Green Chemistry

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Cite this: DOI: 10.1039/c5gc00992h

Received 9th May 2015,Accepted 12th June 2015

DOI: 10.1039/c5gc00992h

www.rsc.org/greenchem

Enzymatic reactive extrusion: moving towardscontinuous enzyme-catalysed polyesterpolymerisation and processing

S. Spinella,a,b,c M. Ganesh,a G. Lo Re,c S. Zhang,c J.-M. Raquez,c P. Duboisc andR. A. Gross*a

This paper demonstrated the feasibility of conducting an enzy-

matic ring-opening polymerisation by reactive extrusion (REX) at

high shear and temperature conditions. Using immobilized

Candida antarctica Lipase B (CALB) as catalyst at temperatures

ranging from 90 to 130 C, -pentadecalactone (PDL) was con-verted (>99%) by REX at 60 RPM for 15 min to PPDL with Mw163 000 g mol1.

The majority of current polymerisation methods use metalcatalysts. Residual metal catalysts are often undesirable inmaterials used for biomedical and electronic applications.1

Immobilized enzyme-catalysts have been shown to have dis-tinct advantages relative to most metal catalysts including: (1)naturally derived, (2) low toxicity, (3) high chemo- and regio-selectivity, (4) activity at relatively low temperatures and (5) noneed for strict exclusion of water and oxygen.26 The most com-monly employed lipase for enzyme-catalysed ring openingpolymerisations (eROP) and polycondensations is the immobi-lized lipase form of Candida antarctica Lipase B (CALB). Of themany lactonic substrates for which CALB is an active poly-merisation catalyst, CALB eciently catalyses ROPs of largerlactones (e.g. -pentadecalactone, PDL).3 The immobilizedCALB catalyst used in ref. 4 and herein is Novozyme 435(N435). ROP of larger lactones are known to be dicult for

many organometallic catalysts because the polymerisations areprimarily entropy-driven.7 Nevertheless, recent progress hasresulted in a number of chemical catalysts that successfullyconvert PDL to high molecular weight polymers. Examples ofthese catalysts are aluminium salen and terdentate phonoxy-imineamine aluminium. Problems encountered with thesecatalysts are as follows: (i) synthesis from expensive ligands,(ii) requiring inert reaction conditions (e.g. performed in aglove box) and (iii) the use of solvents.8,9

The ability of lipases to catalyse ring-opening and conden-sation polymerisations at relatively low temperatures (e.g.7090 C) is advantageous to reduce energy input and to pre-serve thermally sensitive chemical moieties. However, whenhigh molecular weight polymer synthesis is desired, corres-ponding diusional constraints must be overcome by eitherrunning reactions at higher temperatures (e.g. 150220 C),which is generally regarded as not feasible for enzyme-catalyststhat denature under such conditions,10 or by adding solvent.For example, for N435-catalyzed synthesis of high molecularweight (Mn > 50 000 g mol

1) poly(-pentadecalactone), PPDL,and poly(-caprolactone, PCL), the viscosity was lowered by theaddition of toluene. Subsequently, the final polymer productsare obtained by precipitation into a non-solvent such asmethanol.1113 Solvent-based processes reduce the volumetricproductivity of reactions and also require solvent recycling.

Reactive extrusion (REX) is an industrially relevant tech-nique because it combines polymerisation and processing intoa single step.14 REX has been used to overcome the aforemen-tioned problems of bulk polymerisations, mainly reactionmixing and heat transfer, that slow chain growth. In oneexample, poly(-caprolactone) (PCL) with Mn 200 000 g mol1

was produced by REX using aluminium triisopropoxide as thecatalyst with only a 5 minute residence time.15 Enzymes havebeen used in twin-screw extruders for food-related applications(i.e. depolymerisations).1618 However, to the best of our knowl-edge, enzymatic polymerisations have not been performedusing REX.

The objective of this study was to determine whetherenzyme activity for lipase-catalysed polymerisations can be

Electronic supplementary information (ESI) available: MR spectra of Poly-(w-pentadecalactone prepared by e-REX (10% N435, 90 C), molecular weighttime course data as a function of reaction time; linear regression analysis of theeect of N435 concentration; tabulated data from granulometry; tabulatedresidual enzyme activity data. See DOI: 10.1039/c5gc00992hCurrent address: SyntheZyme LLC, 11 University Place, Rensselaer NY, 12144,USA.

aDepartment of Chemistry and Biology, Center for Biotechnology and

Interdisciplinary Studies, Rensselaer Polytechnic Institute (RPI), 4005B

BioTechnology Building, 110 Eighth Street, Troy, New York 12180, USA.

E-mail: grossr@rpi.edubDepartment of Chemical and Biomolecular Engineering, NYU Polytechnic School of

Engineering, Six Metrotech Center, Brooklyn, New York 11201, USAcCentre dInnovation et de Recherche en MAtriaux Polymres CIRMAP, Service des

Matriaux Polymres et Composites, University of Mons, Place du Parc 23, B-7000

Mons, Belgium

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maintained at the high temperatures, confined bulk con-ditions and mechanical shearing created by the co-rotatingscrews and for times required by REX to reach high molecularweight polyesters. The possibility of directly extruding thepolymer out of the die was also examined. Initial e-REX experi-ments were conducted at 90 C with 10% N435 based on opti-mized conditions used for batch solution (toluene)polymerisations.19 An initial mixing speed of 60 RPM waschosen based on previous research.20

Fig. 1 shows the force produced by screw rotation as a func-tion of REX temperature and N435 loading. The viscosity ofthe polymer is directly related to extruder force. In anotherwords, more viscous products requiring more energy to main-tain the same mixing speed.22 The force versus reactiontime graph shown in Fig. 1 has three distinct regions:(1) initial decrease in the force, (2) a plateau and, finally, (3) asharp increase in the viscosity of the product. This can beexplained by that, during the initial stages of the reaction, themonomer is melting, leading to an overall decrease in the vis-cosity of the system. Subsequently, in the plateau region,enzymesubstrate complexes form that initiate chain growth.

Finally, the polymerisation progresses such that highmonomer conversion and large increases in polymer mole-cular weight occur, resulting in high viscosity reaction media.PPDL prepared at 90 C with 10% N435 reached sucientchain length (see below) after 20 min of polymerisation in theextruder, and began to crystallize, thus resulting in highforce.23 The increase in viscosity for N435-catalyzed polymeris-ations performed by REX is similar to chemical catalysed REXpolymerisations.14,22

PPDL, recovered after 20 min (90 C, 10% N435) from theextruder, was analysed directly by SEC and NMR (without frac-tionation, e.g. by precipitation). Its Mn is 90 000 g mol

1 ( =2.0) and PDL conversion reached >99% (Table 1). This is incontrast to PDL polymerisations performed in bulk for 24 hwith 20% N435, which gave PPDL with comparatively low Mn(15 300 g mol1) and higher dispersity ( = 4.4). Furthermore,increasing the reaction time to 72 h for a bulk reactionresulted in a small increase in Mn from 15 300 to 22 100 gmol1 (Table 1).8 Larger values for aforementioned reactionsconducted in bulk are attributed to the formation of oligomerswhich, due to diusion constraints, do not condense withother oligomers to form longer chains.5,21 Polymerisations per-formed in the extruder at 90 C for 15 min gave similar mole-cular weights as well as values similar to those performed intoluene at 85 C for 72 hours (Table 1, entry 4 vs. entry 1).Moreover, proton NMR analyses (shown in Fig. SI-1) showedgreater than 99% conversion of PDL after 15 minutes of extru-sion. The relatively short reaction time required to reach highMn PPDL by e-REX relative to batch solution and bulk N435-catalyzed PPDL reactions is attributed to the confined bulkconditions and mechanical shearing created by the co-rotatingscrew and the ability of the immobilized enzyme to retainactivity under such these conditions.

In the previous example, reactions were conducted at90 C, which is below PPDLs peak melting transition (Tm) at97 C and close to the crystallization temperature (85 C).23

To explore the possibility of continuous manufacturing byreactive eROP extrusion, where the polymer is extruded fromthe die after >99% monomer conversion, and to determine thefeasibility that REX may be performed with shorter residencetimes, the eect of temperature on the enzymatic reactiveextrusion was explored.

Fig. 1 Force as a function of reaction time for reactive extrusion poly-merisations of PDL catalyzed by 2.5, 5 and 10% N435 and performed at90 C and 110 C.

Table 1 Comparison of polymerisations to prepare PPDL that were conducted using enzymatic reactive extrusion (e-REX) and batch polymeris-ations in bulk and in solution

Entry Polymerisation Time (h) Temp (C) N435a (%) Mnb (g mol1) b,c Conversione (%)

1 e-REX 0.25 90 10 90 000 2.0 >992 Bulk21 24 80 20 15 300 4.4 >993 Bulk21 72 80 20 22 100 3.0 >994 Solution,11,d 72 85 10 80 000 2.0 >99

aNovozyme 435, physically immobilized Candida antarctica Lipase B (CALB) immobilized on Lewatit. bMolecular weights were determined bysize exclusion chromatography (SEC) with chloroform as the eluent using narrow dispersity polystyrene standards. c = Mw/Mn.

d Reactionperformed in toluene at 0.15 mol PDL (33.7 g) in 29.6 mL toluene. eDetermined by 1H NMR, signals corresponding to residual monomer werenot observed.

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The temperature was varied from 90 to 130 C with the resi-dence time and mixing rate fixed at 15 min and 60 RPM,respectively (Table 2). In all cases, complete PDL conversionwas observed. The reaction temperature did not have a signifi-cant eect on PPDL Mn, which ranged from 86 000 to 78 000.Complete monomer conversion at these high temperaturesinfers that, as the polymerisation proceeds, the enzyme main-tains sucient residual activity for complete monomer conver-sion. This retention of activity for N435 even at 130 C isimportant since this infers that residence times can be shor-tened and REX polymerisations using N435 can be conductedfor higher melting (e.g. 110 C) polyesters.24 Indeed, based onthe time required to reach 3500 N, the reaction time decreasedfrom 15 to 10 min by conducting e-REX at 90 and 130 C,respectively.

Control experiments were performed without the additionof N435 at both 90 C and 110 C for 60 min, and no conver-sion of PDL to PPDL was observed by 1H NMR. This verifiesthat N435 is active in the extruder and the polymerisation isnot autocatalysed by high shear stresses generated during theextrusion process. By performing the reaction above PPDLsTm, the final product was extruded out of the die. Experimentswere also performed to determine the eect of N435 concen-tration on PDL conversion and PPDL molecular weight. A pro-cessing temperature of 110 C was chosen since this allows thematerial to be extruded out of the die at the end of the poly-merisation. As the %-by-weight N435 relative to monomer wasdecreased from 10 to 5 and 2.5%, the time required to reachan axial force of 6000 N increased in a manner that follows alinear relationship (r2 = 0.967, Fig. SI-2). A study of%-monomer conversion and molecular weight change versustime was performed for polymerisations conducted by eREX at110 C with 2.5% N435. The results plotted in Fig. 2 and listedin Table SI-1 show that, initial chain growth is slow (Mn =1600 at 10 min), and is attributed to an apparent lag period forat least the first 10 min. Origins of the initial lag time areattributed to the time required for enzyme-monomer activationand chain initiation to occur. Indeed, Kobayashi reported thatchain initiation is the limiting step in e-ROP.25 After thisinitial lag time, the Mn increases linearly with PDL %-conver-

sion to 95% (Fig. 2). Thereafter, a large increase in Mn from121 000 to 142 000 g mol1 occurs with a relatively smallincrease in monomer conversion (about 95 to >99%). This isconsistent with previous reports and corresponds to a changefrom chain-growth to a step-growth mechanism as polyconden-sation between chain ends becomes increasingly competitivewith lactone ring-opening from chain ends.26 Furthermore,the general characteristics of this e-REX polymerisation of PDLconducted at 110 C with 2.5% N435 agrees well with themechanism proposed for the N435-catalyzed batch solutionpolymerisation of -caprolactone, CL, to form PCL, in tolueneat 60 C.26

N435 beads were recovered after e-REX (10%-N435, 90 C,15 min, mixing rate of 60 RPM) by dissolving the reactionmixture in xylene and separation of the enzyme beads by fil-tration. Prior to use, the N435 beads are spherical with novisible surface defects (Fig. 3A). Optical microscopy of recov-ered beads from e-REX shows little change in bead integritybut that physical abrasion occurs at bead surfaces (Fig. 3B).

Potential changes in bead dimensions due to the e-REXwere evaluated by granulometry.27 Measurements wererecorded for the mean particle size (D 50, m) and from bothextremes of the particle size window (D 10 and D 90, m).

Fig. 3 Optical microscopy pictures of: (A) an unused N435 bead and (B)a bead recovered after 90 C for 15 minutes at 60 RPM.

Fig. 2 Relationship between molecular weight and monomer conver-sion for the reactive extrusion polymerisation of PDL catalyzed by 2.5%N435 at 110 C with a mixing rate of 60 RPM.

Table 2 Eect of temperature on the reactive extrusion polymerisationof PDL catalyzed by 10% N435 performed at dierent temperatures withthe reaction time and mixing rate xed at 15 min and 60 RPM,respectivelya

Temp (C) Mnb (g mol1) a

90 86 000 1.995 83 000 2.0100 84 000 2.2105 84 000 2.2110 78 000 1.9120 80 000 2.1130 81 500 2.0

a Conversions are >99% from 1H NMR analysis. bMolecular weightswere determined by SEC.

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The results listed in Table SI-2 show that a decrease in thebead size after e-REX is observed but the change in size issmall.

In addition to study of changes in bead physical character-istics, recovered N435 beads after e-REX (10%-N435, 90 C,15 min, mixing rate of 60 RPM) were analysed to determinetheir residual activity. The assay used is described in detailelsewhere.28 In summary, the unused and recovered N435beads were assayed to determine monomer conversion by1H-NMR for -caprolactone polymerization conducted intoluene-d8 at 80 C. Results from this experiment are listed inTable SI-3. Comparing the slopes of CL conversion versustime, the recovered enzyme beads have 25% lower activity. Togain further information on the recovered beads, the proteincontent was determined and compared to unused N435. Thisexperiment is important since N435 is manufactured by physi-cal adsorption of CALB onto and within macroporous poly(methyl methacrylate), PMMA, beads. Presumably, if enzymeleaching occurs, the detached free enzyme migrates into thesurrounding polymer product. Since the bead material is con-structed from PMMA, nitrogen content in N435 is directly pro-portional to the bead protein content. X-ray photoelectronspectroscopy (XPS) was performed on unused and recoveredbeads, %-nitrogen values were converted to %-protein contentin beads and the results are listed in Table SI-4. The control(unused) beads have 8.4 1% CALB whereas N435 recoveredafter e-REX has 4.5 0.5% or 54% of the protein in unusedbeads. The migration of enzyme from the beads into productdoes not imply that the leached enzyme becomes inactive;however, free CALB activity should dier from immobilizedCALB. The extent of this change in enzyme activity is currentlyunknown.

Since the recovered beads have a lower CALB content, thedierence in activity between the unused and recoveredenzyme beads may be due to enzyme leaching and not enzymedenaturation during e-REX. Previous work by our group hasshown that loses in protein due to enzyme-leaching are notproportional to loses in bead activity.29 Furthermore, the%-enzyme lost can greatly exceed the observed activity loss ofthe recovered enzyme beads.29

Conclusions

This paper explored the feasibility of performing lipase-cata-lysed polyester synthesis by REX at temperatures up to 130 C,under confined bulk conditions and with mechanical shearingcreated by the co-rotating screws. Surprisingly, the immobi-lized enzyme withstood these conditions. The ecient mixingby REX shortens reaction times, decreases catalyst require-ments and enables the formation of high molecular weightpolyester under bulk reaction conditions. Furthermore,immobilized lipase e-REX reached high monomer conversionsand polymer molecular weights such that the synthesizedpolymer was extruded without the need for post-reactionproduct purification (e.g. by precipitation). In contrast, N435-

catalyzed PDL polymerisations conducted in bulk using abatch reactor did not exceed Mn values of about 20 000 g mol

1

due to diusion constraints. For reaction temperatures up to130 C (15 min, 60 RPM), N345 retained sucient activity suchthat high PDL conversion and PPDL molecular weight (>99%and Mn 81 500) was achieved. The ability to conduct e-REXpolymerisations up to 130 C suggests that shorter residencetimes, substantially below 15 min, may be sucient to achievehigh molecular weight polyesters. Furthermore, highermelting polymers (e.g. about 110 C, 20 C below the extrusiontemperature) may also be synthesised and directly extruded bythis method. Future work will build on the findings disclosedherein to develop practical processes in which the residencetime is decreased to a few minutes and the enzyme is immobi-lized within the extruder such that stronger interactionsbetween the support and the enzyme prohibit enzyme leach-ing. These improvements will facilitate continuous processesin which the immobilized enzyme can be re-used over multiplecycles. Methods of shortening reaction time currently underinvestigation include using higher activity lipases and cuti-nases from other sources or that are improved by proteinengineering.30,31 Furthermore, improvements in the catalystwill be needed that increase is kinetic and thermal stability.

Acknowledgements

The authors thank the National Science Foundation Partner-ship for Innovation (NSF-PFI) program (Award #1114990)entitled Next Generation Bioplastic Nanocomposites forfinancial support. Financial support from Wallonia and Euro-pean Commission in the frame of SINOPLISS-POLYEST projectand OPTI2MAT program of excellence and from FNRS-FRFCare also gratefully acknowledged.

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