In vitro enzymic synthesis of polymers containing saccharides, lignins, proteins or related compounds: a review

Polymer International 47 (1998) 257È266

In vitro Enzymic Synthesis of PolymersContaining Saccharides, Lignins, Proteins

or Related Compounds: a Review

Michael J. Donnelly

Corporate Technology (Fibres and Chemicals), Courtaulds plc, 101 Lockhurst Lane, Coventry, CV6 5RS, UK

(Received 22 March 1997 ; accepted 12 February 1998)

Abstract : The in vitro use of enzymes for polymer synthesis is considered as ameans of attempting to emulate and extend the range of polymers found innature which are readily biodegradable and have superb control over structureand properties. The issues raised in the use of non-conventional reactionenvironments are discussed. These include (i) the environmental compatibility ofthe synthesised polymer, the (often modiÐed) enzyme, and the other componentsof the system such as organic solvents and additives, (ii) the factors which need tobe considered in order to control the enzyme speciÐcity and stability, and (iii)tests and their validity for assessing the biodegradability of all of the materials inthe system.

Examples of a wide range of synthesised polymers are provided. These includethose from smaller units composed of various saccharides, lignins and proteinsand related compounds. In addition, examples of enzymic modiÐcation of thearchitecture of existing polymers composed of these substances are given. Finally,cases are described where saccharides, lignins and protein-based substances areincorporated into other polymeric materials, either as grafts or by inclusion inthe main chain, using either enzymic or chemicoenzymic procedures. 1998(Society of Chemical Industry

Polym. Int. 47, 257È266 (1998)

Key words : enzymic polymer synthesis ; non-conventional media ; saccharides ;lignins ; proteins ; biodegradability

INTRODUCTION

In vivo versus in vitro synthesis of polymers

In nature a very wide range of polymeric structures aresynthesised utilizing enzymes and, although carried outon a huge scale and for a very long period of time, thesematerials and the enzyme catalysts have not markedlyaccumulated in the biosphere to any great extent.Micro-organisms have evolved the ability to degradethem, and in this manner contribute to the recycling ofelements in the biosphere.1 In addition to recyclabilitythese polymers possess a superbly controlled structurewhich is reÑected in the sophistication and range ofdemonstrated properties. The most abundant of theseare based on (a) sugars, represented, for example, by

microbial polysaccharides or plant polymers such aspectins, (b) amino acids, represented by a huge range ofproteins and enzymes, or (c) derivatives of coniferylalcohol as found in lignins. Mention should also bemade of important materials such as RNA and DNAwhere a sugar is incorporated with other substances inthe polymer chain.

Figures 1 and 2 illustrate two of the structures fromthe natural polymer group. Figure 1 shows a represen-tation of the repeat peptide sequence of the mussel poly-phenolic protein. Unlike man-made adhesives, thispolymer has the remarkable ability to form strong

Fig. 1. Representation of the repeat peptide sequence ofmussel polyphenolic protein.

2571998 Society of Chemical Industry. Polymer International 0959È8103/98/$17.50 Printed in Great Britain(

258 M. J. Donnelly

Fig. 2. Structure of the microbial polysaccharide xanthan.

bonds in the presence of water, thereby anchoring themussel Ðrmly to rocks.2,3 Figure 2 shows the structureof the microbial polysaccharide xanthan. The backbone(which is essentially cellulose) is substituted on everysecond glucose with a trisaccharide unit. Unlike cellu-lose, the polymer is water soluble and has unique andreproducible functionality which has been exploitedtechnologically. Its rheological properties are distinct, inthat solutions are highly viscous at low polymer con-centration, but under shear thin quickly yet can recoverrapidly when the force is removed.4,5

The in vitro use of enzymes is one way to producepolymers from biodegradable substances like sugars,lignins and proteins, which could be expected to mimicto some degree the properties of natural polymers. Fur-thermore the rise, largely over the last 10È15 years, ofenzymic biotransformation technology has demon-strated the viability of enzymes in a wide range of di†er-ing reaction environments. This ability to move awayfrom the predominantly aqueous and relatively lowtemperature systems employed in nature o†ers thepotential to synthesize new structures.

Early biotransformation studies focused mainly onsmall molecules, especially the synthesis and hydrolysisof esters.6 This work has been extended, via the suc-cessful synthesis of oligomeric esters, to high molecularweight polyesters.6h10 The further extension of thisconcept to include the synthesis of sugar-containingpolyesters and a few other types of polymers has beenbrieÑy reviewed.11,12

The purpose of the current review is to describe thetype of polymeric structures that can be modiÐed orsynthesized with enzymes to contain or be producedsolely from sugars, proteins, lignin or related com-pounds ; to discuss issues relating to (i) the advantages,stability and speciÐcity of the enzymes in, and the

environmental compatibility of, the reaction systememployed for synthesis, (ii) the biodegradability of theformed polymers, and (iii) the collation of some con-cepts which may be utilized towards expanding the areaof enzymic synthesis of environmentally compatiblepolymers.

ISSUES RELATING TO THE IN VITRO USE

OF ENZYMES FOR POLYMER SYNTHESIS

Actual and potential advantages of enzymes

The generally perceived advantages in biotransfor-mations of the in vitro use of enzymes is that they areclassiÐed as readily biodegradable catalysts which canoperate under mild conditions with a high degree ofregio- and stereospeciÐcity, yielding products with con-trolled architecture and functionality. In addition,enzyme systems are regarded as being simpler thanthose based on previously or currently living micro-organisms.

On the basis that, if an enzyme is used to synthesise astructure, then it is often assumed that an enzyme willbe able to assist in breaking it down in the environment.This expectation of assistance towards biodegradabilityis most certain if a hydrolytic enzyme has been used forthe synthesis, in which case a reverse reaction can takeplace when the product reaches an aqueous environ-ment. The expectation is, however, less certain for non-reversible enzyme reactions, and for those enzymeswhich generate free-radicals and whose action mayinvolve some degree of relatively uncontrolled chemicalreaction. For the speciÐc case of polymer synthesis thisenzymic breakdown in the environment would mostbeneÐcially produce monomers or components whichare known to be biodegradable, and here the use ofsugars, proteins and lignin is advantageous.

With regard to control of polymer architecture fromthe in vitro use of enzymes, it has been shown that desir-able features are shown for the use of enzymes in thecase of the cellulase catalysed polymerization of aro-matic amide monomers where the polymer products arechiral, of reasonably high molecular weight and lowpolydispersity.13

Features for consideration in the use of enzymes innon-conventional media

A large proportion of the advances made in biotransfor-mation has come about from the realization thatenzymes can be utilized in a wide range of reactionsystems and not just an aqueous one. Examples includeorganic solvents (with water contents varying from nearzero to substantial proportions), gases, supercriticalÑuids, solid-phase, reverse micellar systems, or systemswhich include additives. The versatility may be further

POLYMER INTERNATIONAL VOL. 47, NO. 3, 1998

In vitro enzymic polymer synthesis 259

extended by modiÐcation of the enzyme, for example byimmobilisation, chemical derivatization or cross-linking.In this way synthetic reactions may be performed byreversal of hydrolytic reactions, and chemical reactionscan be achieved which were previously not possible inaqueous systems either due to poor solubility of sub-strate or product in water or to undesired side-reactions.

The use of enzymes in non-conventional media raisesa number of issues regarding their stability and speci-Ðcity, and has lead to numerous studies which attemptto understand and predict these features. The generalproblem is that some of the components of thesesystems can lead to deactivation of the enzyme ordecrease of the reaction rate compared to aqueoussystems. However, development of a suitable system canbe shown to work well, but needs a number of featuresto be considered or examined and optimized. Forexample, the activity of some proteases and lipasesagainst amino acid esters or glycerides in organic sol-vents may be enhanced by prior lyophilization fromaqueous solution containing a ligand or a lyoprotec-tant ;14 reductive alkylation of trypsin improves theesteriÐcation of glucose in dimethylformamide ;15 use ofamphiphilic molecules to produce a reverse micellarsystem improves the substrate concentration near theenzyme and provide a superactivity in isooctane relativeto an aqueous system.16 The activity of horseradish per-oxidase was improved by increasing the enzyme hydro-phobicity in studies involving deglycosylation, benzyl orpolyethylene glycol modiÐcation.17 The speciÐcity ofcertain proteases toward a range of substituted aminoacid substrates has been shown to be reversed whenchanging the solvent from water to octane,18 and theenantioselectivity towards an amino acid ester can becontrolled by the reaction environment because di†er-ent values are demonstrated in di†erent solvents.19Enantioselectivity was also found to depend on themethod of enzyme preparation and temperature forreactions in di†erent organic solvents.20

The type of organic solvent, and the water contentand its location in the reaction system, have proven tobe key features in work involving organic solvents. Forchymotrypsin and laccase in homogeneous solutions ofwater and polar solvents such as alcohols, glycerol andformamide, the maximal rate of amino acid ester hydro-lysis was highly dependent on the percentage volume ofthe organic solvent. Up to relatively high values the ratewas not a†ected, but a further small increase led to adramatic drop in activity.21 The level of water in glyc-erol has also been shown to inÑuence the yield and theregiospeciÐcity of lipase catalysed synthesis of glycerololeates.22 A quantitative description of the e†ect ofwater content and the nature of the organic solvent onthe activity of the enzyme has been developed byKhmelnitsky and co-workers.23 This observation hasbeen extended to use of the “denaturation capacityÏ as a

quantitative criterion for selection of organic solvents.24The water content of some organic solvents can be mea-sured by techniques such as KarlÈFischer reagent, but amore sensitive technique such as NMR is required fordetermination of water bound to proteins suspended innon-polar solvents.25

Some procedures for chymotrypsin, and some generalconsiderations for optimizing the use of enzymes inorganic solvents, have been published.26,27 Strategiesfor obtaining stable enzymes have been reviewed byJanecek28 and investigations into how a wide range ofsolvents a†ect the stability of two glycosidases in oligo-saccharide synthesis have been carried out by Larouteand Willemot.29 Short reviews on solvent selection forbiocatalysis in mainly organic systems, and how thisinÑuences the equilibrium position of a reaction, havebeen carried out by Halling,30 and developments in fun-damental and applied aspects of non-aqueous enzy-mology in the approximate period 1991È1992 have beenreviewed by Dordick.31 Thermodynamic predictions ofbiocatalysis is non-conventional media, in particularusing the concept of water activity, have been exten-sively examined by Halling in an excellent review.32

Biodegradability issues and testing

For a polymer synthesis route to be ideally environ-mentally compatible, consideration needs to be given tothe whole process and not just to the polymer product.This would then mimic nature where all components ofthe reaction system are biodegradable and recyclable.For the in vitro use of enzymes, consideration needs tobe given to features such as (a) whether modiÐcation ofthe enzyme allows retention of the readily biodegrad-able status and, if not, whether the catalyst can berecovered and reused ; (b) whether the other chemicalsused, such as solvents or additives, or reaction by-products, are biodegradable or need to be recoveredand recycled efficiently to minimize environmentalimpact, where this impact is likely to be and how it canbe controlled ; and (c) whether the polymer product canor has been tested in a meaningful way. From a scienti-Ðc viewpoint, for a polymer to be completely environ-mentally compatible it must be proven to be brokendown in a test representative of the likely conditionsfound in the environments in which the products areexpected to end up, and then proven to ultimately andwithin a reasonable timescale be converted to carbondioxide and water. However, an environmentally com-patible product has sometimes been taken to be onewhich breaks down to small particles not readily appar-ent to the naked eye. This approach is exempliÐed bythe Ðlling of synthetic polymers with rapidly and readilybiodegraded materials. Once the latter material hasbroken down, the synthetic polymer particles remain,and the resulting greater surface area and accessibilitymay well enhance the biodegradability rate. These two


260 M. J. Donnelly

features could however, equally lead to the polymer orits degradation products posing an increased environ-mental risk if either were to demonstrate any signiÐcanttoxic e†ect.

DeÐnitions of biodegradability and agreed methodsof testing remain a difficult area, have tended to bebased on a regional rather than a global basis, andsome test methods are more widely adopted thanothers. The status of these concepts in the early 1990shas been examined by EDANA,33 Hirata34 andSteinbuchel35 with the emphasis, respectively, onEurope, Japan, and natural polymers, especially bac-terial polyhydroxyalkanoic acids.

For biodegradability testing of a wide range of chemi-cals including some polymers, a correlation study hasbeen carried out by Gerike and Fischer.36 This com-pares seven widely used test methods, concludes thatthe degree of biodegradability seems to be test speciÐc,that all materials cannot be tested by a single method,and suggests that several methods should be usedinstead. Biodegradability test methods have also beenreviewed by Seal37 because biodegradability is now partof the requirements for (i) the registration of new chemi-cals, (ii) information required on material safety datasheets, (iii) part of the ecotoxicity data used to deter-mine if a material is considered dangerous to theenvironment.

The assessment of the biodegradability of waste-waters and associated materials has been dealt with inthe UK in a series of booklets published byHMSO.38h41 These deal with various aerobic andanaerobic methods in tests which range from verysimple ones to attempts to simulate the ecosystem. Forthose situations where the chemicals used for synthesis,or the polymer product, may end up in a marineenvironment the ECETOC Technical Report is useful.42General background information on the biologicaltreatment of hazardous waste and microbial aspects ofpollution is available in various texts.43,44 Ref. 44includes a section on biodeterioration and biodegrada-tion of synthetic polymers. The pros and cons of variousbiodegradability tests for industrial wastes and intracta-ble substances have been examined by Drews.45 Simi-larly, a proposal by Van Ginkel and Stroo46 has beenmade to extend the closed bottle test to 200 days inorder to determine the biodegradability of “recalcitrantÏand toxic organic compounds. Over these timescalescompounds, including various polymeric materials,have been shown to be inherently biodegradable, incontrast to the results from normal closed bottle tests of28 days duration described in previous test methods.The available test methods have also been furtherextended to include biodegradation tests of packagingmaterials and plastics and other consumer products inaccelerated land-Ðll, short-term controlled composting(up to 45 days) and longer-term composting (up to 6months).47 A very precise method has been developed

by Komarek et al. for the establishment of the biode-gradability of cellulose esters.48 In this work radiolabel-led cellulose acetate and propionate were tested with amixed microbial culture derived from activated sludge.The extent of biodegradation was highly dependent onthe degree of substitution of the polymers, and this wasproven conclusively by the detection of liberated 14CO2indicating the microbial species had utilized thepolymer as a carbon source.

Apart from microbial testing, enzymes have also beenused in vitro to provide information on the degradationor biodegradability of chemicals and polymers. Areview on the use of enzymes for waste treatment byAitkin49 shows the numerous types of chemical struc-tures which can be modiÐed with enzymes either toenhance microbial biodegradation or to remove orimprove the removal of materials from wastewaters.Pkhakadze and Snegirev50 reviewed progress in thedevelopment of enzymically degradable polymers formedical applications up to 1989, covering the pre-parative methods, properties and mechanisms of degra-dation of polymers containing natural units. Polymerswere based on (a) copolymers of amino acids or pep-tides and synthetic polymers, or (b) sugar-containingpolyurethanes. The tissue variables and structural fea-tures of medical polymeric devices which inÑuence theirsusceptibility to biodeterioration and biodegradationhave been reviewed by Williams and Zhong.51 Againthis illustrates the wide range of structures which can behydrolysed enzymically, and emphasizes the need toconsider the neglected area of oxidative enzymes ande†ects of free-radicals. In order to evaluate the biode-gradability of substituted celluloses a rapid and low costenzymic method has been devised by Glasser et al.52 inwhich the rate of reducing sugar generation is mea-sured. The use of commercial cellulases allows anassessment of the e†ects of the degree of substitutionand the number of carbon atoms in the substitutedgroup on the rate of degradation. However, althoughthe enzymes were used in excess, the results will relatenot only to the polymer composition but also to com-position of the particular enzymes used, because com-mercial preparations are known to vary widely in therelative amounts of the di†erent cellulases (and otherenzymes) and in their action towards di†erent cellu-loses.53 Failure in this test method does not thereforenecessarily mean the polymer is not degradable, but canserve as a useful indicator if a positive result is obtained.Once a polymeric material has been shown to be micro-bially degraded, the elucidation of the role of theenzymes involved can be a complex task. This is illus-trated in the thesis of Saki, in which the role of anumber of puriÐed enzymes in the degradation of poly-vinyl alcohol was examined.54

The polymer characteristics which inÑuence enzymicdegradation have been reviewed from a polymerchemistÏs perspective by Timmins and Lenz ;55 this



includes polymer hydrophobicity, composition, conÐgu-ration, morphology, mobility, molecular weight andsample geometry. Techniques for the characterization ofthe e†ects of enzymes on polymers has often involvedmeasurement of weight loss, but can also include moresophisticated methods such as titration to determinecleavage rate of powdered polyester,56 scanning elec-tron microscopy, X-ray photoelectron spectroscopy andadhesion tests to measure topographical, chemicale†ects and performance properties, respectively, forlipase treated PET,57 changes in crystallinity andmolecular weight for microbial PHB treated withdepolymerase,58 or measurement of soluble organiccarbon for either protease-treated polymers containingamino acids,59 or for lipase treatment of certain poly-esters.60,61

POLYMER STRUCTURES SYNTHESIZED OR

MODIFIED WITH ENZYMES

In describing the structures produced by enzymes usingnon-conventional media, there is a difficulty is ascer-taining the degree of novelty which has been achieved.This arises because (a) not all the structures present innature, for example all microbial polysaccharides,62have been determined, and (b) the polymeric structuressynthesized with enzymes in vitro have not always beencharacterized in great detail. The examples given arethose which are thought to contain some degree ofnovelty either in terms of the stereo or positional natureof the bonding holding the “monomersÏ together, thechemical species which are present in the synthesizedpolymer, and the order in which the species are com-bined spatially. In these examples the term polymer isassigned to entities with a degree of polymerization often or preferably higher, and emphasis is placed on thestructure of the polymer with only brief mention ofknown or likely applications.

Structures based on or containing saccharides

Three approaches have been utilized to prepare oligo-meric and polymeric materials from saccharides. Theseare the following

(a) Reversal of the action of a hydrolase enzyme inwhich the equilibrium position is altered bydecreasing the water activity in, removal of pro-ducts from, or an increase in substrate concen-tration of the reaction system.

(b) Utilization of the transferase activity of hydro-lase enzymes.

(c) Use of transferase enzymes.

This area and the features that need to be controlled,together with the type of products which may be

produced have been reviewed by Monsan andco-workers,63,65 Cote and Tao,64 Bucke and Rastall,66and Whitesides and co-workers67 up to about 1990.These examples of polymer synthesis from sucroseinclude levan, a high molecular weight (MW) polyfruc-tan produced by levansucrase and composed ofbranched fructofuranosyl units linked in the b2È6 orb2È1 positions, and high MW (up to 107 daltons)glucans synthesized by dextransucrase, in which thetype of bonding depends on the source of the enzymeused and the acceptor. Work on deÐning the propertiesof transferase enzymes has also shown that the source ofthe enzyme and the reaction conditions inÑuence thedegree of polymerization of the product which isobtained.68,69 This may perhaps be exploited to givedi†ering proportions of speciÐc linkages in the productmixture and di†erent polydispersities to provide a rangeof product properties. For the case of dextran, industrialuses require a lower MW (5000È70 000), and this can beachieved by the addition of maltose or isomaltose as anacceptor molecule, the concentration of which may beused to control the MW. Other acceptor molecules suchas glucose, methyl glucoside and isomaltotriose, can beused to give lower MW materials which are potentiallyuseful as food additives.70 Amino sugars have also beenshown to be acceptors for levansucrase, o†ering anotherroute to di†erent polymer structures. As an exception tothe general view that glycosyl donors are thermody-namically unsuited for polymer synthesis an Escherichiacoli amylomaltase has been shown to produceuncharacterized amylodextrines from maltose.64 Thesynthesis of speciÐc oligosaccharides by enzymes, andtheir attachment by, for example, the reverse reaction ofdebranching enzymes may be one route to branchedpolymers. Alternatively as reviewed by Whitesides andco-workers,67 Pfannemuller et al. have produced arange of comb, linear and star shaped polymers carry-ing amylose side chains using potato phosphorylase.67

Reversal of cellulase enzymes is known71 and hasbeen used elegantly by Kobayashi et al.72 to producesynthetic cellulose with a degree of polymerization ofabout 22 together with cellooligosaccharides from cello-biosyl Ñuoride in acetonitrile/acetate bu†er. Whetherthe polymer is novel is unclear, because it is unknown ifit contains a terminal Ñuorine ; this will depend on themechanism involved. Furthermore, no evidence hasbeen produced for links other than b1È4 found innatural cellulose, although the polymer has been shownto be a cellulose II allomorph.73,74 Interestingly use of apartially puriÐed cellulase has allowed the productionof the cellulose I allomorph by this procedure.75

Oxidative enzymes have recently been shown to actboth on oligosaccharides and polymers.76,77 In theformer case maltooligosaccharides containing up to atleast seven glucose units were substrates for glucooligo-saccharide oxidase from Acremonium strictum ; this reac-tion may be useful as a route for their incorporation as


262 M. J. Donnelly

side-chains. In the latter case Avicel, Whatman CF 11,a-cellulose and carboxymethyl cellulose were substratesfor a cellobiose oxidizing enzyme from a Cytophagaspecies and the reaction may be a route to productswith novel properties or new structures after furtherchemical modiÐcation using the oxidized group(s) as areaction site.

Starch polymer has been shown to be modiÐable intwo ways to form new structures with di†erent rheologi-cal properties. It may be partially debranched with iso-amylase or a novel pullulanase from a Bacillus sp.78Alternatively, the polymer may be chain-extendedusing glucosyl Ñuoride in the presence of inorganicphosphate and the enzyme potato or sucrose phos-phorylase.79 In particular, this approach extends theshort outer amylopectin chains to give an enhancedamylose-like character.

Some enzyme mediated reactions which produce(where characterized) species having 7È14 repeat units,may potentially have applicability to the synthesis (orbranching) of high polymers, because of the smallamount of work that has been done on controlling thewater activity or devising e†ective methods of mixingreaction systems which are often highly viscous syrups.Such reaction include reverse hydrolysis with glucoamy-lase or a-mannosidase to produce novel hetero-oligosaccharides,80,81 highly branched oligosaccharidesarising from transglycosylation reactions on starch withneopullulanase,82 and novel inositol derivatives produc-ed by the addition of up to ten glucose units withCGTase ;83 modiÐcation of b-galactosidase with smalllevels of glutaraldehyde increased the yield of trisaccha-ride and higher levels commenced the production oftetrasaccharides from lactose,84 whilst immobilizationhad a similar e†ect,85 and the addition of diethyleneglycol diethyl ether or triethylene glycol dimethyl etherinitiated the formation of complex oligosaccharidesfrom glucose with glucoamylase, a reaction notobserved in water alone.86 ModiÐcation of cyclodextrinproperties can be achieved by the production of novelstructures in which side-chains of varying length areadded by the synthetic reaction of pullulanase.87h89CGTase acting on starch in the presence of cellobioseor trehalose acceptors has been used to produce oligo-mers with novel sugar compositions.90 Similarly, inbu†er and acetonitrile or methanol, cellulase has beenshown to accept non-glucose substrates such asmannose, xylose or substituted glucoses, o†ering thepossibility of synthesis of oligosaccharides containingdi†erent sugars using one enzyme;91 cellulases havealso been shown to catalyse transglycosylation oflactose to form galactooligosaccharides.92 Increasingthe concentration of mannose lead to an increase in theconcentration of di- and trisaccharides formed, and alsothe onset and increase in tetrasaccharide formation.93

One unusual approach from Seeman and co-workersis described as nanotechnology. This involves the use of

DNA and RNA strands, which contain the sugardeoxyribose or ribose, respectively, to enzymically pro-duce structures and topologies such as catenated mole-cules, knots, cubes and octahedrons.94h96

The enzymic modiÐcation of polysaccharides hasbeen reviewed by Gacesa.97 In this paper examples aregiven of tailoring the architecture and mostly the rheol-ogical properties, of alginates, agar and galactomannanswith a variety of enzymes. When changing the mannoseto galactose ratio of guar gum with a-galactosidase,Critchley98 has pointed out that reaction conditions,such as the guar concentration, may also be used toinÑuence the rate of enzymic side-chain removal ;unusually, very high concentrations of guar (up to 70%)form a particulate material which may be processed infood mixers and extruders. Guar is a relatively poor vis-cosifying agent, but increased viscosity or gels can beobtained by modiÐcation with galactose oxidase so thata crosslinking reaction takes place.99 Enzymic treat-ment of sugar beet pectin with a partially puriÐedAspergillus niger enzyme preparation leads to areduction of arabinose units, some deacetylation anddemethoxylation, producing an improvement in thegelling properties, whereas acid treatment produces adi†erent polymer and gel properties.100 Similarly, di†er-ent pectin structures can be obtained from plants usingdi†erent protopectinases.101 Daicel ChemicalIndustries, in collaboration with the Osaka MunicipalTechnical Research Institute, have recently discoveredmicrobes which modify cellulose acetate (CA) that willbe utilized to develop new biodegradable plastics.102These enzymic systems are presumably used to controlthe structure and properties of cellulose acetates, sinceKamide and co-workers have devised an explanation ofthe solubility properties of CA based on supermolecularconcepts such as degree of hydrogen bonding and con-formation of the glycosidic links ; these properties are inturn dependent on the total degree of substitution andthe distribution of glycosidic links.103

Extensive work has been carried out on the acylationand deacylation of low molecular weight carbohydratesand some other hydroxyl-containing compounds in awide range of non-conventional environments, whichhas lead to a reasonable understanding of the control ofthe selectivity of the reactions.104h108 Little of this tech-nology appears to have been applied to polymers,although some examples are known. Deacetylation ofxylan with acetyl esterase can modify solubility andrender the material insoluble ;109 more complex xylanmodiÐcations are achievable with the range of accessoryenzymes available for the various substituents present inxylan.110 Deacetylation of chitin to various chitosansyielding products of wide-ranging applicability is wellknown.111

A wide range of polymers have been prepared byDordick, Lindhardt, Rethwisch and their co-workers112h115 which contain sugars either in the main



chain or as side-chains. These include examples wherethe polymer is prepared directly with enzymes, or thespeciÐcity of enzymes is used to make monomers withcontrolled functionality which are then chemically poly-merized. Thus sucrose has been reacted with the diacidderivative bis(2,2,2-triÑuoroethyl)adipate in pyridinecatalysed by protease enzymes112 to yield polymerstypically of and a polydispersity of 1É31 ;Mw B 2100some higher molecular weight materials can also be pre-pared by this technique which has been extended to useraffinose, lactose and fructose sugars.114 Methyl, phenylor nitrophenyl galactosides can be transesteriÐed withvinyl acrylate in pyridine catalysed with lipase to form6-acryloyl esters which are then chemically polymerizedto yield high polymers113 and these may be slightlycross-linked to give materials which absorb many timestheir own weight of water. Some general details on theuse of this technology to produce swellable polymershave been reviewed114 and this paper also describes themethodology for preparing poly(sugar acetylenes) inwhich the glycosyl donor, propargyl alcohol, is used ina transglycosylation reaction with lactose, maltose orcellobiose catalysed by glycosidases. Other method-ologies have been described115 using other sugars suchas methyl glucoside or for preparing poly(sugarmethacrylates). These materials show di†ering solu-bilities in water and organic solvents which are struc-ture dependent, and information has been generatedthat these polymers can be broken down enzymically inaqueous environments, indicating the potential for bio-degradability.

Structures based on lignin and related compounds

Lignins are a group of complex three-dimensional phe-nolic network polymers, mainly formed from coniferyl,coumaryl and sinapyl alcohol covalently linked in anumber of ways. Postulate on the mechanism of ligninbreakdown have included the involvement of hydroxylradicals, ligninperoxidases (ligninases) and also laccaseenzymes, but the formation of phenoxy radicals indi-cates the potential for spontaneous repolymerization, inaddition to a depolymerization reaction. Some bio-chemical properties and the potential role of theseenzyme systems in the incompletely resolved under-standing of lignin degradation/polymerization havebeen published.116h120

In vitro enzymic reactions using laccase, lignin per-oxidases (LiP) and the more readily commercially avail-able horseradish peroxide (HRP) in the presence ofhydrogen peroxide have been carried out in bothaqueous and organic phase reaction systems ; the natureof the product is dependent on the system and thesubstrate used. Organosolve lignin treated withlaccase in dioxane/water showed a large increase inmolecular weight and an increase in phenolic groups to-gether with other chemical changes.121 Although the

degradation pattern of treatment of spruce milled-woodlignin with LiP and HRP showed similarities, treatmentwith LiP in 10% dimethylformamide resulted incomplex changes the hydrodynamic properties, consis-tent with a signiÐcant increase in molecular weight,which were not shown with HRP.122 HRP in aqueoussystems has been used to (a) attempt degradation ofsoluble sodium lignosulphonates (but often poly-merization is the observed reaction), and (b) to poly-merize some phenolic structures123 in 85% dioxane toproduce polymers of average molecular weight rangingfrom about 400 to well over 26 000. For a particularreaction system the molecular weight may be controlledbecause it is highly dependent on slight changes in thewater content of the media.

Copolymerization of lignin has also been achieved.Thus HRP catalyses the grafting, probably via involve-ment of phenoxy radicals, of p-cresol onto milled woodlignin, kraft lignin or a selectively O-demethylatedlignin in aqueous dioxane producing high MW poly-mers.124 The molecular weight of Organosolv andIndulin lignin can be increased by reaction in the pres-ence of laccase. This system can be used to copolymer-ize lignin with vanillic acid, diisocyanate or acrylamide ;in this case, evidence was gathered for the presence of asuperoxide radical in the reaction mixture when laccaseand lignin were incubated.125 These enzyme systemshave been used to form polymers from a wide range ofphenolic structures for applications ranging from theproduction of conducting polymers to removal of aro-matics from wastewater. Examples of the monomersused include hydroquinone, which is Ðrst enzymicallymonoglycosylated in aqueous solution to confer watersolubility before peroxidase polymerization ;126 p-ethylphenol in a reverse micellar system, in which theconcentration of surfactant is used to control MW;127p-cresol, in which the reaction media can be used tocontrol product distribution ;128 mono- or dichlorinatedphenols or monomethylated phenols, in which tem-perature and enzyme concentration have proved impor-tant factors,129 and various phenols and aromaticamines used for polyphenol synthesis without involvingtoxic formaldehyde used in the current chemicalsynthesis of these types of polymer.130 Vanillic acid,catechol and other phenols have been treated withlaccase and the products precipitated onto thermome-chanical pulp. Paperboards prepared from this enzymetreatment route had 2È3 times the plybond strength ofmaterials which did not involve an enzyme treat-ment.131

Mechanistic studies on these enzymes involvingdimer formation provide useful information, especiallyon regioselectivity ; again these reactions may be a routeto formation of side-chains, and include HRP couplingof methyl sinapate,132 oxidative coupling of mono-lignols,133 and the dimerization of urushiol withlaccase.134


264 M. J. Donnelly

Structures based on, or containing, proteins or aminoacids

This area has had to compete with the chemical synthe-sis of polymers containing amino acids135h137 andrecombinant DNA technology,138,139 and much lessprogress has been reported on the enzymic synthesis ofsuch polymers. Polymers containing amino acids (forexample glycine, leucine or phenylalanine and ethanediol and adipic acid) have been shown to be readilysolubilized by the action of proteolytic enzymes.59,140

Numerous examples of peptide formation withenzymes in organic environments have been reported.The products are mostly low MW oligomers, but anumber of noteworthy features have emerged. Proteo-lytic enzymes can be highly stable in polar organic sol-vents,141 non-protein amino acids can be incorporatedinto peptides,142 the use of solvent can be dispensedwith by use of eutectics formed from the peptide precur-sors,143 and the type of L-a-amino acid methyl esterused deÐnes the chain length of the peptide formed in aone-pot reaction catalysed by papain.144 A promisingexample is the use of polyethylene glycol modiÐedpapain catalysis in toluene, in which the water level iscritical in deÐning the extent of polymerization ; pro-ducts containing up to ten residues can be formed.145

As a potential contribution to the synthesis of glyco-proteins with possible medical applications, the enzymicsynthesis of saccharideÈamino acid conjugates has beenachieved.146,147

Immobilization of enzymes onto “carrierÏ polymers iswell known, but attachment of enzymes to polymers foranother role warrants mention. Thus subtilisin, aftermodiÐcation with polyethylene glycol to enhance solu-bility and stability in water/organic solvents, can beincorporated into poly(methyl methacrylate) duringfree-radical polymerization. The enzyme retains itsactivity when used as a co-monomer, and variation ofthe reaction conditions allows control of polymerproperties such as porosity and molecular weight.148 Asimilar approach has been taken for the controlledrelease of biologically active molecules from self-biodegradable polymers : papain was covalently boundto a glutamic acid based polymer and the polymerÈenzyme conjugate crosslinked by radical copolymer-ization with acrylamide.149

CONCLUSIONS

Enormous progress has been made in the last few yearsin the in vitro use of enzymes for polymer synthesis. Thearea is complex due to the need to balance often contra-dictory factors. However, a remarkable range ofmaterials has been produced, of both technological andacademic interest. Further opportunities exist in thisarea, both for the production of simple analogues of

natural polymers and for more controlled structures,especially for sophisticated medical and other applica-tions.

ACKNOWLEDGEMENTS

Drs Nigel Briggs and Steve Rannard are thanked forhelpful technical discussions, and Mrs Dot Kettell forsecretarial support.

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Documents

In vitro enzymic synthesis of polymers containing saccharides, lignins, proteins or related compounds: a review