MsAcT in siliceous monolithic microreactors enables quantitative ester synthesis in water

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Introduction
The acyltransferase from Mycobacterium smegmatis (MsAcT) catalyses ester synthesis in water (Scheme 1). 1,2Its unique active site is rather apolar, thus creating a local situation in which the course of reaction is not dominated by thermodynamics of the aqueous media but hydrophobicity of the active site.This asset makes MsAcT an excellent candidate for application in ester syntheses and transesterifications (such as biodiesel production) that are typically performed in aqueous or biphasic media. 38][9] To enable reaction engineering with MsAcT the aim was to immobilise the enzyme.The octameric MsAcT (72x72x60 Å, Fig. 1) needs to be tightly bound to ensure its long-term use, 2 and hydrophobic carriers (e.g.CNT) should be avoided since they may result in unfavourable partition effects and non-specific interactions of the enzyme or the products, detrimental for its catalytic properties and overall performance. 10,11he most recent study of MsAcT synthetic properties shed light on its application prospects to catalyse reactions in aqueous media. 1 But the reported experiments made use of cell free extracts with low enzyme concentrations and were carried out in a batch system.Thus while indicating the potential of MsAcT, yields were modest and useful application limited to primary alcohols.From an application perspective continuous-flow systems with very high performance are desirable, in particular so since they offer a new handle to reaction engineering. 12or this reason we deemed it important to undertake studies of the MsAcT behaviour in a continuous-flow transesterification of neopentylglycol (NPG) in a microchannel (micro)reactor.NPG is the substrate employed for the activity assay of MsAcT.It is particularly suitable for this study as it is a symmetric diol with two primary hydroxyl groups.This allows to follow two conversion steps, mono and diesterification.Additionally it is a good model for other primary diols and even for glycerol that also has two primary hydroxyl groups. 13Thus under these conditions the enzyme and the reactor should demonstrate their full capabilities, while at the same time offering an effective processing system.To preclude nonspecific hydrophobic interactions between the enzyme and its support the microreactor was made of (hydrophilic) silica monolith with a hierarchical pore structure in micro-and nanometric size scales and meandering flow-through channels.This enables facile flow of reactants and results in intensive mixing and mass transport to the enzyme.The latter was immobilised in very large mesopores using two distinctly

Synthesis of silica monoliths (MH).
The general procedure was as follows 14,15 : first polyethylene glycol 35000 (PEG, 9.09g) was dissolved in 1M HNO 3 (104.6mL), after which tetraethoxysilane (TEOS, 87.1 mL) was added slowly under stirring (500 rpm) to the PEG solution in an ice bath followed by the addition of cethyltrimethylammonium bromide (CTAB, 4.016g).The solution was mixed, then left to gel in polypropylene tubes at 40 ○ C and aged for 10 days.Next the alcogels obtained were impregnated with ammonia solutions: 1 M for 9 h at 90 ○ C. The samples were washed with water, dried at room temperature and then calcined at 550 ○ C for 8 h (ramp of 1 ○ C/min) to obtain silica rods 50 mm in length and 6 mm diameter.The monoliths were functionalised and clad with polymer resin (L285MGS-H285MGS type) to obtain singlerod continuous-flow microreactors.Functionalisation of silica monoliths (MH) -aminofunctionalised carrier.Pristine silica monoliths were modified to attach the enzyme non-specifically by means of amino-glutaraldehyde linkage [16][17][18] (Scheme 2A).In short, oven-dried (overnight, 150 ○ C) silica monoliths (1 g), was gently stirred and refluxed with 3-aminopropyltrimethoxysilane (0.27 mL) in 35 mL dry toluene for 72 h.After taking out it was washed with toluene and dried.Functionalisation of silica monoliths (MH) -metalfunctionalised carrier.In alternative approach for immobilisation via adsorption by the His linkage Ni and Co were incorporated 19,20 (Scheme 2B).Silica monoliths (1 g, oven-dried at 150 ○ C overnight) were gently stirred and refluxed with 3-iodopropyl trimethoxysilane (0.41 mL) in 30 mL dry toluene for 10 h under Ar atmosphere in the dark.After 10 h the toluene was removed and the monolith was washed in MeCN (acetonitrile).Next cyclam (1,4,8,11tetraazacyclotetradecane, 0.2 g) and excess potassium carbonate (0.37 g) were added and the mixture refluxed in MeCN (30 mL) for a further 21 h.After separating the monolith, it was refluxed with the excess of nickel (II) chloride or cobalt (II) chloride (0.35 g) in H 2 O (80 mL) for 3 h.Finally, it was filtered off and washed with H 2 O to remove any unattached nickel/cobalt.
Enzyme immobilisation -specific adsorption.Prior to MsAcT adsorption, the Ni/Co functionalised monolith (6 mm x 40 mm) was washed with ethanol and then distilled water for 45 min each (flow rate of 0.5 mL/min) and then with 0.1 M phosphate buffer (Na 2 HPO 4 /KH 2 PO 4 , pH 7.5).A solution of MsAcT (1.1 mg MsAcT (CEF)/mL, 5.5 mL) in the buffer was pumped (0.2 mL/min, 2.5 h) through the reactor under recycling to immobilise the protein.Excess protein was removed by washing the columns with a 0.05 M phosphate buffer (pH 7.5).The amount of the attached enzyme was determined using the Lowry method.

Enzyme immobilisation -nonspecific covalent method
Before the attachment of MsAcT to the amino-functionalised silica monolith (6 mm x 40 mm), the carrier was washed with ethanol and then distilled water for 45 min each (0.5 mL/min), followed by 0.1 M phosphate buffer (Na 2 HPO 4 /KH 2 PO 4 , pH 7.0).To attach aldehyde groups onto the monolith's surface a 2.5 vol.% glutaraldehyde solution in 0.1 M phosphate buffer (pH 7.0) was circulated (0.5 mL/min) through the reactor for 45 min, whereupon it was washed with water and 0.1 M phosphate buffer (pH 7.5).Finally, 5.5 mL of MsAcT (1.1 mg MsAcT (CEF)/mL) solution in the phosphate buffer was passed (0.2 mL/min, 2.5 h) through the reactor as described above.Excess protein was removed by washing as was described in ref. [15][16][17][18] Prior to activity assays, the monolith was washed with 0.05 M phosphate buffer (pH 7.5).The amount of immobilised enzyme was determined using the Lowry method.

Characterisation of enzyme carriers
To date MsAcT was immobilised only on carbon nanotubes (CNTs) and used for its perhydrolase activity in latex paint. 10,23,24Here we propose application of hydrophilic macro/mesoporous monoliths with meandering/twisting flowthrough channels, stimulating a chaotic movement (perturbations) of fluids and additionally enhancing mass transport. 25Employing nonhydrophobic carrier would help avoiding nonspecific interactions between the surface of the supports and the MsAcT active site.
10 Microscopic (SEM) and low temperature nitrogen adsorption studies of silica monoliths revealed the prevalent presence of through-pores of 30 -50 µm sizes and also mesopores of about 20 nm, and a total pore volume of over 4 cm 3 /g, determined by Hg porosimetry measurements, as reported earlier 15 (see Supporting Information).This very open structure can also be seen from the images displayed in Fig. 2. Equally important, the modification of the monoliths appeared to have no effect on porosity in macro scale.The specific surface area (S BET ) 26 of silica monolith measured by nitrogen adsorption was ca.300 m 2 /g, after surface modification a slight decrease in this parameter was observed.Thus not surprisingly, the measurements of backpressure vs. flow rate of water through the monoliths in the flow rates range of 0.03 -3 mL/min gave a backpressure drop of only 0.028 -2.8 kPa, that is significantly less than in capillary reactors or packed bed columns filled with beads of micrometric sizes. 27,28These experiments also corroborated very good stability of the monoliths structure during at least two weeks of continuous flow operation, observed in our earlier studies. 18 0][31][32][33] As cyclam is known as a stable ligand for several metals, MH-cyclam-Ni and MH-cyclam-Co were prepared as described previously 20 (after minor modification).
ICP mass spectrometry measurements gave the values of metal loadings of 8.96 mg Co/g silica ±16 % and 7.81 mg Ni/g silica ±10 %.After metal incorporation, originally white silica monolith turned to yellow or blue, for Ni and Co, respectively (Fig. 2).The same colour intensity, despite considerable size (6 mm x 4 mm) of the monolith indicated a homogenous distribution of the metal over its volume.To the best of our knowledge it is the first time that metal ions were homogenously incorporated into the monolithic column of that size, as to date only capillary or packed bed column with metals were used.

29-33
For non-specific MsAcT immobilisation the same silica monolith but functionalised with amino groups was used, and presence of the latter was confirmed by FT-IR, as shown earlier, 15 and elemental analysis (LECO CHN analyser).The amino group loading was 1.32 mmol/g of silica (1.85 wt % of nitrogen).Before enzyme immobilisation the amino groups were activated with glutaraldehyde, after which the column changed the colour uniformly to brown over the whole length of the monolith.

Performance of the monolithic microreactor
The recent extensive report on MsAcT-catalysed transesterification of NPG in buffer/ethyl acetate aqueous solutions in batch reactor using the native enzyme 1 provide an excellent platform to compare its behaviour with that of continuous-flow monolithic microreactors with the immobilised enzyme (Scheme 1).While an effective immobilisation of the enzyme in the silica monolith's mesopores remained a critical factor, our studies primarily centered on the synthetic potential of the immobilised enzyme/microchannel reaction system.Two immobilisation methods were applied: (i) conventional covalent bonding tends to give more stable catalysts, but could reduce enzyme activity 11,18 , and (ii) specific adsorption on to the Ni/Co modified support via the His-tag, 19,20,[29][30][31][32][33] since the His-tag is remote from the active site (Fig. 1).At first, activity, stability and enzyme loadings were studied using porous silica monoliths as enzyme support.Monolithic silica supports were functionalised with amino groups activated with glutaraldehyde (GLA), to produce a Schiff's base for covalent attachment, and with Ni or Co for specific adsorption.However, we started with the evaluation of enzyme loadings obtained from different immobilisation methods which, appeared to be fairly similar: 2.96 mg/g for covalent, 3.15 and 3.07 mg/g carrier for adsorption on Ni and Co, respectively.That gives 60 -70 % of immobilisation yield.As it is not possible to predict whether the octameric MsAcT will be immobilised via one metal or one lysine amino group or rather by a multipoint attachment it was considered important to ensure that an excess of both metal and aldehyde groups was available for binding.This clearly was the case (Table 1).Performance of the biocatalysts in 50/50 % v/v biphasic conditions of 0.05 M phosphate buffer (Na 2 HPO 4 /KH 2 PO 4 , pH 7.5) and ethyl acetate also appeared to be much the same (Fig. 3).We believe that this similarity in the enzyme loading and activity indicates a similar orientation of the enzyme attached This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins by means of different immobilisation procedures, and a free access to its active site.Bearing in mind that silica is hydrophilic by nature, it is likely that in a nonspecific covalent immobilisation hydrophilic regions of the protein were involved, and hence its active center located in a very hydrophobic region (Fig. 1) was free and not distorted by immobilisation.As shown in Fig. 1, the His-tag residues are surrounded by hydrophilic amino acids, implying that the same region of protein was involved in both immobilisation procedures.
Closer inspection of data given in Fig. 3 indicates that the NPG transesterification in microchannel reactor qualitatively portrays all trends observed earlier in the batch reactor studies. 1 But it also reveals a huge quantitative discrepancy between the operating characteristics of batch reactor and performance of the continuous-flow microchannel reactor with immobilised enzymes.The results displayed in Fig. 3 (B, D,  F) demonstrate that the reaction of monoester formation in a biphasic system was exceedingly fast, in strong contrast to the following reaction of diester formation, the concentration of which grew steadily but slowly with time.Already after about 30 sec of mean residence time the concentration of monoester achieved 90 mM, whereas those of substrate and diester were very low.Thus, very shortly after the reaction start up (reactants' entry into the microreactor) substrate conversion exceeded 90 %.After further 15 sec (longer residence time owing to application of lower flow rate) the substrate was fully converted and the concentration of monoester reached a maximum of over 90 mM.For longer reaction/residence times (lower flow rates) the concentration of diester at exit continued to grow as before, at the expense of monoester.For the longest residence time, of ca. 12 min, the diester yield was 50 -60 %, for a single-pass flow.Thus, the rate of the enzymecatalysed reaction appeared to be significantly enhanced in the proposed microchannel reactor compared to batch reactor with the native enzyme 1 , in which full substrate conversion was not achieved even during 7 hours, irrespective of the enzyme loading (cf.Fig. 1 and 2 in ref. 1 ), and mono-to-diester molar ratio was also much lower in the latter system.These results not only show exceptional performance of the proposed monolithic microchannel reactor in boosting enzyme catalyzed reaction, which may be of major practical interest, but also that the course of transesterification was fully controlled by the catalytic properties of MsAcT confined in well defined, functionalised mesoporous environment.That again may be a major asset of the proposed solution, seen from both fundamental and most practical point of view.We believe that this performance can be explained by two factors: (i) the flow-induced reaction effects typically observed in microfluidic/microchannel reactors, which in the case under study boosted monoester formation, additionally enhanced by (ii) large concentration of the enzyme embedded in the mesoporous environment most favourable for expressing its activity.Similar effects were recently reported for the consecutive reactions carried out in microchannel reactors, but without heterogeneous catalysts. 12Asano et al. showed that the application of microreactor technology could increase the yield of monosubstitution product from 58.6 % to 98.6 % in case of bromination, from 77 % to 86.3 % (nitration) and from 25.2 % to 38.1 % (ester reduction) in comparison with batch method. 12Very high activity of enzymes embedded in large mesopores of silica monoliths and in particular mesoporous materials was also observed by other groups and us before.17,18,28,34   Remarkably, at least 50 % conversion to the diester is achieved under these aqueous conditions, independent of the carrier.Equally remarkably, up to a flow rate of 1 mL/min no diol was observed.A residence time of 1 min was thus sufficient to achieve complete esterification of at least one alcohol group of NPG.This implies that this catalytic system has a great potential for the rapid ester synthesis (including bio-diesel) under aqueous conditions.
For practical applications the stability of immobilised biocatalysts is a factor of major importance.In this respect, both the enzyme coupled by His-tag adsorption and by covalent binding showed very good performance during almost 50 h continuous operation (Fig. 4), and only small decrease in activity was observed after 100 h (cf.Supporting Information).Also intermediate washing of the reactor and storage of the reactor in buffer did not change the activity.
Another factor of significance for this reaction system is the water phase present.Typically this contains buffers leading to a significant salt load that has to be separated after the reaction and that increases corrosion.Therefore, stability and the catalysts performance at different flow rates were studied for MsAcT immobilised on Ni-modified silica in the absence of a buffer (Fig. 5).
Also in this case the increase of monoester and decrease in diester formation were observed when the flow rate increased (Fig. 5A).However, while in a biphasic system the largest concentration of monoester, at full substrate conversion, was observed for the flow rates in the range of 0.7 -1.0 mL/min (Fig. 3), in the non-aqueous situation (Fig. 5A) it was only about 0.35 mL/min.This is partly due to the higher NPG leading (no dilution by buffer), but also evidence of a significant activity drop.Moreover, the non-aqueous system also detrimentally effected enzyme stability, as seen from a rapid decline in the enzyme activity after 4 h of continuous steady operation.MsAcT not only catalyses the formation of the esters but also some hydrolysis of ethylacetate leading to acetic acid and consequently a pH drop.After washing the enzyme with the buffer initial activity could be restored (Fig. 5B), indicating that the deactivation was reversible.
A destructive effect of the absence of a water phase was also observed by Mathews et al., 2 who found that below 2.5% of water content MsAcT activity significantly decreased.The relatively high activity of the immobilised enzyme in the organic solvent system (Fig. 5) might be ascribed to water adsorbed to the silica carrier.Acetic acid formation and loss of water slowly led to a deactivation that could be reversed by treatment with a buffer (Fig 5B).This demonstrates the great gain in enzyme stability achieved by immobilisation.
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Conclusions
Our studies demonstrated that the metal ions and amino groups can be uniformly introduced on to silica monoliths surface, and that post synthesis treatment has no detrimental effect on their macroporous morphology.Functionalised silica monoliths were successfully used to immobilise MsAcT and demonstrate its exceptional properties in a continuous-flow reaction.Complete ester synthesis could be achieved in aqueous media within very short reaction times.No differences in activity were observed for the enzyme attached using nonspecific covalent immobilisation and very specific chelation procedure.This highlights the importance of a judicious selection of the carrier.The microrector demonstrated very high activity and stability of catalytic and structural properties in the continuous esterification of NPG with ethyl acetate at room temperature and in biphasic conditions.Please do not adjust margins Please do not adjust margins Fig. 1 The octameric MsAcT (stereoview) displays an overall mixed hydrophilic surface: hydrophilic residues are highlighted in blue; grey residues are neither hydrophobic nor hydrophilic; hydrophobic residues are highlighted in red.The His-tag is located at the C-terminal (highlighted in green; please note that the His-tag is not included in the structure); the arrow indicates the entrance of the active site.Structural data from PDB: 2Q0S; picture was generated using the PyMOL Molecular Graphics System.

ARTICLE
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Fig. 4 10 |
Fig. 4 Catalytic stability of MsAcT covalently immobilised on amino modified monolith (A), adsorbed by His-tag on: Ni-modified monolith (B) and on Co modified monolith (C).For covalent immobilisation and His-tag adsorption on Co cumulative flow rate 0.1 mL/min (buffer/ethyl acetate 50/50 % v/v) for His-tag adsorption on Ni cumulative flow rate 0.06 mL/min (buffer/96 mM neopentylglycol in ethyl acetate 50/50 % v/v).Vertical lines show the interruptions in measurements, the immobilised enzyme was washed and stored in buffer: (black) diester, (blue) monoester.

Fig. 5
Fig. 5 Transesterification of neopentylglycol in absence of buffer by MsAcT adsorbed by His-tag on Ni-modified monolith (A) and process stability (flow rate 0.1 mL/min) (B).Conditions: 96 mM neopentylglycol in ethyl acetate; room temperature.(red) substrate, (black) diester, (blue) monoester.Vertical lines show the interruptions in measurements, the immobilised enzyme was washed and stored in buffer.
was separated into water and organic phase, and products in the organic phase were determined by GC (Scheme 1).Concentrations of the substrate and products were determined by GC analysis on an Agilent Technologies model 6890N Network GC System gas-chromatograph equipped with a flame ionisation detector (FID) and a capillary column HP-5, Agilent Technologies (diameter 0.32 mm, length 30 m, thickness 0.25 µm).The following temperature program was applied: initial oven temperature 70 ○ C held for 9 min, next to 135 ○ C (45 ○ C/min) and held in this temperature for 1 min, then from 135 ○ C to 140 ○ C (45 ○ C/min) held in 140 ○ C by 1 min and finally to 300 ○ C (50 ○ C/min).The injector and the detector temperature were 320 ○ C and 350 ○ C, respectively.The carrier gas was helium with a flow rate of 2.5 mL/min.The retention times of the NPG and its esters under these conditions were equal to: NPG 4.5 min, monoester 8.1 min and diester 11.5 min.The concentrations were calculated from the peak areas using calibration curves.All the samples were analysed twice.
temperature.Two pumps (one for buffer and one for organic phase) and a mixing unit, located immediately before microreactor inlet, were applied.For experiments with organic solvent (Fig.5) only one pump was employed.Reaction Please do not adjust margins Please do not adjust margins mixture