than

ε-CL. However, polymerization of the

macrolides using lipase PF catalyst proceeded much faster than that of

ε-CL.

This speciﬁc polymerizability by lipase catalyst was quantitatively evaluated by
Michaelis–Menten kinetics (160,168,170–172). For unsubstituted lactones in the

344

ENZYMATIC POLYMERIZATION

Vol. 2

Fig. 10.

Postulated mechanism of lactone polymerization catalyzed by lipase.

range of ring-size from 7 to 17, linearity was observed in the Hanes–Woolf plot
for the formation of the acyl-lipase intermediate, indicating that the polymer-
ization followed Michaelis–Menten kinetics. V

max(lactone)

m(lactone)

and V

max(lactone)

values increased as a function of the ring-size; on the other hand, K

m(lactone)

values

were not so different from each other. These data imply that the enzymatic poly-
merizability increased as the ring-size increased, and the large polymerizability
of macrolides through lipase catalysis is mainly due to the large reaction rate
(V

max

), but not to the binding abilities, ie, the ring-opening reaction process of

the lipase–lactone complex to the acyl-enzyme intermediate is the key step of the
polymerization.

Fluorinated lactones in the ring-size from 10 to 14 were enantioselectively

polymerized using lipase catalyst (173). The lipase CA-catalyzed polymerization
of 10-ﬂuorodecan-9-olide (10-membered) produced the optically active polymer
with positive rotation. Interestingly, the corresponding oxyacid gave an optically
inactive polyester.

Enzymatic synthesis of aliphatic ester copolymers was achieved by lipase-

catalyzed polymerization of two lactones. The copolymerization of

δ-VL and ε-

CL catalyzed by lipase PF produced the corresponding copolymer having random
structure of both units (174). In the copolymerization of OL with

ε-CL or DDL,

random copolyesters were also formed (162), suggesting the frequent occurrence
of transesteriﬁcations between the polyesters. On the other hand, the copolymer
from

ε-CL and PDL was not statistically random (166).

Polyesters with high optical purity were synthesized by the lipase CA-

catalyzed copolymerization of racemic

β-BL with ε-CL or DDL (175). (S)-β-BL was

preferentially reacted with DDL to give the (S)-enriched optically active copoly-
mer with ee of

β-BL unit = 69%. δ-CL was also enantioselectively copolymerized

by the lipase catalyst to give the (R)-enriched optically active polyester with ee up
to 76%.

Frequent occurrence of transesteriﬁcation between polyesters chains was

expanded to synthesis of random ester copolymers by the lipase-catalyzed

Vol. 2

ENZYMATIC POLYMERIZATION

345

polymerization of lactones in the presence of poly(

ε-CL) (176). Intermolecular

transesteriﬁcations between poly(

ε-CL) and poly(PDL) also took place through

lipase catalysis.

Ester copolymers were synthesized by lipase-catalyzed copolymerization of

lactones, divinyl esters, and glycols (177). The

C nmr analysis showed that the

resulting product was not a mixture of homopolymers, but a copolymer derived
from the monomers, indicating that two different modes of polymerization, ring-
opening polymerization and polycondensation, simultaneously take place through
enzyme catalysis in one pot to produce ester copolymers.

Immobilized lipase showing high catalytic activity toward the enzymatic syn-

thesis of polyesters was demonstrated (178). Only a small amount of immobilized
lipase PF adsorbed on a Celite was effective for the polymerization of lactones.
The catalytic activity was further enhanced by the presence of a sugar or PEG at
the immobilization. Surfactant-coated lipase efﬁciently catalyzed the ring-opening
polymerization of lactones in organic solvents, in which the modiﬁed enzyme was
soluble (179).

Enzymatic

synthesis

end-functionalized

polymers

such

macromonomers and telechelics has been achieved by initiator and terminator
methods. An alcohol could initiate the ring-opening polymerization of lactones by
lipase catalyst (“initiator method”). In the lipase CA-catalyzed polymerization of
DDL using 2-hydroxyethyl methacrylate as initiator, the methacryloyl group was
quantitatively introduced at the polymer terminal, yielding the methacryl-type
polyester macromonomer (180). In the lipase-catalyzed polymerization of

ε-CL in

the presence of functional alcohols (181), end-functionalized poly(

ε-CL) as well as

the cyclic by-product was formed.

Polyesters bearing the sugar moiety at the polymer terminal was synthe-

sized by lipase CA-catalyzed polymerization of

ε-CL in the presence of alkyl glu-

copyranosides (182–184). In the initiation step, the primary hydroxy group of the
glucopyranoside was regioselectively acylated. Poly(

ε-CL) monosubstituted ﬁrst

generation dendrimer was synthesized using lipase CA as catalyst. The monoa-
cylation of the initiator took place at the initial stage (185).

Polymeric hydroxy group also initiated the enzymatic ring-opening polymer-

ization of

ε-CL (186). The polymerization was performed using thermophilic lipase

as catalyst in the presence of hydroxyethyl cellulose (HEC) ﬁlm to produce HEC-
graft-poly(

ε-CL) with degree of substitution from 0.10 to 0.32.

Single-step synthesis of polyester macromonomers was achieved by lipase-

catalyzed polymerization of lactones in the presence of vinyl esters acting
as terminator (“terminator method”) (187,188). A methacryl-type poly(DDL)
macromonomer was obtained using vinyl methacrylate (12.5 or 15 mol% based
on DDL) and lipase PF as terminator and catalyst, respectively. By the addition
of divinyl sebacate, the telechelic polyester having a carboxylic acid group at both
ends was synthesized.

Cyclic Diesters.

Cyclic diesters were subjected to the lipase-catalyzed ring-

opening polymerization. Lactide, cyclic dimer of lactic acid, was polymerized
by lipase PC in bulk at high temperature (80–130

◦

C) to produce poly(lactic

acid) with M

up to 2.7

× 10

(189,190). Protease (proteinase K) also in-

duced the polymerization of lactide; however, the catalytic activity was relatively
low.

346

ENZYMATIC POLYMERIZATION

Vol. 2

Ring-opening polymerization of cyclic diesters obtained from diacid and gly-

col, ethylene dodecanoate, ethylene tridecanoate, and 1,4,7-trioxa-cyclotridecane-
8,13-dione, took place through lipase catalysis (191,192). The former two
monomers were polymerized by lipase CA, PC, or PF catalyst (191). The en-
zyme origin affected the polymerization behaviors; the polymerization of these
bislactones using lipase PC catalyst proceeded faster than that of

ε-CL and DDL,

whereas the reactivity of these cyclic diesters was in the middle of

ε-CL and DDL

in using lipase CA.

In Vitro PHA Polymerase-Catalyzed Polymerization to PHA.

Alcaligenes

eutrophus

has

been

used

for

industrial

production

poly(hydroxyalkanoate)s (PHAs). PHA is prepared from acetyl CoA in three
steps and the last step is the chain growth polymerization of hydroxyalkanoate
CoA esters catalyzed by PHA polymerase, yielding PHA of high molecular
weight, which has been in vitro examined, leading to synthesis of PHAs with
well-deﬁned structure. This synthetic process obeys the biosynthetic path-
ways (see P

OLY

(3-

HYDROXYALKANOATES

)).

The growing polymer chain was covalently attached to a highly conserved

cysteine residue (Cy319) of the polymerase (193). The granules of the precipi-
tated polymer were quickly formed when the puriﬁed polymerase was exposed
to (R)-hydroxybutyryl CoA (HBCoA) (194,195). The artiﬁcial PHB granules were
spherical with diameters of up to 3

µm, signiﬁcantly larger than the native ones.

The polymerization of the CoA monomers of (R)-hydroxyalkanoate was of

chain growth in a living fashion; each molecule of the polymerase initiated and
catalyzed the formation of one molecule of the polymer (196–198). By utilizing
this property of polymerase, random and block copolyesters were synthesized.
The resulting polymer had high molecular weight (

>10

). In the polymerization of

racemic HBCoA, only the R monomer was polymerized. Furthermore, the presence
of the S monomer did not reduce the polymerization rate of the R isomer. These
data indicate that the S monomer does not act as competitive inhibitor for the
polymerase.

Recombinant PHA synthase from Chromatium vinosum showed different

catalytic behaviors in comparison with that of A. eutrophus (199). In combi-
nation of this synthase with puriﬁed propionyl-CoA transferase of Clostridium
propionicum, a two-enzyme in vitro PHB biosynthesis system was established,
which allowed the PHB synthesis from (R)-hydroxybutyric acid as substrate
(200).

Hydrolase-Catalyzed Modiﬁcation of Polymers.

Terminal hydroxy

group of poly(

ε-CL) was reacted with carboxylic acids using lipase CA catalyst

to give end-functionalized polyesters (181). Lipase MM catalyzed the regioselec-
tive transesteriﬁcation of the terminal ester group of oligo(methyl methacrylate)
with allyl alcohol (201).

The lipase-catalyzed acetylation of high molecular weight methacrylic poly-

mers containing racemic hydroxy groups in the side chain was achieved with a
maximum conversion of 40% (202,203). The optical rotation was low (up to

−1.2

◦

suggesting a low enantioselectivity of this esteriﬁcation. The enzymatic transes-
teriﬁcation of amylose ﬁlm with vinyl caprate in the isooctane solution containing
solubilized subtilisin Carlsberg produced an amylose derivative regioselectively
acylated at the C-6 position (204).

Vol. 2

ENZYMATIC POLYMERIZATION

347

Enzymatic epoxidation of polybutadiene was demonstrated (205). Lipase CA

catalyzed the oxidation of polybutadiene using hydrogen peroxide as oxidizing
agent in the presence of acetic acid.

Polycarbonates

Polycondensation.

Oligocarbonate with molecular weight of less than

×10

was formed by lipase CC-catalyzed polycondensation of carbonic acid

diphenyl ester with bisphenol A (206). Diethyl carbonate was polymerized with
1,4-butanediol by lipase CA catalyst (207,208). The successive two-step polymer-
ization, the prepolymerization under ambient pressure, followed by the polymer-
ization under vacuum (0.5 mm Hg), produced poly(tetramethylene carbonate) with
M

of more than 4

×10

Activated dicarbonate, 1,3-propanediol divinyl dicarbonate, was used as new

monomer for enzymatic synthesis of polycarbonates (209). Lipase CA catalyzed
the polymerization with

α,ω-alkylene glycols under mild reaction conditions and

the M

value reached 8

×10

. Aromatic polycarbonate was enzymatically obtained

from the activated dicarbonate and xylylene glycol (132).

Ring-Opening Polymerization.

1,3-Dioxan-2-one, six-membered cyclic

carbonate, was polymerized in the presence of lipase catalysts (210–212). Under
mild reaction conditions (

≤70

◦

C), lipase CA efﬁciently catalyzed the polymeriza-

tion to give the corresponding polycarbonate with M

more than 1

×10

(211,212).

No ether bond was observed in the nmr spectrum of the product, indicating that
elimination of carbon dioxide did not occur during the enzymatic polymerization.
The polymerization in the presence of a small amount of PPL (0.1 or 0.25 wt%
for the monomer) at 100

◦

C produced the high molecular weight polymer (M

1.6

×10

) (210).

The enzymatic polymerization of 5-methyl-5-benzyloxycarbonyl-1,3-dioxan-

2-one produced the corresponding polycarbonate (213). Lipases PF and CA
showed high catalytic activity for the polymerization. Debenzylation by cat-
alytic hydrogenation led to the water-soluble polycarbonate with pendent carboxyl
group.

Lipase CA catalyzed the polymerization of cyclic dicarbonates, cyclo-

bis(hexamethylene carbonate) and cyclobis(diethylene glycol carbonate), to give
the corresponding polycarbonates (214). The enzymatic copolymerization of cy-
clobis(diethylene glycol carbonate) with DDL produced a random ester-carbonate
copolymer. Enzymatic synthesis of poly(ester-carbonate) was also achieved by the
copolymerization of 1,3-dioxan-2-one and lactide (215). The PPL-catalyzed copoly-
merization at 100

◦

C produced the copolymer with M

higher than 2

×10

Besides polyesters and polycarbonates, lipase-catalyzed synthesis of poly-

mers from cyclic monomers has been reported. 3(S)-Isopropylmorpholine-2,5-
dione, six-membered depsipeptide, was polymerized by lipase PC and PPL cat-
alysts to give poly(ester-amide) (216,217). High temperature (100 or 130

◦

C) was

required for the polymerization, yielding biodegradable poly(depsipeptide) with
maximum M

= 3×10

. During the polymerization, the racemization of the va-

line residue took place. PPL-catalyzed synthesis of poly(phosphate) was demon-
strated (218). The ring-opening polymerization of ethylene isopropyl phosphate,

348

ENZYMATIC POLYMERIZATION

Vol. 2

ﬁve-membered cyclic phosphate, took place at 40–100

◦

C to give the polymer with

molecular weight of ca 1

×10

Polyaromatics

In living cells, various oxidoreductases play an important role in maintaining the
metabolism of living systems. So far, peroxidase containing Fe-active site, laccase
containing Cu-active site, tyrosinase (polyphenol oxidase, Cu-active site), bilirubin
oxidase (Cu-active site), etc, have been reported to act as catalyst for oxidative
polymerization of phenol and aniline derivatives and for polymer modiﬁcation via
oxidative coupling.

Enzymatic Oxidative Polymerization.

Polyphenols.

For enzymatic oxidative polymerization of phenol deriva-

tives, peroxidase has been often used as catalyst. Catalytic cycle of peroxidase is
shown in Figure 11. Peroxidase catalyzes decomposition of hydrogen peroxide at

Fig. 11.

Catalytic cycles of peroxidase for polymerization of phenols.

Vol. 2

ENZYMATIC POLYMERIZATION

349

Fig. 12.

Peroxidase-catalyzed oxidative polymerization of phenol.

the expense of aromatic proton donors in living cells. In some cases, the peroxidase-
catalyzed oxidation of these donors, eg, phenols, yields water-insoluble polymeric
materials, which had not been characterized yet.

In 1987, enzymatic synthesis of a new class of polyphenols has been ﬁrst

reported. An oxidative polymerization of p-phenylphenol using horseradish per-
oxidase (HRP) as catalyst was carried out in a mixture of water and water-miscible
solvents such as 1,4-dioxane, acetone, DMF, and methyl formate to give powdery
polymeric materials (219). The reaction medium composition greatly affected the
molecular weight, and the highest molecular weight (2.6

×10

) was achieved in

85% 1,4-dioxane.

In the case of phenol, the simplest and most important phenolic compound in

industrial ﬁelds, conventional polymerization catalysts afford an insoluble prod-
uct with noncontrolled structure since phenol is a multifunctional monomer for
oxidative polymerization. On the other hand, the peroxidase catalysis induced the
polymerization in an aqueous organic solvent to give a powdery polymer consist-
ing of phenylene and oxyphenylene units showing relatively high thermal stability
(Fig. 12) (220–224). HRP and soybean peroxidase (SBP) were active as catalyst
for the polymerization in the aqueous 1,4-dioxane (220–222). However, the result-
ing polymer showed low solubility; the polymer was partly soluble in DMF and
dimethyl sulfoxide, and insoluble in other common organic solvents. The solubility
was much improved by using a mixed solvent of buffer and methanol, producing
the DMF-soluble polymer with molecular weight of 2100–6000 in good yields.
Furthermore, the unit ratio (regioselectivity) could be controlled by changing the
solvent composition; the polymer in the range of the phenylene unit from 32 to
66% was obtained (223,224).

So far, various phenol derivatives have been polymerized through peroxidase

catalysis in the aqueous organic solvent (225–227). For the case of a combination
of p-n-alkylphenols and HRP, the polymer yield increased as the chain length of
the alkyl group increased from 1 to 5 (228,229). Polymer formation was observed
in using all cresol isomers by HRP catalyst (230). The polymer was obtained in a
high yield from p-i-propylphenol, whereas ortho and metaisomers were not poly-
merized under the similar reaction conditions. Poly(p-n-alkylphenol)s prepared in
the aqueous 1,4-dioxane showed low solubility toward common organic solvents,
and the molecular weight was in the range of several thousands. On the other
hand, soluble oligomers with molecular weight less than 1000 were formed in
using an aqueous DMF as solvent (231).

Enzymatically synthesized polyphenols showed biodegradability (232), al-

though the degradation rate was not high. Antioxidant effects of the polymers ob-
tained from various phenols through the enzyme catalysis were evaluated (233).
Pronounced improvement for the autooxidation of tetralin was observed.

350

ENZYMATIC POLYMERIZATION

Vol. 2

As to meta-alkyl substituted phenols, the soluble polyphenols were obtained

by HRP or SBP catalyst in the aqueous methanol (234). Enzymatically synthe-
sized poly(m-cresol) had glass-transition temperature (T

) of higher than 200

◦

The enzyme origin strongly inﬂuenced the polymer yield; HRP could readily poly-
merize the monomer having a small substituent, whereas in the case of large
substituent monomers, the high yield was achieved by using SBP as catalyst.

The enzymatic reaction kinetics on the HRP-catalyzed oxidation of p-cresol

in aqueous 1,4-dioxane or methanol showed that the catalytic turnover number
and Michaelis constant were larger than those in water (235). Numerical and
Monte Carlo simulations of the peroxidase-catalyzed polymerization of phenols
were demonstrated (236). The simulations predicted the monomer reactivity and
polymer molecular weight, leading to synthesis of polymers with speciﬁc molecular
weight and index. In an aqueous 1,4-dioxane, the formation of monomer aggregate
was observed (237), which might elucidate the speciﬁc polymerization behaviors
in such a medium.

Effects of the monomer substituent and substituted position on the initial

reaction rate in the HRP-catalyzed polymerization of substituted phenols were
examined (238). Substrates with the electron-drawing group or ortho-substituted
substrates showed low polymerizability. Lactoperoxidase also showed catalytic
activity for the polymerization of phenols.

Four interfacial systems, micelles, reverse micelles, a biphasic, and Lang-

muir trough systems, have been examined for preparation of the enzymatic syn-
thesis of polyphenols. In the polymerization in micelle solution consisting of surfac-
tant and buffer, the obtained polymer from p-phenylphenol had narrow molecular
weight distribution in comparison of that in the aqueous 1,4-dioxane (239).

HRP and p-ethylphenol were encapsulated in the reverse micelle, which was

a ternary system composed of isooctane, water, and bis(2-ethylhexyl) sodium sul-
fosuccinate (AOT). The introduction of hydrogen peroxide into the system induced
the polymerization to produce the polymer particles in the diameter range from
0.1 to 2

µm quantitatively (240–242). Similar particles were obtained by pouring

the solution of enzymatically prepared polyphenol into a nonsolvent containing
AOT (243).

HRP-catalyzed polymerization of p-alkylphenols proceeded in a biphasic sys-

tem consisting of two mutually immiscible phases (isooctane and water) (226). The
molecular weight increased as a function of the carbon number of the alkyl group.

Enzymatic polymerization of phenol derivatives in a monolayer form was

demonstrated (241,244,245). A monolayer was formed from p-tetradecyloxyphenol
and phenol at the air–water interface in a Langmuir trough, which was polymer-
ized by HRP catalyst in the subphase. The polymerized ﬁlm could be deposited
on silicon wafer with a transfer ratio of 100% for the Y-type ﬁlm. The monolayer
thickness determined by Eppipsometric and AFM was 27.8 ˚A.

Poly(2,6-dimethyl-1,4-oxyphenylene) [poly(phenylene oxide), PPO] is widely

used as high-performance engineering plastics, since the polymer has excellent
chemical and physical properties, eg, a high T

(ca 210

◦

C) and mechanically

tough property. PPO was ﬁrst prepared from 2,6-dimethylphenol monomer us-
ing a copper/amine catalyst system (246,247). 2,6-Dimethylphenol was also poly-
merized through HRP catalysis to give the polymer consisting of exclusively 1,4-
oxyphenylene unit (248). On the other hand, a small amount of Mannich-base

Vol. 2

ENZYMATIC POLYMERIZATION

351

Fig. 13.

Enzymatic synthesis of PPO derivative from syringic acid.

and 3,5,3

-tetramethyl-4,4

-diphenoquinone units are contained in commercially

available PPO. The polymerization also proceeded in the presence of laccase de-
rived from Pycnoporus coccineus under air without the addition of hydrogen per-
oxide.

HRP, SBP, and laccase catalysis induced a new type of oxidative polymeriza-

tion of 4-hydroxybenzoic acid derivatives, 3,5-dimethoxy-4-hydroxybenzoic acid
(syringic acid) and 3,5-dimethyl-4-hydroxybenzoic acid. The polymerization in-
volved elimination of carbon dioxide and hydrogen from the monomer to give PPO
derivatives with molecular weight up to 1.8

×10

(Fig. 13) (110,249–251).

Polymerization of p-alkoxyphenols regioselectively proceeded by HRP cat-

alyst to give PPO (252). Peroxidase-catalyzed synthesis of poly(catechol) was
achieved and the iodine-labeled polymer showed low electrical conductivity in
the range of 10

− 6

–10

− 9

·cm

− 1

(253).

Thiol-containing polyphenol was synthesized by peroxidase-catalyzed

copolymerization of p-hydroxythiophenol and p-ethylphenol in reverse micelles
(254). CdS nanoparticles were attached to the copolymer to give polymer–CdS
nanocomposites. By a similar procedure, polyphenol–iron oxide composites were
synthesized (242). The reverse micellar system was also effective for the enzy-
matic synthesis of poly(2-naphthol) showing a ﬂuorescence characteristic of the
naphthol chromophore (255). nmr, ir, and uv analyses showed the formation of the
polymer with quinonoid structure.

Bilirubin oxidase (BOD), a copper-containing oxidoreductase, catalyzed the

oxidative polymerization of 1,5-dihydroxynaphthalene to give the polymer show-
ing low solubility (256,257). The polymerization proceeded regioselectively to pro-
duce the polymer ﬁlm having a long

π-conjugated structure. This monomer was

also polymerized by HRP catalyst (258). The polymerization in the presence of
porous silicon (PS) wafer produced the polyphenol–PS composite showing opto-
electronic properties.

Bisphenol A was polymerized by SBP catalyst to give a soluble polymer with

molecular weight of several thousands in good yields (259). Interestingly, the poly-
mer was subjected to thermal curing at 150–200

◦

C. 4,4

-Biphenol was polymerized

by HRP catalyst in an aqueous 1,4-dioxane to give the polymer showing high ther-
mal stability (260).

The mechanistic study of the HRP-catalyzed oxidative polymerization was

performed by using nmr spectroscopy (261,262). In the initial stage of the poly-
merization of 8-hydroxyquinoline-5-sulfonate, the oxidative coupling took place at
carbons of the 2-, 4-, and 7-positions of the monomer. Polymerization and copoly-
merization of 8-hydroxyquinoline also took place through HRP catalysis (263).

352

ENZYMATIC POLYMERIZATION

Vol. 2

Fig. 14.

Chemoenzymatic synthesis of poly(hydroquinone).

Peroxidase catalysis induced the oxidative polymerization of glucose-

β-

hydroquinone (arbutin) in a buffer to produce a water-soluble polyphenol (264).
The acid treatment of the polymer led to the quantitative deglycosylation of the
polymer, yielding poly(hydroquinone) soluble in polar organic solvents (Fig. 14).
The resulting polymer was used as a mediator for amperometric glucose sensors
(265). Another route for chemoenzymatic synthesis of poly(hydroquinone) from 4-
hydroxyphenyl benzoate was demonstrated (266), whose structure was different
from that obtained from arbutin. Enzymatically synthesized phenolic copolymer
containing ﬂuorophore (ﬂuorescein or calcein) was applied as array-based metal-
ion sensor (267).

A polynucleoside with unnatural polymeric backbone was synthesized by

SBP-catalyzed oxidative polymerization of thymidine 5

-p-hydroxyphenylacetate

(268). Chemoenzymatic synthesis of a new class of poly(amino acid), poly(tyrosine)
containing no peptide bonds, was achieved by peroxidase-catalyzed oxidative poly-
merization of tyrosine ethyl esters, followed by alkaline hydrolysis (269). The am-
phiphile higher alkyl ester derivatives were also polymerized in micellar solution
to give the polymer showing surface activity at the air–water interface (270). The
polymerization was monitored by the quartz crystal microbalance (271).

HRP catalysis induced a chemoselective polymerization of a phenol deriva-

tive having methacryloyl group (272). Only the phenol moiety was polymerized
without involving vinyl polymerization of methacryloyl to give a polymer having
the methacryloyl group in the side chain (Fig. 15). The resulting polymer was
subjected to thermal and photochemical curings (273).

A phenol with an acetylenic substituent in the meta position was also chemos-

electively polymerized to give the polyphenol having the acetylenic group (274).
The resulting polymer was converted to carbonized polymer in a much higher yield
than enzymatically synthesized poly(m-cresol).

Cardanol, a main component obtained by thermal treatment of cashew nut

shell liquid, is a phenol derivative mainly having the meta substituent of a C-15
unsaturated hydrocarbon chain mainly with one to three double bonds. A new
cross-linkable polymer was synthesized by the SBP-catalyzed polymerization of
cardanol (110,251,275). The polymerization in an aqueous acetone produced oily
polymeric materials having the carbon–carbon unsaturated group in the side
chain. The curing by cobalt naphthenate gave the cross-linked ﬁlm with high gloss
surface. The hydrogenated cardanol derivative was also oxidatively polymerized
by HRP (276).

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ENZYMATIC POLYMERIZATION

353

Fig. 15.

Chemoselective polymerization of a phenol derivative having a methacryloyl

group.

Fluorinated phenols, 3- and 4-ﬂuorophenols and 2,6-diﬂuorophenol, were

subjected to peroxidase-catalyzed polymerization, yielding ﬂuorine-containing
polymerizations. During the polymerization, elimination of ﬂuorine atom partly
took place to give the polymer with complicated structure (277).

Morphology of the enzymatically synthesized polyphenol was controlled un-

der the selected reaction conditions. Monodisperse polyphenol particles in the
submicron range were produced by HRP-catalyzed dispersion polymerization of
phenol using poly(vinyl methyl ether) as stabilizer in an aqueous 1,4-dioxane (278–
280). The particle size could be controlled by the stabilizer concentration and sol-
vent composition. Thermal treatment of these particles afforded uniform carbon
particles. The particles were also formed from m-cresol and p-phenylphenol.

Bienzymatic system (glucose oxidase

+ HRP) was used as catalyst for

the polyphenol synthesis. This system induced the polymerization of phenol in
the presence of glucose without the addition of hydrogen peroxide to produce the
polymer in a moderate yield (281). Hydrogen peroxide was formed in situ by the
oxidation of glucose catalyzed by glucose oxidase, which acted as oxidizing agent
for the polymerization.

354

ENZYMATIC POLYMERIZATION

Vol. 2

In vitro synthesis of lignin, a typical phenolic biopolymer, was claimed by

the HRP-catalyzed terpolymerization of lignin monomers, p-coumaryl alcohol,
coniferyl alcohol, and sinapyl alcohol (14:80:6 mol%) in an extremely dilute aque-
ous solution at pH 5.5 (282). Dialysis membrane method was applied to the poly-
merization of coniferyl and sinapyl alcohols, yielding insoluble polymeric ma-
terials (283). In the HRP-polymerization of coniferyl alcohol in the presence of
a small amount of lignin component, the molecular weight distribution became
much broader than that in the absence of lignin (284).

Peroxidase-catalyzed polymerization behavior of coniferyl alcohol has been

compared with that by laccase (285). Peroxidase oxidized the substrate faster than
laccase in the presence of hydrogen peroxide. As to the laccase-catalyzed poly-
merization, the oxidation rate and reaction mechanism depended on the enzyme
origin.

Enzymatic polymerization of soluble lignin fragments (lignin oligomer) was

demonstrated. In the polymerization catalyzed by HRP, or polyphenol oxidase
(potato), brown precipitates were formed (286). The increase of the molecular
weight was observed in the laccase-catalyzed treatment of the lignin oligomer
(287).

Peroxidase-catalyzed grafting of polyphenols on lignin has been attempted by

HRP-catalyzed polymerization of p-cresol with lignin in the aqueous 1,4-dioxane
or reverse micellar system. (288–290). The monomer was incorporated into lignin
by the oxidative coupling between the monomer and the phenolic moiety of lignin.

Low molecular weight coal (4 kDa) was polymerized by HRP or SBP catalyst

in a mixture of DMF and buffer (291). The resulting product was partly soluble
in DMF and the DMF-soluble part had a larger molecular weight than that of the
starting substrate.

A novel system of enzymatic polymerization, ie, a laccase-catalyzed cross-

linking reaction of new “urushiol analogues” for the preparation of “artiﬁcial
urushi,” has been demonstrated (Fig. 16) (292,293). Single-step synthesis of the
urushiol analogues was achieved by using lipase as catalyst. These compounds
were cured in the presence of laccase catalyst under mild reaction conditions
without the use of organic solvents to produce the cross-linked polymeric ﬁlm
with high gloss surface and good elastic properties. Catechol derivatives directly

Fig. 16.

Laccase-catalyzed curing of urushiol analogues to “artiﬁcial urushi.”

Vol. 2

ENZYMATIC POLYMERIZATION

355

connecting an unsaturated alkenyl group at 4-position of the catechol ring were
also cured by laccase to give the cross-linked polymeric ﬁlm showing ideal dynamic
viscoelasticity (294).

Polyanilines.

Oxidoreductases also catalyze oxidative polymerization of

aromatic amines. HRP induced the polymerization of aniline. In the HRP-
catalyzed polymerization under neutral conditions, the polymer with complicated
structure was obtained in low yields (295). The resulting polymer showed good
third-order nonlinear optical properties (296).

On the other hand, the polymerization using sulfonated polystyrene (SPS) as

template produced the electroactive form of polyaniline (297–299). The resulting
polymer was soluble in water and the conductivity reached 5

×10

− 3

·cm

− 1

with-

out doping. Besides SPS, a strong acid surfactant, sodium dodecylbenzenesulfonic
acid, provided suitable local template environments leading to the formation of
conducting polyaniline. Aniline was also polymerized by BOD catalyst to give the
polyaniline ﬁlm, which was electrochemically reversible in its redox properties in
acidic solution (300).

HRP-catalyzed oxidative polymerization of o-phenylenediamine in a mix-

ture of 1,4-dioxane and phosphate buffer produced a soluble polymer consisting
of an iminophenylene unit (301). From para and meta isomers, the polymer with
well-deﬁned structure was not obtained (302). Enzymatic polymer formation was
observed from p-aminobenzoic acid (303), p-aminophenylmethylcarbitol (304), 2,5-
diaminobenzenesulfonate (305), and p-aminochalcones (306). Cytochrome c cat-
alyzed oxidation of o-phenylenediamine to give oligomeric products (307). Mono-
layer of aniline/p-hexadecylaniline prepared by LB technique at the air–water
interface was polymerized through HRP catalysis to give polymeric monolayer
(244,245).

A new class of polyaromatics was synthesized by peroxidase-catalyzed oxida-

tive copolymerization of phenol derivatives with anilines. In case of a combination
of phenol and o-pheneylenediamine, ftir analysis showed the formation of the cor-
responding copolymer (308).

Polymer Modiﬁcation by Oxidoreductases.

Tyrosinase (polyphenol

oxidase, a copper-containing monooxygenation enzyme) was used as catalyst for
modiﬁcation of chitosan. The enzymatic treatment of chitosan ﬁlm in the presence
of tyrosinase and phenol derivatives produced a new material of chitosan deriva-
tive (309). During the reaction, unstable o-quinones were formed, followed by the
reaction with chitosan to give the modiﬁed chitosan. In the enzymatic treatment
of p-cresol with a low concentration of chitosan (

<1%), the reaction solution was

converted into a gel (310).

The similar treatment in the presence of chlorogenic acid produced the

modiﬁed chitosan soluble under both acidic and basic conditions (311). On the
other hand, the tyrosinase-catalyzed reaction of 3,4-dihydroxyphenylethylamine
(dopamine) provided water-resistant adhesive properties to chitosan (312). A chi-
tosan derivative modiﬁed with hydroxy or dihydroxybenzaldehyde was cross-
linked by tyrosinase to give stable and self-sustaining gels (313). Phenolic moiety
of a synthetic polymer, poly(p-hydroxystyrene), was also subjected to tyrosinase-
catalyzed oxidation (314).

Peroxidase-catalyzed treatment of soy proteins (315) and wheat gliadins

(316) was demonstrated, in which tyrosine residues of the protein were subjected

356

ENZYMATIC POLYMERIZATION

Vol. 2

Fig. 17.

Regioselective polymerization of 4-phenoxyphenol catalyzed by a tyrosinase-

model complex to unsubstituted PPO.

to the enzymatic oxidative coupling, yielding a network of peptide chains. The
treatment increased the tensile strength of the materials. In the oxidative poly-
merization of bovine pancreatic ribonuclease A catalyzed by lignin peroxidase, the
coupling between tyrosine residues was conﬁrmed (317).

Oxidative Polymerization Catalyzed by Enzyme-Model Complexes.

Metal complexes containing a catalytic site of enzymes or its modiﬁed moiety have
been used as catalyst for oxidative polymerizations. Regioselective oxidative poly-
merization of 4-phenoxyphenol was achieved by using tyrosinase model complexes
as catalyst, leading to crystalline 2,6-unsubstituted PPO (318–321) (Fig. 17). The
complex also catalyzed the regioselective polymerization of 2,5-dimethylphenol to
give the crystalline PPO derivative with melting point higher than 300

◦

C (322).

From o- and m-cresols, polymers consisting mainly of 1,4-oxyphenylene unit were
also formed (323,324).

Catalytic site of peroxidase is a heme, which is rapidly oxidized in its free form

to hematin. p-Ethylphenol was polymerized using hematin as catalyst in an aque-
ous DMF (325). Iron-N,N

-ethylenebis(salicylideneamine) (Fe-salen) also can be

regarded as model complex of peroxidase. Fe-salen catalyzed an oxidative polymer-
ization of various phenols such as 2,6-dimethylphenol, bisphenol A, cardanol, and
urushiol analogues (251,293,326–329). The polymerization of 2,6-diﬂuorophenol
by Fe-salen produced a crystalline ﬂuorinated PPO derivative (330).

Vinyl Polymers

Some oxidoreductases have been reported to induce polymerization of vinyl
monomers. HRP, xanthin oxidase, alcohol oxidase, chloroperoxidase, and laccase
could catalyze the polymerization of acrylamide and hydroxyethyl methacrylate
(331,332). Glucose oxidase mediated the initiation of vinyl polymerization in the
presence of Fe

and dissolved oxygen (333).

A novel initiating system for vinyl polymerization, HRP/hydrogen

peroxide/

β-diketone, was demonstrated (334–336), in which the catalytic ac-

tion of HRP generates carbon radical, a real initiating species, from hydrogen

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ENZYMATIC POLYMERIZATION

357

peroxide and

β-diketone through an oxidoreductive pathway. A combination of

laccase and organic peroxide initiated the polymerization of acrylamide in the
presence of lignin, yielding lignin-graft-polyacrylamide (337).

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HIRO

OBAYASHI

IROSHI

YAMA

Kyoto University

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