39 icalEngineering, and has been a 20,40–42 This paper details various ddin,SchoolofChem 43,44 2011 Society of Chemical Industry Universitas Lampung, Bandar Lampung c When an enzyme is on the membrane, 45,46 and Subhash Bhatia ∗ a and suspended enzymes; hydrolysis reactions; esterification; Correspondenceto:AzlinaHarun Kamaru Universiti Sains Malaysia, 14300 Nibong Tebal, PulauE-mail: Pinang, [email protected] Malaysia. School ofMalaysia, Chemical 14300 Nibong Tebal, Engineering, Pulau Pinang, Malaysia Engineering Campus,Department of Chemical Universiti Engineering, Sains 35145, Lampung, Indonesia Work on two separate phase-enzymatic membrane reactors ∗ a b ve process since it has a large interfacial area and exchange ts. Many factors influence its successful operation, and these es were resolved primarily by kinetic resolution, but dynamic n hydrolytic reactions, but esterification processes were also determining factors of thein TSP-EMR, development which should of be thetechnology examined technology. with Different its different applications characteristics of are described. the employed in plant-scale capacities. (TSP-EMR or biphasic EMR) were started many years after Rony published a pioneeringhollow report fibre membranes on (HFM).still Up enzymes to uses immobilization now, the the in HFMand TSP-EMR, as which achiral the enzymes compounds dock,Production such has of processed the as racemic single drugs,used enantiomers this acids, EMR from type. an esters Thekinetic achiral technology resolution and of was substrate the encouraged racemic oils. compounds, in dynamic TSP-EMR: BASIC NEEDS Immobilization of enzymesnecessary at for EMR organic–aqueousat implementation. these interfaces Many interfaces. is enzymes are active eration, membrane material and size, enzyme preparation and uld be specified. The immobilization site, reactor arrangement, tion and immobilized or suspended enzyme(s) are determined The The 1–12 30–32 25–27 23,24 13–15 Compared 28,29 to US$ 225 billion 16 thus selective removal of -blockers, the distomers give β 21,22 : 1032–1048 www.soci.org Azlina Harun Kamaruddin 18–20 86 a,b 2011; Studies show that one of the best approaches 33 enzymatic membrane reactors; operational factors; immobilised Many methods have been applied to generate single Therefore, it is an attractive alternative to the enzymatic 37,38

17 34–36 -blocking effects, reduce the overall drug selectivity, produce

β 2011 Society of Chemical Industry Enzymatic resolution of racemic compounds in membrane c to improveEMR. economic and technical competitiveness is the products from reactioninhibited sites or increases thermodynamically unfavourable conversion reactions. of product- processes. reactors (enzymatic membranebenefits. reactors Conversion and ortake separation place EMR) in of give a the single many operation, enantiomers can need for single enantiomericfrom the synthetic world market, where drugs consumption of canincreased single rapidly enantiomers be from US$ 74.4 observed billion in 1996 EMR overcomes disadvantages of batch stirred tank reactors, J Chem Technol Biotechnol They could provideenantiomers. an efficient and cleaner route to single consumes low energy and is easy to scale-up. side-effects as the eutomers or possibly cause adverse effects. no These facts emphasize they usuallytions. do not Hence, contribute enantiomers to should medica- be used individually. INTRODUCTION Enantiomers of drugs oftenpharmacodynamic have properties. As different in pharmacokinetic and Abstract A two separate phase-enzymatic membrane reactor is an attracti phase operations Joni Agustian, surfaces, simultaneous reaction and separationinclude and characteristics other of benefi the enzyme, membrane,are circulating fluids the and reactor main operations. factor, Althoughdevelopment, the the other solubility operating of substrates conditions factors and products, must type ofloading op be procedure, and considered cleanliness of before, the recirculated during fluids or sho after itslater. application. Some factors At still the needcapacity initial further to studies. stage process Utilization racemic of of and achiral the reactor kinetic compounds. technology resolution The is has racemat described been for exploited.exploited. use The from technology multigram- focused to o plant-scale Keywords: the determining factors in two separate Enzymatic membrane reactors: (wileyonlinelibrary.com) DOI 10.1002/jctb.2575 Review Received: 30 August 2010 Revised: 10 December 2010 Accepted: 10 December 2010 Published online in Wiley Online Library: 19 January 2011 dissolved or no-solvent operation, classic or emulsion opera kinetic resolution in 2005. enantiomeric synthetic drugs. with other technologies,stability, better control the of single enantiomers EMR production, enriched and has concentrated high products productivity and decreased and reaction times.

1032 1033 Xin but It is 97 93,94 89–92 Although higher 57,101–103 wileyonlinelibrary.com/jctb 36,37,50 For the TSP-EMR, temperature, 71 Many lipases show high activity in hy- 98 99,100 Although the reactor design for the biphasic 92,96 suggested an aqueous-organic biphasic system or a water 95 . In the biphasic system, characteristics of the organic solvent Operating conditions of the TSP-EMR are described in Table 1. pH of the aqueousimmobilized phase, enzyme, concentration transmembrane of pressure thethe and substrate(s) organic and flow and aqueous phase rate are of conditionedMost during operation. of thethe factors optimum could performance;activity however, be during the the managed processes immobilized is externallyby not enzyme evaluating controlled; to enzyme this deactivation. is achieve determined system has severaldrug complications, solubility problem the and system forms overcomes irreversible the reactions. are important since thevaries enzymatic significantly. activity in the organic phase The technology isrun mostly in applied in batchperformed hydrolytic at recirculation mild reactions, temperatures, mode.low and transmembrane relatively pressures In and long medium flow reaction rates general,and in times, the lumen-side shell- operations to give are lowhigh selectivities. to The organic high and aqueous conversions phases are andboth fed through medium sides to ofduring the experiments. HFM modules. All parameters are measured Fluids characteristics Enzymes are stable and active in organic solvents, drophobic solvents with low polarities. saturated organic solvent reactiondrugs. in Lipases hydrolysis are ofinterfaces. found the to racemic be effective at the organic–water of gas phase,issue, but etc. other sensitive (Fig. factors 2). should alsoduring be Operating considered and before, after conditions application. are the main Operating conditions Operatingconditionsisoneofthemostimportantfactors,involved in scale-up of a reaction process. a certain amountneeded to of keep them water in catalytically active bound conformation. on to enzyme surfaces is et al undeniable that the solvent andon water enantioselectivity. content may have effects 2011 Society of Chemical Industry c as illustrated andreduction Use of HFM to 29,47 49,50 They also have high The modules have 50,56 ) has to be moved to 35,70 S 32,57–59 and enzyme conformational 54,55 50,57,60–62 This system is of interest when Use of this biphasic EMR is highly : 1032–1048 48 64,67 ) has to be transported from the 86 P 51–53 2011; Other membrane configurations having only one However, they have several drawbacks, for instance diffusion resistance 68,69 Materials transportation in an enzymatic membrane reactor. 63 62–66 The TSP-EMR is formed by two immiscible fluids, a continuous The HFM modules have four separate openings, an inlet and Two separate phase enzymatic membrane reactor www.soci.org enzyme sites, and productreaction sites ( to the other side of the membrane in Fig. 1. Catalytic activity ofapplicable the to enzyme the on two the separate interface phase makes membrane-based it system. organic phase and a continuous aqueousenzyme-membranes phase, separated interfaces. by the attractive when HFM are employed. an outlet forwhich each allow side continuous ofTransport flows could the be across achieved membrane using the hydrophobicpositive as membranes pressure membrane and on shown the surfaces. in aqueous sideand Fig. or 1, positive hydrophilic membranes pressure on the organic side. high interfacial area, absenceflow of rates, emulsion, no no unloadingdensity flooding difference at between at phases. low high flow rates and do not require J Chem Technol Biotechnol OPERATING CONSIDERATIONS: THE DETERMINING FACTORS Development ofenzyme the loading TSP-EMR procedure, involves membrane type operational and strategy, material, use Figure 1. in order for reaction to occur, substrate ( of down-stream processes. house the enzymes hasimmobilization advantages methods. Large over exchange conventional surface per enzymes unitis volume provided by these membranes. changes. products are insoluble inand the separation organic can phase besimplifiedprocess,reductioninproductioncost, since performed the simultaneously, reaction leading to a solutions must be free of particulate materials,of use of fibre, specific type connection to downstream/permeate-sidewound are such not suitable for as the application. the spiral- volumetric productivity, simultaneouseasily products reutilized, separation, cheap enzyme are fixation,catalytic little activity, high loss substrate of concentration, theactivity. and enzyme stable lipases : 1032–1048 108 This reduces 86 63 2011; called the TSP-EMR with 40 Only a small conversion difference 48 J Chem Technol Biotechnol suggested using an extremely pure liquid to Hydrolysis of oils also proceeded in a no-solvent 107 56 . 1 The increase of olive oil concentrations led to the − 76,77 min µ Cleanliness of the liquid/air phase is also an important factor. system. The solution must be free of particulate materials. key reactant(s) feda through ‘multiphase a EMR’; water-immisciblereactant(s) organic while is phase the referredin to aqueous Table as 1, phase the mostSince dissolving operations ‘extractive’ TSP-EMR key are type. operations based As areand on separation, described based the the reactant(s) on multiphase must simultaneousThis type. not means that mix reaction in the with multiphase TSP-EMR, the the substrate(s) product(s). should have high solubility in the organicaqueous solvent and phase. not In dissolve in contrast,in the the the product(s) aqueous phase, mustproblems. so dissolve Hence, the that easily solubility they behaviour canproduct(s) of must the be be substrate(s) considered, transported and since without many commercial organic Multiphase or extractive TSP-EMR In dissolved operation, substratesorganic can or be supplied aqueous either phase. in Matson the membranes fouling caused by unwanted materials thus increasing the membrane service life. Installation ofunits filters before and the reactor usematter. of Breslau non-corrosive materials prevents particulate avoid blockage or contamination of the membranes. Dissolved- or no-solvent operation Operating the TSP-EMRsolvents for to drugs dissolve the resolutionphase racemic uses (Table drugs 1). the and Dissolved-solvent tothe organic operations form are substrates the applied organic areoperations when solid have been compounds developedorganic or with phase. viscous no For solvent liquids. example,porcine added Some to ethyl liver the butyrate esterase was9600 in hydrolysed several by hours at the reaction rate of was found when this biphasicof EMR an was organic operated solvent in (60%) the or presence with the pure oil (50%). increase of catalytic efficiency. 73 . used Its re- )shifts et al 106 43 w . a 104 et al Hydrophobic 105 www.soci.org J Agustian, AH Kamaruddin, S Bhatia 2011 Society of Chemical Industry c Hydrophobic solvents 103 These results show that different This solution reduced the inhibitory 75 44 Albeit phosphate buffers were a good 56,85,86 found similar results in which the aqueous phase 44 . Factors in TSP-EMR operation. et al As described in Table 1, a common aqueous phase is phosphate are not dissolved easily in thein aqueous the phase, HFM so good modules interfaces based-on can strongly hydrophobic be solvents formed. suchTable Thus, as 1. The many those moisture content shown TSP-EMRs plays a in key are roleesterification in in enzyme-mediated organic media since high water activity ( product, shortened reaction time,and increased improved the the enantioselectivity. rate of reaction the chemical equilibrium towards the reverse reaction. aqueous phase, they occasionallya could proper not aqueous be environment. usedmethyl-methoxy As to phenylglycidate provide in (MMPG), hydrolysis the ofled phosphate to the buffer base-catalysed hydrolysis racemic of the substrateby-product, to form which aldehyde inhibitedhydrolysis, enzyme researchers activity; used hencealdehyde in contamination. a MMPG bisulphite solution to prevent solvents have lower capability to remove water fromso the that enzymes, in these solvents the enzymes have higher activity. buffer solution at pH values of 5.5–8.5. Kamaruddin wileyonlinelibrary.com/jctb concluded that the pHprocesses of greatly the influenced aqueous the phasephosphate lipase buffer during at enantioselectivity, pH hydrolytic 8 and resulted a in theenantioselectivity. highest optical During purity and esterificationthe of conversion racemic increased ketoprofen, but as pH it was dropped changed at from pH 6.0 to 8. 7.0, enantioselectivity is found insolvents hydrophilic provide solvents, higher hydrophobic reaction rates. Figure 2. quirement may vary as the solvent is changed. bisulphite solution pH 8.5, whichin produced enantioselectivity, a although pH significant 8.0 increase showed higher productivity. Dodds at pH 8 gavepH high 7 enantiomeric produced excess,of greater although the the hydrolysis. aqueous buffer Because at pH,added of to sodium the the hydroxide importance aqueous solutionbuffer phase was condition. during frequently TSP-EMR runs to maintain reactions require a different aqueous phase. Lopez

1034

1035

Recirculation

4 ufrp . 0 DN DBatch- ND ND ND 500 8.0 pH buffer 40,74 ty uyaeN,N-ovn S L pnePN5 .010Ehlbtrt 0 PO 500 butyrate Ethyl 0.80-1.0 50 PAN Sponge PLE NSO No-Solvent ND, butyrate Ethyl

Flow

Recirculation Counter substrate

4 56 Batch- Horizontal, ND 45 500 8 pH buffer ty uyaeN-ovn,05Lpure L 0.5 No-Solvent, butyrate Ethyl S L pneN D10Ehlbtrt 0 PO 500 butyrate Ethyl 1.0 ND ND Sponge PLE NSO

Recirculation

4

-lcdlbtrt D oSletNOPLSog A D08 lcdlbtrt 5-5 PO 250-350 butyrate Glycidyl 0.85 ND PAN Sponge PPL NSO No-Solvent ND, butyrate )-glycidyl ( ufrp . 5-5 DN DBatch- ND ND ND 250-350 7.8 pH buffer 40,56 ±

Recirculation wileyonlinelibrary.com/jctb

4 75 Batch- ND 40 ND ND 7 pH buffer -eorfn2-5m PCL pneP 0005Dclrpoae-hxn 0PO 40 hexane - Dichloropropane 0.015 10 PS Sponge CALB MP mM 20-35 )-ketoprofen ( ±

Recirculation

4 ufrp . 0 DN DBatch- ND ND ND 300 5.5 pH buffer 40 -bpoe PCLSog A D08 etnl30PO 300 Pentanol 0.85 ND PAN Sponge CCL MP M 2 )-ibuprofen ( ±

Recirculation

4 -bpoe se 0 ME epoeSog A 0N ylhxn 0 PO 400 Cyclohexane ND 50 PAN Sponge Seaprose EX mM 500 ester )-ibuprofen ( ufrp . 0 DN DBatch- ND ND ND 400 7.0 pH buffer 74 ±

Recirculation

4

ufrp . DHxn DN TN Batch- ND RT ND ND Hexane ND 7.0 pH buffer PO 1.90 18 Cellulose Lumen 6 Prozyme EX 41 -bpoe se 25 L g 32.51 ester )-ibuprofen (

±

1 −

Recirculation

4

XPoye6LmnPNN .5Ccoeae-oun DPO ND -Toluene Cyclohexane 0.85 ND PAN Lumen 6 Prozyme EX ufrp . DN 0N Batch- ND 30 ND ND 7.0 pH buffer 41 -bpoe se 6 L g 161 ester )-ibuprofen (

±

1 −

Recirculation

4

-bpoe se 46 L g 64.60 ester )-ibuprofen ( rzm pnePN5 . ylhxn DPO ND Cyclohexane 1.0 50 PAN Sponge 6 Prozyme CCL EX ufrp . DN 0N Batch- ND 20 ND ND 7.0 pH buffer 41

± +

1 −

Recirculation

4 -bpoe se D oSletNOCLSog A D08 ( 0.85 ND PAN Sponge CCL NSO No-Solvent ND, ester )-ibuprofen ( -bpoe se 0-5 PO 400-450 ester )-ibuprofen ufrp . 0–5 DN DBatch- ND ND ND 400–450 7.8 pH buffer 40,41 ± ±

Flow

Recirculation Counter

4 32,33,73 Batch- Horizontal, 40 40 50–450 8 pH buffer -bpoe se 510m PCLSog,LmnPN5 .9 sotn 020PO 50-250 Isooctane 0.093 50 PAN Lumen Sponge, CRL MP mM 25–100 ester )-Ibuprofen ( ±

Flow

Counter Recirculation

2011 Society of Chemical Industry

4 -arxnetrN PCLSog A5 DIocaeN PO ND Isooctane ND 50 PA Sponge CRL MP ND ester )-naproxen ( ufrp . DN DHorizontal, ND ND ND 7.0 pH buffer 72 Batch-

± c

Flow

Recirculation Counter

4 Batch- Horizontal, 30 25 330 7.0 pH buffer 65 -arxnetrM R pne ue AP 03 .0/.0 sotn 3 PO 330 Isooctane 0.005/0.004 10/30 PA/PS Lumen Sponge, CRL MP ester )-naproxen ( ±

Flow

Counter Recirculation

4 ufrp . D03 Vertical, 30 0 ND 7.0 pH buffer -arxnetrN PCLSog A5 .1 sotn,Euso DPO ND Emulsion Isooctane, 0.015 50 PA Sponge CRL MP ND ester )-naproxen ( Batch- 60 ±

Recirculation

4 ufrp . 0 74 0N Batch- ND 30 17-41 300 7.0 pH buffer 52 -arxnetrN PCLSog,LmnP 0N sotn 0 PO 300 Isooctane ND 50 PA Lumen Sponge, CRL MP ND ester )-naproxen ( ±

Recirculation

4

ufrp .03040N DN Batch- ND ND ND 300–400 8.50 pH buffer 40,41 -arxnetr07 PCLSog A N PAN Sponge CCL MP M 0.75 ester )-naproxen ( .5Mty sbtlKtn 0–5 PO 400–450 Ketone Isobutyl Methyl 0.85 D ±

Flow : 1032–1048

Counter

Recirculation 86

4 -arxnetrN PCLSog A5 .7 sotn,Euso DPO ND Emulsion Isooctane, 0.076 50 PA Sponge CRL MP ND ester )-naproxen ( ufrp . D5 0Horizontal, 30 50 ND 7.0 pH buffer Batch- 21 ±

2011;

yeCnetainLcsTp ieMaterial Site Type Locus Concentration Type )Shell (m (kDa) m min (mL )Lumen min (mL oeRef. Mode Orientation C) ( (kPa) )

2 1 1 − − ◦

WCO MW Area lwRate Flow lwRate Flow Operational Reactor T TMP

usrt nyeMmrn Medium Membrane Enzyme Substrate

al 1. Table prtn odtoso hrldusadnnrcmcsbtae eouini h TSP-EMR the in resolution substrates non-racemic and drugs chiral of conditions Operating Two separate phase enzymatic membrane reactor www.soci.org J Chem Technol Biotechnol Flow

: 1032–1048

hnlglycidate phenyl Counter Recirculation

86

26-3(/)M F30Sog A 007-0TleeN iufiep . D3-11-9Horizontal, 15-19 31-51 ND 8.5 pH Bisulfite ND Toluene 0.75-60 30 PAN Sponge 360 OF MP (w/w) 12.6%-23 thoxy )-methyl-me ( 43 Batch- ±

Recirculation

3 0 DN DBatch- ND ND ND 300 40,74 mlaeaeN,N-ovn S add iaeSpong lipase Candida NSO No-Solvent ND, acetate Amyl A 008-. mlaeae5-5 .501MNaHCO M 0.05-0.1 50-150 acetate Amyl 0.80-1.0 50 PAN e 2011;

ester ester Recirculation

3 -74Sog A 008-. hnxaeaemethyl Phenoxyacetate 0.80-1.0 50 PAN Sponge L-1754 Candida NSO No-Solvent ND, methyl acetate Phenoxy 5 . NaHCO M 0.1 150 0 DN DBatch- ND ND ND 300 40,74

Recirculation propionate

4 40 Batch- ND ND ND 300 7 pH buffer .3mls oSletNOCniaSog A D08 uy -hoopoint 0 PO 300 propionate 2-chloro Butyl 0.85 ND PAN Sponge Candida NSO No-Solvent moles, 3.03 2-chloro )-butyl ( ±

cy ester octyl propionate Recirculation

4 2 0 .5MK M 0.05 400 pionate )-chloropro ( 0.85 50 PAN Sponge lipase Candida NSO No-Solvent moles, 0.96 2-chloro )-octyl ( 0 DN DBatch- ND ND ND 400 PO 40,41 ± ±

Flow

Recirculation Counter propionate

4 80 Batch- Horizontal, ND ND ND Heptane ND 7.2 pH buffer 0 MM .pLmnP 0003 PO 0.0030 50 PA Lumen P.Sp MP mM 100 ethyl )-1-phenyl ( ±

J Chem Technol Biotechnol

-propylacetate

Flow )-1-phenyl-1 ( ±

Recirculation Counter acetate,

4 79 Batch- Horizontal, 30 ND 5-25 8 pH Buffer --hnlethyl )-1-phenyl ( 0m PPS pneP 0001 etn -0PO 5-20 Heptane 0.0017 50 PA Sponge P.Sp MP mM 50 ±

Flow

Co-current cdester acid Recirculation

4 ufrp D2,2,53 Horizontal, 30 5 20, 20, ND 7 pH buffer 0 MM C pnePN E,P 03,N .09000 sotn rHxn DPO ND Hexane or Isooctane 0.0009-0.0007 ND 30,30, PE PES, PAN, Sponge PCL MP mM 100 octanoic )-2-hydroxy ( 78 Batch- ±

Recirculation cdester acid

4 ufrp DN 0N Batch- ND 30 ND ND 7 pH buffer 53 0 0 MM R,PLSog A,PS1,3 .00003 sotn DPO ND Isooctane 0.0030-0.0036 30 10, PES PAN, Sponge PCL CRL, MP mM 100 20, octanoic )-2-hydroxy ( ±

Recirculation substrate

4 ufrp 0 4N DBatch- ND ND 34 400 8 pH buffer 77 lv i oSlet . pure L 0.2 No-Solvent, oil Olive S R pneP 0N lv i 0PO 80 oil Olive ND 50 PA Sponge CRL NSO

Flow

substrate Co-current Recirculation

4 lv i oSlet . pure L 0.5 No-Solvent, oil Olive S M pneP,R 611 . PO 1.0 1.1, 66 RC PS, Sponge MML NSO ufrp lv i . 54 Vertical, 40 15 2.5 oil Olive 3 7 pH buffer 76 Batch-

Flow

Recirculation Co-current

4 ufr353 0Horizontal, 40 30 335 buffer Batch- 48 lv i DM R pneMPN DOieoil Olive ND ND MPP Sponge CRL MP ND oil Olive etn 5PO 85 Heptane + Recirculation

www.soci.org J Agustian, AH Kamaruddin, S Bhatia

lv i D oSletNOCLLmnR D08 lv i 0Bfe H642 DN DBatch- ND ND ND 20 6.4 pH Buffer 40 oil Olive 0.80 ND RC Lumen CCL NSO No-Solvent ND, oil Olive 40

2011 Society of Chemical Industry

Recirculation c

lv i D oSletNOCLSog A D10Oieol2030Bfe H648 DR-7N Batch- ND RT-37 ND 80 6.4 pH Buffer 250-340 oil Olive 1.0 ND PAN Sponge CCL NSO No-Solvent ND, oil Olive 40

Flow

substrate Co-current Recirculation

4 amolN-ovn,05Lpure L 0.5 No-Solvent, oil Palm S M pneP,R 611 . PO 1.0 1.1, 66 RC PS, Sponge MML NSO ufrp amol251 0Vertical, 40 15 2.5 oil Palm 3 7 pH buffer 76 Batch-

Flow

Co-current Recirculation

4 amol5-2 MM R ue P510PO 1.0 5 CP Lumen CRL MP mM 59-326 oil Palm ufrp .52. sotn 175 D3 Vertical, 30 ND 11.7–50 Isooctane 2.55-20.5 7 pH buffer Batch- 35,38

yeCnetainLcsTp ieMaterial Site Type Locus Concentration Type )Shell (m (kDa) m min (mL )Lumen min (mL oeRef. Mode Orientation C) ( (kPa) )

2 1 1 − − ◦

WCO MW Area lwRate Flow lwRate Flow Operational Reactor T TMP

usrt nyeMmrn Medium Membrane Enzyme Substrate

al 1. Table ) Continued ( wileyonlinelibrary.com/jctb

1036

1037

P utpae X xrcie S:N-ovn Operation. No-Solvent NSO: Extractive, EX: Multiphase; MP:

:ga,w egt :Lte :mole/Litre. M: Litre, L: weight, w: gram, g:

TE -ezy--yoieEhlEtr TE -ctlTrsn ty Ester, Ethyl N-Acetyl-Tyrosine ATEE: Ester, Ethyl N-Benzoyl-L-Tyrosine BTEE:

A:Plarlntie A oymd,P:Plsloe E:Polyether PES: Polysulfone, PS: Polyamide, PA: Polyacrylonitrile, PAN: ufn,P:Plehln,R:RgnrtdCluoe P:MdfidPolyp Modified MPP: Cellulose, Regenerated RC: Polyethylene, PE: sulfone, oyee P urpae WO oeua egtCut-Off. Weight Molecular MWCO: Cuprophane, CP: ropylene,

sp., Pseudomonas P.Sp: lipase, miehei Mucor MML: esterase, liver Pig PLE: lipase, pancreatic Porcine PPL: lipase, cepacia Pseudomonas PCL: lipase, 360 OF OF-360: B, fraction lipase antarctica Candida CALB: lipase, rugosa Candida CRL:

Flow

current

Counter- Recirculation

wileyonlinelibrary.com/jctb 4 ufrp . D5 0Vertical, 30 50 ND 7.0 pH buffer 88 Batch- raei DM C hl A 007 oun DPO ND Toluene 0.75 50 PAN Shell CCL MP ND Triacetin

ester

cd -hnlalanine L-phenyl acid,

carbonyl)-L-aspartic Flow

(benzyloxy ctt p 5) (pH acetate Co-current buffer Acetic Recirculation

N- DE hrolsnLmnN 007 ctcbuffer Acetic 0.70 50 ND Lumen lysin Thermo EX ND butyl 0 96 0Vertical, 40 19.60 200 acetate Butyl 200 87 Batch- + +

Recirculation

4 2 0 DN DBatch- ND ND ND 400 PO 40 -TE28 MM hm rpi DPNN . cao 0 . K M 0.1 400 Octanol 1.0 ND PAN ND trypsin Chimo MP mM 2.86 )-ATEE ( ±

Recirculation

4 -TE02m PCyotysnSog A D10Slcn i DPO ND oil Silicone 1.0 ND PAN Sponge trypsin Chymo MP mM 0.2 )-BTEE ( ufrp . 00N DN Batch- ND ND ND 1000 7.8 pH buffer 40,74 ±

Recirculation

4 ufrp . 5-0 DN DBatch- ND ND ND 250-500 7.0 pH buffer 40,74 -TE1-0m PCiotysnSog A D10Ay ctt/cao 050PO 10-500 acetate/Octanol Amyl 1.0 ND PAN Sponge trypsin Chimo MP mM 10-40 )-BTEE ( ±

Recirculation

4 ufrp 0 DN DBatch- ND ND ND 400 6 pH buffer 40 -ezy yoie1 mlE hm rpi DPNN . cao 0 PO 400 Octanol 1.0 ND PAN ND trypsin Chimo EX mmol 17 tyrosine N-Benzoyl

Flow

icarboxylate di Co-current Recirculation

4 3 MM L pneP 004 eae20PO 270 Hexane 0.49 30 PS Sponge PLE MP mM 230 -1,2 -4-ene -cycloxex cis ufrp 0 02 Vertical, 25 30 102 7 pH buffer 86 Batch-

Flow

Recirculation Co-current 12d carboxylate -1,2-di

2011 Society of Chemical Industry

4 85 Batch- Vertical, 25 ND ND 7 pH buffer DM L pneP 002Hxn DPO ND Hexane 0.2 30 PS Sponge PLE MP ND -4-ene -cycloxex cis

c

Recirculation

eaocai DM R ue PN .7Strtdsrio ouin2 eadcn 0N 0N Batch- ND 30 ND 30 Hexa-decane 20 solution sorbitol Saturated 0.77 ND CP Lumen CRL MP ND acid Decanoic 84

Flow

Co-current Recirculation

eaocai 0-00m PCLSog C1 . eae30Ar09 D2 Horizontal, 20 ND 0.90 Air 3.0 Hexane 1.0 10 RC Sponge CRL MP mM 100-1000 acid Decanoic Batch- 83

Flow Continuous

Co-current Recirculation,

eaocai 0 MM R pneR D07 . eae45ArN D2 Horizontal, 25 ND ND Air 4.5 Hexane 1.0 0.7, ND RC Sponge CRL MP mM 100 acid Decanoic Batch- 82

Flow

Co-current Recirculation hnlglycidate phenyl marcescen

3

H85N DN Vertical, ND ND ND 8.5 pH Batch- 81 -ehlm thoxy me )-methyl ( DM Serratia MP ND pnePN5 .5Tlee10NaHSO 150 Toluene 0.75 50 PAN Sponge

± : 1032–1048

hnlglycidate phenyl aaaeM Palatase

Recirculation 86

4 -ehlm thoxy )-methyl-me ( 08-,4 PMP OF, MAP, MP g -2,340 20.8 pnePNN .51 oun 0-5 PO 400-450 Toluene 0.75-12 ND PAN Sponge ufrp . 0-0 DN DBatch- ND ND ND 400-500 7.0 pH buffer 44 ±

2011;

yeCnetainLcsTp ieMaterial Site Type Locus Concentration Type )Shell (m (kDa) )Lumen min (mL oeRef. Mode Orientation C) ( (kPa) ) min (mL

2 1 1 − − ◦

WCO MW Area lwRate Flow lwRate Flow Operational Reactor T TMP

usrt nyeMmrn Medium Membrane Enzyme Substrate

al 1. Table ) Continued ( Two separate phase enzymatic membrane reactor www.soci.org J Chem Technol Biotechnol 40 85 122 the In the 117 found that : 1032–1048 Combination 66 86 121,123–125 Candida rugosa 86 In this method, 74 2011; From the economic 48,122,126 The physical entrapment of 40,74 found that the biphasic reactor with Membranes have high surface area for 79 J Chem Technol Biotechnol 116 For high enzyme attachment, a suitable However, for immobilization of 118 33,115 As described in Table 1, most TSP-EMR use hydrophilic 121 49 119,120 The strongest immobilization method is obtained by the Several enzyme loading procedures as shown in Fig. 3 were lipase on different membrane materials, Hollownia immobilization strategy ishave required. been Thousands reported. of procedures covalent bond. the enzyme flows diffusively toflowing the the enzyme solution. HFM Immobilization surfaces of thechemically without enzymes modified membrane cross- onto surfaces has also been conducted, in which covalent bondsare of formed. the enzyme–chemical–membranes ResultsEMR of are shown enzyme in immobilization Tableattached 2. inside Relatively to low the enzyme the TSP- concentrations processes compared HFM with the surfaces initial solution occur concentration. after the immobilization HFM. Sakaki and Itoh TSP-EMR, the preferredadsorption method through cross-flow to filtration. immobilize This methodan the recirculates enzyme enzymes solution from is (shell- one side to to lumen-side the oradsorb other to the side the of other membrane the layers. way)immobilisation HFM A causing diffusive method the mode was of enzyme the also to enzyme developed. the adsorption methodthan the provided chemical higherhigher binding enzyme process method, catalytic efficiencies as activity. but These the the results earlier latter were produced observations. the same compared to evaluatesurfaces, protein i.e. the distribution axial andtheenzymeactivitydependedonitsdistributionalong on radial distribution the of the membrane thefibres. enzyme since These membranes accommodatehydrophobic high enzyme membranes load. arestable, Although more create high physically affinity and and open-state chemically lipases, The procedures (3a) and (3b) gavedistribution. higher axial However, and radial enzyme enzyme by immobilization recirculating the could enzymeHFM. be solution through done both sides of the Membrane type Since enzymes arethan more in effective organic solvents, in hydrophilic membranesMajor an are aqueous preferred. differences environment betweenmembranes are the the hydrophobic thicknesssmaller of and reaction in layers, hydrophilic of which the is hydrophilic hydrophobic much membraneshence membranes, the to and immobilized increase enzymesactivities. the could the retain capability their enzymes full activity, catalytic Enzyme immobilization and loading procedure The quantity ofis enzymes attached important. to the membrane surfaces hydrophilic type providesinterfaces, a higher thick enzyme contactand lower zone, activity, enzyme water-wetted lower concentration. enzyme desorption the enzyme ontoof the the HFM enzymelonger surfaces process, activity, which led whileactivity. led to the to higher a covalent lower retention bond retention required of a the enzyme of hydrophilic–hydrophobic membranes was also tested. hydrophobic microfiltration membranes wasoptical not resolution well suited bypalm to and lipase-catalyzed olive oil hydrolysis.hydrophobic hydrolysis, However, adsorption HFM of than in lipase hydrophilic was membranes. higher in enzyme loads and provide strong covalent attachment. 112 This whereas Although 49,82,109 113 115 Besides, shell- higher catalytic 52 109 65 www.soci.org J Agustian, AH Kamaruddin, S Bhatia 33,65 2011 Society of Chemical Industry c Although the specific 21 roductivity/effectivity than )-naproxenmethylester,andfrom S It is considered that HFM modules , R compared a TSP-EMR using purified 31,110 and higher loading capacity. 52 . 114 et al However, formation of the emulsion environment 21 but this orientation requires a higher pressure input. 111 The initial source of the enzymes could influence TSP-EMR on the membrane surfaces may complicate workup, lipase with a TSP-EMR basedwere on prepared crude by lipase, where the bothcatalyzed above lipases by method. the They crude foundconcluded the lipase that reactions had the lower crudelow enantioselectivity, lipase or and contains no some enantioselectivity, which hydrolasesAnother observation act was with that to surfactant-coated lipase reduce gave higher productconversions purity. (1.4 to 2 times) than the native type. Immobilisation site Either the shell-side (spongycan be layers) used or as asurface lumen-sideare to usually of dock employed the the enzymes. as HFM the The described spongy enzymes in layers Table on 1. theand Immobilization enantiomeric shell-side of excess gave than higher the lumen-side. protein attachment the classic biphasic EMR offers simple operation. 96% (TSP-EMR) to 100% (TSP-E-EMR)ester,andtheoverallmasstransfercoefficientoftheTSP-E-EMRwas for racemic naproxen butyl larger by 58%. activity of the enzyme withthe and enantiomeric without excess the of the emulsion product wasEMR)to97%(TSP-E-EMR)for( increased same, from 74% (TSP- with vertical fibre orientation are betterfouling, at controlling membrane wileyonlinelibrary.com/jctb Enzyme preparation The TSP-EMR enzymesmethod. First, were the generally biocatalystsbuffer were prepared dissolved pH by in thethe a 7–8 phosphate solution simple with was(3000–5000 centrifuged gentle rotation per to minute, steering 5–15 removeenzymes min). were After immobilized. for the separation, the insoluble 45–120 matter min. Then performance. Most enzymes wereenzymes). used directly Sakaki (crude or native Classic or emulsion TSP-EMR Recently, the TSP-EMR integratedto improve the performance use of the of immobilizedan emulsion enzyme. emulsion In of in this oil-in-water case, order (O/W)technique prepared is by mixed the with emulsification antransferred enzyme to solution. the The HFM mixture modules isan to then emulsion form enzymatic environment reactors on with EMR the with membrane surfaces. emulsion Theenzyme optimized TSP- improved distribution of the the mass immobilized transfer. compounds have low solubility in the aqueous phase. immobilization of the enzymes onspecific the activity, lumen-side it led showed to lower higher enantioselectivity. activity and stability, Reactor arrangement Even though differentthe specifications TSP-EMR of was HFM primarilyflow or have developed vertical co-current-flow been as arrangement (Table a used, horizontal 1). type horizontal In is fact, counter- preferred, the as itwhile the was opponent modules found were useful to in resolve oil hydrolysis. racemates, Counter- current flow reactors showed higher p factor must be considered in the first stage of development. the co-current TSP-EMR. side immobilization allowed higher conversion,

1038 1039 70 78 35,38 . 21 52 60 . . . 85 . 1nm). et al 48 . 79 < et al et al et al 32 33 73 75 . . . . et al et al et al et al et al et al Koter Sousa Knezevic Xua 2 2 2 ± enzyme Ref wileyonlinelibrary.com/jctb 5.2 mg/m mg/m units/cm Immobilized C) T ◦ ( ) d ( TMP (kPa) and will be immobilized easily and no leaks of If the enzyme molecules are large compared 66 86 m/s 35 RT 0.10-0.17 g Kamaruddin 30 2 − m), ultrafiltration membranes (10–100 nm), nanofil- ND 50 ND 8.1, 35 mg Giorno ND 50 ND ND Sakaki and Itoh 10 rate µ Flow × 5 . 40 25 RT 0.05 mL/mL Aboul-Enein 1–10 . ) ) h c ( enzyme will occur from the HFM modules. Enzymes with moleculartra sizes filtration 10–500 membranes, with kDa a(MWCO)of10 are typical kDa.Sincethesizesofmanyenzymesare10–80 molecular retained weight by cut-off ultrafiltration membranes ul- with MWCO kDa, of 1–100frequently kDa used. are the most Membrane and enzyme size Membranes are classified intosizes 0 microfiltration membranes (pore tration (1–10 nm) and reverse osmosis membranes ( higher process efficiency than the polyamide for chemicalof binding the same lipase. with the membrane poremembrane sizes, layers, they cannot diffuse through the NDND 1 300 mL/min 35 RT 0.13-0.17 g Kamaruddin NDND ND 300 mL/min 35 50-70 RT ND 3.38 0.17 mg g Giorno Kamaruddin permeate volume permeate volume permeate 120 mL 80% 24 ND ND 30 ND Ceynowa and 4ND40ND8.1–13mgGiorno 0.5∼ 30 mL/min ND ND 154.8 ∼ 1 h & 50% 2011 Society of Chemical Industry 1 c − 2 − 1 − ND 1 ND 120 RT 1.28 ND 2 ND ND 25 68.68–230.32 1 Initial − 1 1 1 1 1 1 [enzyme] Time ( − − − − − − ) b ( 0.05 mg cm The highest process 2gL 0.86-17.12 mL L 1gL 0.03 & 3gL 2gL 2gL 0.25, 1 g L 4 65,115 4 4 4 4 4 4 4 4 4 buffer 2 g L : 1032–1048 4 buffer buffer buffer pH 7 buffer, pH 7.2 buffer pH 7 buffer pH 7 buffer buffer pH 8 buffer pH 7 buffer pH 7 86 50 mM PO There may be a significant advantage Higher enantioselectivity was obtained 2011; Further observation proved that the PES ) 48 79 a 53 ( 30, PE - PS 10 50 mMPAN PO 30, PS PA 50 20 mM PO PS 30 100 mM PO PA 50PAN 50 PO 50 mM PO PA 50PAN 50 50 mM PO 50PAN mM 50 PO 50CP mM 5 PO MPP Distilled water 7.5 gPA L 50 50 mM PO 50 mM PO The TSP-EMR enzymes immobilization characteristics Various procedures for the enzyme loading. 127 adsorption adsorption Bonding adsorption adsorption adsorption adsorption adsorption adsorption adsorption adsorption adsorption A comparison between polyacrylonitrile (PAN) and polyether- ND: No Data. Physical Physical Covalent Physical Physical Physical Physical Physical Physical Physical MethodPhysical MembranePhysical Solvent Table 2. Two separate phase enzymatic membrane reactor www.soci.org produced higher reaction rates than thelectivities PAN, were but similar. the enantiose- sulfone (PES) HFMtioselectivity, showed but that the PES theunit membrane developed PAN area. higher gave reaction higher rates per enan- J Chem Technol Biotechnol perspective, hydrophilic membranesenzymes are are preferred required. since fewer Figure 3. efficiency was found withadsorption polypropylene of membrane enzyme, during while the the cellulose membrane produced by polyamide than polysulfone HFM. in employing relatively thinthe membranes membrane thickness with is high aloads. loads critical when factor for optimum enzyme 134 : 1032–1048 The racemic 3 86 74 CH 3 lipase. CH 2011; Candida cylindracea modules, which gave O C. A commercial-scale 2 ◦ O Organic Phase hyl ibuprofen ester, had higher Aqueous Phase This process was completed after 74 Serratia marcescens and Lopez and Matson. Using toluene and bisulphite solution The ester dissolved in isooctane was 41 . J Chem Technol Biotechnol 43 O et al 33,73 of 99% enantiomeric excess of the ester. Similar . 2 OH − et al m membrane effective area), the TSP-EMR produced O 1 2 Matson 3 − N C 3 CH 40 3 C CH 3 C and 40 K Pa transmembrane pressure to give conversion ◦ CH H C COOH 3 3 O H Several patents on resolution of racemic ibuprofen esters Racemic ibuprofen ester resolution was also studied by results were obtained using through hydrolysis and esterificationMatson, reactions were granted to enantioselectivity of 24.6, product yield of 42.9% and enantiomeric excess of 82% (product) in 22 h at 16–19 Kamaruddin 46 h. at pH 8.5,enantiomeric excess the of process 84.4%pilot-plant under study gave optimized (100-fold scale-up 42.6% conditions.with ratio, In conversion 10 7.5 a pilot-scale m and modules product of the TSP-EMR. MMPG hasCompound been resolved resolution in was an industrialOF started facility. at 360 bench-scale was where utilized. lipase lipase (CCL) under no-solventphate operation buffer using at potassium pHyields phos- 7.8 were and obtained conditions after asibuprofenacidwasthendevelopedusingthesamelipaseadsorbed 76 stated h in operating Table time. 1.onto Esterification polyacrylonitrile Low multiphase of TSP-EMR. Pentanol wasthe organic used phase as and alcohol asmetric donor. After analysis 68 indicated h, that spectrophoto- the estersulfomethyl ibuprofen was ester formed. using Prozyme Resolution 6 of protease in antive extrac- TSP-EMR produced 1.76 g of resolved ibuprofenthe from substrate 32.3 after g of 17 h. Later,to Seaprose an enzyme extractive was cross-linked polyacrylonitrileibuprofen HFM sulfomethyl ester. to hydrolyse the racemic ibuprofen trifluoroethyl ester was resolved by circulated continuously inproducts were the transported to phosphate membrane bufferthe at lumen-side. pH shell-side, The 8 polyacrylonitrile reactor flowing was and in operated forat 10 the 40 h facility was then set-up using twelve 60 m 55 kg year up to 31% and product enantiomericalkyl excess length of 90%. of The the effectshort-chain of ester substrate, compounds 2-ethoxy et was alsoenantiomeric investigated. ratio The thanof 1-heptyl the racemic ester. cyanomethyl Previously, ibuprofen hydrolysis ester was obtained in CH O 3 CH 3 CH as illustrated O O O 2 H www.soci.org J Agustian, AH Kamaruddin, S Bhatia 33,43 2011 Society of Chemical Industry 70 c OH O N H 3 This occurs when membrane CH , is one important application -methylmethoxyphenylglycidate C 3 H 128,129 O trans During these processes, the enzyme acts Multiphase operation of the TSP-EMR has OH They could also be diminished by a new O 72,73 N C 3 C CH 78,132,133 3 30,128,131 The fouling and polarization of concentration during H C 3 H 130 Kinetic resolution of a racemate in the TSP-EMR. 78 Hydrolysis of racemic considerable advantages for enzymaticthe optical substrates are resolution poorly water-soluble when andin the water. products dissolve as an enantioselective converting systemas and the a HFM functions barrier that separates the enantiomers, module design and fluid-dynamic operation. wileyonlinelibrary.com/jctb Figure 4. Drugs and Intermediates Kinetic resolutions(NSAIDs) of give medium non-steroidal to highand selectivities anti-inflammatory long at mild drugs reaction temperatures almost maximum times. resolution values, some Although conversions are these low. processes(MMPG), achieved a diltiazem intermediate Resolution of racemic compounds Resolution of racemicester drugs, molecules organic haveesters acids, or taken their amino place. long-chain compounds acidsreactions, Either are and resolved but short-chain by hydrolytic esterification racemic developed. Performance of of the hydrolysis the inenantioselectivity terms is racemates of increased with activity decrease and has ofthe aliphatic chains alcohol also of group. been APPLICATION OFENZYMES TSP-EMR: IMMOBILIZED Fouling control Generally a majorperformance limitation due of to membrane fouling. operation is loss of flux decreases as a functionresistance. of time due to increase in hydraulic EMR operations could shorten membraneeconomic service life benefits, and reduce butimproving flows these at limitations theboundary can membrane layers) surfaces, be by (whichmodifications. reduced disrupt introducing the by Dean vortices or by surface in Fig. 4. Commonlyphase one as isomer product is andorganic the transported phase. unreacted to enantiomer the remains aqueous in the

1040 1041 - - 1 )- 79 n n − S , was R 1 .with on the − 1 − was fed to 4 2.86 mmol L and a constant PO 2 1 40 K − 1 phosphate buffer at − A lower reaction rate Pseudomonas sp 1 BTEE in amyl acetate . Later, the BTEE in phosphate buffer at pH − 1 1 40 phosphate buffer at pH 1 − − − 1 )-1-phenylethyl propionate − wileyonlinelibrary.com/jctb S , R C. These multiphase operations ◦ (ATEE) was conducted using the -benzoyl tyrosine (BT) was also mole min N After immobilization of the enzyme µ 82 applied the TSP-EMR to select particular 40 ) run in the shell-side at 500 mL min )-1-phenyl ethanol by 1 )-octyl 2-chloro propionate was done with- solution was flowed in the lumen-side at the 79,80 R -octanol was flowing at 400 mL min S − 4 , 210 g of the racemate was recirculated at n ). Finally, the same enzyme was cross-linked to R 1 -Chymotrypsin was immobilized by cross-linking − PO α 2 in the shell-side of the PAN HFM, which housed )-enantiomer) was transported across the HFM to -heptane and phosphate solution at pH 8.0 were . The reaction was started by introducing a solution phosphate buffer at pH 7. K S 1 n 40,41 1 1 1 − − The − − L1754 immobilised by the cross-flow filtration method. 40 mole min µ )-1-phenyl-1-propyl acetate were hydrolysed to their alcohols ) as illustrated in Fig. 6. S Hydrolysis of ( Esterification of racemic , W -acetyl-L-tyrosine ethyl ester -Chymotrypsin and polyacrylonitrile module. a R it with 2.5% glutaraldehyde in 50 mmol L of 4.85 g of BT in phosphatesame buffer at flow pH rate. 6 in The the lumen-side BTEE120 at h. was the produced at 10% conversion after Other racemic esters Ceynowa and Koter pressure of 6.5 psi, and 200 mmol L thelumen-sideatthesameflowrate.Cross-linkingoftheenzyme with 2.5% glutaraldehyde in 50 mmol L than the previous experiments was obtained. Hydrolysis of ( shell-side of the module, while 100 mmol L Candida 50 mmol L surfaces of the PAN HFM50 mmol with L a 2.5% glutaraldehyde solution in Reactions of non-chiral substrates Esterification of decanoic acid The gas phase wasthe used esterification instead process. of Constantthe the shell-side humidity of aqueous air cellulose phase was( dialyzer during flowed HFM in to control water activity An innovative step was performedcompound in (( the process: thethe unreacted aqueous phase.( Later, racemicin 1-phenylethyl polyamide acetate membranes at and 30 ATEE dissolved in produced 40–60% conversion of their (R)-esters. out solvent. pH 7 on the shell-side of the polyacrylonitrile HFM. 200 mL octanol mixed with 87 mL ethanol400 mL was min flowed in the shell-side at studied. 7 produced a low reaction rate. hydrolysed by thethe same same enzyme TSP-EMR module attached byalbumin cross-linking to and it the glutaraldehyde. with bovine A shell-side(700 serum high of reaction rate was obtained 400 mLmin was recirculated in the shell-side at 10 mL min enantiomers of racemic alcohol. ( enzyme (immobilized byHFM the module. cross-flow filtration In method) this and case, 10 mmol L 7.8 flowed ingave a the reaction lumen rate side of of 45 the TSP-EMR. This process octanol (40 mmol L sameflowrate.41%oftheesterwashydrolysedduringeration. 6.8 A daysop- higher result was obtained whenpropionate racemic butyl was 2-chloro hydrolysed using theimmobilization same method. module, enzyme and in the lumen-side, decanoic acid and dodecanol in hexane were N α dissolved in circulated inside polyamide HFM as shownwas in Fig. hydrolysed 5. to The ( substrate 55% conversion and 99% enantiomeric excess of the substrate. . 1 C. − ◦ 2011 Society of Chemical Industry c Cusing mol h ◦ -benzoyl- µ N Subtractive )-ester with R Initially, a high -Chymotrypsin α 40,56 Similar results to 65,72 31 of substrate dissolved .Itwasfoundthatlower 1 1 − lipase B immobilized on − )-ketoprofen acid dissolved The process, conducted in R lipase (CRL) immobilized on 53,78 : 1032–1048 86 mole min -benzoyl-L-tyrosine ethyl ester (BTEE) C, gave enantioselectivity of 12–86% µ 200 mmol L N ◦ )- S 2011; , and silicone oil was maintained in the , 77 lipase immobilized on the spongy layers esterified racemic ketoprofen acid using an 1 40,74 R )-ester reacted faster, and more than 98% of − S 75 )-naproxen acid obtained excellent product Candida rugosa . S )-ketoprofen acid. ( )-naproxen ester, dissolved in isooctane at 30 Candida antarctica S S , et al For 36 hs, the hydrolytic rates were 9–14 R 40 21,52,60,65,72 C. The TSP-EMR also gave better performance than a batch )-enantiomer was converted. ◦ S 10%). Aboul-Enein Production of ( A cardiovascular drug intermediate, racemic glycidyl butyrate, < Two separate phase enzymatic membrane reactor www.soci.org immobilized on the shell-sidethe of polyacrylonitrile diffusive (PAN) method. HFM by 96.7% enantiomeric excess after 9.3 h operating at 28 spongelayersofpolyamidemembraneswasoperatedtohydrolyse the substrate, ( polyacrylonitrile HFM. During the process, both enantiomershydrolysed, were but the ( of three different membranes. Organic acids The enzymaticenantiomeric resolution excess, but of the technology hasRacemic organic rarely been 2-hydroxy applied. acid octanoicPseudomonas esters cepacia acid gave esters high were hydrolysed by the ( enantiomeric excess( (74–100%), but low conversion values those of Kamaruddin were found. Thedeactivationupto16 reactor showed no days.Further,observationofthehydrolysisof enzyme racemic cyanomethyl-[2-(4-isobutylphenyl) propionate] indicated that reduction of the immobilized enzyme activitythe compared free-enzyme was with mostly due to low massacross transport the of membranes. reagents washydrolysedinanextractiveTSP-EMRby Amino acids Various strategies have beentyrosine compounds. developed These to involve study typeor extractive of resolution operation, reaction, cross-flowing of multiphase or cross-linkingand the the enzyme solvent type. ( and product recovery ofacid 30–67% butyl in which ester themethyl led esters. hydrolysis to of the higher enantioselectivity than the acid in buffer solutionside at at pH 1000 7.8 mL min was pumped through the lumen- J Chem Technol Biotechnol polyamide membranes, which housed CCL. resolution of the racemate recovered 60% of the ( was treated with porcine pancreatic lipase. stirred tank free-enzyme system. isooctane or hexane at 30 concentration of racemic naproxen methylCCL ester in was a polyacrylonitrile resolved multiphase TSP-EMR by at highboth flow rates phases. on alcohol to make ( in a dichloropropane-hexane mixture (20to : 80 v/v) an was converted estersponge layers by of polysulfoneenantiomeric excess HFM. of High product (87.8%) conversionat were 40 obtained (73%) after and 24 h substrate flow rate producedEMR lower was activity. also A used multiphase to TSP- hydrolyse racemic BTEE using the same Although the conversions were low, the effectspressure of transmembrane andbatch emulsion stirred were tank free-enzymeexhibited revealed. higher reaction, activity the per Compared mass enzymatic of with reactor protein. the L-tyrosine acid, recovered inthe module the activity aqueous was 80 phase indicated that shell-side at a constant pressure of 9 psi. The product, of 76 oil 1 1 Song − − h 76 3 − : 1032–1048 Cfor12h. ◦ 40 Mucor miehei 86 2011; -cycloxex-4-ene-1,2- )-cyclohex-4-ene-1,2- of oleic acids when R cis 1 During operation, the ,2 − S h 56 3 lower fatty acids resulted. It − ctivity after 20 operating cycles When the oil was hydrolysed by This multi-gram scale operation 77,114 )-1-Phenylethyl acetate )-1-Phenylethyl 48 fatty acids were obtained using CRL S 38 Phosphate Buffer ( 1 − 85,86 Complete conversion of substrate was h buffer at pH 8 was pumped in the Compared with the batch stirred tank 3 4 − 118 35 J Chem Technol Biotechnol PO membrane area. The reactor productivity was approximately 1 2 − − lipase adsorbed on the polysulfone HFM. m 1 136 40,56,74 − Lower results (40–50% yields) were achieved using the obtained 7.1–23.45 mol m 38 108 . The hydrolysis of corn oil generated 50% yield of linoleic Hydrolysis of palm oil gave approximately 6 mol m )-Phenyl S , same HFM and lipase afterwas 2 h stable reaction time, for although 120 the h. lipase concentrations by employing CRLHFM at attached a reactor to residence time the oflipase Cuprophane 0.067 retained h 85% of where its the original immobilised a (137 h). was found that, using theproduced same higher HFM conversion module, than the the cross-link cross-flow CCL. CCL Previously, 0.074 mol m using hexane yieldedenantiomeric 100% excess. conversion Activity25 days. of and Higher reaction the more rates were enzymethe obtained than batch in was stirred the 97% tank TSP-EMR free-enzyme retained than reactor. for Hydrolysis of ethyl butyrate and amyl acetate Conversion of ethylsolvent butyrate operation. to The ethyl butyric butyrateside acid of was the circulated TSP-EMR used where in porcine the a liver esteraseand shell- was no-added immobilized, 200 mmol L free-enzyme system, oil hydrolysis in theconversion TSP-EMR and showed reduced higher back reaction, butachieved faster in equilibrium the was batch system. acid afteroil 4 h in of thefatty reactor acids shell-side production. time, of where polypropylene the membranes presence increased of the palmitic acid, but the quantity of oleic acid was almost negligible. CRL on polyamide membranes, obtained when babassulipase oil immobilized was onmembranes. hydrolysed the by sponge layers of polyetherimide Yields of 51.8–99% were obtained with 0.059–0.326 mol L Chiral product preparation Hydrolysis of a stereoisomer molecule, Mucor miehei lumen-side. immobilized onto modified polypropylene surfaces, which was still stable after 10 successive runs. presence of butyric acid in the aqueous phase decreased pH; hence the CRL on the polyacrylonitrile was used at 30 450 kg year et al dicarboxylate, to an enantiomer, (1 dicarboxylate, was catalysedpolysulphone membranes. by an esterase immobilized on Ethyl Acetate R -Heptane n 50 mL 0.05 mol/L ( did not of oleic ,theester W 3 a W − The organic a 84 )-Phenyl Ethanol )-Phenyl Ethyl Acetate )-Phenyl Ethyl -Heptane R R ( n ( www.soci.org J Agustian, AH Kamaruddin, S Bhatia 2011 Society of Chemical Industry 83 c control produced 84% Decanoic acid ester Decanoic acid Dodecanol Hexane Olive oil, palm oil, corn W Saturated salt tank a 135 ofpalmiticacidin15husing 3 = 0.75 − W a )-1-phenylethylacetate. S , of 0.75 caused by diffusion limitation at the Buffer pH 8.0 R W Air pump a 50 mL Phosphate The TSP-EMR for esterification of decanoic acid. Hydrolysis of ( Previously, a unique strategy was developed. Recent olive oil hydrolysis produced 225 mol m Decanoic acid Dodecanol Hexane acid and less than 50 mol m yield. Under continuous operation, the yield decreased to less50% than at a constant affect mass transfer coefficients significantly. wileyonlinelibrary.com/jctb reacted where the formed moisture was delivered toand the gas freed phase into saturated salt solution. By controlling Figure 6. Figure 5. membranes. Increasing the enzyme load or varying product dissolved in the organic phasein was 120 obtained h, at while 97% the yield experiments without Hydrolysis of oils Hydrolysis of oils in theconsidered two-liquid a phase more membrane cost reactor effective was tool. phase (decanoic acid inphase hexadecane, (water lumen-side) andcontinuously containing aqueous inside D-sorbitol, Cuprophaneimmobilised HFM shell-side) in where were the thesorbitol contacted enzyme flowed lumen-side. the lipase-decanoic was acid The complex.was The reaction sorbitol transported occurred fromsurfaces when and the the ester aqueous productbut was phase the moved moisture to to dissolved the inimmobilized organic the enzyme the was phase, stable aqueous after membrane 570 phase. h. Activity Theplace esterification at of took higher the reaction rates compared with thefree-enzyme batch stirred system. tank oil, butter oil and babassu oilacids were in successfully the converted biphasic-EMR. to fatty

1042 1043 CALB, ETOH )-Acid + S , )-Acid )-Ester S R R ( ( ( Organic Phase Catalytic activity of 33,75 wileyonlinelibrary.com/jctb Racemase )-Acid )-Acid S and gives higher observed activity , S ( R ( Aqueous Phase However, the free enzymes generally 50,52 51 but in chiral hydrolysis the free enzyme processes Higher free enzyme loading was required for the 75 )-Acid + 52,115 Schematic diagram of mandelic acid DKR. S , )-Acid )-Ester 24,51 S R R ( ( ( Organic Phase CALB, ETOH Many TSP-EMR processes recover the target molecule in the One of the focuses of TSP-EMR operations is distribution of Compared with batch stirred tank processes using free enzymes, AlthoughtheDKRhasbeenintroduced,theracemiccompounds have higher enantioselectivityenzymes. than the TSP-EMR immobilized aqueous phase. Sincetransmembrane pressure, the controlling the productimportant. materials Some transport flow optional is strategies determines most can be the observed in Table 3. materials. All theinterfaces, existing thus reactions the takeorganic substrates place phase. are After at frequently theare the circulated usually reaction, organic in distributed the the in substratesracemic the compounds and are reaction used, products unreacted medium; enantiomermedium. however, exists Hence, racemic in when resolution the in the TSP-EMR is morethan difficult with the non-racemic substrates. Thebe final either product(s) should the reactionthe product output or should the be chosen unreacted in enantiomer, the beginning. i.e. the TSP-EMR performs better.overall It lipase has activity better operational andenzyme stability, half-life product and enantiomeric lower enzyme excess, load. longer are generally resolvedstill by concentrates kinetic its resolution KRThe applications (KR). on hydrolytic The the kinetic TSP-EMR hydrolytic resolutionsracemates. reactions. utilize Hence, the preparation esterchemical forms esterification of of of the the before the raw racemates they materials shouldesterification-based are be through TSP-EMR has conducted been fed introduced,between a comparison to the hydrolysis the andstated clearly. esterification resolution results reactions. has not Although been Figure 7. teristics, type of reactionduring and the type development of of the operations TSP-EMR.have Several are been innovative focused introduced steps such on as the use of emulsion and DKR. the TSP-EMR immobilizedthat enzymes of is the free-enzymes much more stable than per mass of protein. use lower enzyme quantity to achieve 100% productexcess. enantiomeric esterification process to achievethe TSP-EMR, performance comparable with 1 − 2011 Society of Chemical Industry c acetic buffer 1 − . 100 mmol L )-mandelic acid, 1 S − used two enzymes through the lumen- 42 . 1 -benzyloxycarbonyl-L- − N et al Choi 41 : 1032–1048 )-mandelic acid. The product, the S 86 , . The organic phase was recirculated 1 R The racemate dissolved in sodium − 2011; 41 40,74 )-ibuprofen. The aqueous phase, 50 mmol L S , 87 R C by adding 3 g Prozyme 6 to the aqueous phase. C. ◦ ◦ and ( sodium hydroxide solution was added continuously. . 3 g of thermolysin was added to the aqueous phase to 2 vertical Hemophan HFM at 60 mL min 1 40 − )-mandelic acid with ethanol occurred in the organic 2 − L1754 enzyme. R )-mandelic acid ester, was obtained in 65% isolated yield and -benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester, a R Two separate phase enzymatic membrane reactor www.soci.org phase; therefore the racematethe reaction should sites. cross The the unreactedwas enantiomers, membranes transported ( to back tothe the racemase aqueous to phase give and the racemised ( by 98% enantiomeric excess after 48 h. saturated with butyl acetatethe pH module 5, at was 200 mL fed min to the lumen side of Similar to this process, hydrolysissolvent of operation amyl using acetate the was polyacrylonitrileCandida also membrane a to no- dock phosphatebufferatpH7wasfedtotheshell-sideofacelluloseHFMmodule, while hexane flowedreaction in took the place lumen-side. at The(Prozyme room 6) hydrolytic was temperature dissolved when inquantity 2 the of g ibuprofen aqueous of acid phase. recovered protease After into 6.3 the 50% h, organic conversion phase of the the related racemic ester.polyacrylonitrile Another module where attempt a used mixture the of cyclohexane–toluene (80 : 20, v/v) wasits pumped lumen-side through was the filled with membrane sodium shell-sideof phosphate buffer and 48.3 consisting g racemic sulfomethylfor 6.75 ibuprofen. h The at 30 hydrolysis was run Dynamic kinetic resolution of racemic mandelic acid Dynamic kinetic resolution (DKR)TSP-EMR of is still racemic a compounds novelTSP-EMR technology. in However, to the the resolve idea racemic toand use mixtures the co-workers was who proposedethyl by ester described Matson the DKR of racemic naproxen start the reaction. The process yieldedand more the than product 70% purity conversion of 91.7% after 24 h. Preparation of aspartame precursor N precursor of aspartame, was formed from The reaction gave 48.3% conversion of the racemic ester. J Chem Technol Biotechnol FURTHER NEEDS Aspects of thestrates operating and conditions, product fluid properties, enzyme characteristics, and sub- membrane charac- phosphate buffer at pHand 7.2 substrate containing was the pumped mandelate at racemase 30 mL min Kinetic resolution of racemic ibuprofen ester An extractive type ofsulfomethyl TSP-EMR was ibuprofen. used to resolve the racemic APPLICATION OFENZYMES TSP-EMR: SUSPENDED 6.0 mol L suspended in each phase to carry-out the DKRacid of racemic as mandelic described in Fig. 7. ThewasusedtodissolvetheCALBandcirculatedthroughtheshell-side organic phase, ethylene dichloride, of 1.05 m in the shell-side at the0.2 same kg flow cm rate and a constant pressure of side. Both phasesof were the flowed ( counter-currently. Esterification ( aspartic acid and L-phenylalanineTSP-EMR methyl using ester in aoperated at an 40 Sepracor extractive MBR-500 model 10 HFM module : 1032–1048 86 2011; 140 The ionic liquid allowed the organic interfaces phase, preferably not dissolved in organic phase organic interfaces solvent. High solubility in organic solvent is preferable. organic interfaces organic solvent 139 With or without organic Preferable: high solubility in Ionic liquids increased the selectivity and recovery Remark(s) Products J Chem Technol Biotechnol 132 Organic phase Organic phase organic phase organic phase Aqueous phase or Aqueous phase or Aqueous phase or Aqueous phase or → → → → Organic Organic Phase Main substrate is moved to Organic Organic Phase Reagent is moved to the Organic Organic Phase Organic Organic Phase Organic Organic phase Transport substrates to the Aqueous Aqueous Aqueous Aqueous phase Aqueous Aqueous phase Aqueous Aqueous phase Aqueous Aqueous phase High solubility in aqueous Aqueous Aqueous phase Aqueous Aqueous phase Aqueous Aqueous → → → → → → → → → → → → → → → The cross-flow filtration technique is commonly used to adsorb pressure activity of the enzymatic membrane process. enzymes onto theenzymes HFM solution only surfaces. in The one direction:lumen-side technique from or the circulates shell-side vice to the the made versa to as immobilize enzymes shown on bothis in HFM given sides. as Fig. to 4. No whether explanation both Effortshigher sides have immobilized immobilization enzyme would been quantity. develop a enantioselective transport of the ibuprofen enantiomer acrossmembrane the reactor. These solvents have been associatedA with resolution supported in liquid theemployed EMR. to membrane resolve racemic ibuprofen. containing an ionic liquid was CONCLUSIONS Compared with batch stirred tankthe processes using TSP-EMR free-enzymes, gavea better successful results. TSP-EMR Manysolvents, factors including moisture content contributed characteristics and to since pH, of and they the type influence of organic substrates the aqueous and phase, activity product(s) ofmany should enzymatic enzymes. reactions be The take place taken solubility inthe the into product(s) of organic solvent is account while generally since of recovered in no-added the organic aqueous solvents,and phase. emulsion Use environment the operation gasinvestigated. phase The preferred instead method of to aqueous immobilize the phase enzyme have all been Transmembrane Organic or Aqueous Organic or Aqueous Organic or Aqueous or Aqueous Organic Organic Organic Aqueous Aqueous Organic Organic 137,138 XAqueous XOrganic XOrganic √ √ √ phase Aqueous Solubility X X X √ √√ √ √ √√ √√ www.soci.org J Agustian, AH Kamaruddin, S Bhatia this innovation still Main Reagent Aqueous phase 2011 Society of Chemical Industry Organic Reagent Main Aqueous c 29,113 nterfaces) Shell Lumen Organic ntation requires a high pressure no-solvent operation directly. Use dissolved operation Description Hydrophobic Shell Lumen Organic Distribution Both X Organic Liquid: Dissolved or Optional strategies in the TSP-EMR operation As can be observed in Table 1, vertical reactors required a 108 Combinations of reactor orientation and fluid direction have It was proved that enzymes immobilized in the TSP-EMR could It is likely that some factors need further study. Although the Solvent-based operation is preferable as many products are Immobilizedenzyme(attheorganici Membrane Hydrophilic Shell Lumen Organic Product(s) Substrates Solid: Cannot be used Table 3. requires more investigation. been investigated. Vertical orie input. wileyonlinelibrary.com/jctb The substrates, productsdevelopment of and the flow membrane direction. characteristics affect be used for many cyclesstill of retaining TSP-EMR high operation catalytic with activity. the Thisthe enzymes is, operation, which of overcomes course, the problem a of enzymes benefit recycling for faced by the batch stirred tank free-lipase system. TSP-EMR has used emulsion in its operation, whichbetter indeed showed performance than the classic type, similarTMPtothehorizontaltype,butnoTMPhasbeenoperatedinthe vertical arrangement and furtherbe investigation considered. of this A should comparisonbe of studied the further: fluidproductivity, although direction the counter-current co-current should TSP-EMR flow was also stillcompounds gave favoured and for upflow-type higher achiral reactors. available in crystal and liquid form. However, no-solventcould operation reduce the operational cost, although itto can only liquid be applied non-viscous substrates.frequently used Although as buffer the aqueous solutionsdevelop TSP-EMR phase, are based the on opportunity ionic existsalternative liquids to media since for they have enzymatic become enantioselective reactions.

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