Facile modulation of enantioselectivity of thermophilic Geobacillus zalihae lipase by regulating hydrophobicity of its Q114 oxyanion

Enzyme and Microbial Technology 93–94 (2016) 174–181

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Facile modulation of enantioselectivity of thermophilic Geobacillus zalihae lipase by regulating hydrophobicity of its Q114 oxyanion Roswanira Abdul Wahab a,∗ , Mahiran Basri b,f,∗∗ , Raja Noor Zaliha Raja Abdul Rahman c,f,g , Abu Bakar Salleh d,f,g , Mohd Basyaruddin Abdul Rahman b,f , Thean Chor Leow e,f,g a

Dept. of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81310, Johor Bahru,Malaysia Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia c Department of Microbiology, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia d Department of Biochemistry, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia e Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia f Enzyme and Microbial Technology Research Center, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia g Institute of Bioscience, Universiti Putra Malaysia, 43400, UPM Serdang, Selangor, Malaysia b

a r t i c l e

i n f o

Article history: Received 26 April 2016 Received in revised form 20 August 2016 Accepted 30 August 2016 Available online 30 August 2016 Keywords: T1 lipase Geobacillus zalihae 2DSN Site-directed mutagenesis Enantioselectivity Oxyanion

a b s t r a c t Site-directed mutagenesis of the oxyanion-containing amino acid Q114 in the recombinant thermophilic T1 lipase previously isolated from Geobacillus zalihae was performed to elucidate its role in the enzyme’s enantioselectivity and reactivity. Substitution of Q114 with a hydrophobic methionine to yield mutant Q114M increased enantioselectivity (3.2-fold) and marginally improved reactivity (1.4-fold) of the lipase in catalysing esterification of ibuprofen with oleyl alcohol. The improved catalytic efficiency of Q114L was concomitant with reduced flexibility in the active site while the decreased enantioselectivity of Q114L could be directly attributed to diminished electrostatic repulsion of the substrate carboxylate ion that rendered partial loss in steric hindrance and thus enantioselectivity. The highest E-values for both Q114L (E-value 14.6) and Q114M (E-value 48.5) mutant lipases were attained at 50 ◦ C, after 12–16 h, with a molar ratio of oleyl alcohol to ibuprofen of 1.5:1 and at 2.0% (w/v) enzyme load without addition of molecular sieves. Pertinently, site-directed mutagenesis on the Q114 oxyanion of T1 resulted in improved enantioselectivity and such approach may be applicable to other lipases of the same family. We demonstrated that electrostatic repulsion phenomena could affect flexibility/rigidity of the enzyme-substrate complex, aspects vital for enzyme activity and enantioselectivity of T1. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Lipases (triacylglycerol hydrolases E.C.3.1.1.3) are industrially important enzymes that catalyze a broad range of reactions such as esterification, transesterification, organic synthesis in waterrestricted environments, and stereospecific hydrolysis of racemic esters [1,2]. Due to their wide industrial applications [1–3], many efforts to further optimize enzymatic properties of lipases through advances in enzyme immobilization on specific surfaces have been explored [4–7]. It has been indicated that enzymes immobilized

∗ Corresponding author. Tel.: +607 551 3613; fax: +607 556 6162. ∗∗ Corresponding author at: Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. E-mail addresses: [email protected], [email protected] (R. Abdul Wahab), [email protected] (M. Basri). http://dx.doi.org/10.1016/j.enzmictec.2016.08.020 0141-0229/© 2016 Elsevier Inc. All rights reserved.

onto suitable support matrices and confined in cavities exhibited higher activity than in their free forms [5,6,8,9]. This approach involves stabilization of the multimeric enzymes on surfaces by methods of entrapment, covalent immobilization and physical adsorption [5,10], thereby altering properties viz. selectivity and specificity [8] as well as operational stability and catalytic efficiency of the immobilized enzymes [11,12]. Aside from enzyme immobilization, molecular protein manipulation strategies viz. site-directed mutagenesis and directed evolution have been indicated [13–15]. In view of improving catalytic properties of current lipases viz. tolerance to solvents, high temperature and denaturants as well as to increase enantioselectivity [13–17] using molecular approaches, exploration into the effects of different amino acid substitutions in creating more efficient, enantioselective and catalytically competent lipases merit scientific relevance. We previously cloned the thermoalkalophilic extracellular lipase T1 isolated from Geobacillus zalihae into a

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prokaryotic system, E. coli BL21 DE3 pLys for high-yield expression of the recombinant enzyme [18–20]. The oxyanion hole of T1 is formed by the two residues Q114 and F16, as inferred from the crystal structure of the lipase BTL2 from Geobacillus thermocanulatus (96% sequence identity with T1). BTL2 residue F17 is highly conserved among bacterial lipases of 1.5 family, and the F17 side-chain contributes to shaping the oxyanion-binding pocket [21]. Considering that both residues Q114 and F16 of T1 are similarly located, therefore their mutation may substantially change the topology of the catalytic pocket [22], which can affect T1 enantioselectivity as well as expectedly less damaging and more tolerable [16]. Furthermore, review of literature has indicated that mutations within 11 Å of the active site may affect enantioselectivity more strongly than distant mutations [22–24]; at distances greater than 11 Å, E-values i.e., reflecting the enantioselectivity of a reaction are reduced by at least a factor of three [25]. In fact, significant increases in enantioselectivity following mutations close to the active site have been reported in horseradish peroxidase [26], phosphotriesterase [27], an esterase [23], and a lipase [28]. Since such mutations may lead to topological changes of the active site [22] and affect efficacy of lipase-catalyzed reactions [22–23,25]; their enhanced tolerance toward various organic solvents, denaturants, and high temperatures as well as enantioselectivity may therefore, be plausible. It has been described in literature that a combination of chemical modification [5] and genetic strategies [3,16,22] can be used to increase substrate affinity and decrease activation energy [22,29]. Chemical modification on enzymes using various reagents (cross-linkers) [28], for instance bifunctional reagents such ethylene-glycol-N-hydroxysuccinimide (EGNHS) and glutaraldehyde [14] and 1-ethyl-3-(3-di-methylaminopropyl)carbodiimide [28] have been described. Similarly, enzyme stabilization by in vivo methods such as methylation of certain amino acids and posttranslational modification of N-terminal cysteine [28] as well as in vitro modification using a non-crosslinking citraconic anhydride [14] and acetone treatment [15] have also been reported. Pertinently, the common objective of such approaches are to further increase the stability of enzymes by enhancement of their conformational stability. Since genetically tailoring properties of biocatalysts for industrial applications may provide a more expedient means to creating a more efficient enzyme and abolish downstream processing of reaction products altogether, such factor was considered in our efforts to improve the catalytic properties of T1 lipase. In our earlier work, we mutated the oxyanion Q114 of T1 lipase with 19 other amino acid homologs and it was discovered that mutations with more hydrophobic amino acids (leucine, methionine, valine and isoleucine) were generally well tolerated than substitutions with hydrophilic ones. The catalytic activity of the mutant lipases Q114L, Q114M, Q114V and Q114I, respectively, were found either comparable or improved over that of T1 lipase [29]. In this context, it is pertinent to indicate here that selection of lipases to be further investigated was based upon several satisfactory enhanced catalytic aspects seen in our previous work [29], as we found that Q114L and Q114M being the more catalytically efficient ones. Considering that mutational studies to improve enantioselectivity are yet to be explored for T1 and its mutants, and the fact that both Q114M and Q114L lipases mutants were catalytically more competent than T1 [29]; appropriate assessments with regards to these aspects may prove interesting. In this study, we substituted the oxyanion Q114 with the hydrophobic residue Met or Leu to modulate T1 lipase activity and enantiorecognition. Since T1 has not been investigated with respect to enantioselective biosynthetic reactions, we used the esterification of ibuprofen with oleyl alcohol as a model reaction to evaluate the enantioselectivity of lipase

175

T1 and its mutants Q114L and Q114M. The conditions evaluated included time, temperature, enzyme and desiccant loading. 2. Experiments The components for the growth media were purchased from Difco Laboratories (USA). The culture harboring the recombinant T1 lipase gene was obtained from the Enzyme and Microbial Technology Laboratory, Universiti Putra Malaysia. The BL21 (DE3) pLysS cultures were purchased from Invitrogen (Groningen, The Netherlands). The Quick-ChangeTM site-directed mutagenesis kit was purchased from Stratagene (La Jolla, USA). Agar plates were prepared by addition of tributyrin to Luria-Bertani agar (Oxoid, UK), and antibiotics, chloramphenicol (35 ␮g/mL), and ampicillin (50 ␮g/mL) were supplied by Amresco (Ohio, USA). The Bradford reagent was also from Amresco (Ohio, USA). Oleyl alcohol was purchased from Fluka (Switzerland). Ibuprofen was purchased from Sigma-Aldrich (USA), and HPLC grade isooctane was obtained from Merck (Germany). 2.1. Substitution of Q114 residue by site-directed mutagenesis Q114L and Q114 M lipase proteins were engineered using the QuickChange method. The PCR reaction mixture was prepared according to the manufacturer’s protocol. Primers were designed so that the sequence change was positioned in the center of the primer with approximately 10–15 bases on both sides. DNA was incubated with the restriction enzyme Dpn-1 at 37 ◦ C for 1 h to digest parental methylated DNA. The Dpn-1-digested DNA was introduced into competent E. coli BL21 (DE3) pLysS cells, and the cultures were grown on Luria Bertani–tributyrin agar media supplemented with ampicillin and chloramphenicol. Each mutation was confirmed by DNA sequencing. Glycerol stocks of T1, Q114L and Q114M were then prepared and kept at −80 ◦ C. 2.1.1. Preparation of stock and working culture The stock culture was prepared by inoculating a colony of lipase producing bacteria into aliquotes of Luria Bertani broth. The cultures were incubated for 12–16 h. Aliquots of Luria Bertani broth (800 ␮L) were added to sterile 15% glycerol (200 ␮L) in 1.5 mL Eppendorf tubes, vortexed, and stored at −80 ◦ C. For the working culture, one loop of culture from the glycerol stock was streaked onto tributyrin-nutrient agar-ampicillin plates and incubated for 12–16 h at 37 ◦ C. Only a single colony was chosen and inoculated aseptically into a 10 mL bottle of sterilized Luria Bertani broth and incubated for 12–16 h at 37 ◦ C, shaking at 200 rpm. The bacterial culture was pelleted down (10,000 rpm, 10 min) and at stored at −80 ◦ C. 2.1.2. Partial purification of T1, Q114L and Q114 lipases The culture containing recombinant T1, Q114L and Q114 M lipases (1000 mL) was harvested by centrifugation, re-suspended in 40 mL phosphate-buffered saline (PBS; pH 7.4) containing 5 mM dithioreitol and sonicated. The cell lysate was cleared by centrifugation at 12,000 × g for 30 min and filtered with a 0.45-␮m membrane filter (Sartorius). Glutathione-Sepharose HP (10 mL) was packed into an XK 16/20 column (GE Healthcare) and was equilibrated with 10 column volumes of PBS. The cleared cell lysate was loaded on the glutathione-Sepharose HP column at a flow rate of 0.25 mL/min. The column was washed with PBS until no protein was detected. The bound lipase was eluted with a buffer containing 100 mM Tris-HCl, 100 mM NaCl, and 0.33 mM CaCl2 , pH 8.0. The enzymecontaining fractions were determined by SDS-PAGE, pooled, and concentrated using Amicon Ultra-15 centrifugal filter (Millipore, Bedford, USA) and the activity of the enzyme was determined according to the method of Bradford (1976) [30]. The concentrated

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solution was subjected to gel filtration on Sephadex G25 in an XK16/20 column, pre-equilibrated with PBS. The solution was run in the same buffer at flow rate of 1 mL/min. The enzyme-containing fractions were collected and concentrated with an Amicon Ultra-15 centrifugal unit. The homogeneity of the partially purified protein was confirmed by SDS-PAGE. The protein was lyophilized and stored at −20 ◦ C. 2.2. Standard lipase assay and protein concentration Lipase activity was determined using the standard assay according to Leow et al. [18]. The protein content was determined by the Bradford method (Bradford) [30]. Amresco assay reagent and bovine serum albumin (BSA: Sigma, USA) were used as standards. The different concentrations of BSA were prepared by using a stock solution (0.1 mg BSA in 10 mL of deionized water and the absorbance was measured at 595 nm in a spectrophotometer (HITACHI U-3210) using preparations without BSA as blank.

2.3. Enantioselective esterification of (R,S)-Ibuprofen with oleyl alcohol The enantioselective esterification of (R,S)-ibuprofen with oleyl alcohol by lipases T1 and its mutants, Q114L and Q114 M, was performed in triplicates. Lyophilized lipase powder (4 mg), (R,S)ibuprofen (0.8252 g, 4 mmole) and oleyl alcohol (1.872 mL, 6 mmol) were stirred at 200 rpm in isooctane (8 mL) in screw-capped flasks (25 mL) in an oil bath at 50 ◦ C. At the indicated time points, samples (100 ␮L) were collected in triplicates and put on ice to stop the reaction. The mixtures were diluted appropriately for analysis by chiral HPLC. Additionally, 0.5 mL of the reaction mixture was withdrawn, mixed with hexane (2 mL), and titrated with NaOH (0.03 M) to determine the remaining (R,S)-ibuprofen acid.

2.4. Effect of reaction conditions We evaluated the effect of incubation time (4–24 h) on the enantioselectivity of the esterification reaction. The mixtures containing (R,S)-ibuprofen (0.8252 g, 4 mmol), oleyl alcohol (1.872 mL, 6 mmol) and lyophilized lipase (4 mg) were suspended in (8 mL) isooctane, continuously stirred at 200 rpm at 50 ◦ C, and reaction samples taken at the indicated time points, following chiral HPLC analysis to determine enantioselectivity. To study the effect of reaction temperature, the previously described reaction mixtures were incubated at temperatures ranging from 40 to 70 ◦ C for up to 24 h, and enantioselectivity was determined by chiral HPLC analysis. To examine the effect of enzyme loading on enantioselectivity, esterification was carried out in mixtures containing lyophilized lipase (4 mg), which ranged from 0.5 to 2.5% (w/v). Reactions without enzymes were used as the negative control.

2.5. Analysis and determination of ibuprofen esters The reaction mixture was analyzed on an Agilent 1200 HPLC equipped with a chiral phase column: (R,R)-Whelk-O1 chiral column (Regis Technologies, Morton Grove, USA) and an ELSD detector. A mixture of hexane, isopropanol, and acetic acid (98:2:0.5, v/v/v) was used as eluent. The flow rate was maintained at 0.75 mL/min and the column temperature was set at 25 ◦ C. Samples (10 ␮L) were injected using an autosampler and the respective retention times of [standard] (R)-ibuprofen acid, (S)-ibuprofen acid, (R)-ibuprofen ester and (S)-ibuprofen ester were 9.2, 10.5, 5.4, and 5.7 min.

The conversion (C) of ibuprofen was determined by measuring its decrease using titration with NaOH. The conversion of ibuprofen ester was calculated using the following Eq. (1): X(%) =

C0 − Ci × 100 C0

(1)

where X is the overall conversion, C0 is the initial amount of racemic ibuprofen (mM), and Ci is the amount (mM) of racemic ibuprofen at a particular reaction time. The enantiomeric excess of the product, eep%, and enantiomeric ratio, E-value, are described using Eqs. (2) and (3) below: E=

In[1 − (X/100)(1 + (eep /100))] In[1 − (X/100)(1 − (eep /100))]

eep (%) =

[R]ester − [S]ester × 100 [R]ester + [S]ester

(2) (3)

3. Results 3.1. Effect of incubation time on ester formation and enantioselectivity of T1 lipase and its mutants (R,S)-ibuprofen is an unnatural substrate for T1 lipase and its mutants, Q114L and Q114M, and their ability to enantioselectively catalyse the esterification of racemic ibuprofen has never been evaluated. The effect of time on ibuprofen conversion and enantiomeric excess (eep%) were monitored over a period of 24 h, and evaluated at hold values of 50 ◦ C, mmol of oleyl alcohol:ibuprofen (2:3), enzyme loading of 4 mg and stirred in 8 mL isooctane at 200 rpm. The effect of incubation time on ester formation and enantioselectivity by T1 lipase, Q114L, and Q114 M mutants is summarized in Table 1a. The time course esterification profile showed that ester formation was not observed in the initial 4 h, but increased from then on, with all three lipases achieving the highest amount of ibuprofen conversion at 12–16 h. T1 lipase and Q114 M converted 15.8% and 16.9% of the substrate after 12 h, whereas Q114L reached 12.9% conversion after 16 h. Q114 M exhibited a delayed 50% reduced conversion only after 20 h compared with T1 and Q114L, and the concentration of ester was constant thereafter at 7.9% (Fig. 1a). Activity of T1 and Q114L dropped after 12 h, whereas conversion decreased slightly for Q114L after 16 h, before reaching equilibrium after 20 h. The slow inactivation of Q114L was anticipated because its optimal working temperature of 80 ◦ C is much higher compared with Q114M at 60 ◦ C. Therefore, the delayed inactivation recorded for Q114L in this study too prove common, inferring higher structural stability of the lipase and its protein was not adversely affected under such prolong reaction time. It was indicated in Fig. 1b and c that lipases T1, Q114L, and Q114M showed similar e.ep% profiles with all three lipase preferentially catalyzed the formation of the R-enantiomer of the ibuprofen ester. All lipases attained maximum e.ep% prior to 12 h, followed by a decline in enantioselectivity. Q114M was the most enantioselective enzyme (e.ep > 90% at 12 h, E-value 24.6). Lipases T1 and Q114L achieved an e.ep of 80% and 70%, respectively, with highest E-values at 16 h (E-values 7.2 and 6.0, respectively). In view of the high conversions, we therefore chose 12–16 h as sampling times for the subsequent experiments. 3.2. Effect of temperature on ester formation and enantioselectivity of T1 lipase and its mutants Temperature is a crucial parameter that affects both enzyme activity and enantioselectivity [29,31,32]. Lowering the reaction temperature increases enantioselectivity of lipase-catalyzed resolution of racemates [33]. The effect of temperature on ibuprofen conversion and enantiomeric excess (eep%) were evaluated at hold

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177

Table 1 The effect of (a) incubation time and (b) reaction temperature on ester formation and percentage conversion (C%) in the esterification of racemic ibuprofen with oleyl alcohol catalyzed by T1 lipase, Q114L and Q114M mutants. The preferred product enantiomer formed in the reaction is also indicated.

Lipase T1 Q114L Q114M

(a) Incubation Time

(b) Reaction Temperature

Highest Conversion (%,) Time (h) 15.8 (12) 12.9 (12) 16.9 (16)

Preferred enantiomer

Highest Conversion (%), Temperature (◦ C) 20.3 (70) 18.8 (70) 18.7 (60)

R R R

Conversion (%)

a)

Preferredenantiomer R R R

25 20 15 T1

10

Q114L

5

Q114M

0 30

e.ep (%)

b)

40

50 60 o Temperature ( C)

70

80

100 75 50 25 0

c)

40

50

60 70 o Temperature ( C)

80

40

50

60 70 o Temperature ( C)

80

60

E-value

45 30 15 0

Fig. 1. The effect of incubation time monitored for 4–24 h in the enantioelective esterification of (R,S)-ibuprofen with oleyl alcohol catalyzed by T1 lipase, Q114L and Q114M mutants under hold values of 50 ◦ C, mmole of ibuprofen:oleyl alcohol (2:3), enzyme loading of 4 mg and stirred in 8 mL isooctane at 200 rpm.

values of 50 ◦ C, mmol of oleyl alcohol:ibuprofen (2:3), enzyme loading of 4 mg and stirred in 8 mL isooctane at 200 rpm and sampled at durations of 12–16 h. In this investigation, temperature was found to have a noteworthy effect on the activity of the lipase variants, observable in the increased lipase activity when the reaction temperature was elevated. The highest activity for Q114M was obtained at 60 ◦ C with 18.7% conversion under incubation time of 12–16 h (Fig. 2a). The highest conversion of ibuprofen by T1 and Q114L (20.3 and 18.8%, respectively) occurred at 70 ◦ C (Table 1). In this study, ester production was improved with increasing temperature from 40 to 70 ◦ C, except for Q114M which revealed its product formation declined from 70 ◦ C onwards. Conversion amount remained almost constant for T1, when temperature was increased from 60

Fig. 2. The effect of temperature monitored for 12–16 h in the enantioelective esterification of (R,S)-ibuprofen with oleyl alcohol catalyzed by T1 lipase, Q114L and Q114M mutants under hold values of 50 ◦ C, mmole of ibuprofen:oleyl alcohol (2:3), enzyme loading of 4 mg and stirred in 8 mL isooctane at 200 rpm.

to 70 ◦ C, whereas reaction product formation for Q114L continued to increase at temperatures above 70 ◦ C. This finding supports our previous report that the optimal temperature for Q114L is higher than 70 ◦ C to improve lipase activity [29]. Usually, over long periods of time, the ability of the lipase variants to distinguish between ibuprofen acids enantiomers decreases. Such phenomenon has been numerously reported attributable to the loss of enzyme activity and selectivity during the course of the reaction, and are associated to various causes [34]. The study found that E-values for T1 and Q114L decreased with temperature increase and all three lipases catalyzed formation of the R-enantiomer of the ibuprofen ester. The e.ep% and E-values of reactions with respect to temperature are depicted in Fig. 2b and c, respectively. An increase from 40 to 50 ◦ C caused

R. Abdul Wahab et al. / Enzyme and Microbial Technology 93–94 (2016) 174–181

e.ep% of T1 and Q114L to significantly decline and resulted in Evalues ranging from 10.9 to 6.0 for the latter, and 5.8–2.0 for the former. The e.ep% of Q114M remained fairly constant (91.8–94.2%) at 40–50 ◦ C, whereas at 50–70 ◦ C e.ep% declined sharply from 91.8 to 57.9%. At 60–70 ◦ C, Q114L, Q114M and T1 almost completely lost enantioselectivity (E = 1). Q114M showed significantly reduced Evalues at 50–70 ◦ C, T1 and Q114L at 60 ◦ C to 70 ◦ C. Based on these observations, 50 ◦ C was maintained as reaction temperature for the following experiments since at this temperature, enzyme activity, enantioselectivity and reaction time yielded acceptable E-values.

a) Conversion (%)

178

20 T1

15

Q114L Q114M

10 5 0 0

0.5

1 1.5 2 2.5 Enzyme loading (w/v%)

3

0

0.5 1 1.5 2 2.5 Enzyme loading (w/v%)

3

0

0.5

3.3. Effect of enzyme loading on ester formation and enantioselectivity of T1 lipase and its mutants

3.4. Effect of the presence of desiccant on ester formation and enantioselectivity of T1 lipase and its mutants Desiccants improve the enantioselectivity of reactions enzymes by removing reaction-generated water that hydrolyzes the preferentially formed product. They thus improve e.ep% of enantioselective reactions [32,36]. The effect of the addition of desiccant on ibuprofen conversion and enantiomeric excess (eep%) were evaluated at constant conditions of 50 ◦ C, mmol of oleyl alcohol:ibuprofen (2:3), enzyme loading of 2 2.0% (w/v), stirred at 200 rpm in 8 mL isooctane and sampled at durations of 12–16 h (Table 2b). Surprisingly, the amount of ibuprofen conversion was highest in mixtures without added molecular sieves (Fig. 4a). Lipases T1, Q114L, and Q114M showed highest substrate con-

e.ep (%)

b) 100 80 60 40

c) E value

The optimum enzyme amount is crucial when using short reaction times, to ensure a high-yield and cost-effective process [35]. Hence, a proper balance between high yield and good enantioselectivity should be established. The profile for the effect of enzyme loading on ibuprofen conversion and enantiomeric excess (eep%) were obtained at constant values of 50 ◦ C, mmol of oleyl alcohol:ibuprofen (2:3), enzyme loading of 4 mg and stirred in 8 mL isooctane at 200 rpm and sampling duration between 12 and 16 h. Enzyme loading of 2.0–2.5% (w/v) resulted in highest ester conversion for T1, Q114L, and Q114 M corresponding to 15.8, 10.9, and 18.9%, respectively (Table 2). The lowest conversion observable for all three lipases occurred with the lowest enzyme loading 0.5% (w/v) (Fig. 3a) No ester formation was detected in the absence of enzyme (data not shown). The amount of ester conversion for Q114M continued to increase, even when the enzyme load exceeded 2.0% (w/v) and remained constant when enzyme load was increased from 2.0 to 2.5% (w/v), which suggests that increasing the enzyme amount will not improve yield. In this study, it can be seen that substrate diffusion inhibition for lipases T1 and Q114L will be increased when the enzyme load exceeded 2.0% (w/v) beyond which ester conversion began to decline. In our investigation, lipase selectivity was notably enhanced with increased enzyme loading. The effects of enzyme loading on enantioselectivity of T1, Q114L, and Q114M lipases are shown in Fig. 3b and 3c. This effect was most pronounced in Q114M, and observed to a lesser degree in T1 and Q114L. The highest eep% for T1 and Q114L (78 and 68%, respectively) were obtained at 2.0% enzyme load (w/v), with E-values of 7.8 and 5.2, respectively. The E-value of Q114M was highest at an enzyme load of 2.5% (w/v) being 48.5. E.ep% of T1 and Q114L declined, however, when enzyme loading increased 2.0% (w/v). Lowest E-values for all lipases (E values 3.9–18) were recorded at 0.5% enzyme load (w/v) and all three lipases preferentially catalyzed formation of the R-ibuprofen ester. Considering the high ester conversions and enantioselectivities attained in this examination, the subsequent reactions for lipases T1, Q114L and Q114M reported in this study were performed at an enzyme load of 2.0% (w/v).

60 45 30 15 0 1 1.5 2 2.5 Enzyme loading (w/v%)

3

Fig. 3. The effect of enzyme loading monitored between 12–16 h in the enantioelective esterification of (R,S)-ibuprofen with oleyl alcohol catalyzed by T1 lipase, Q114L and Q114M mutants under hold values of 50 ◦ C, mmole of ibuprofen:oleyl alcohol (2:3), stirred in 8 mL isooctane at 200 rpm.

version at 12 h (15.2%, 12.0%, and 18.1%, respectively) (Fig. 4a). However, with increasing amounts of molecular sieves added into the reaction mixture resulted in decline in production of the ester, consequently the eep% and E values of in T1, Q114L, and Q114M also deteriorated from 16.2 to 14.0, 13.9 to12.5, and 52.5–33.1, respectively (Fig. 4b and c). 4. Discussions The initial ascending trend of ester conversion may be attributed to the increased in the overall kinetic energy when the temperature of the system is elevated. Such aspect would promote effective collisions between enzyme and substrate molecules, hence yielding higher concentration of the ester product [37,38]. By elevating the reaction temperature, the equilibrium constant would be further increased [39], conferring other benefits such enhancing the mass transfer between the catalyst and reactant [40], solubility of the reactants [41] in addition to decreasing the viscosity of the medium [42]. Such observation is consistent with previous reports that suggested increased kinetic energy of the system promotes effective collisions between enzyme and substrate molecules. On the other hand, the observed susceptibility of T1, Q114L, and Q114M to undergo denaturation when the reaction temperature is elevated

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179

Table 2 The effect of (a) enzyme loading and (b) molar ratio (olely alcohol:ibuprofen) on ester formation and percentage conversion (C%) in the esterification of racemic ibuprofen with oleyl alcohol catalyzed by T1 lipase, Q114L and Q114M mutants. The preferred product enantiomer formed in the reaction is also indicated. (a) Enzyme loading

(b) Molar ratio (olely alcohol:ibuprofen)

Lipase

Highest Conversion% (w/v%)

Preferred enantiomer

T1 Q114L Q114M

12.8 (2) 9.5 (2) 18.9 (2.5)

R R R

a) Conversion (%)

20

T1 Q114L Q114M

15 10 5 0 0

b)

1 2 3 Amount of molecular sieves (mg)

4

100

e.ep (%)

95 90 85 80 75 70 0

1 2 3 4 Amount of molecular sieves (mg)

0

1 2 3 4 Amount of molecular sieves (mg)

c) 60

E value

45 30 15 0

Fig. 4. The effect of the presence of desiccant monitored between 12–16 h in the enantioelective esterification of (R,S)-ibuprofen with oleyl alcohol catalyzed by T1 lipase, Q114L and Q114M mutants under hold values of 50 ◦ C, mmole of ibuprofen:oleyl alcohol (2:3), enzyme loading of 2.0% (w/v) and stirred in 8 mL isooctane at 200 rpm.

and performed under prolong incubation times can be directly associated with such conditions that tend to aggravate protein denaturation [43–45]. Such circumstances increase propensity of temperature-induced excessive unravelling of the protein tertiary [3] attributable to excessive vibrations and movement of the lipase molecule [45] which consequently leads to the loss of their catalytically competent forms [3]. Likewise, the significantly reduced conversion for Q114M at 70 ◦ C compared with 60 ◦ C is most likely owing to increased Q114M lipase deactivation at the higher temperature which has exceeded its reported optimal temperature [29]. Apart from deformation of the lipase structure, alcohols have

Highest C% (olely alcohol:ibuprofen) 12.2 (2) 11.8 (2) 14.5 (2)

Preferred enantiomer

R R R

been described to act as terminal inhibitors and their inhibitory effects on the lipases can be intensified at high temperatures owing to increased solubility of the alcohol in the medium [46,47]. In contrary, Q114L activity was still increasing beyond 70 ◦ C which could be ascribed to Q114L being more thermostable. The lipase is yet to adopt its catalytically most active conformation, and thus, not having reached its full catalytic potential. A noteworthy aspect to mention here, the excess build-up of water is prevalent under conditions of high temperature and long incubation time. In this context, it is vital that water an unwanted byproduct of esterification reaction is removed from the reaction as it leads to the hydrolysis of the ester. Therefore, the addition of molecular sieves in this study was expected to shift the equilibrium toward the formation of product [46,48]. The molecular sieves function to absorb the water by-product liberated in the esterification reaction and avert the counterproductive hydrolysis of ibuprofen ester back into its starting components. Interestingly, their addition did not improve the percentage conversion of the ester, possibly due to increased bulk density of the reaction medium that interfered with the effective collisions between the catalysts and the substrates. We revealed that T1, Q114L, and Q114M lipase selectivity was strongly dependent on incubation time, and that their enantioselectivity varied over time. This is because when enzyme deactivation is time-dependent, deterioration of enantioselectivity is usually associated with thermal denaturation [49]. A matter of fact, stability of an enzyme, as any kind of catalyst, is important for its application in industry [50]. Thermal deactivation is one the few known shortcomings encountered when enzymes in their free forms [50] are used for catalyzing synthetic processes. While the selectivity of the lipases observable here was increased to a certain extent with the increase in incubation time, presumably due to longer contact time that favorably promoted effective collisions between the enzyme and substrate molecules [42], the undesirable effect of temperature on the active structure of the lipases was also elevated [11,42]. Aside from thermally induced structural alterations, the free forms of lipases T1, Q114L, and Q114M, the enzymes were found susceptible to the deactivating effects of substrates over time [11], indicative of their low operational stability that may restrict their biotechnological applicability [5]. A possible observation for this fact is the hypothesis that is consistent with increased unravelling the lipase protein structure due to gradual loss of internal hydrogen bonds or weak van der Waals forces that stabilise its structure, following longer incubation time at elevated temperatures. Subsequently, the loosened protein strands on the surface of the lipase are exposed to the inherently denaturing high concentrations of the acid and alcohol substrates as well as solvent present in the mixture, further deactivating the lipase. In this context, the decline in selectivity of the T1, Q114L, and Q114M lipases seen here with the increase in reaction time was amply justifiable. Moreover, considering that the enantioselectivity of a reaction (Evalue) is dependent on the conversion rate and enantiomeric excess [35], therefore a change in e.ep% has a more profound effect on the E-value than a change in conversion rate. Therefore, while this present study concurs that the use of free forms of the T1, Q114L

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and Q114M lipases in catalyzing enantioselective esterification of (R,S)-ibuprofen with oleyl alcohol as feasible; their immobilization onto appropriate supports could prove helpful in conferring higher selectivity as well as stability [4,5,10] in the lipases. Hence, further investigations into identifying a suitable immobilization protocol to improve selectivity of these lipases may constitute an interesting future study. In this study, enantioselectivity of ibuprofen esterification by T1 lipase significantly decreased with increasing reaction temperature. Similar dependences have been reported for other lipase-catalyzed hydrolysis of esters of secondary alcohols, enol esters of ketones, or for esterification of carboxylic acids [51]. A possible reason for the deteriorating enantioselectivity of T1, Q114L, and Q114M lipase was due to protein unfolding and denaturation, caused by the breakdown of weak ionic and hydrogen bonds, particularly the within the catalytic site [52]. Moreover, as mutations of the oxyanion Q114 of T1 proceeded with substitution with moderately hydrophobic amino acids (leucine and methionine), conceivable changes in enzyme conformations can be translated also into stabilization/destabilization of the three dimensional structures of the lipases. It must be emphasized that enantioselectivity of the mutated lipases is determined by a stabilized three-dimensional structure in the active site [52]. However, in lipases T1, Q114L and Q114M, at elevated reaction temperatures regardless of residues substituted into the oxyanion Q114–resulting in over flexibility within the active site [44], occurred on relatively large surface area and it could compromise ability of the lipases to discriminate enantiomers interacting with the active site. The findings are in agreement with reports of decline in lipase selectivity owing to deformation of the active site [44] and loss of catalytically competent conformation [52,53] under elevated temperatures. A noteworthy point to highlight here, reactions performed utilizing higher enzyme loading could switch from being rate limited and to mass transfer limited, a well-known counterproductive phenomenon in any esterification reaction that results in the lowering of lipase activity [54]. Diffusion limitations partially contributed to the differences in enantioselectivity as a result of a poorer access of the substrates to the active site of the lipases. Pertinently, it was clear that substitution of Q114 with Met positively affected enantioselectivity in T1. It is believed that Met reduced the flexibility at the bottom of the catalytic pocket and thus possibly stabilized its topology. A previous study suggested that polar residues can induce localized amino acids flexibility and disorder [55], but can also impart structural specificity that supports enzyme selectivity [56]. Under certain circumstances, however, overly flexible residues for instance, large polar or charged hydrophilic residues such as glutamic acid, glutamine, arginine and lysine, as well as serine, at the bottom of the catalytic pocket may compromise enzyme enantioselectivity. Since hydrophilic amino acids have been reported to contribute substantially to the apparent flexibility in surrounding are of the mutation site [29,57], modulation of substrate enantiorecognition is possible by adopting a slightly more rigid catalytic pocket that allows access of only a single enantiomer. In this regard, substituting the hydrophilic glutamine in T1 with the more hydrophobic methionine could have reduced such flexibility and the catalytic pocket of Q114M can better retain its active form under elevated temperatures or prolong reaction duration. This is a very important technological premise since transient hydrophobicity of the oxyanion-114 might be essential until the maximum enantioselectivity is achieved. On the contrary, Leu introduction adversely affected T1 lipase enantioselectivity, which could be ascribed to decreased electrostatic repulsion of the carboxylate substrate ion [58]. Reduction of electrostatic hindrance consequently results in poorer enantioselectivity of the Q114L lipase mutant [55]. In this perspective, the

changes of ester formation and enantioselectivity in the lipase variants observed here may have originated from alterations in the mobility of residues and hydrogen bond networks between amino acid residues surrounding the catalytic pocket, as well as from different spatial degrees of freedom in this pocket. Conclusively, the present study shows that genetic tailoring of T1 lipase by substituting the hydrophilic Q114 with methionine (Q114M) may prove to be a practical means to producing a more efficient enzyme as compared to the wildtype. Furthermore, the conventional method of bioprospecting for microbes producing enantioselective lipases can be time consuming and incurring high costs. Hence, by developing new mutant lipases from existing ones using the approach of rational site-directed mutagenesis in the region of the active site may present a facile and rapid means to engineering effective reaction-specific enzymes.

Acknowledgments This study was supported by grants, Green Chemistry from the Ministry of Science, Technology and Environment, Malaysia (Grant no. 5487729) and Fundamental Research Grant Scheme from the Ministry of Education (Q.J130000.7826.4F649), respectively.

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