giorno & drioli_ 2000

Biocatalytic membrane reactors combine selectivemass transport with chemical reactions, and theselective removal of products from the reaction

site increases the conversion of product-inhibited orthermodynamically unfavourable reactions. Membranereactors using biological catalysts can be used in production, processing and treatment operations. Therecent trend towards environmentally friendly tech-nologies makes these membrane reactors particularlyattractive because they do not require additives, are ableto function at moderate temperature and pressure, andreduce the formation of by-products. The catalyticaction of enzymes is extremely efficient and selectivecompared with chemical catalysts; these enzymesdemonstrate higher reaction rates, milder reaction con-ditions and greater stereospecificity. Their potentialapplications have lead to a series of developments inseveral technology sectors: (1) the induction of micro-organisms to produce specific enzymes; (2) the devel-opment of techniques to purify enzymes; (3) the development of bioengineering techniques for enzymeimmobilization; and (4) the design of efficient productive processes.

Since the 1950s, when protease was first immobilizedon diazotized polystyrene, many enzymes and micro-organisms have been used in membrane reactors tocatalyse bioconversions (Table 1). The use of biocata-lysts for large-scale production is an important appli-cation because it enables biotransformations to be inte-grated into productive reaction cycles. Biocatalysts (e.g.enzymes, microorganisms and antibodies) can be used:(1) suspended in solution and compartmentalized by amembrane in a reaction vessel or (2) immobilizedwithin the membrane matrix itself. In the first method,the system might consist of a traditional stirred tankreactor combined with a membrane-separation unit. Inthe second method, the membrane acts as a support forthe catalyst and as a separation unit (Fig. 1).

The membrane can have a flat-sheet shape, assem-bled in a plate-and-frame module (Fig. 2a) or a spiral-wound module (Fig. 2b), or tubular-like, assembled intube-and-shell modules (Fig. 2c); it can also have a sym-metric (Fig. 2d) or an asymmetric (Fig. 2e) structure.

The biocatalyst can be flushed along a membrane mod-ule, segregated within a membrane module, or immo-bilized in or on the membrane by entrapment, gelifi-cation, physical adsorption, ionic binding, covalentbinding or cross-linking (Fig. 3).

The choice of the reactor configuration depends onthe properties of the reaction system. For example, bioconversions for which homogeneous catalyst distri-bution is particularly important are optimally performedin a reactor with the biocatalyst compartmentalized bythe membrane in the reaction vessel. The membrane isused to retain large components (i.e. the enzyme andthe substrate), while allowing small molecules to passthrough (i.e. the product). Concentration-polarizationphenomena and fouling strongly affect the perfor-mance of this type of reactor. Appropriate fluid-dynamic conditions and reactor design are necessary tocontrol performance at a steady state. Examples of newmembrane module design and fluid-dynamics opera-tions include the rotary disk1 and the use of baffles tocreate a Dean vortex2, respectively. Typical reactionsthat require biocatalysts to be suspended in a solution(in order to be in direct contact with both substrate andbiocatalyst, thus limiting diffusional resistance) include:(1) the hydrolysis of starch (by a- and b-amylase); (2)the fermentation of sugars (by yeast); (3) the hydrolysisof pectins (by pectinase); (4) the hydrolysis of K-casein(by endopeptidase); (5) the hydrolysis of collagen (byprotease); and (6) coenzyme-dependent reactions3.

A reactor with the biocatalyst segregated in the mem-brane module (either in the lumen or the shell) is suit-able for enzymes and cells that can be deactivated byshear stress. Reactors with segregated cells are suitablefor therapeutic applications4,5; for example, the bio-artificial pancreas, the bioartificial liver and the extra-corporeal detoxification device4,5. In addition to func-tioning as a selective separation barrier, membranes canalso provide structural support for the biocatalyst; thebiocatalyst can be loaded within the porous structureor on the surface of the membrane. The advantages of immobilizing enzymes include increased reactor stability and productivity, improved product purity andquality, and reduction in waste.

The enzymes and cells can be entrapped within thepolymeric matrix during the preparation of the mem-brane by phase-inversion. Alternatively, they can beentrapped by filtration within the porous layer of theasymmetric hydrophilic membrane and the symmetric

TIBTECH AUGUST 2000 (Vol. 18) 0167-7799/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(00)01472-4 339

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Biocatalytic membrane reactors: applicationsand perspectivesLidietta Giorno and Enrico Drioli

Membranes and biotechnological tools can be used for improving traditional production systems to maintain the sustainable

growth of society. Typical examples include: new and improved foodstuffs, in which the desired nutrients are not lost during

thermal treatment; novel pharmaceutical products with well-defined enantiomeric compositions; and the treatment of waste-

water, wherein pollution by traditional processes is a problem.

L. Giorno ([email protected]) and E. Drioli ([email protected])are at the Research Institute on Membranes and Modelling of ChemicalReactors, IRMERC-CNR, University of Calabria, Via Pietro Bucci17/C, Rende-CS, Italy.

hydrophobic membrane. When the biocatalyst is withinthe membrane, in order for the reaction to occur, thesubstrate has to be transported across the membrane tothe catalyst and the product has to be transported fromthe reaction site to the other side of the membrane,

where it is recovered as a permeate. In general, it is themass-transport resistance that primarily influences theperformance of these reaction systems. In order for a reactor to function at its optimal performance, it should work in a reaction-limited regime rather than a

340 TIBTECH AUGUST 2000 (Vol. 18)

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Table 1. Industrial applications of enzymes

Type of industry Enzyme Application

Detergent Proteases To remove organic stainsLipases To remove greasy stainsAmylases To remove residues of starchy foodsCellulases To restore a smooth surface to the fibre and restore the

garment to its original colours

Food Proteases and lipases To intensify flavour and accelerate the aging processLactases To produce low-lactose milk and related products for special

dietary requirements

Wine b-Glucanase To help the clarification processCellulase To aid the breakdown of cell wallsCellulase and pectinase To improve clarification and storage stability

Fruit juices Pectinases To improve fruit-juice extraction and reduce juice viscosityCellulase To improve juice yield and colour of juice

Oils and fats Lipases The industrial hydrolysis of fats and oils or the production of fatty acids, glycerine, polyunsaturated fatty acids used to produce pharmaceuticals, flavours, fragrances and cosmetics

Alcohol a-Amylases Liquefaction of starch or fragmentation of gelatinized starchAmiloglucosidase Saccharification or complete degradation of starch and

dextrins into glucose

Starch and sugar a-Amylases Enzymatic conversion of starch to fructose: liquefaction, saccharification and isomerization

Liquefaction of starchGlucoamylase and pullulanase SaccharificationGlucose isomerase Isomerization of glucose

Animal feed b-Glucanases The reduction of b-glucans

Brewing industry b-Glucanases The reduction of b-glucans and pentosans

Fine chemical Lipases, amidases and nitrilases Enantiomeric intermediates for drugs and agrochemicalsHydrolysis of esters, amides, nitriles or esterification reactions

Leather Lipases To remove fats in the de-greasing process

Textiles Amylases and cellulases To produce fibres from less-valuable raw materials

Pulp and paper Xylanases Used as a bleaching catalyst during pre-treatment for the manufacture of bleached pulp for paper

Figure 1Main configuration types of membrane reactors: (a) a reactor combined with a membrane operation unit, (b) a reactor with the membraneactive as a catalytic and separation unit.

trends in Biotechnology

ReactorReactants

Permeate

(a)

Enzyme

Immobilized enzymeMembraneMembrane

(b)

Reactants Retentate

Permeate

diffusion-limited regime. The parameter that can givea measure of this condition is the Thiele modulus6,which is given by Eqn 1,

(1)

where L is length, Vmax is the maximum velocity of thereaction, Km is the Michaelis–Menten constant and Deffis the effective diffusivity. This has the physical mean-ing of a ratio between the reaction rate and the diffusionrate. For f<1, the system is essentially controlled bykinetics and the mass-transfer limitation is negligible7.

Immobilization can also be obtained by gelification.When an enzyme solution is flushed through an ultra-filtration membrane that rejects the enzyme molecules,the enzyme will accumulate on the membrane surfaceand deposit as a thin gel layer characterized by enzy-matic catalytic activity. The actual gelation of enzymeproteins and their dynamic immobilization on themembrane surface occurs when the protein concen-tration at the membrane–liquid interface reaches thegel-concentration value. When the biocatalyst is immo-bilized on the surface, flushing the substrate solutionalong the enzymatic gel also causes the conversion of substrate into product in the retentate stream. If the enzyme is inhibited by the product, the reactor performance at steady state is decreased.

Enzyme attachment can take place by: (1) ionic bind-ing to ion-exchanger supports (e.g. DEAE cellulose,DEAE sephadex and carboxymethyl cellulose), (2)adsorption through van der Waals interactions tohydrophobic supports (e.g. polypropylene and teflon)

or (3) covalent binding between the amino or carboxylgroups of amino acids and the support membrane. Thecovalent bond is usually formed by active bridge mol-ecules, such as CNBr, and bi- or multifunctionalreagents such as glutaraldehyde. This immobilization ishighly stable, but has the disadvantage of denaturingthe native enzyme during the binding process; furthermore, it is difficult to replenish the denaturedenzyme. Therefore, this technique is only useful if the initial denaturation step is negligible. Both the typeof immobilization and the reactor configuration thatenables one to obtain these objectives have to be inves-tigated on a case-by-case basis because currently thereare no simple rules that enable the selection a priori ofa particular biocatalytic membrane reactor. Kineticmodels have been developed for specific immobilizedsystems operating on a laboratory scale that help us tounderstand the parameters that most influence the performance of a reactor8–12.

In addition to the type of immobilization, biocata-lytic membrane reactors are commonly distinguishedon the basis of their operation mode, for example,ultrafiltration membrane reactors, biphasic (organic andaqueous) membrane reactors, and so on. Ultrafiltrationmembrane reactors are used when the substrate has ahigher molecular weight compared with the product,and both substrate and product are soluble in the samesolvents. Here, by choosing a membrane with theappropriate pore size, the substrate is transported to theenzyme immobilized in or on the membrane, but itcannot pass through the membrane, whilst the productcan freely pass through and can be recovered from theother side of the membrane (Fig. 4a). If the substrate

f = L

Vmax

Deff • Km

12

TIBTECH AUGUST 2000 (Vol. 18) 341

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Figure 2The different types of membrane and membrane modules: flat-sheet membranes assembled in (a) plate and frame, and (b) spiral woundmodules; (c) a hollow fibre membrane assembled in a tube-and-shell module; (d) a symmetric membrane: a cross section of a flat membranemade of polyetheretherketone (PEEK-WC); and (e) an asymmetric membrane: a cross section of a capillary membrane made of polyamide.


Feed

Retentate

Spacers

Permeate Hollow fibreShell

Out In

Cover leaf

Filtrate

Porousmembrane

support

Feedsolution

Spacerscreen

Membrane

Permeateflow path

Membrane

(d)

(a) (b) (c)

(e)

and product have the same molecular size, they bothpass through the membrane; thus, it is necessary tomatch the transport rate with the reaction rate to ensurethat as the substrate reaches the enzyme, it is converted,and the product is transported to the other side.

If the substrate has a different solubility to the prod-uct (e.g. an ester and its hydrolysis products), a biphasicmembrane reactor can be used. In this type of system,the enzyme-loaded membrane is located between twoimmiscible liquid phases, an organic and an aqueousphase (Fig. 4b). The organic phase contains the sub-strate, which is flushed along one side of the membrane;the substrate is transported (by diffusion) to the enzyme,where the reaction takes place, and the product isextracted into the aqueous phase and flushed along theother side of the membrane. If the biocatalyst is selec-tive for only one of the two enantiomers present in aracemic mixture, a biphasic system is particularly use-ful for producing pure enantiomers. This has recentlybeen highlighted because of the importance of usingbioactive molecules as pure isomers when they have tobe administered to humans and animals.

Applications of biocatalystsBiocatalysts are particularly useful for certain appli-

cations, specifically in terms of energy consumption,safety, pollution prevention and the high quality ofproducts produced. However, the use of biocatalysts onan industrial scale is not yet fully established. Only afew examples have been described: (1) the productionof L-aspartic acid with Escherichia coli cells entrapped inpolyacrylamides13; (2) lactase (b-galactosidase) entrappedin the fibres of cellulose acetate and used for thehydrolysis of milk and whey lactose14; (3) the synthesisof the dipeptide aspartameTM using termolysin15; (4)the production of L-alanine using Pseudomonas dacunhaeimmobilized with glutaraldehyde16; (5) the glucose-isomerase reticulate with glutaraldehyde used in the

production of fructose-concentrated syrups17; (6) L-amino acids produced from racemic mixtures usingan amino acylase immobilized on DEAE-Sephadex18; (7)the production of L-malic acid by Brevibacterium ammo-niagenes entrapped in polyacrylamide19; and (8) the pro-duction of the (2R,3S )-trans isomer of methyl ester of4-methoxyphenylglycidic acid (a chiral intermediate ofdiltiazem – an important calcium-channel blocker usedin the treatment of hypertension and angina)20.

The major technological difficulties of using biol-ogical immobilized systems on an industrial level are:(1) the availability of pure enzyme at an acceptable cost(often the commercial enzymes are mixtures of severalproteins); (2) difficulties in immobilizing enzymes thatoften need expensive cofactors; (3) the necessity forbiocatalysts to operate at low substrate concentrations;and (4) microbial contaminations.

These aspects indicate that immobilized systems arenot generally reliable, and each system requires indi-vidual consideration in order to overcome their tech-nological difficulties. The processes wherein interdis-ciplinary investigation has guided the research since thestart of development have been shown to be more likelyto be effective20. Several recent studies have describedthe preparation of biocatalytic membrane reactors andtheir optimization for agro-food, pharmaceutical andbiomedical applications21–26.

The use of biocatalytic membrane reactors inthe agro-food sector

The integrated use of biocatalytic reactors and mem-brane processes, such as microfiltration, ultrafiltration,reverse osmosis, membrane extraction and so on, is par-ticularly important for products obtained by fermen-tation processes, such as organic acids and antibiotics,and in the processing of food and beverages (e.g. wine,fruit juices and milk). The main applications of bio-catalytic membrane reactors (Table 2) in the agro-food


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Figure 3Examples of biocatalytic membrane reactors with enzymes immobilized using different methods.


Biocatalytic membrane reactors

Support binding Cross-linking

Membrane serves asa catalytic and separation unit

Membrane serves asa separation unit

Biocatalystcontinuously flushed

along membrane

Biocatalystsegregated withinmembrane module

Biocatalystentrapped withinmembrane pores

Biocatalystgelified onmembrane

Biocatalystbound to

membrane

Physicaladsorption

Covalentbinding

Ionicbinding

sector are for: (1) reducing the viscosity of juices byhydrolysing pectins; (2) reducing the lactose content inmilk and whey by its conversion into digestible sugar;(3) the treatment of musts and wines by the conversionof polyphenolic compounds and anthocyanes; and (4)the removal of peroxides from diary products.

The hydrolysis of pectins in fruit juicesPectins are linear polymers of D-galacturonic acid,

characterized to a certain extent by the methylation oftheir carboxylic groups. The interactions betweenpectins and sugars (e.g. galactose, arabinose and rham-nose) are primarily responsible for the high turbidityand viscosity of fruit juices. Pectinases (able to hydro-lyse the polygalacturonic chain) and pectinesterases(able to hydrolyse the ester bonds of pectins to producepectic acids) are predominantly used to reduce the vis-cosity of fruit juices. The use of pectinases immobilizedin membrane reactors increases the possibility of reusingthe enzymes and controlling membrane fouling duringthe clarification process by cross-flow ultrafiltration27.Using the enzyme immobilized on the membrane (thepolysulfone spiral-wound module), the permeate fluxwas increased by ~30% compared with that obtainedwhen the enzyme was freely suspended in solution27.However, the steady-state permeate flux needs to beimproved for large-scale applications. Other importantparameters affecting the permeate flux are: (1) the amountof immobilized biocatalyst; (2) the transmembranepressure; (3) the axial velocity; and (4) the operationmode22,28,29.

The treatment of winePolyphenolic compounds in must (the pressed juice

of grapes) contribute to the characteristic organoleptictaste and colour of wine. To stabilize the must, laccaseis used to oxidize polyphenols in solution30. Anthocyaneshave also been hydrolysed by anthocianase immobilizedon synthetic and natural polymers, in order to stabilizemust, white and rosé wine (A. Martino et al., unpublished).

During the maturation process, a secondary fermen-tation occurs that converts malic acid into lactic acid(Fig. 5). Control of this reaction will enable the pro-duction of a product with good organoleptic proper-ties. Cells of Leuconostic oenos immobilized in a micro-porous membrane have been used to carry out themalolactic fermentation in white wine. The cells areimmobilized by cross-flow filtration through poly-propylene capillary membranes, the wine is recycledalong the membrane module and the permeate is col-lected aside. It is possible to precisely control this con-version by manipulating the appropriate cell density onthe membrane and the residence time of wine throughthe membrane. The increase of wine pH from 3.4 to 4.2results in improved taste because the slightly higher pHvalue prevents precipitation of proteins in the mouth.

The treatment of milk or cheese wheyThe use of membrane reactors as continuous systems

for the hydrolysis of lactose (present in whole milk orcheese whey) is an effective technique. Both lactoseconversion and the recovery of high-molecular-weightproteins can be accomplished in a single step and thehydrolysis of lactose has been carried out using b-galactosidase immobilized in membrane reactors31,32.

Currently, there is considerable interest in the produc-tion of hydrolysed milk for people who are intolerantto lactose and other dairy products. Producing hydro-lysed cheese whey is also of considerable interest forboth economic and environmental reasons33,34. Cheesewhey is a highly polluting product, consisting ofapproximately 0.7% protein, 5% lactose, 93% water andsalts. It is possible to increase the cost effectiveness ofcheese-making processes and reduce waste simply byrecovering and reusing these compounds. The wheyproteins (such as a-lactalbumin), which have excellentfunctional properties, can be recovered by ultrafiltrationand hydrolysed to produce many useful pharmaceu-tical intermediates. In addition, permeates from the


REVIEWS

Figure 4A schematic representation of the transport mechanism of (a) an ultrafiltration membranereactor, and (b) a biphasic membrane reactor.


Retentate

Product

Permeate

Substrate

Substrate

(a)

(b)

Organicphase

(R )-RCOOR1

(S )-RCOOR2 (S )-RCOOH

Membrane

Aqueousphase

Product +Unconvertedsubstrate

Enz

yme

Enz

yme

Aqueousphase

Organicphase

ultrafiltered milk and whey contain lactose, which canbe converted to glucose and galactose syrup.

The treatment of oils and fatsThe treatment of oils and fats has recently been inves-

tigated in order to evaluate the possibility of replacingtraditional chemical processes35 with biotechnologicalprocesses36. The use of lipase immobilized in membranereactors is well documented37–39 (Table 2). The perfor-mance of the different reactors depends on the physico-chemical properties of the reaction mixtures. Giornoet al.40 compared the efficiency of lipase to hydrolyse vegetable oil triglycerides into fatty acids and glycerolusing different reactor configurations: (1) a traditionalemulsion stirred tank reactor (STR); (2) an emulsified

organic–aqueous enzyme membrane reactor (E-EMR;where the reaction occurred in emulsion and the aque-ous phase was ultrafiltered through a membrane, thusseparating the product); and (3) a biphasic organic–aque-ous enzyme membrane reactor (B-EMR) (where thetwo phases were separated by the membrane that alsocontained the immobilized enzyme). The results showedthat the apparent volumetric reaction rate of the freeenzyme was higher compared with the immobilizedenzyme (6.8 and 4.5 mmol l-1 h-1, respectively), but thecatalytic activity of the immobilized enzyme was con-siderably more stable. The catalytic activity of lipase asa function of time in an emulsioned tank reactor or immobilized in a biphasic membrane reactor41 isshown in Fig. 6. Although the catalytic activity of the


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Table 2. Applications of biocatalytic membrane reactors in the agro-food industry

Reaction Membrane bioreactor Purpose

Hydrolysis of lactose to glucose and Axial-annular flow reactor Delactosization of milk or whey for human b-galactose (b-galactosidase) consumption

Hydrolysis of high-molecular-weight Asymmetric hollow fibre Production of baby foodprotein in milk (trypsin and with gelified enzymechymotrypsin)

Hydrolysis of raffinose Hollow fibre reactor with Production of monomeric sugars(a-galactosidase and invertase) segregated enzyme

Hydrolysis of starch to maltose CSTR with UF membrane Production of syrups(a-amylase, b-amylase, pullulanase)

Fermentation of sugars (yeast) CSTR with UF membrane Brewing industry

Anaerobic fermentation (yeast) CSTR with UF membrane Production of alcohol

Hydrolysis of pectines (pectinase) CSTR with UF membrane Production of bitterness and clarification of fruit juice and wine

Fermentation of Lactobacillus bulgaricus CSTR with UF membrane Production of carboxylic acids

Removal of limonene and CSTR with UF membrane Production of bitterness and clarification of naringin (b-cyclodextrin) fruit juice

Hydrolysis of K-casein (endopeptidase) CSTR with UF membrane Milk coagulation for dairy products

Hydrolysis of collagen and muscle CSTR with UF membrane Meat tenderizationproteins (protease, papain)

Conversion of glucose to gluconic acid Packed bed reactor Prevention of discolouration and off-flavour of (glucose oxidase and catalase) egg products during storage

Hydrolysis of triglycerides to UF capillary membrane Production of foods, cosmetics and fatty acids and glycerol (lipase) reactor emulsificants

Hydrolysis of cellulose to cellobiose and Asymmetric hollow fiber Production of ethanol and proteinglucose (cellulase and b-glucosidase) reactor

Hydrolysis of malic acid to lactic acid MF capillary membranes Improve taste in white wine(Lactobacillus oenos) with entrapped cells

Hydrolysis of fumaric acid to UF capillary membrane Production of food additivesL-malic acid (fumarase) reactor

Hydrolysis of olive oil triglycerides Hydrophobic plate-and- Treatment of oils(lipase) frame membrane reactor

Hydrolysis of soybean oil (lipase) Hydrophilic hollow fiber Treatment of oilsmembrane reactor

Hydrolysis of butteroil glycerides (lipase) Hydrophobic flat-sheet Treatment of oils and products for themembrane reactor cosmetics industry

Hydrolysis of milk fat (lipase) Spiral-wound polypropylene Treatment of fats and oilsmembrane reactor

Abbreviations: CSTR, continuous stirred tank reactor; UF, ultrafiltration; MF, microfiltration.

immobilized enzyme decreased, its stability increasedshowing no activity decay over two weeks of continuousoperation.

The production of pharmaceuticals usingbiocatalytic membrane reactors

The different systems that have been used for the production of amino acids, antibiotics, anti-inflamma-tories, anticancer drugs, vitamins, and so on, are sum-marized in Table 3. In addition, there are many systemsthat are also used for the production of optically pureenantiomers, and membrane technology strategies usedin the production of optically pure isomers have beenrecently described42. Many studies have also focused onthe production of amino acids, arylpropionic acids,amines and carboxylic acids43–48. The major problemsin the production of these compounds on a large scaleinclude: (1) the requirement for expensive cofactors;(2) the low water-solubility of the substrates; and (3)the separation and purification of the products fromcomplex solutions. However, studies carried out mainlyon a laboratory scale indicate that in many cases the useof the appropriate type of membrane and membrane-reactor design can overcome these difficulties.

In reactor systems using coenzyme-dependent reac-tions, negatively charged membranes are used to retainthe cofactor in the reaction vessel. Retention is obtainedby electrostatic repulsion between the negatively chargedcofactor and the membrane. Good rejection coeffi-cients (Rj 5 12Cp/Cf, where Cp and Cf are the con-centration in the permeate and feed, respectively) havebeen reported using the NTR7400 series of membranes.For example, for the retention of NADP(H), Ikemi etal. obtained an Rj value of 0.87 with an NTR-7410membrane49 and Giorno et al. obtained an Rj value of0.95 using an NTR-7450 membrane (L. Giorno et al.,unpublished); for the retention of NAD(H), Nidetzkyet al. obtained an Rj 5 0.99 with NTR-7430, an Rj5 0.89 with Y05 membranes and an Rj 5 0.98 withDS5 membranes50,51.

When the substrates have a low water solubility, theheterogeneous reaction is carried out in an emulsionedorganic–aqueous system where the organic phase con-tains the substrate, the product is extracted in the aque-ous phase and the enzyme is adsorbed at the interface.However, one problem associated with these systems istheir scale-up, and relates to the realization of stableemulsions and the separation of phases after the reac-tion has reached thermodynamic equilibrium. The useof biphasic membrane systems that contain the enzymeand keep the two phases separated20,52 (but in contactwith the membrane) can solve most of the problemspresented by the traditional systems (Fig. 4b). The mostfrequently used enzymes are hydrolases, in particular,lipases. In general, the enzyme activity and enantio-selectivity of immobilized enzymes are preserved if the transport of reagents through the enzyme-loadedmembrane is not limiting and the organic–water inter-face is obtained at the level where the catalyst is immo-bilized. For example, the production of S-naproxen ina biphasic membrane reactor demonstrated an enan-tiomeric excess of 92% with no decay of activity overa period of two weeks of continuous operation.

In addition to the use of enzymes in organic–aqueoussystems, their use in pure organic solvents is becoming

more widespread. Enzymes in organic media are ableto work in microenvironments that contain very littlequantities of water (usually less than the solubility limit)and several studies have confirmed that it is possible tocarry out biotransformations in organic media53,54.Immobilized enzymes operating in organic media shownovel properties such as enhanced stability and alteredsubstrate specificity. Several studies carried out usinglipases immobilized on DEAE-Sephadex55, chitosan56,zirconia57 have demonstrated these effects. The use ofa hydrophilic matrix can help to protect the biocatalystbecause it helps to maintain the water molecules aroundthe enzyme, and inorganic membranes are currentlyavailable that are resistant to the majority of organic sol-vents. An investigation of a transesterification reactionin anhydrous tetrahydrofuran carried out with free andimmobilized lipase demonstrated that an enzymeimmobilized on a zirconia membrane is more activethan an enzyme that is suspended and the selectivitytowards a reaction intermediate changes, resulting inthe production of a non-naturally available flavonoid57.

In many instances, when the product is obtained byfermentation it is present as a component of a complexsolution from which it needs to be separated and purified.In these cases, integrated membrane systems can beused for continuous production and downstream separation. For example, the production of L-lactic acid


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Figure 5The time course of L-malic acid (closed diamonds) and L-lactic acid(open squares) during malo-lactic fermentation in an immobilizedmembrane bioreactor.

Figure 6The productivity of free lipase (open squares) and immobilized lipase(closed squares) as a function of time operation. (Reproduced, withpermission, from Ref. 75.)


6543210C

once

ntra

tion

(g l–1

)

0 100 200 300 400

Time (h)

500 600 700 800


Pro

duct

ivity

(µm

ol h

–1)

0

50

100

150

200

0 5 10 15 20

Time (days)

is obtained by continuous fermentation in a membranefermentor. This consists of a traditional fermentor com-bined with an ultrafiltration unit. During operation ofthe bioreactor, its volume is maintained by adding freshmedium at the same rate as it permeates through theultrafiltration membrane. The solution recovered aspermeate contains the product, L-lactic acid, togetherwith other small molecules that are not retained by themembrane, whilst the cells and macromolecules arerecycled back to the bioreactor. The product thenneeds to be further purified and concentrated. Thepurification process can take place by membrane-basedsolvent extraction carried out through two membranemodules58. This operation is based on the transport ofthe solute from an aqueous solution at acidic pH (feed)to another at basic pH (stripping) via an organic phase(extracting). The phases are kept in contact at the poreentrance of the membrane, which is situated betweenthem. When specific carriers are used as the extractantphase, the separation can be highly selective.

The use of biocatalytic membrane reactors inbiomedical applications

The use of membrane reactors in the developmentof artificial organs is attracting significant attention59,60.

Most of the recent research activity has been focusedon organ replacement and extracorporeal treatment. Inmany medical tools, membranes act as separationdevices. For example, during haemodialysis through anartificial kidney, the patient’s arterial blood passesthrough a dialyser along one side of the membranewhile a buffer solution circulates counter-currently onthe other side of the membrane at a higher flow rate(approximately two-fold higher) in order to ensure theefficient removal of metabolic toxins through the mem-brane59. In artificial lungs, membranes are used forblood oxygenation. Blood and oxygen are flushedcounter-currently along hydrophobic membranes,which are permeable to gas but not to liquids. Bothextracorporeal and intracorporeal membrane oxygena-tors have also been investigated60. The extracorporealdevice is more efficient in terms of oxygenation,whereas the intracorporeal device is safer in terms ofsanitary conditions60.

Membrane bioreactors (using mammalian cells, frag-ments of tissues or enzymes) have important applications,for example, membrane bioartificial pancreas areformed by segregating isolated islets of Langerhans inmembrane devices. The islets have to produce insulinand regulate the patient blood-glucose levels. Therefore,


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Table 3. Applications of biocatalytic membrane reactors in pharmaceutical and biomedical treatments

Reaction Membrane reactor Purpose

Conversion of fumaric acid to L-aspartic acid Entrapment in polyacrylamide gel Pharmaceuticals and feed additives (Escherichia coli with aspartase)

Conversion of L-aspartic acid to L-alanine Entrapment in polyacrylamide gel Pharmaceuticals(Pseudomonas dacunhae)

Conversion of cortexolone to hydrocortisone Entrapment in polyacrylamide gel Production of steroidsand prednisolone (Curvularia lunata/Candida simplex)

Conversion of acetyl-D,L-amino acid to Ionic binding to DEAE-sephadex Production of L-amino acids for L-amino acid (aminoacylase) pharmaceutical use

Synthesis of tyrosine from phenol, Entrapment in cellulose triacetate Production of L-amino acids for pyruvate and ammonia (tyrosinase) membrane pharmaceutical use

Hydrolysis of a cyano-ester to Entrapment in biphasic hollow fibre reactor Production of anti-inflammatories ibuprofen (lipase)

Production of ampicillin and amoxycillin Entrapment in cellulose triacetate fibers Production of antibiotics(penicillin amidase)

Hydrolysis of a diltiazem precursor (lipase) Entrapment in biphasic hollow fibre reactor Production of calcium-channel blocker

Hydrolysis of 5-p-HP-hydantoine to Entrapment in UF polysulfone membrane Intermediate for the production of D-p-HP-glycine (hydantoinase and carbamylase) cephalosporin

Dehydrogenation reactions Confination with UF-charged membrane Production of enantiomeric amino (NAD(P)H-dependent enzyme systems) acids

Hydrolysis of DNA to oligonucleotides (DNase) Gelification on UF capillary membrane Production of pharmaceutical substances

Hydrolysis of hydrogen peroxide Entrapment in cellulose triacetate Treatment in liver failure(bovine liver catalase) membrane

Hydrolysis of whey proteins (trypsin, Polysulfone UF membrane Production of peptides for medical chymotrypsin) use

Hydrolysis of arginine and asparagine Entrapment in polyuretane membrane Care and prevention of leukaemia and (arginase and asparaginase) cancer

Abbreviation: UF, ultrafiltration.

the membrane can enable the transport of moleculesfrom the segregated cells to the blood and vice versa.Cells can be segregated in the lumen or in the shell ofhollow-fibre membranes, while blood flows along theopposite side of the membrane61 (for immunoprotec-tive reasons). Membrane bioartificial pancreas representan alternative approach to whole-organ transplantationin patients with insulin-dependent diabetes62,63.Recently, various investigations have been directed tothe protection of cells from immunorejection64, tomaintain cell viability and function, and to reduce thediffusion resistance of nutrients and metabolites65,66.

Isolated hepatocytes supported on membranes canfunction as a bioartificial liver for the temporary treat-ment of patients with acute liver failure. The hepato-cytes are segregated in hollow-fibre membranes64 or ina modular flat-sheet bioreactor26. They are mainly usedas extracorporeal devices where the membrane acts asa support for cell adhesion and as an immunologicalbarrier between the patient’s blood and the isolatedhepatocytes. The first clinical report of a bioartificialmembrane liver was reported by Matsumura et al.67 andan extensive analysis of these applications has beenrecently reported by De Bartolo and Drioli68.

The use of biocatalytic membrane reactors inwaste-water treatment

In recent years, membrane technology has also attractedconsiderable attention for the treatment of water andwaste-water69. Many studies have been carried outinvestigating the use of membranes and membranebioreactors for the treatment of activated sludge70 andthe removal of nitrogen from waste-water71,72. Lu et al.combined highly concentrated activated sludge pro-cesses with a rotary-disk ultrafiltration membrane forthe treatment of high-strength fermentation waste-water73. The performance of the system over the long-term (~130 days) demonstrated that the system isamenable for the treatment of fermentation waste-water.

Zoh et al. combined a membrane bioreactor with aceramic cross-flow ultrafiltration module for treatingsynthetic waste-water containing hydrolysis by-products of hexahydro-1,3,5-trinitro-1,3,5-triazine74

(RDX, which is highly explosive). Amongst the highlyexplosive compounds that are manufactured, RDX isthe most common and is also classified as potentiallycarcinogenic. The hydrolysis by-products of RDXconsist of acetate, formate, formaldehyde and nitrite;nitrate can be removed by using a denitrifying (anoxic)biological process that converts the hydrolysates toharmless end-products, such as N2 and CO2. A ceramicmembrane was used to recycle the cells back to thebioreactor while removing the treated water as a cleareffluent in the permeate74.

Mourato recently described an immersed hollow-fiber membrane system for water and waste-watertreatment75. The immersed membrane operates in anopen tank environment under a slight vacuum (20.15to 20.55 bar) to draw water through the membrane.This operating system therefore reduces membranefouling because contaminants are not forced into themembrane pores under high pressure. The membranesystem uses a reinforced membrane assembled in indi-vidual modules that can be combined to form cassettes(8–10 modules per cassette). The integration of a

biological reactor with the immersed membraneenables one to combine clarification and filtration ofan activated sludge process into a single-step process.The membranes form a barrier to solids and bacteria,and retain them in the process tank. The system canoperate at high levels of biomass (10 000 mg l21

to 15 000 mg l21) and high sludge retention time(20–50 days).

In addition to waste-water, waste-gas treatment hasalso recently been highlighted76–79. Biological wastegases having a low air–water partitioning coefficient canbe treated in membrane reactors where the contami-nating gases are transported through a membrane to aliquid phase and then converted by microorganismsinto less-dangerous products. Microorganisms such asMycobacterium, Pseudomonas putida and Hyphomicrobiumdegradate trichloroethylene, ethylene and dimethylsul-phide (I. De Bo et al., unpublished).

ConclusionsThe potential advantages of membrane reactor tech-

nology over more conventional approaches include itshigher efficiency and reduced costs owing to the inte-gration of bioconversion and product purification, thusreducing equipment costs and the number of process-ing steps. Enzymatic membranes will also contribute tothe growth of new research areas, such as non-aqueousenzymology, the use of antibodies as highly specific catalysts, the use of non-biological catalysts (such ascyclodextrines) and the development of novel biosen-sors for diagnostic purposes. However, in order to fullyestablish the use of biocatalytic membrane reactors,studies on the design of bioprocesses, particularly forlarge-scale production, the control of the reaction andkinetic mechanisms, and immobilization procedures,need to continue.

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REVIEWS

RNA, unlike DNA, folds into a myriad of tertiarystructures that are responsible for its diverse func-tions in cells. In addition to its role as a mediator

of genetic information from DNA to protein, RNAserves as genetic material (in some viruses), as a struc-tural component of many ribonucleoprotein (RNP)particles and in some cases as a catalytic subunit ofRNPs1. In most instances, RNA is associated withRNA-binding proteins (RBPs) that protect, stabilize,package or transport RNA, mediate RNA interactionswith other biomolecules or act catalytically on RNA2,3.The structural information obtained for RNA alone4,5

and RNA–protein complexes3 has elucidated a varietyof RNA tertiary structures and diverse modes forRNA–protein interaction6. The specific interaction ofproteins with highly structured RNAs should make it possible to target unique RNA motifs with small

molecules, thus making RNA an interesting target fortherapeutic intervention. The interaction of a naturalcompound or a chemically designed drug with anRNA molecule might influence the biological activityof the RNA by preventing or enhancing the bindingof effector proteins, by inhibiting RNA catalysis or byforcing an alternative conformation on the RNA7,8.

The ability of small molecules to interact with RNAhas been recognized for some time. Antibiotics are anexample of a class of naturally occurring compoundsthat interact with RNA. Many antibiotics are knownto target the ribosomal RNA (rRNA) of prokaryotes,thus affecting translation9,10. These antibiotics arechemically and structurally diverse, several examples ofwhich are shown in Fig. 1. The site and mechanism ofaction of some of these antibiotics are summarized inTable 1. Aminoglycoside antibiotics, the most well-characterized RNA-binding antibiotics, target the 16SrRNA near the A-site in the 30S ribosomal subunit(decoding of the mRNA takes place at the A-site,where the anticodon of a tRNA forms base pairs with

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67 Matsumura, K.N. et al. (1987) Hybrid bioartificial liver in hepaticfailure: preliminary clinical report. Surgery 101, 99–103

68 De Bartolo, L. and Drioli, E. (1999) Membranes and membranereactors in artificial organs. In Biocatalytic Membrane Reactors(Drioli, E. and Giorno, L., eds) pp. 191–206, Taylor & Francis

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brane separation for collective human excreta treatment plant. WaterSci. Technol. 25, 241–251

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74 Zoh, K-D. et al. (1999) Treatment of hydrolysates of the high explo-sives hexahydro-1,3,5-trinitro-1,3,5-triazine and octahydro-1,3,5,7-tetrazocine using biological denitrification. Water Environ. Res. 71(2),148–155

75 Mourato, D. (2000) Water reuse with the immersed membrane andthe membrane bioreactor. Desalination & Water Reuse 9, 27–30

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78 Reij, M. et al. (1998) Membrane bioreactor for waste gas treatment.J. Biotechnol. 59, 155–167

79 Mark, H. (2000) Environmental Processes I – Wastewater Treatment(Winter, J., ed.), Wiley–VCH

TIBTECH AUGUST 2000 (Vol. 18) 0167-7799/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(00)01464-5 349

REVIEWS

RNA as a drug target: methods forbiophysical characterization and screeningK. Asish Xavier, Paul S. Eder and Tony Giordano

RNA folds into complex structures that can interact specifically with effector proteins. These interactions are essential for

various biological functions. In order to discover small molecules that can affect important RNA–protein complexes, a thorough

analysis of the thermodynamics and kinetics of RNA–protein binding is required. This can facilitate the formulation of high-

throughput screening strategies and the development of structure–activity relationships for compound leads. In addition to

traditional methods, such as filter binding, gel mobility shift assay and various fluorescence techniques, newer methods such

as surface plasmon resonance and mass spectrometry are being used for the study of RNA–protein interactions.

K.A. Xavier ([email protected]), P.S. Eder and T. Giordanoare at Message Pharmaceuticals, 30 Spring Mill Drive, Malvern, PA19355, USA.

giorno & drioli_ 2000

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