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Cite this: DOI: 10.1039/c1gc15178a

www.rsc.org/greenchem CRITICAL REVIEW

Enzyme immobilization on/in polymeric membranes: status, challenges andperspectives in biocatalytic membrane reactors (BMRs)

Peter Jochems,a,b Yamini Satyawali,*a Ludo Dielsa,b and Winnie Dejonghea

Received 17th February 2011, Accepted 14th April 2011DOI: 10.1039/c1gc15178a

Immobilization of enzymes is beneficial in terms of improving the process economics by enablingenzyme re-use and enhancing overall productivity and robustness. Increasingly, membranes arethought to be good supports for enzyme immobilization. These resulting biocatalytic membranesare integrated in reactors known as biocatalytic membrane reactors (BMRs) which enable theintegration of biocatalysis and separation. Often the available commercial membranes requiremodifications to make them suitable for enzyme immobilization. Different immobilizationtechniques can be used on such suitable membranes, but no general rules exist for making a choicebetween them. Despite the advantages of BMR application, there are some issues which need to beaddressed in order to achieve up-scaling of such systems. In this review, the different aspects ofenzyme immobilization on membranes are discussed to show the complexity of this interdisciplinarytechnology. In addition, the existing issues which require further investigation are highlighted.

1. Introduction

Enzymes are becoming increasingly important in industrialprocesses due to their ability to work at milder pH, temperature

aSeparation and Conversion Technology, Flemish Institute forTechnological Research (VITO), Boeretang 200, 2400, Mol, Belgium.E-mail: Yamini.satyawali@vito.be; Fax: +32 14 321186; Tel: +32 14335690bUniversity of Antwerp, Department of Bioscience Engineering,Groenenborgerlaan 171, 2020, Antwerp, Belgium

ir. Peter Jochems

ir. Peter Jochems studied atthe University of Antwerp andGhent University, where in 2009he graduated as bio-scienceengineer specialized in celland gene biotechnology. Duringhis master thesis, he workedon enzymatic degumming ofcrude vegetable oils in coop-eration with Desmet Ballestra.He is currently working as aPhD student at the Depart-ment of Bioscience-engineering(University of Antwerp) and is

performing his research at VITO. Within the enzyme immobiliza-tion team, he is investigating process intensification by means ofbiocatalytic membranes.

Dr Yamini Satyawali

Dr Yamini Satyawali is cur-rently working at VITO as a re-search scientist. She is special-ized in membrane bioreactor(MBR) development for envi-ronmental applications, biore-mediation, groundwater andwastewater treatment. In 2009she completed her PhD fromTERI University, New Delhi(India) in environmental sci-ence and engineering. She wasa recipient of PhD fellowshipgrant from University Grants

Commission (UGC), Government of India and Department ofHigher Education and Scientific Research, Flemish Government,Belgium. She has 12 peer-reviewed publications to her credit.She is currently working on immobilization of hydrolases andoxidoreductases for food and environmental applications.

and pressure conditions, limited by-product formation, highactivity, and unparalleled selectivity. This growing importanceis resulting in a growing world-wide enzyme business, whichis believed to be worth around $2.7 billion by 2012. Thisgrowth is mainly driven by a general need for environmentallyfriendly technologies and sustainable production methods.1–3

Despite all the advantages, the implementation of enzymesis not always straight-forward, because a lot of issues ariseduring implementation. One of the major problems with theindustrial application of enzymes is their lack of stability notonly in temperature and pH extremes, but also under mechanical

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stress and in the presence of salts, alkalis and surfactants.1,4

Furthermore, enzyme re-use is almost impossible and productquality is often adversely affected by enzyme inactivation stepsand possibly also by difficult product purification schemes.5,6

Some of these issues can be addressed by the use of immobilizedenzymes.7

Enzyme immobilization is mainly performed to enable en-zyme re-use and thereby changing the economics of the processtowards a viable situation.7 This seems rather obvious, but atthe beginning of the 20th century the first trials of enzymeimmobilization were not promising.8 Some of these trials wereperformed by adsorption of invertase, which converts sucroseinto glucose and fructose, on charcoal. These immobilizedinvertases had a variable and rather low activity, which is notappropriate for industrial application.8,9 However, in the 1940s,invertase was the first immobilized enzyme to be used on anindustrial scale.10 This cost effective (at that time) productionmethod was used by Tate & Lyle, but this method had to beabandoned due to the limited flavor quality of the end product,Golden Syrup, which is an invert sugar syrup.11 In the 1960s,immobilized aminoacylase was used by the Tanabe Seiyaku Co.in Japan to produce L-methionine from a synthetic, racemicmixture of DL-methionine. After this successful application ofimmobilized enzymes, other applications started emerging.11,12

Currently the production of high fructose corn syrup is prob-ably the largest industrial process which uses immobilizedenzymes.3,11 Glucose isomerase, which is the enzyme used in thisprocess, is tremendously stabilized by immobilization. Improvedstability and re-usability of the enzymes are the two mostimportant advantages of enzyme immobilization, that enablethe use of otherwise cost-prohibitive enzymes.7,11

Apart from allowing the use of more expensive enzymes,enzyme immobilization also, in many cases, reduces the com-plexity of the production process, allowing continuous oper-ation as well as better control of the catalytic process.5,7,11,13

Some enzymes, mostly lipases, even show an increased activitywhen immobilized on the right support with the appropriatetechnique.14,15 Reportedly, despite all these advantages, only 20%

of industrial biocatalytic processes utilize immobilized enzymesin one of their processes.7 To enhance the acceptability ofimmobilized enzymes in industrial processes, the major chal-lenge is to find the immobilization techniques and operationalconditions that suit the ground requirements. Also, it is essentialto highlight the technological and economic benefits of theprocess.

Enzyme immobilization technology comprises several tech-niques, all of which have their own benefits and drawbacks.Furthermore, in terms of support, enzyme immobilization canbe done on carriers such as synthetic resins, biopolymers,inorganic polymers, etc. or on membranes.16,17 The conventionalenzyme immobilization techniques on various carriers, excepton membranes, have already been extensively reviewed byvarious authors.6,7,16,18–22 Therefore, this review will focus onthe use of polymeric membranes as a support for enzymeimmobilization. This focus is chosen because of the currentimportance of polymeric membranes, even though ceramic,metal and liquid membranes are gaining more importance.Nevertheless, polymer materials allow the design of membraneswith a wide variability of barrier structures and properties.Polymeric membranes will therefore also remain very impor-tant in the future.23 Enzyme immobilization on membranesis advantageous as some level of product separation couldalso be achieved along with biocatalytic conversion in enzymemembrane reactors (EMRs). Typically, membrane separationshave the advantage of requiring only a limited amount ofenergy, because there is no phase change involved in suchprocesses.24 Furthermore, the scale-up of this type of processes isrelatively easy and critical operational parameters can readily bemodified.25,26 Easy scale-up was demonstrated by Sepracor (now:Sunovion Pharmaceuticals Inc.) and Tanabe (now: MitsubishiTanabe Pharma Corporation), who worked on the productionof a diltiazem intermediate and successfully implemented thison an industrial scale (a plant with a capacity of over 75metric tons product per year).26 The continuous removal ofthe products can increase the productivity of product-inhibitedenzymes. Moreover, this continuous removal can shift the

Prof. Dr Ludo Diels

Prof. Dr. Ludo Diels is Sci-entific Manager for Sustain-able Chemistry. Previously, hewas the manager of VITO’sEnvironmental Technology De-partment for 15 years. Hehas a background in microbi-ology and biotechnology and isa professor at the Universityof Antwerp, Belgium (environ-mental stress, environmentalengineering, separation tech-nology, green chemistry andsustainable development). He

has more than 30 years experience in environmental biotechnol-ogy, biotechnology, engineering and chemistry. He has a wideexperience in environmental technology and has more than 150publications and 7 patents.

Dr ir. Winnie Dejonghe

Dr ir. Winnie Dejonghe is work-ing as a project manager inVITO in the unit separation andconversion technology and is in-volved as partner/coordinatorin several EU projects, nationalresearch projects and feasibilitytests. She is specialized in theremediation of contaminatedsediments and aquifers throughbiotic and abiotic techniquesand the study of the microbialcommunity structure by the useof molecular techniques ((Q)-

PCR-DGGE). Since 2007 she started to work in the field ofsustainable chemistry. Currently she coordinates projects on theimmobilization of enzymes on membranes/electrodes for processintensification and the microbial production of bioplastics.

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equilibrium of a reaction towards the product side and therebyincreasing the productivity of the whole process, which is aremarkable advantage of EMRs. This increase is clearly visiblein cases where chemical equilibrium affects the reaction yieldor where the reaction is thermodynamically unfavorable.5,27 It ismainly the decline in by-product formation and the low energyrequirements which make EMR technology an environmentallyfriendly production method.

This review analyzes the research in enzymatic membranereactors from the perspective of (a) membrane modificationmethods to make them amicable to enzyme immobilization and(b) different techniques for enzyme immobilization on mem-branes. Finally, the limitations and challenges in the existingprocesses are summarized and the potential areas for furtherinvestigations are discussed. This review aims to provide adetailed overview of the synergies that are possible by combiningmembrane and enzyme technology.

2. Enzyme membrane reactors

In an enzymatic membrane reactor, the membrane governs themass transport across itself thus also retaining the enzymesinside the reactor and achieving some level of product separationas well. This enzyme retention can be achieved by using differentmethods that are divided into two categories (Fig. 1). The firstcategory uses the membrane as a selective barrier thus retainingthe enzyme. The second category immobilizes the enzymes byphysical or chemical interactions in or on the membrane.27–29

Fig. 1 Enzymatic membrane reactors: looking at the membrane. (A)–(F):Membranes only used as selective barrier. Membrane retains enzyme byenzyme size (A), due to enlargement of the enzyme by its immobilizationon a carrier (B), due to enlargement of enzyme by cross linking (C), dueto electrostatic repulsion (D–E) or the enzymes are retained betweentwo membranes due to differences in IEP-point. (G)–(I): Biocatalyticmembranes enzymes can be covalently linked to membrane (G), non-covalently linked to membrane (H) or entrapped in the membrane (I).

2.1. Membrane as a selective barrier

The enzyme can be retained by the membrane in various waysviz. size exclusion, which implies that the enzymes are largerthan the membrane pores.29 This difference in size enables theretention, but in some cases the enzymes need to be enlarged, i.e.when the product is larger than the enzyme. Such an enlargement

of enzymes can be achieved by linking the enzymes together bycreating cross-linked enzyme crystals (CLECs) or cross-linkedenzyme aggregates (CLEAs) or by binding them onto a carrier.30

Retention can also be achieved by repelling charged enzymeswith charged membranes of the same sign, which is also knownas electrostatic repulsion. This method, however, is mainly usedto retain cofactors (NAD+, NADP+) in the reaction vessel.27,31

Yet, another method is isoelectric trapping, where the enzyme isretained between two membranes in the presence of an electricfield. These membranes have isoelectric point values far apartfrom each other and the isoelectric point of the enzyme lies inbetween those of the membranes.32 All these types of retentionhave their own advantages and have been included in otherreviews.5,33,34 However, this category falls outside the scope ofthis review.

2.2. Membrane as a selective barrier and support for enzymeimmobilization - the biocatalytic membrane reactor (BMR)concept

In this category, the membrane is used as a carrier or matrixfor enzyme immobilization as well as a selective barrier. Suchreactors are referred to as biocatalytic membrane reactors(BMRs) hereafter in this article. One of the interesting advan-tages of BMRs is the occurrence of transport phenomena, thatare governed by convective flows. Convective flows are indeedthe combination of diffusive and advective flows and thus aremore intense than only diffusive flows. In case of conventionalsupports/carriers such as beads, only such diffusive flows governthe process rendering it less effective.35 Unfortunately, to the bestof our knowledge, this has not been proven in a straightforwardlaboratory experiment. An important feature made possible byBMRs is process intensification or the reduction in equipmentsize. Process intensification in BMRs is achieved due to theintegration of biocatalytic conversion and product separation.The level of product separation depends upon the selectivity ofthe membrane used and this level will increase drastically withthe development of highly selective membranes.23,36

In BMRs, there is generally a close contact between the mem-brane and the enzymes as a result of the enzyme immobilization.Such a close contact affects the properties of the immobilizedenzymes depending both on the properties of the membraneand the enzyme itself.3,37,38 One of these enzyme properties,the pH optimum, often shifts after the immobilization on acertain carrier. Predicting the direction of such a shift in pHoptimum is sometimes possible by looking at the charge ofthe used carrier, but such predictions are uncertain and merelyindicative.39,40 Furthermore, enzyme properties are affected bythe immobilization technique used, which also will significantlydetermine the performance of the system.3,37,38 Therefore, thechoice of immobilization technique and membrane is vital toobtain a high performing system and both need attention whendesigning a new BMR system.

Immobilization techniques/chemistries exist in a wide varietyand those used on membranes are briefly summarized in section2.2.2. However, before an enzyme can be immobilized on amembrane, the appropriate membrane needs to be chosen. Suchmembranes suitable for enzyme immobilization are, however,very rare and often membranes need to be modified to make

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them suitable for enzyme immobilization. A brief summary ofthe different membrane modification techniques is given in thenext section.

2.2.1. Membrane modification for enzyme immobilization.Membranes are required to have some specific properties such asthe need to withstand certain mechanical stresses, temperatures,pH extremes, etc. These requirements are very specific for aparticular process and sometimes they can be easily met by asimple polymeric membrane. The characteristics of such a poly-meric membrane are mainly determined by the polymers used formembrane preparation. However, for certain processes, these re-quirements can only be met when the membranes are either madefrom polymer blends, or that they consist of multiple polymericlayers, or that they are made from polymers and (in)organicfillers (mixed-matrix membranes).40–43 In order to further renderthem suitable for a specific application, membranes can bemodified after their preparation. Similarly, for a specific applica-tion like enzyme immobilization, modification of the membranesurface is sometimes necessary. In fact, enzyme immobilizationis only possible on a surface that has enzyme binding sites whichare the regions where the membrane is able to form chemicalbonds with the enzymes. Essentially such enzyme binding sitesare not always present on the native membranes, but they canbe introduced by different surface modification techniques.

Membrane modification techniques therefore aim to intro-duce functional groups on the membrane surface and therebyimproving its surface properties without actually affecting thebulk properties.44 Some of these functional groups enable theimmobilization of enzymes on the membranes. Moreover, con-trolling the amount of functional groups, which are introducedon the surface, is crucial for maintaining good enzyme activity.40

The requirements for these modifications are largely dependenton the enzyme, because enzyme–membrane interactions play animportant role in maintaining the enzyme activity. Membranemodification can have an additional advantage in terms ofreducing membrane fouling by reducing undesired (secondary)interactions.23,45,46

Surface modifications on membranes can be achieved inthree different ways, grafting, etching and coating. Graftingmethods can in general be divided in two classes, i.e. “grafting-to” and “grafting-from” processes. The grafting-to process is theincorporation of polymers, which can have a very well controlledstructure, and can be tailor-made. The grafting-from process isthe incorporation and polymerization of monomers using aninitiator on the membrane surface.23 Etching, in fact, is thedeliberate introduction of defects on the polymer surface, whichresults in the formation of new functional groups. On the otherhand, coating is the addition of an extra layer on top of theoriginal polymer surface.

In the following section, various studies have been reportedwhere membrane modification was conducted with the aim toimmobilize enzymes on the modified membrane surface. Basedupon the methodologies used, the studies are classified here inthe four major categories (Table 1), which are elaborated in therest of this section.

Wet chemical methods. The wet chemical modification tech-niques often use liquid chemical reagents to introduce functionalgroups, such as amino, epoxy, azido, etc. (Table 1), throughout

Table 1 Surface modification techniques used for enzyme immobiliza-tion on membranes

Type of technique Functional group Enzyme

Wet chemical Amino, amidoxyme,amide, hydrazide

Urease45

Amide Cellulase140

Epoxy Urease47,141

Amino b-Galactosidase142

Amide, amino Catalase + GOx109

Polyaniline GOx143

HRP80

Azido GOx60

Cibacron Blue a-Amylase68

Cibacron Blue & Fe(III) Catalase70

UV-induced Epoxy Lipase50

Invertase48,49

a-Amylase131

Urease144

g-Irradiation Amino Lipase52

Plasma Amino GOx53

AChE56

Glucoseisomerase55

Hydroxyl Glucoseisomerase54

pAG, pSG Lipase57,145

AChE = Acetylcholinesterase; GOx = glucose oxidase; HRP =horseradish peroxidase; pAG = poly(a-allyl glucoside); pSG = poly(g-stearyl l-glutamate)

the membrane. These methods are considered effective in intro-ducing the functional groups throughout the porous structureof the membrane. The introduction of such functional groupsgenerally affects the hydrophilicity of the membrane surface.Carboxylic, amino, or thio groups of the polymer, grafted to themembrane, can be further used for enzyme immobilization. Forexample, the epoxy groups grafted onto the membrane surface,directly react with sulfhydryl, amino and carboxyl groups toform stable covalent bonds with biomolecules.47

The grafting of various chemical groups via wet chemicaltechniques (Table 1) can be easily conducted because no specificequipment is required. Despite the ease of operation, these wetchemical techniques for membrane surface modification are notsuitable for large scale, industrial applications because they lackspecificity beyond a certain degree. The repeatability of suchtechniques between polymers of different molecular weight,crystallinity or physical properties is generally considered quitelow. This results in a heterogeneous distribution of functionalgroups and unpredictable results. In addition, the environmentalburden of these techniques is fairly high due to the productionof hazardous waste and the use of concentrated corrosivesolutions.40

UV and Gamma radiation methods. In enzyme immobiliza-tion, UV irradiation has been used to manufacture mem-branes containing epoxy groups and commonly for this typeof membranes copolymers of poly(hydroxyethy methacrylate)–poly(HEMA) and an epoxy group containing monomer areused. Poly(HEMA) is a hydrophilic and biocompatible syntheticpolymer which is very stable against microbial contaminationand resistant to the attack by a large number of chemicals. Thepoly(HEMA) and epoxy group such as glycidyl methacrylate

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(GMA) undergo polymerization in the presence of UV radiationand an initiator such as a-a¢-azobisisobutyronitrile (AIBN).Such resulting membranes have been used for the immobiliza-tion of invertase.48,49 UV initiated photopolymerization was usedto coat a polyurethane (meth)acrylate and GMA blend on apolypropylene membrane to have a better surface hydrophilicity,biocompatibility, strength and chemical stability. This mem-brane, with a good protein binding capacity, was used forlipase immobilization.50 UV-induced modification techniquesare applicable to small as well as large scale applications.51

These techniques require only simple equipment, but are onlyable to modify a shallow region near the membrane surface.Furthermore, these techniques are usually cheap, rather fast anddo not damage the bulk properties of the membrane.44

As compared to UV, gamma radiation has high penetra-bility and has an advantage of also modifying the inter-nal surface of the membrane while retaining its mechani-cal properties. Application of gamma irradiation has beeninvestigated for inducing graft polymerization of GMA ona polypropylene hollow fiber membrane for lipase immobi-lization and the obtained membrane was used to hydrolyselipids.52

Plasma methods. Plasma surface modifications exhibit com-plex, multifunctional chemistries including oxidation, degra-dation, changes in the carbon/hydrogen ratio which leads tocross-linking and structural changes occurring in the surfacelayer without substantially changing the bulk structure. Thus,these chemistries occur only at the contact point betweenplasma and surface of the solid materials resulting in changedphysicochemical properties of the surface. The complexity ofthe chemistries is due to the complexity of plasma, whichcontains electrons, ions, radicals, metastable excited species andvacuum ultraviolet radiation.53 The plasma induced changes onpolymer surfaces are mainly determined by the type of gasesused. When using inert gases (Ar, He), the material is etchedand radicals, which can create various polar groups (peroxides,hydroperoxides) after contact with air, are introduced at thesurface. When using more reactive gasses (N2, O2, NH3, CO2),functional groups can be introduced at the membrane surface.Apart from these processes, plasma containing an organic mayform a deposit at a surface called plasma polymer. All theabove processes occur often simultaneously in the plasma,which doesn’t make it easy to predict which groups will prevailafter treatment. The processes can be directed by altering theused gas and the plasma conditions. However, these techniquesremain relatively nonspecific and not very suitable to obtainmonofunctionalized surfaces.54,55

The investigated applications of plasma for enzyme im-mobilization include modification of the polymeric materials’surface such as cellulose acetate, polypropylene and polysulfone,etc. for the immobilization of lipase, glucose oxidase, acetyl-cholinesterase, etc.53,56,57 All the studies described here introducebinding sites on the membrane surface. Although it has neverbeen used on a membrane, there is also the possibility of directimmobilization of biomolecules via plasma. In that case, theenzyme along with an organic precursor (e.g. acetylene, pyrrole,etc.) and a carrier gas (e.g. helium) are introduced into anatmospheric plasma. In this plasma, a polymer coating startsto form on the surface of the membrane and the enzymes

are entrapped within this coating. The main advantage of thistechnique is that it is simple and direct.58

2.2.2. Enzyme immobilization techniques/chemistries onmembranes. Every enzyme reacts in its own way towards animmobilization technique, therefore it is difficult to identify auniversal method for obtaining a good immobilized enzyme.Furthermore, there is currently no systematic approach todetermine the best immobilization technique, therefore a trialand error approach is implemented but at the same time thisalso implies that it is almost impossible to determine if theimmobilization protocol is optimal. It is therefore believed thatmany immobilized enzymes work in suboptimal conditions.6

Immobilizing enzymes on a membrane makes the choice be-tween different enzyme immobilization techniques even moredifficult because at that time the enzyme activity and membraneproperties both need to be safeguarded and complementaryto each other. Since the immobilization processes cannot begeneralized for a specific enzyme-membrane matrix, a widerange of investigations are required. Some practical guidelinesfor describing biocatalytic reactions including those of biocat-alysts immobilized on a carrier, are provided by the EuropeanFederation of Biotechnology Section on Applied Biocatalysis.59

However, there are no guidelines for reporting the membranespecific parameters nor is there a uniform reporting method inliterature.

The immobilization techniques used to make biocatalyticmembranes can be divided in several categories. In this review,the studies have been divided in three major categories Thefirst category includes the techniques which bind the enzymecovalently to the membrane. The second category contains thetechniques in which the enzyme is linked to the membrane in amanner other than covalent linkage. The third category containsthe techniques where the enzyme is not bound to but entrappedin the membrane.

Covalent binding of enzymes onto membranes. For the covalentbinding of an enzyme, presence of binding sites on the mem-branes is a prerequisite. These binding sites are the functionalgroups, which are already present or are introduced on themembrane surface (section 2.2.1). As evident from the studiessummarized in Table 2, various functional groups can be used tocovalently link enzymes with the membrane. The immobilizationis usually performed by immersion of the membrane in differentsolutions and sometimes by filtrating these solutions throughthe membrane. Depending on the used binding chemistry, thereis sometimes a need for an external energy source. In the case ofphotolinker mediated methods, light is used as an energy sourceto start the binding reaction. One of these methods uses UV lightto convert an azido-group into a highly reactive nitrene-group.60

Spacers are sometimes introduced between the enzyme andthe membrane to increase the distance between them. Increasingthis distance can be beneficial, because the direct immobilizationof enzymes on a membrane can cause conformational changesor decrease the accessibility of the active sites.61 The longer thelinker, the more the immobilized enzyme will react like the freeenzyme.37 However, the introduction of a long spacer does notalways imply a high retained activity, which can be deducedfrom Table 2. The spacer length is only a small factor comparedto the binding chemistry and the bulk characteristics of the

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Tab

le2

Enz

ymes

cova

lent

lyim

mob

ilize

don

am

embr

ane

Opt

ima

Stab

ility

Enz

yme

Mem

bran

eF

unct

iona

lgr

oup

Spac

er/c

ross

linke

rIm

mob

iliza

tion

proc

edur

eR

etai

ned

acti

vity

(%)

pHT

pHT

Stor

age

Per

mea

bilit

y(%

ofin

itia

l)R

efer

ence

s

a-A

myl

ase

p(H

EM

A-G

MA

)E

poxy

/Im

mer

sion

67–7

6→

↑+

++

/13

1A

LP

Mod

ified

nylo

nA

nhyd

ride

/F

iltra

tion

1/

//

//

/12

7A

myl

osuc

rase

Mod

ified

PP

Exo

py/

Filt

rati

on2–

4/

//

//

/95

b-G

alac

tosi

dase

Mod

ified

Nyl

onE

xopy

Hyd

razi

ne+

GA

Imm

ersi

on6

↓↑

→+

//

37C

2-di

amin

e+

GA

Imm

ersi

on18

↓↑

→+

//

37C

6-di

amin

e+

GA

Imm

ersi

on83

→↑

→+

//

37G

Ox

Mod

ified

nylo

nA

nhyd

ride

/F

iltra

tion

2/

//

//

/12

7PA

N-M

MK

-DC

PM

ID

ichl

orop

heny

l/

Imm

ersi

on68

–72

↑→

++

+/

43C

ellu

lose

Nit

rene

/U

Vin

duce

d19

→↑

++

+/

60A

Ac-

g-P

VD

FA

mid

eC

arbo

diim

ide

Imm

ersi

on29

↓↑

++

+/

84G

Ox

+ca

tala

seM

odifi

edPA

NA

mid

e/am

ino

GA

Imm

ersi

on/F

iltra

tion

22–8

8↑

→+

++

18–9

810

7–10

9M

odifi

edPA

Am

ide/

amin

oG

AIm

mer

sion

/Filt

rati

on36

–58

//

//

+66

–90

107–

109

HR

PA

AM

P-P

PA

min

oG

AIm

mer

sion

75/

//

/—

/79

Inve

rtas

ep(

MA

-alt

-H-1

)A

nhyd

ride

GA

+B

SAIm

mer

sion

15–6

5↑

→+

+/

/14

6p(

HE

MA

-GM

A)

Epo

xy/

Imm

ersi

on40

↑↑

++

+/

49L

acca

seM

odifi

edP

PA

min

oC

2-di

amin

e+

GA

Imm

ersi

on34

–78

→↑

++

//

83A

min

oC

2-di

amin

e+

GA

Imm

ersi

on/

↑↑

++

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membrane.37 A recent review on the use of epoxy supports forenzyme immobilization even suggests to minimize the spacerlength. Such short spacers do not compromise the activity of theenzyme, but stabilize the enzyme by several magnitudes becausea short spacer implies that the groups involved in immobilizationare quite fixed to their relative positions. However, if a highstabilization is obtained, the activity of the enzyme is usuallydecreased.62

In general, covalent binding leads to strong and stable enzymesupport attachment but at the same time may also reduceenzyme activity due to changes in the enzyme’s native structurewhich might change after covalent binding.63 From Table 2 it israther clear that regardless of the binding chemistry used, theenzyme activity normally decreases after immobilization, butthe immobilization of lipase is often an exception. A possibleexplanation can be found in the structure of lipases, which oftenhave a lid that blocks the active site. Due to interfacial activation,conformation changes occur and the active site is accessiblefor substrates. Hydrophobic supports are believed to cause thisinterfacial activation.6

Non-covalent binding of enzymes onto membranes. A widerange of non-covalent binding mechanisms such as hydropho-bic, hydrophilic or electrostatic interactions occur between anenzyme and a membrane. Adsorption is one of the mostsimple and common mechanisms that can be used to bindan enzyme non-covalently to a membrane. Adsorption as aphenomenon is mainly attributed to van der Waals forces,entropy changes and hydrogen bonds which are formed dueto the hydrophobic and hydrophilic interactions between theenzyme and the membrane.6

Adsorption can be performed on unmodified membranes(Table 3). However, in most cases specific membrane modifica-tions are required to achieve better immobilization and optimalenzyme activity. For example, lipases can exhibit a higher activityon hydrophobic surfaces. In a study, a chitosan membrane wasgrafted with stearic acid to increase its hydrophobicity and theoptimal grafting degree was found to be approximately 30%.Due to the increased hydrophobicity, as a result of grafting,the immobilized lipases retained ±25% more activity comparedto the native chitosan membrane. It is important to note thatonly a 30% grafting degree was appropriate to achieve optimalenzymatic activity, thus implying that the most hydrophobicmembrane does not necessarily result in the most activebiocatalytic membrane. The authors explain this by reducedbiocompatibility of the membrane, but another explanationcould be that too hydrophobic membranes cause higher enzymeloadings.64 Such high enzyme loadings are known to causemolecular crowding, which results in enzyme deactivation.19,35,65

Although adsorption is caused by rather weak interactions,a lot of enzymes are stabilized by these interactions and theiroptimal process conditions can be changed by them (Table 3).Stronger interactions can occur when charged compounds areintroduced in the membrane. These charged compounds canform ionic bonds with the enzyme. Depending on the isoelectricpoint of the enzyme and the pH of the reaction mixture, themembrane needs to be negatively (e.g. carboxyl groups) orpositively (e.g. protonated amino groups) charged.6 A very clearexample of introducing charged compounds in the membrane isthe introduction of Fe(III) ions in a modified chitosan membrane.

In that study, catalase was immobilized on a metal ion chelatedchitosan membrane to achieve a reversible immobilization byinteraction of a chelated metal ion and an electron acceptorgroup on the enzyme surface. The immobilization resulted in1.2 fold enhancement in residual enzyme activity at 55 ◦Cthus indicating that the thermal stability increases. Also theenzyme storage stability in phosphate buffer was increasedas the immobilized catalase lost only 23% of its activity ascompared the free enzyme which lost its complete activity in20 days.66 In addition, introduction of various dyes has alsobeen reported to enhance enzyme binding. Dye ligands forma special group of compounds due to the presence of variousreactive groups (a mono- or dichlorotriazine ring), which enablethe binding of a wide variety of proteins on this membrane.Furthermore, these dyes are often inexpensive and commerciallyavailable.67 Cibacron Blue F3GA, Brilliant green and Procionred are some dyes that have been successfully incorporatedin membranes.68–71 These dyes can easily be incorporated incellulose based membranes due to the hydroxyl groups presentin cellulose.67 These hydroxyl groups interact with the chlorideor fluoride atoms present on the triazinyl groups in the dyes.But with appropriate functionalization, it is also possible toincorporate these dyes in a lot of other membranes (e.g. PE, PPand nylon).72–74

Non-covalent bindings of enzyme are easier in terms ofpreparation of support and enzyme prior to binding, however,the risks of enzyme leaching from the support are higher in non-covalent linkages due to weaker enzyme support interactions.75

The enzymes bound to a membrane through non-covalentbindings can be linked together with glutaraldehyde (GA) tomake sure that the enzymes are not released from the membrane.This, however, is not a general practice as revealed by Table 3.69

Entrapment of enzymes into membranes. Entrapment of en-zymes has the advantage of wider applicability to many enzymesand relatively small changes in enzyme structure and functionafter immobilization.63 Entrapment of enzymes within a mem-brane can be achieved in two ways. Firstly, the enzyme can beincorporated during the membrane manufacturing. Secondly,an enzyme solution can be filtered through a membrane in orderto get it entrapped inside the membrane pores. As evident fromthe studies summarized in Table 4, the incorporation of enzymesduring membrane manufacturing results in low retained enzymeactivity compared with the retained activity of enzymes filteredinto the pores of the membrane. In general, no cross-linkingagents are used during entrapment. However, two exceptionsare reported in Table 4. In the first case, the cross-linking agent,GA, is used to interlink the different fibers which build upthe membrane, but this will also result in a limited amountof covalent bonds between the enzyme and the membrane.76 Inthe second case, the enzymes are cross-linked by GA inside thepores.77

The division in different types of immobilization techniquesviz. covalent, non-covalent and entrapment shows no clearindication that could help to determine the best category ofimmobilization techniques. However, Tables 2, 3 and 4 show aninteresting trend about the influence of covalent bonding. Thedecrease in enzyme activities are much more prevalent in thecase of covalent binding. At the same time, the highest retainedactivity is also seen for an enzyme immobilized covalently on

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Table 3 Enzymes immobilized by non-covalent interactions on a membrane

Optima Stability

Enzyme Membrane Functional groupImmobilizationprocedure

Retainedactivity (%) pH T pH T Storage

Permeability(% of initial) References

a-Amylase Nitrocellulose Cibacron Blue F3GA Immersion 124–250 / / / / / / 68Modified PVDF / Immersion / / / / / / 77 110PES / Immersion / / / / / / 25 110Modified PVDF / Filtration / / / / / / 37 110PES / Filtration / / / / / / 11 110

b-Galactosidase Anion exchange Amino Filtration 45 / / / / / / 151b-Xylosidase PA / Immersion 88 ↑ ↓ → → / / 86Catalase pHEMA-CB Cibacron Blue F3GA Immersion 75 → ↑ + + + / 70

Fe(III) Immersion 91 → → + + + / 70Modified CH Fe(III) Immersion 32 → ↑ + + + / 66

GOx Modified PAN Polyaniline Immersion 60 ↑ ↑ + + + / 143HRP PP / Immersion / / / / / — / 79

AAMP-PP Amino Immersion / / / / / — / 79Modified PP Polyaniline Immersion / / / / / + / 80

Lipase CH Amino Immersion 67 ↑ ↑ → + / / 64Modified CH Amino + stearyl Immersion 72–84 ↑ ↑ / + / / 64Modified PP DEA Immersion / / / + + + / 52Modified CA Cibacron Blue F3GA Immersion 3–86 → → + + / / 71Polysulfone PVP/PEG Immersion 12 ↓ ↑ → + / / 152PANCMA CH Immersion 54 ↓ ↑ + + / / 153PP / Immersion 55–60 ↑ ↑ → + / / 154Modified PP PAPs Immersion 71–87 ↑ ↑ → + / / 154

PAG Filtration 47–53 / / / + / / 57,145PSLG Filtration 69–76 / / / + / / 57,145

PP / Filtration 33–89 / / + + / / 155/ Filtration 55–60 / / / + / / 57,145

PGA Ethyl cellulose Brilliant green* Immersion 39 → → + + / 50 69Proline* Immersion 72 / / / / / / 69

+ = improved compared to free enzyme; - = deteriorated compared to free enzyme; → = steady state; ↑ = higher; ↓ = lower; / = no appropriatedata available; * = GA as linker; AAMP-PP = allylamine plasma treated polypropylene; CA = cellulose acetate; CH = chitosan; DEA = diethylamine; GA = pentane-1,5-dial (glutaraldehyde); GOx = glucose oxidase; HRP = horseradish peroxidase; PAG = poly(a-allyl glucoside); PANCMA =poly(acrylonitrile-co-maleic acid); PAPs = phospholipid analogous polymers; PEG = poly(ethylene glycol); PGA = penicillin G acylase; pHEMA-CB =poly(2-hydroxyethyl methacrylate) Cibacron Blue F3GA derivatized; PSLG = poly(g-stearyl l-glutamate); PVDF = poly(vinylidene fluoride); PVP =poly(n-vinyl-2-pyrrolidone).

Table 4 Enzymes immobilized by entrapment in the membrane

Optima Stability

Enzyme MembraneImmobilizationprocedure

Retainedactivity (%) pH T pH T Storage

Permeability(% of initial) References

AChE PVA* Incorporation duringpreparation

40 → / — / + / 76

b-Galactosidase pHEMA Incorporation duringpreparation

4 → → → → / / 39

b-Glucosidase Polysulfone Filtration 131 → → + + / 57 15Dextranase Polysulfone + activated carbon Incorporation during

preparation/ / / / / / / 87

Fumarase Polysulfone Filtration 100 / / / / / / 2Lipase PVA Incorporation during

preparation90–100 / / / + + / 156

PAN Filtration 70 / / / / / / 88PTFE* Formation of CLEAs

in the pores/ / / / / / / 77

PA Filtration 9–1583 / / + + / 4–7 90–92Composite membrane (CA & PTFE) Filtration 60–100 / / + + / / 89

PLE Polysulfone Filtration 62–63 / / / / / / 93,94Urease Cellulose acetate Incorporation during

preparation7–43 / / / / + / 106

+ = improved compared to free enzyme; - = deteriorated compared to free enzyme; → = steady state; ↑ = higher; ↓ = lower;/ = no appropriate dataavailable; * = GA as linker; AChE = Acetylcholinesterase; CA = cellulose acetate; CLEA = cross-linked enzyme aggregates; PA = polyamide; PAN =polyacrylonitrile; pHEMA = polyhydroxyethylmethacrylate; PLE = pig liver esterase; PTFE = polytetrafluorethene; PVA = polyvinyl alcohol.

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a membrane.78 This implies that the effect of the covalentimmobilization is much stronger than the effect of the otherimmobilization techniques. Among most of the immobilizationtechniques, higher activity of the enzyme is often seen forimmobilized lipases, but also b-glucosidase entrapped in apolysulfone membrane shows a higher activity compared to thefree enzyme.15

In general, it is believed that immobilization can improve thestability of an enzyme. In the case of covalent binding, a lotof the articles confirm this, however, exceptions can be found.For instance, immobilization of horseradish peroxidase ontoplasma treated polypropylene membrane via glutaraldehydelinkage resulted in low storage stability.79 However, horseradishperoxidase can have an improved stability, when immobilized onpolypropylene membrane grafted with polyaniline.80 Similarly,laccase immobilization on modified polyvinylidene fluoride(PVDF) membrane showed that the immobilized enzymeactivity dropped by 30% in the first three months of storagewhereas in case of free enzyme no significant activity decreasewas observed in the first two months.81 The data for the otherimmobilization techniques is much more limited, but for allthe techniques some of the enzymes show a decreased stability.Acetylcholinesterase, which is entrapped in a PVA membrane,showed a decreased pH stability.76

The optimum pH of the immobilized enzyme can change afterimmobilization, but this change cannot be predicted easily. Thiscan be illustrated with a laccase of two different organisms. Whenthese two enzymes were immobilized in almost the same way ona modified polypropylene membrane, the immobilization causedno change in pH optimum for one enzyme, while for the otherthe optimal pH increased from 4.6 to 5.5.82,83 Another example isthe covalent immobilization of glucose oxidase on three differ-ent membranes, respectively: a mixed polyacrylonitrile (PAN)and methylmethacrylate dichlorophenyl maleimide (MMA-DCPMI) membrane, a photoreactive cellulose membrane anda modified PVDF membrane (Table 2). The results indicatedthat the first immobilization method causes an increase of thepH optimum, the second a decrease and the third no change atall.43,60,84 The optimum temperature of the immobilized enzymecan change after immobilization and in most of the casesthis optimum is equal or higher than that of the free enzymedepending upon the enzyme and immobilization technique.For example, the temperature optimum of glucose oxidaseand lipase stayed similar to that of the free enzyme afterimmobilization on a modified polyacrylonitrile membrane anda polypropylene membrane, respectively.50,60 However, in otherstudies, the optimum temperature for glucose oxidase andlipase increased 1.1 to 1.2-fold after immobilization.84,85 This,however, is not always the case and one of these exceptionsis b-xylosidase immobilized on a polyamide membrane. Thetemperature optimum of this immobilized b-xylosidase is 25 ◦Clower than that of the free enzyme.86

2.2.3. BMRs and separation. Often the articles dealingwith BMRs state that one of the advantages of BMRs is thecombination of separation and conversion, which cannot beobtained by using other supports. This is most certainly true,however, most of these articles do not report any data onproduct purity or how membrane performance is influenced

by the enzyme immobilization. The most common methods toachieve product separation in BMRs are filtration and extraction(membrane contactors). The principle of these two methods isshown in Fig. 2.

Fig. 2 Filtration versus contactor. A: dead-end filtration, B: cross flowfiltration, C: membrane contactor.

Membrane filtration can only be used to separate substratesand products when their size is different from each other. In thecase of hydrolysis of biopolymers, the product is smaller thanthe substrate and relatively pure products can be obtained in thepermeate. Dextranase immobilized in a mixed-matrix membranewas used to hydrolyse dextran into oligodextrans. The mem-brane’s molecular weight cut-off (MWCO) was 8 kDa, however,the products in the permeate ranged from 200 Da to 40 kDa. Thisis due to the membrane characteristics and the same membranewithout enzymes gives similar results. It was however possible toalter the average product size by altering the enzyme loading inor on the membrane.87 In the other case, the products are largerthan the substrate and the product remains in the retentate.An example is the conversion of a soluble product into aninsoluble one, which causes the formation of particles, which insome cases can be retained by a microfiltration membrane. Theconversion of N¢,N¢-(dimethyl)-N-(2-hydroxyphenyl)urea intoan insoluble oxidation product by laccase is such an example.This process was investigated for waste water treatment.81 Thesetypes of separations can be used for the purification of chemicals,however, for the synthesis of fine chemicals the use of membranecontactors is often more appropriate.

Membrane contactors separate products based on theirdifference in distribution coefficient. In most cases, there is asolvent at one side of the membrane and an aqueous solutionon the other side of the membrane. Lipases are often usedto convert a non-polar product into a polar product, whichallows a rather easy separation in a membrane contactor.Racemic ibuprofen-esters have already been hydrolysed bydifferent lipases to achieve the pure (S)-ibuprofen acid, whichis of pharmaceutical value.88,89 Another pharmaceutical, whichis formed in a similar way, is naproxen.90–92 Also an esterasecan be used to catalyze an enantioselective hydrolysis and thiswas used for the production of (1S,2R)-cyclohex-4-ene-1,2-dicarboxylic acid monomethyl ester from cis-cyclohex-4-ene-1,2-dicarboxylate.93,94 Unfortunately, all these studies only report

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partial information about the product purity. In most cases,only the optical purity of the obtained product is stated, butno information is given about the amount of substrate presentin this product stream. The ultimate goal should always be tomake a comparison of a new technology with the best availabletechnology.

Apart from product purity, product formation often needsto be controlled especially in those processes where over-processing of the product is possible. Changing operationalparameters (flow rate, pH, . . . ) can alter the obtained product.Maltooligosaccharides produced with amylosucrase can havea different average length depending on the flux through themembrane.95 Similar effects are thought to take place duringdextran hydrolysis. Optimizing the ratio between the separationand reaction processes is vital to obtain a productive system.87

3. Existing issues emphasizing the future researchperspectives in BMRs

An appropriate immobilization technique is always the centre ofresearch efforts in case of enzyme immobilization and is indeedan important issue. Since every enzyme has a unique way ofresponding to an immobilization technique, generalizations areoften difficult. Moreover, in order to compare different tech-niques that are used for enzyme immobilization on membranes,investigation on a complete set of parameters is required. Theseparameters often include enzyme activity, stability, intrinsicproperties such as optimum pH of the enzyme and the efficiencyof the immobilization process. In addition, the values of suchparameters, in the case of immobilized enzymes, are oftendifferent from those of the free enzymes. Many observed changesin the enzyme’s characteristics are believed not to be due to theinteractions between the enzyme and the membrane or becauseof the conformational changes of the enzyme, but due to the factthat the micro-environment in the vicinity of the membrane iscompletely different from the bulk environment.37,96

Apart from the issue of appropriate enzyme immobilization,the BMRs also encounter some general problems. The mostimportant problems, which are encountered in enzymatic mem-brane reactors, are discussed in the following section.

3.1 Changes in membrane performance due to enzymeimmobilization

3.1.1 Flux and permeability of membranes. Membraneperformance in terms of flux (or permeability) and selectivity,is often hindered by membrane fouling. Such fouling occurs invirtually all membrane processes and especially pressure-drivenmembrane processes. In general, this leads to a declined perme-ate flux, increased hydraulic resistance and an overall decline inmembrane performance. The phenomena, which occur duringthe fouling process, are caused by complex interactions betweenthe membrane and the feed solution.

Fortunately, the basic mechanisms are known and this helpsto understand and predict membrane fouling. The first basicmechanism is the reversible formation of a gel layer dueto concentration polarization. The second mechanism is theirreversible absorption of soluble material on the membrane(surface and inside the pores). This leads ultimately to blocking

of the pores by high molecular weight products and it mayalso form a layer on top of the membrane.97–99 Although thebasic mechanisms are known, prediction of membrane foulingis difficult and especially in those cases where natural products,i.e. starch or proteins, are a part of the feed. This is mainly dueto the complex nature of the occurring interactions and in thecase of natural products also due to the variability of feedstockmaterial. Still a lot of research has to be performed to understandthese interactions between membrane and complex feed streams.In practice, this variability of the feed stock causes the optimaloperational values to shift depending upon the feedstock.25,97

Knowing the optimal operational values is important, becausemembrane fouling can be postponed by changing the processparameters. However, the only real possibility, to maintain highenough productivity, is to regularly clean or eventually replacemembranes. This cleaning and replacing of membranes is acostly operation and therefore a lot of studies are performed toavoid membrane fouling phenomena.98,100–102 One of the methodsto control fouling is to alter membrane characteristics becausefouling largely depends upon the interactions of membrane sur-face with the reaction mixture.23,101,103 Even when the membraneis only used to retain the enzyme in the reaction vessel, it may beinteresting to modify its surface. One advantage of this method isthat changing the membrane surface does not necessarily requirechanges in the process parameters.23,101,103 This is advantageous,because in EMRs, the enzymes can limit the possible operationalchanges. The membrane modification discussed in section 2.2.1for enzyme immobilization are also used for altering membranesurfaces to combat fouling.103

While it is normally not recommended to use membranes inconjunction with viscous and concentrated streams, it has beenreported that immobilizing enzymes on the membranes, whichcan reduce the size of these high molecular weight products,can positively affect the permeate flux. For example, in casesof some specific feeds such as the filtration of fruit juices, ithas been reported that in membrane filtration with immobilizedpectinase the flux achieved was 1.3-fold higher as compared tofiltration with the free enzyme in the feed.104 Another positiveeffect also observed in a case of pectinase immobilization forjuice clarification was prolonged operation without cleaning,because the permeate flux remains longer at an acceptablelevel.105 In the previous examples, the foulant is broken downby the enzyme, but even more striking is that the immobilizationof urease in a cellulose acetate membrane resulted in a decreasedprotein absorption. Such a decrease results in reduced fouling,however, this reduced absorption was only proven for bovineserum albumin (BSA).106 Despite these positive results, there arealmost no studies reporting the effect of immobilized enzymes onmembrane performance. Since it is expected that immobilizingenzymes on a membrane will at least change its hydrophobicity,its surface charge and morphology, such studies would bebeneficial for further advancement of BMRs.5,104,105

Apart from fouling induced by filtration, the flux of amembrane also often declines upon enzyme immobilization,however, sometimes the immobilization hardly influences theflux of the membrane.107–110 As obvious from Tables 2–4 (section2.2.2) only a few studies have reported the changes in flux of themembrane upon enzyme immobilization. The argument in favorof such a practice could be that the information on the change in

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productivity is much more important than the changes in flux.Unfortunately, such values are also not often reported. In orderto better understand the differences between immobilizationprocesses, it would be interesting to know how these processesinfluence the flux of the membrane.

3.1.2. Selectivity/rejection of individual compounds by mem-branes. Membrane selectivity can be defined as the ability ofa membrane to separate two products/molecules from eachother. Such a separation of two products is only possible ifthe difference in rejection by the membrane is large enough.Membrane selectivity is not solely depending on membranecharacteristics, but also on feed characteristics and differentprocess parameters. Therefore it can be considered as applicationor even process dependent and due to this large numberof parameters, it is difficult to predict the rejection of asingle component in complex process streams. The rejectionof such a component is influenced by its molecular weight,shape, charge and hydrophobicity together with a lot of otherfactors.99,111,112 Enzyme immobilization on a membrane oftenalters its selectivity and the complexity of the system increases,which makes any prediction about membrane behavior evenmore difficult. First of all, enzyme immobilization changes themorphology of the membrane and its average pore size. It hasbeen previously reported that immobilization of the enzymessignificantly reduces the molecular weight cut-off (MWCO) ofa membrane. Indeed, a 90% decline in the pore diameter ofpolysulfone membrane (50 kDa MWCO) was reported afterlipase immobilization.113

It is also interesting to note that changing buffer concentra-tions or the addition of stabilizing salts can affect membraneselectivity and therefore should be carefully considered beforeimplementation in existing processes. For example, in the caseof nanofiltration membranes, a decrease in the retention ofneutral solutes is observed in the presence of charged ones.This decrease becomes even larger when the concentration ofthe charged molecules increases, but also depends on the usedmembrane. This phenomenon can be explained by differenthypotheses, however, still a lot of research has to be performed tounderstand the underlying mechanisms.114,115 Also the oppositephenomenon is mentioned in the literature. A decreased saltretention was observed in the presence of high concentrationsof sugars.116 Such a decreased retention is often not desiredin BMRs, because they can oblige the addition of extra saltsto stabilize the used enzymes. Alternatively, such an additioncan also be desired when more open membranes are used,because such membranes cannot retain these stabilizing salts.117

Regardless of which phenomenon occurs, it is essential that theseeffects are taken into account.

In addition, other process parameters such as temperatureand product concentration influence reaction efficiency as wellas membrane selectivity. In terms of membrane selectivity,increasing the temperature often leads to reduced retentions,probably because the water layer adsorbed on the pore wallsbecomes thinner and this results in a larger effective porediameter.118 High feed concentrations also affect product re-tention negatively, because the back diffusion is hindered inthe concentration polarization layer present at the membranesurface. This effect is even more intense when the MWCO of

the membrane is higher and the products are smaller. Apartfrom the feed concentration, the concentration polarizationlayer also depends upon the applied pressure, which eventuallyaffects the membrane compaction and permeate flux. Therefore,the optimal applied pressure will have to be a compromisebetween solute rejection and permeate fluxes.119,120 Furthermore,the separation will improve, when the membrane has a narrowpore size distribution.36

Rejection of neutral solutes during nanofiltration is notonly affected by simple sieving effects, but also by interactionbetween solute and solvent. This allows the fractionation of twouncharged products with almost the same molecular weight.Also, a difference in molecular conformation can result indifferent retention efficiencies.118 In general, uncharged solublemolecules are rejected by membranes in such a way that asigmoid curve is obtained when the rejection is plotted as afunction of the molecular size. Therefore, a single membraneseparation is often not enough to separate two molecules, whichare only slightly different in size, charge or hydrophobicity. Tocomplicate things, also process parameters, such as pressure andtemperature, can alter the selectivity of a membrane. Especiallythe rejection of small uncharged molecules is easily affected bychanges in process parameters.99,119,121,122

Furthermore, specific separation problems arise for particularapplications such as protein purification. Proteins, that needto be purified, must differ at least 10 times in size from theimpurities to enable an effective separation with conventionalultrafiltration processes. Fortunately, high performance tangen-tial flow filtration (HPTFF) may lower this limit. HPTFF isa conventional tangential flow filtration linked to a co-flowarrangement, which results in a transmembrane pressure that isfairly constant across the whole membrane. In such an assembly,it is also very important that the pH and the ionic strength ofthe feed is controlled very precisely.123,124

3.2. Changes in enzyme performance due to enzymeimmobilization

Enzyme performance is determined by its activity, stability,selectivity and specificity and all of them can be improvedby enzyme immobilization.20 Although in section 2.2.2 it hasalready become clear that in many cases immobilization affectsvery negatively the activity of enzymes, in some cases it alsoaffects them in a positive way.15,78 In addition, it became clearthat choosing the right immobilization technique and the rightmembrane is critical for optimal activity retention. Furthermorein section 2.2.2 the stability of immobilized enzymes is discussedand in most cases the stability improves or at least remains thesame compared to the free enzymes. However, several exceptionswhere the enzyme stability declines after immobilization wereobserved.76,79,81 Such a decline in stability is striking, butunfortunately the reasons for these declines are unknown and agood hypothesis is also missing. The activity and stability of theimmobilized enzyme will determine largely the productivity ofthe biocatalytic membrane.

Apart from activity and stability, selectivity and specificityplay important roles in enzyme performance. Specificity andselectivity are two very closely related terms. Normally enzymeshave a very high specificity towards their natural substrate,

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however in industrial situations the enzyme often needs toperform unnatural reactions. Thus altering the enzyme substratespecificity is often desired. Another factor, which industryregularly wants to tune, is enantioselectivity and its importanceincreases when the enzymes are used to produce fine chemicalsor pharmaceuticals. For the production of such products highenantioselectivity is often a prerequisite and is also one of themain reasons to use enzymes.20

The enzymes which are mostly used in organic synthesis arelipases. Different lipases have already been used to producea wide variety of products by stereoselective hydrolysis andesterification reactions. Kinetic resolution of racemates for theproduction of optically pure isomers is one important processfor which they are used. The kinetic resolution of (S)-naproxen-acid was performed in a biocatalytic membrane reactor andcompared with the free enzyme reaction. The free lipasehad a higher enantioselectivity compared to the immobilizedlipase. However, by altering the immobilization procedure, itwas possible to reach almost the same selectivity with theimmobilized enzyme.125 Another example is the hydrolysis ofthe racemic ester of ibuprofen to produce (S)-ibuprofen. Inthis case the enantioselectivity of the lipase improved afterimmobilization. Furthermore, there was a certain tradeoffbetween activity and selectivity. Fortunately, the stability of theenzyme improved more than 3 times due to the immobilization.By tuning the process conditions it was possible to improve theenantioselectivity of the enzyme even further.89

Predicting the behavior of enzymes after immobilization isvery difficult and the enzyme characteristics can be affectedpositively or negatively. A lot of trial and error experimentsare needed to find the desired behavior.6 The optimizationof the enzyme characteristics should be done together withoptimization of the membrane characteristics to achieve goodbiocatalytic membranes.

3.3. Productivity, lifetime & stability of biocatalyticmembranes

BMRs have a huge industrial potential, however, industry willalways look at three important aspects of these systems, e.g.productivity, lifetime and stability. All these aspects can onlymeet the high standard demands of industry if both membraneand enzyme performance are optimized. Since the weakest factorwill always determine the overall performance of the system,extreme optimization of one factor is pointless. Depending uponthe application, the main optimization efforts will be eithertowards the enzyme or either towards the membrane.

Productivity of the biocatalytic membranes implies the pres-ence of fairly active enzymes. Thus one important parameter ofbiocatalytic reactions is the total enzyme activity, which dependslargely on the enzyme load. It seems logical that the totalenzyme activity increases when more enzymes are immobilizedon a membrane. In many cases, however, it is observed thatenzyme activity decreases when enzyme loadings are increasedabove a certain threshold concentration, which can be explainedby molecular crowding. In this case, crowding refers to highconcentrations of macromolecules on the membrane surface.These high enzyme concentrations may alter the propertiesof an enzyme and in the worst case, it can result in enzyme

denaturation or active site blocking.19,35,65 Thus, a part of activityloss is due to the inaccessibility of the immobilized enzymefor the substrate.23 However, it is also possible that the enzymeactivity declines during process operation and this can often beattributed to improper immobilization of the enzymes, whichresults in enzyme leaching into the permeate or retentate,depending on the size of the enzyme and the MWCO ofthe membrane. Such enzyme leaching is undesirable and forcontinuous operation the enzyme should be well bound to themembrane in addition to a high enzyme activity.

Furthermore, the presence of enzymes in the final product isoften problematic, because this may lead to the over processingof the product when the enzyme stays active. Even inactiveenzymes can be seen as an undesired contamination of thefinal product and thus implying extensive purification steps.126

Product purity can also be an issue towards productivity, becausepurification usually causes losses. In addition, the purificationprocess can be very time consuming. Membrane permeabilityand membrane selectivity therefore play an important role inthe productivity of BMRs.

Similarly, the lifetime of biocatalytic membranes is deter-mined both by membrane properties as well as by enzymestability. The extent to which membrane properties or enzymestability acts as a determining factor for the membrane lifetime,largely depends on the applied immobilization technique (re-versible or irreversible). In general, the reversible immobilizationtechniques allow the regeneration and thus the re-use of thebiocatalytic membranes. However, the economics of the regen-eration process will determine its feasibility. Using irreversibleimmobilization techniques implies in general that regenerationis impossible or much harder than in the case of reversibletechniques. In addition, the process conditions also influencethe lifetime of biocatalytic membranes. Especially, the usedsolvent system greatly affects membrane lifetime and limits thetype of membranes that can be used.99 Furthermore, membranelifetime is also influenced by every operation that is performedon a membrane. Backwashing and cleaning operations are moreharsh for the membrane than normal filtration operations.Therefore, reducing membrane fouling and thus minimizingthe need for cleaning operations can extend membrane life.Regeneration processes are often just as harsh as cleaningprocesses, which should be used with caution and this impliesalso the importance of enzyme stability.127,128 Maximization ofmembrane lifetime is not the most economical solution andtherefore process conditions have to be chosen in such a waythat an optimal balance between productivity and membranelifetime is obtained.129

The operational lifetime of biocatalytic membranes is animportant economical aspect, but the storage stability ofbiocatalytic membranes is equally important. Storage stabilityis different from operational lifetime, because the conditionsare different, i.e. during operation the enzyme is stabilized bythe substrate. For example, the presence of substrate positivelyaffected Protex (Genencor R©), a bacterial alkaline protease,against thermal inactivation.130 Moreover, as compared to freeenzymes, their immobilized counterparts have been reportedto be more temperature resistant.131 The storage stability ofbiocatalytic membrane also depends upon the basic membranecharacteristics, but the enzyme stability is in most cases the

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determining factor. This storage stability and operational life-time of biocatalytic membranes can be improved by using theright immobilization technique.131 The dry-storage stability ofbiocatalytic membranes is an important factor for the economi-cal use of these membranes. This is particularly important in caseof shipping and transportation of biocatalytic membranes.106

Apart from storage stability, the BMR needs to be stablein different environments. Different impurities can be presentin the feed solutions and these can have an inhibiting ordestructive effect on the biocatalytic membrane. Furthermore,the possibility arises that during processing, shifts in pH ortemperature occur and the biocatalytic membrane should beable to withstand such fluctuations.

3.4. Sterility of enzymatic membrane reactors

A lot of enzymatically produced products find application infood and pharmaceuticals, thus implying the importance ofworking under sterile conditions. Furthermore, working in non-sterile conditions can lead to the formation of byproducts,membrane fouling, or in general a loss of productivity. Despite itsimportance, the information on maintaining sterility in BMRsor EMRs is only reported by a few authors in the literature.132–134

Sterility in enzymatic membrane reactors poses differentchallenges. A first challenge is finding an appropriate membrane,which can be sterilized preferably in situ. Ceramic membranescan be easily steam sterilized, but currently only a limitedamount of polymeric membranes meet the steam sterilizeddemand.123,135 Furthermore, applying the sterilization on biocat-alytic membranes is even more challenging due to the fragilityof enzymes. The sterilization methods, which can be used insuch cases, are limited due to the persistence of microbialcontaminants combined with the limited stability of enzymes.For pharmaceutical applications the number of methods iseven more limited due to regulations.136 Another challenge isto maintain sterility during the production process.

In the biotechnological industry, this sterility problem, duringthe production process, is often solved by steam sterilizing theproduct at each stage and by controlling the microbial levels withsteam sterilizable 0.2 mm filters.123 Heat treatment is however notalways beneficial for the quality of the final product and thereforethe duration of such a treatment needs to be minimized.137 Someauthors claim that the use of ultrafiltration membranes is enoughto ensure a sterile product, but even then there is still the risk oflosing some productivity.117,138 Other studies report the operationat elevated temperature to avoid microbial growth, but they alsohave to consider membrane and product stability.139

4. Conclusions

Integration of immobilized biocatalysts on membranes leads toan interesting combination in the form of BMRs which exhibita huge potential in food, beverages, pharmaceuticals, organictransformations, etc. Such potential is particularly beneficial inthe production of various chemicals and tailor made products.The development and implementation of these systems at anindustrial scale requires a broader as well as in depth understand-ing of the core processes. The knowledge currently available inthe domain comes from rather diverse research activities being

conducted by various research groups. A relatively uniformtrend in reporting enzyme related parameters is hard to find.Furthermore, in this domain, the resulting changes in membranecharacteristics such as selectivity and pressure-flux behavior arealso of crucial importance. Therefore, a better comparison andmore rational choices can only be made when both the mem-brane and enzyme aspects are systematically investigated andreported. For the application based studies, however, in depthinformation on every parameter is not particularly important,because here, the end result is the key issue. Important selectioncriteria for immobilized enzyme applications are the purity of theobtained product, the biocatalytic activity per unit membranesurface, system robustness/stability and the overall productivityof the whole system. However, such selective data collection orthe focus on these selection criteria in application based studieslead to the loss of important information. This makes it fairlyimpossible to compare different techniques and to develop agood selection tool. Therefore, it is essential to realize that acomparison between these different studies is only possible whena complete description of all the biocatalytic parameters is giventogether with detailed information about the immobilizationprocess and the filtration capabilities of the membrane. Dueto this interdisciplinary character, well coordinated efforts arerequired by membrane technologists and biochemists in orderto comprehend the underlying principles.

To achieve utmost productivity in BMRs it is essential thatboth aspects i.e. membrane and enzymes, are maximally opti-mized because the weakest factor would eventually determinethe overall performance. Therefore, extremely optimizing onefactor will not result in high productivity. Achieving this balancehas proven not to be easy due to the complexity and lack ofcomparability between different studies. Although some authorshave found a certain balance in reporting both membrane andenzyme performance, this is unfortunately still far from generalpractice. Though there are still a lot of issues associated withBMRs that need to be addressed, nevertheless a standardizationin reporting the findings at an early stage would benefit theprogress and help to steer the research.

Acknowledgements

This work is supported by VITO’s strategic research fund. PeterJochems gratefully acknowledges the PhD scholarship grantfrom VITO. The authors also want to thank Sandra Van Roy,Wim Doyen, Anita Buekenhoudt, Annelies Verstraete and othergroup members for their useful comments on the manuscript.

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