Î±-Tetralol by Immobilized - ACS Publications - American Chemical

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Kinetic resolution of (R,S)-#-tetralol by immobilized Candida antarctica lipase B : Comparison of packed bed over stirred tank batch bioreactor Manoj P. Kamble, and Ganapati D. Yadav Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.6b03401 • Publication Date (Web): 23 Jan 2017 Downloaded from http://pubs.acs.org on February 1, 2017

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Industrial & Engineering Chemistry Research

Kinetic resolution of (R,S)-α-tetralol by immobilized Candida antarctica lipase B : Comparison of packed bed over stirred tank batch bioreactor Manoj P. Kamble; Ganapati D. Yadav* Department of Chemical Engineering Institute of Chemical Technology Nathalal Parekh Marg, Matunga Mumbai 400019 India * Corresponding author Tel.: +91-22-3361-1001; Fax: +91-22-3361-1002, +91-22-3361-1020 E-mail address: [email protected], [email protected]

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Keywords: Continuous flow packed bed reactor; kinetic resolution; Novozym 435; αtetralol; Lineweaver-Burk plot; Enzyme kinetics

ABSTRACT: Here, we have demonstrated the flow chemistry approach for kinetic resolution of (R,S)-α-tetralol using packed bed reactor of immobilized Candida antarctica lipase B (Novozym 435). We have investigated the performance of different enzymes including Lipozyme RM IM, Lipozyme TL IM, and Novozym 435 and compared their activity and enantioselectivity in non-aqueous media. The kinetics of transesterification of (R,S)-α-tetralol using vinyl acetate as the acylating agent was studied in a packed bed of immobilized enzyme. Continuous flow packed bioreactor shows better behavior in terms of enantioselectivity and conversion vis-à-vis stirred tank reactor. A conversion of ~50% of (R,S) mixture with ~100% selectivity (E ) for (R)-α tetralol (eep ≥ 99.99%) was obtained at 65 °C with a 3 min residence time, using Novozym 435 as the catalyst in continuous flow packed bed reactor, whereas 43.6% conversion of racemic mixture (eep ≥ 99.99%) was obtained in a stirred tank batch reactor in 8 h under identical conditions. There was no back mixing in the fixed bed bioreactor having laminar/plug flow. The enzyme remains stable up to 7th reuses, which show viability of a continuous operation over a longer period. Inhibition by vinyl acetate with Ping Pong bi-bi mechanism was proposed using Lineweaver-Burk plots.


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INTRODUCTION Currently ~25% active pharmaceutical ingradients (API) sold globally are the single enantiopure compounds and the use of racemic mixtures is avoided due to adverse effects of one of the racemates. Separation of enantiopure drug from the racemic mixture and its commercialization is highly sought after. In this regard, conventional methods such as synthon approach, use of chiral stationary phases and crystallization have their own limitations in terms of cost and environmental concern. As a result, various strategies have been adopted for production and separation of pure enantiomers in which use of enzymes has gained significant importance.1,2 Biotransformation has found immense applications in various chemical, pharmaceutical and fine chemical industries. Among biotransformation, kinetic resolution approach by an enzyme is a very well accepted method for production of chiral compounds. In this approach, one enantiomer is selectively converted over the other with high enantiomeric purity and is attractive for production chiral compounds. 3,4 In comparison with chemical catalysts, biocatalysts are achieving significance on commercial scale due to easy scale up, high catalytic turnover numbers; very high selectivity towards single enantiomer-, chemo- and regio-selectivity; broad substrate range; and ability to facilitate reactions at ambient temperature and pressure.5-7 In particular, Candida antarctica lipase B (CALB) with catalytic triad (Ser105His224-Asp187) is a much-used enzyme for kinetic resolution of chiral compounds. The attributes of CALB are as follows: (i) it is commercially available in different forms and preparations, (ii) it shows catalytic activity and great stability in both non-aqueous and aqueous media, (iii) it possess flexibility and plasticity for various substrates with stereoselectivity, (iv) is able to distinguish the chiral centers, (v) there is no need of the additional chemical component (cofactor) to initiate reactions, and (vi) of their high recyclability in immobilized form.3,4,8-10 Immobilized CALB is a robust catalyst, which has 3 ACS Paragon Plus Environment


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have been used to accelerate rate of a variety of reactions in conventional media like esterification, transesterification, amidation, hydrolysis, hydrazinolysis, and epoxidation.9,11 Synergism of biocatalysis and flow chemistry are widely considered to be industrially valuable technologies essentially compatible with sustainability. The use of flow chemistry approach for the enzymatic reaction has increasingly gained importance.12-15 Continuous flow systems are favoured over batch processes in the case of enzymatic reactions due to their precise control, high yield along with selectivity and purity. Packed-bed reactors (PBR) are the most common, amongst all types of continuous-flow reactors, due to their own advantages like low cost, ease of fabrication and operation, and high performance. PBR provide a lower ratio of substrate to enzyme than the stirred tank reactor resulting in increase rates of reaction. Stirred tank batch reactor for immobilized enzymes is not a long term option for industrial scale production since immobilized enzyme particles are prone to abrasion and susceptible to breaking due to shear stress, intense stirring and particle-particle collision.14,1619

Key advantages associated with the use of the PBR are better rates of heat transfer (due to

high surface to volume ratio) and mass transfer (short diffusion path).13,20 Microreactor engineering might play crucial role to develop continuous biotransformation processes at commercial level.18,21 Recently, we reported enantiopure α-tetralol commercially valuable API which is used in treatment of cardiovascular diseases.22 Different approaches have been used to produce enantiopure (R)-α-tetralol using synthetic and enzymatic routes.22 Chemical synthetic procedures have their own limitations such as high energy costs, hazardous environmental impact. To overcome limitations of chemical catalysis, process intensification using biocatalytic approach is on the rise.2,4,8,9 Lipases. particularly CALB, show higher enantioselective preference toward secondary alcohol compared with primary and tertiary alcohols. This phenomenon can be considered to obtain enantiopure (R)-α-tetralol through esterification by using continuous flow packed bed reactors of immobilized CALB.15,22-25 4 ACS Paragon Plus Environment

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In this work, we have investigated effects of various process parameters on the kinetic resolution of (R,S)-α-tetralol with vinyl acetate by using continuous flow packed bed microreactor of immobilized CALB. Our results provide critical insight into use of microreactor in continuous flow mode over stirred tank batch reactor. There is a dearth of literature on immobilized CALB catalyzed kinetic resolution of (R,S)-α-tetralol with vinyl acetate in non-aqueous media in a continuous flow mode.

Experimental Section Enzyme and chemicals All enzymes were used as earlier reported by Shinde et al.2 All chemicals used in the study were of AR grade, purchased from renowned firms and used as such with no further purification (supporting information).

Experimental set up Stirred tank batch reactor Stirred tank batch glass reactor set-up used in this study was the same as reported earlier by us.22 (R,S)-α-tetralol and vinyl acetate were dissolved in toluene with a mole ratio of 1:3 to make the volume 15 mL The reaction was performed at 65 °C using 6.88 mg Novozym 435. Samples were periodically withdrawn to analyze the progress of the reaction. All the experiments were performed in triplicate to determine the experimental variability (within a standard deviation of ± 5%). Continuous flow packed bed column microreactor The continuous flow reactor consisted of a borosilicate glass column (Omnifit BenchMark™ Microbore Chromatography Column having 3.0 mm i.d., 100 mm length) packed with 100 mg of Novozym 435. Experiments were conducted by pumping a pre5 ACS Paragon Plus Environment


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mixed reaction solution in upward direction through the packed-bed microreactor fitted in commercially available Asia flow chemistry system of Syrris Ltd, UK, with proper temperature control system with accuracy of ± 0.5 °C. All the experiments were performed in triplicate to determine the experimental variability (within a standard deviation of ± 5%). Continuous transesterification process A typical reaction mixture consisted of (R,S)-α-tetralol and vinyl acetate dissolved in toluene as a solvent with a mole ratio of 1:3. A premixed reaction mixture was introduced in the high temperature packed bed microreactor (65 °C) with desired flow rate so that residence time in the column varied from 1 to 5 min. The effect of different process parameters was studied considering outcomes of preliminary experiments and operational limitations of setups. Samples were collected only after one column volume of eluent passed through the column for each change in residence time ensuring that the steady state was achieved and analyzed by HPLC. One-factor-at-a-time experimental approach was used to

optimize

parameters. The experiments were performed in a series. For each series the reactor was packed with fresh catalyst (Novozym 435). Method of analysis The progress of reaction was monitored by HPLC-system as reported by Yadav et al.25 (supporting information). Product formation was confirmed by GC-MS analysis using Shimadzu QP-2010 (Figures S1 and S2, Supporting Information). Conversion efficiency (c), enantioselectivity (E) and enantiomeric excess of substrate (ees), enantiomeric excess of product (eep) were calculated by using following formulae,23

ees 

[ S ]  [ R] [ S ]  [ R]

c

ees ees  ee p

E

ln[(1  c)(1  ees )] ln[(1  c)(1  ees )]

Kinetic model for continuous flow process in Novozym 435 packed bed microreactor 6 ACS Paragon Plus Environment

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Enzyme kinetic analysis was carried out to understand mechanism of kinetic resolution of (R,S)-α-tetralol using vinyl acetate as a acyl donor. However, the objective of current study was to determine the rate of reaction and how it varies in response to changes in substrate concentration (supporting information).

Results and Discussions Effects of various biocatalysts Esterification reaction between (R,S)-α-tetralol and vinyl acetate catalyzed by various biocatalysts

was carried out in continuous flow microreactor (Scheme 1). (R)-1,2,3,4-

tetrahydro-1-naphthyl acetate with eep ≥ 99.99% was obtained with Novozym 435 amongst all biocatalysts screened.

Scheme 1. Kinetic resolution of (R,S)-α-tetralol with vinyl acetate

The purpose of screening different commercial immobilized lipase enzymes for enantiomeric separation of (R,S)-α-tetralol was to investigate if any noteworthy change in enantio preference or activation could be attained due to the use of continuous flow reactor system, despite their well-known applications.24,25 Almost all lipases were able to catalyze (R)-selective kinetic resolution of α-tetralol with different reaction rate and conversions. These three supported enzymes, Novozym 43526 (Candida antarctica lipase B from), Lipozyme TL IM (Thermomyces lanuginosus)27 and Lipozyme RM-IM (Rhizomucor 7 ACS Paragon Plus Environment


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miehei)28 were used which differ not only in terms of origin of enzyme but also support for immobilization (supporting information). Hence, considerable variance in catalytic activity, selectivity, and operational parameter (in term of stability) were expected.29 Due to the following unique characteristics of Candida antarctica lipase B, enantiopure separation of (R,S)-α-tetralol with vinyl acetate was the best with Novozym 435: 1) No interfacial activation is observed like other lipases. 2) Very narrow active site pocket which is responsible for high selectivity 3) Stable and active in non aqueous media. 4) Definite activity preference towards the R-enantiomer. 5) Functions across a wide temperature range (30-80 °C). 6) Suitable for both stirred batch tank and continuous fixed bed reactors.

Thus, Novozym 435 showed the highest catalytic activity in comparison to Lipozyme RM IM and Lipozyme TL IM for continuous flow reactor approach (Figure 1). Versatile and thermo stable Novozym 435 attributes higher conformational flexibility toward R-isomer than S-isomer of α-tetralol due to proper interaction in non-aqueous media while the flapping lid of Lipozyme RM IM and Lipozyme TL IM project into the pocket thereby creating steric hindrance in binding of (R)-α-tetralol at the active site.2,4,8,11,24,25,29,30 Thus, all further reactions was carried out using Novozym 435 as a biocatalyst.


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Figure 1. Effects of various biocatalysts (Reaction Condition: (R,S)-α-tetralol- 3 mM, vinyl acetate- 9 mM, solvent- toluene, temperature- 65 °C, biocatalyst-100 mg). Effect of residence time on conversion Continuous flow packed bed microreactor for enzymatic reactions has attracted attention of biotechnologists due to several advantages over batch reactor in terms of high productivity, economy and operating safety. Residence time of reactants plays a crucial role in continuous flow operation since the flow is not back-mixed and typically laminar or plug flow. To facilitate higher conversion (R,S)-α-tetralol, adequate residence time was needed to ensure substrate was interacting with CALB’s active sites. Hence, adjustment of flow rate was done in a manner that the residence time of reaction mixture was between 1 and 5 min. There is a substantial increase in conversion with increase of residence time of reactants; maximum conversion of 50% from the racemic mixture was obtained with a residence time 9 ACS Paragon Plus Environment


of 3 min (flow rate: 0.1036 ml/min) at 65 °C (Figure 2A). This residence time of 3 min was maintained continuously for 35 h in the packed bed microreactor containing 100 mg Novozym 435 catalyst without any appreciable loss in the activity. A total reaction volume of 217.76 ml was processed in the 35 h. Thus, the enzyme was utilized effectively at 0.045% w/v of the total reaction volume processed. To maintain similar conditions in the stirred tank batch reactor, the enzyme loading was kept 0.045% w/v at 65 °C, with agitation at 400 rpm in 15 ml reaction and the conversion efficiency is depicted in Figure 2B. To get 50% conversion of (R,S)-α-tetralol in stirred batch reactor we required ~162 min vis-a-vis ~3 min in packed bed reactor on the basis of normalized residence time.

ees

eep

110

110

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0

0 0

1

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3

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5

ees (%) eep (%)

Conversion

Conversion (%)

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6

Residence time (min)

Figure 2A. Effect of residence time on conversion in continuous packed bed (Reaction Condition: (R,S)-α-tetralol- 3 mM, vinyl acetate- 9 mM, solvent- toluene, temperature- 65 °C).


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As showed in Figure 2A at 65 °C, the rate of reaction is much faster in the Novozym 435 packed microreactor compared to the stirred tank batch reactor. This can be attributed to three factors: 1) Enhancement of mass transfer: Formation of the laminar flow stream in microreactor system shortens the diffusion time and reactants are forced to be in contact with active sites of Novozym 435. 2) Rapid heat transfer: Surface to volume ratio in Novozym 435 packed bed microreactor (20,000 m2/m3) is much higher than that of stirred tank batch reactor (1,000 m2/m3) which dramatically enhances the heat transfer and exposes more active site to reactant. 3) Absence of back-mixing: There is no back mixing of reactants and particles vis-à-vis stirred batch reactor.13-15,19-21 Whereas to get similar performance in stirred tank batch reactor it is required to increase loading of immobilized enzyme (to increase active site) which is not feasible in term of economy.

ees

eep

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0

ees (%) eep (%)

Conversion

Conversion (%)

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0 30

90

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390

450

Time (min)



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Figure 2B. Effect of residence time on conversion in stirred tank batch reactor (Reaction Condition: (R,S)-α-tetralol- 3 mM, vinyl acetate- 9 mM, solvent-toluene up to 15 ml, temperature- 65 °C). Effects of various solvents The influence of reaction media on transesterification of (R,S)-α-tetralol by Novozym 435 has been studied in terms of logP values (Figure 3). There are no correlations till date to establish quantitatively relation between the impact of the reaction media on the catalytic activity and selectivity of enzyme-catalyzed reactions.8,31 As the behavior of the substrate, the enzyme or the enzyme-substrate complex changes according to solvents which affect the initial rate of reaction. Hence selection of solvent is crucial in the case of biotransformation. An optimum amount of water molecule is essential for Novozym 435 to open its catalytic traid to show catalytic activity in the reaction media. Therefore, solvents which have logP value range of 2-4 are more preferred in nonaqueous enzymatic transesterification than hydrophilic solvents. Polar solvents pull out essential water molecules around enzyme which leads to losing its catalytic activity. The highest catalytic activity of Novozym 435 was found to be in toluene with a conversion of 50% with eep ≥ 99.99%. 2,4,8,11,22,24,25,30


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Figure 3. Effects of various solvents (Reaction Condition: (R,S)-α-tetralol- 3 mM, vinyl acetate- 9 mM, temperature- 65 °C).

Effect of mole ratio of vinyl acetate The effect of concentration of vinyl acetate was studied, keeping concentration (R,S)α-tetralol, catalyst loading and toluene volume constant. Figure 4 shows that the high rate of reaction and maximum conversion (49.99%) were obtained with a 1:3 mole ratio.



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Figure 4. Effect of mole ratio of vinyl acetate (Reaction Condition: (R,S)-α-tetralol-3 mM, vinyl acetate- 3-12 mM, solvent-toluene, temperature- 65 °C). Whereas the subsequent increase in vinyl acetate concentration led to decrease in the rate of reaction and also conversion suggesting that there was inhibition of the active site of Novozym 435. In case of sluggish reaction (α-tetralol resolution), efficiency of esterification reaction is assured by irreversibility of reaction. Hence, to secure the complete conversion of the reactive enantiomer, non-racemic compound (vinyl acetate) typically is used in a molar excess over the α-tetralol. This was further confirmed by kinetic modeling studies.


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Effect of temperature A number of experiments were carried out in the wide range of temperature (40-70 °C) under identical conditions. Temperature had a dramatic effect on conversion (Table 1). Maximum conversion of ~50% was obtained at 65 °C with ~100% selectivity and enantiomeric purity. Effect of temperature on selectivity of Novozym 435 catalyzed kinetic resolution has already been reported by our laboratory25,32 and that temperature had no significant effect on enantioselectivity. Increasing temperature have positive effect on kinetic rate constants, However, it deactivates the enzyme and so it is mandatory to optimize the temperature for biotransformation. At higher temperature there was: 1) reduction in external mass transfer limitation (by improving diffusion) 2) reactant molecule gain desired kinetic energy to overcome the energy barrier (rate of the reaction increases).4,33 The activation energy of 11.25 kcal/mol. was obtained from the Arrhenius plot (Figure S3, supporting Information), which is a reasonable value for enzymatic reactions.

Table 1. Effect of temperature on the kinetic resolution of (R,S)-α-tetralol in a continuous flow packed bed microreactor. Temperature (°C)

40

50

55

60

65

70

Residence Time

C

ees

C

ees

C

ees

C

ees

C

ees

C

ees

1

31.37

33.18

33.20

48.88

35.17

53.16

37.75

59.56

42.27

72.06

44.59

79.28

2

35.25

41.11

36.28

56.09

39.27

63.50

39.56

64.34

48.98

94.67

48.84

94.15

3

38.76

49.15

40.72

67.76

44.94

80.34

46.22

84.70

50.00

100.00

50.00

100.00

4

45.56

67.66

47.28

88.63

48.44

92.57

48.85

94.18

50.00

100.00

50.00

100.00

5

47.85

74.97

48.15

91.81

49.96

98.41

50.00

100.00

50.00

100.00

50.00

100.00

(Min)

C- Conversion All the values mention in Table are on % basis. All the experiments were performed in triplicate to determine the experimental variability (within a standard deviation of ± 5%).



Time on stream study The operational stability and activity of the Novozym 435 in packed bed bioreactor were investigated by time on stream study (TOS) up to 35 h, during which continuous transesterification of (R,S)-α-tetralol was carried out using vinyl acetate as acyl donor. Novozym 435 was stable and active up to 35 h without much loss in catalytic activity and enantioselectivity (Figure 5). Novozym 435 undergoes gradual deactivation that might be occurring due to striping of water molecule which protects catalytic triad of the enzyme by reaction solvent used. 4,8,31 Indeed, optimum amount of water molecules or water activity is of great importance, in a continuous flow process. So experiments were done in 7 lots using fresh enzymes to see that activity remained practically the same.

Conversion

ees

eep

100 90

Conversion, ees,eep (%)

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80 70 60 50 40 30 20

10 0 5

10

15

20

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30

35

Time (h)

Figure 5. Time on stream study (Reaction Condition: (R,S)-α-tetralol- 3 mM, vinyl acetate- 9 mM, solvent-toluene, temperature- 65 °C).


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Regeneration and reusability of Novozym 435 in a continuous flow microreactor At optimal conditions, (R)-1,2,3,4-tetrahydro-1-naphthyl acetate yield was maintained at approximately 50%; this result indicated that CALB was stable and could be reused for at least 7 times without losing its catalytic activity (Figure 6). Total volume of 217.76 ml was processed in the 35 h through the catalyst bed. Thus the catalyst was utilized effectively at 0.045% w/v of total reaction volume at a fixed residence time of 3 min. There was a slightly decrease in conversion (up to 1%) after 35 h of the reaction. But during the continuous flow process, the essential amount of water needed to maintain native conformational and active form of Novozym 435 was consumed, which resulted in a slight decrease in activity.31 The Novozym 435 catalyst bed was removed after every 35 h of reaction, and rinsed with toluene saturated with water followed by drying at room temperature. Novozym 435 regained their catalytic activity during the washing process by absorption of water from toluene and, showed nearly constant conversion for next consecutive 7 cycles of 35 h. There was an unavoidable loss of Novozym 435 beads during these operations, almost 8-10% less than the previous batch.

55 50 45 40

Conversion (%)

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35 30 25 20 15 10 5 0 Fresh

1st Reuse

2nd Reuse

3rd Reuse

4th Reuse

5th Reuse

6th Reuse

7th Reuse

Reusability study

Figure 6. Reusability study (Reaction Condition: (R,S)-α-tetralol- 3 mM, vinyl acetate- 9 mM, solvent-toluene, temperature- 65 °C). 17 ACS Paragon Plus Environment


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Kinetic parameter in a packed bed microreactor The investigation of kinetics in packed bed columns for transesterification in continuous flow process can easily be described by modified form of the integrated Michaelis rate equation. It aims to find the effect of flow rate on the variability of apparent kinetic parameters (Km(app)). The data obtained from the flow rate and (R,S)-α-tetralol concentration studies were used for evaluation of the influence of flow rate on Km(app) for the packed bed CALB reactor systems. The experimental data were well fitted with the Lilly–Hornby model.34 Following is the modified form the integrated Michaelis rate equation:

f .[ A0 ] 

C  K m ( app ) .In(1  f ) Q

(1)

The values of Km(app) was obtained by plotting f [A0] against ln (1-f) (Figures S4-S7 supporting information file). Over the tested flow rate range, the Km(app) values for the packed bed microreactor were similar or slightly different (Table 2). In general, mass transfer rate and nature of enzyme are important factors which influences apparent Michaelis constant. As expected, Km(app) values were constant with subsequent increased flow rates, suggesting that the apparent kinetics of the packed bed microreactor is independent of mass transfer resistance. This dependence of Km can be explained on the basis of relationship between flow rates and diffusion layer dimensions (around the particle). There will be inversely proportional relationship between mass transfers of (R,S)-α-tetralol and thickness of layer.


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Table 2. Kinetic constants for Novozym 435 catalyzed kinetic resolution of (R,S)-α-tetralol using a continuous flow packed bed microreactor at 65 °C.

Km(aap) (mM)

R2

311

0.009

0.965

155.54

0.009

0.978

103.69

0.009

0.991

77.77

0.011

0.998

Flow rate (µL/min)

(All the experiments were performed in triplicate to determine the experimental variability (within a standard deviation of ± 5%).

The rate of reactions was determined by varying concentration of both substrate (supporting information) and Lineweaver-Burk plots (Figure 7) were made to investigate reaction mechanism. The equitation obtained with Michaelis-Menten mechanism is: 22

V  Vmax

[ A][ B] [ B] K m ( A ) [ B](1 )  K m ( B ) [ A][ A][ B] Ki ( B )

(2)

The values of kinetic parameters for above mechanism were estimated by nonlinear regression analysis using the software package Polymath 6.0. The values for kinetic parameters were as follows: Vmax, 3.2x10-5 mM/min; Km(A), 0.4 mM; Km(B), 2.2x10-3 mM and Ki(B) 51.4 mM. The proposed kinetic model was validated through the parity plot between experimental and calculated rates (Figure S8, supporting Information file).



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Figure 7. Lineweaver–Burk plot, 1/[Initial rate] vs. 1/[A] at different concentration of [B]. Conclusions In this work, we have demonstrated a novel efficient continuous process for kinetic resolution of (R,S)-α-tetralol by Novozym 435 in a packed bed bioreactor. To our information, so far only stirred tank batch reactors have been used for the same. There was a dramatic reduction in reaction time from the batch process to a packed-bed flow reactor. Additionally, it had better CALB activity compared with batch reactor. A conversion of ~50% with ~100% selectivity for (R)-α-tetralol (eep ≥ 99.99%) was obtained at 65 °C in 3 min, using Novozym 435 as the catalyst. The continuous flow microreactor approach was reusable 7 times with no loss of catalytic activity of Novozym 435. A ping-pong bi-bi model with inhibition by vinyl acetate was proposed for the transesterification process.

Acknowledgements MPK received SRF from UGC under its Meritorious Fellowship BSR program (CAS in Chemical Engineering Department). GDY received support from R.T. Moody Distinguished Professor Endowment and J. C. Bose National Fellowship of Department of Science and Technology, Government of India. The authors thank Novo Nordisk, Denmark for the gifts of an enzyme. 20 ACS Paragon Plus Environment

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Supporting Information Experimental section, Experimental Set-Up, Method of analysis, Results and Discussions (effect of various biocatalysts and kinetic parameters in a packed bed microreactor) Conflict of Interest Statement The authors declare no conflict of interest. Nomenclature [A]

concentration of (R,S)-α-tetralol, mM

[A0]

initial concentration of (R,S)-α-tetralol , mM

[B]

initial concentration of vinyl acetate, mM

[C]

reaction capacity of the continuous flow packed bed microreactor, mol/min

[F]

fraction of (R,S)-α-tetralol converted during the reaction

[Q]

flow rate, mL/min

K m(app)

apparent Michaelis constant, mM

Ki(B)

inhibition constant of vinyl acetate, mM

Km(A)

Michaelis constant of (R,S)-α-tetralol, mM

Km(B)

Michaelis constant of vinyl acetate, mM

M

mol/L

V

initial rate of the reaction, mM/min

Vmax

maximum rate of the reaction, mM/min

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TABLE OF CONTENTS (TOC) GRAPHIC

Kinetic resolution of (R,S)-α-tetralol by immobilized Candida antarctica lipase B : Comparison of packed bed over stirred tank batch bioreactor Manoj P. Kamble; Ganapati D. Yadav* Department of Chemical Engineering, Institute of Chemical Technology, Matunga, Mumbai 400019 India.*Corresponding author Tel.: +91-22-3361-1001; Fax: +91-22-3361-1002, +91-22-3361-1020 E-mail address: [email protected], [email protected]


Î±-Tetralol by Immobilized - ACS Publications - American Chemical

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