- Email: [email protected]
High pressure enhances activity and selectivity of Candida rugosa lipase immobilized onto silica nanoparticles in organic solvent
Accepted Manuscript Title: High pressure enhances activity and selectivity of Candida rugosa lipase immobilized onto silica nanoparticles in organic solvent Author: Zhengyang Zhao Daniela Herbst Bernd Niemeyer Lizhong He PII: DOI: Reference:
S0960-3085(15)00108-X http://dx.doi.org/doi:10.1016/j.fbp.2015.08.006 FBP 640
To appear in:
Food and Bioproducts Processing
Received date: Revised date: Accepted date:
10-6-2015 20-8-2015 21-8-2015
Please cite this article as: Zhao, Z., Herbst, D., Niemeyer, B., He, L.,High pressure enhances activity and selectivity of Candida rugosa lipase immobilized onto silica nanoparticles in organic solvent, Food and Bioproducts Processing (2015), http://dx.doi.org/10.1016/j.fbp.2015.08.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Short Communication
Title: High pressure enhances activity and selectivity of Candida rugosa lipase
ip t
immobilized onto silica nanoparticles in organic solvent
Zhengyang Zhao1, †
cr
Daniela Herbst2, †
us
Bernd Niemeyer2,* Lizhong He1,*
Department of Chemical Engineering, Monash University, Clayton, VIC3800, Australia
2
Institute of Thermodynamics, Helmut-Schmidt-University/University of Federal Armed
an
1
M
Forces Hamburg, Holstenhofweg 85, Hamburg, D-22043, Germany
* Correspondence: Bernd
Niemeyer,
Institute
of
Thermodynamics,
Helmut-Schmidt-
Ac ce p
Professor
d
These authors contributed equally to this work.
te
†
University/University of the Federal Armed Forces, Hamburg, Holstenhofweg 85, D-22043 Hamburg, Germany;
Email: [email protected] and Dr Lizhong He, Department of Chemical Engineering, Monash University, Clayton, VIC3800, Australia.
E-mail: [email protected]
Keywords: chiral access, enzyme immobilization, high pressure, lipase, silica nanoparticle
1
Page 1 of 19
Abbreviations:aw, water activity; CRL, Candida rugosa lipase; I-CRL, immobilized CRL; PP, 1-phenylpropan-2-ol; VA, vinyl acetate; SNP, silica nanoparticle; (R)-PPA, (R)-1phenylpropan-2-yl acetate; (S)-PPA, (S)-1-phenylpropan-2-yl acetate; TEOS, Tetraethyl
Ac ce p
te
d
M
an
us
cr
ip t
orthosilicate
2
Page 2 of 19
Abstract
High hydrostatic pressure has been increasingly utilized to improve functions of enzymes, and most of such studies are currently focused on free enzymes in aqueous solution or
ip t
organic solvent. In this work, Candida rugosa lipase (CRL) was immobilized onto silica nanoparticles and its activity and enantioselectivity in organic solvent were evaluated at high
cr
pressures under different water activities. The application of high hydrostatic pressures (50-
us
200 MPa) led to improved activities of immobilized CRL for transesterification of (R)-1phenylpropan-2-ol with vinyl acetate by 4-6 folds. Additionally the immobilization of CRL
an
resulted in a significant change of selectivities, shifting the of enantiomeric excess from the (R)- towards (S)-1-phenylpropan-2-yl acetate product at atmospheric pressure. The
M
application of high pressures led to either enantiomeric excess toward (R)-1-phenylpropan-2yl or no enantiomeric selectivity, depending on the water activities in the organic solvent and
d
the level of pressures. The interesting behaviour of immobilized CRL under high pressures
te
offers new opportunities to modulate enzyme functions through combination of high
Ac ce p
pressures and enzyme immobilization.
3
Page 3 of 19
1 Introduction Enzymatic synthesis has been increasingly applied in fine chemical industry to produce
ip t
enantiomeric pharmaceuticals (Stinson, 1998). Various chiral intermediates, including amino acids (Drauz, 1997), peptides (Gardossi et al., 1991; Sicard and Brennan, 2013), esters
cr
(Boland et al., 1991) and penicillin (Liu et al., 2011; Volpato et al., 2010) have been manufactured via enzymatic synthesis route. Sustainable application of enzymes for such
us
productions require enzymes with high stability and selectivity under industrial processing
an
conditions (Bornscheuer et al., 2012), and ideally, enzymes should be re-useable for a long period in order to keep the cost low (Mateo et al., 2007). Protein engineering and enzyme
M
immobilization are two classical approaches to engineer enzymes with these desired features (Bornscheuer et al., 2012; Schmid et al., 2001). Alternatively, the performance of enzymes
d
can also be enhanced through altering the reaction conditions such as utilizing organic
te
solvents as reaction medium (Klibanov, 2001), or employing high hydrostatic pressures
Ac ce p
(Eisenmenger and Reyes-De-Corcuera, 2009b).
Recently high hydrostatic pressure processes have shown potentials in enhancing performance of enzymes by increasing their stability and selectivity (Eisenmenger and Reyes-De-Corcuera, 2009b; Mozhaev et al., 1996b; Herbst et al., 2012; Herbst et al., 2014). Although it is well known many proteins denature upon exposure to high hydrostatic pressures (Heremans, 1982; Mozhaev et al., 1996a), more than 25 enzymes have been reported with improved performances under extreme pressures (Eisenmenger and Reyes-DeCorcuera, 2009b). Despite these progresses, applications of bio-catalysis under high pressures are still limited, in part, due to poor knowledge of enzymes at extreme conditions (Berheide et al., 2010). Particularly, there is little understanding about how high pressures affect the
4
Page 4 of 19
function of enzymes in their immobilized form that is important for re-use of enzymes in
ip t
industrial application (Eisenmenger and Reyes-De-Corcuera, 2009a).
Lipase catalysis in organic solvents is frequently used for enantiomeric products synthesis
cr
(Klibanov, 2001). Recently, high pressure has shown the ability to alter properties of free lipase in solvent including activity, stability and enantioselectivity. (Eisenmenger and Reyes-
us
De-Corcuera, 2009a, b; Mozhaev et al., 1996b; Herbst et al., 2012; Herbst et al., 2014). The
an
work by Herbst et al (2012, 2014) has evaluated high pressure effects on Candida rugosa lipase (CRL), one of the most used lipase for industrial application (e.g. transesterification
M
reactions). However, the effect of high pressure on lipase under its immobilized form has not yet been studied. Thus, in this work, we aim to investigate the effect of high hydrostatic
d
pressure on immobilized CRL in organic solvent at different water activities. Solid silica
te
nanoparticle was selected as carrier for CRL immobilization, based on the following considerations: 1) solid silica nanoparticles are stable under high pressures; 2) they have low
Ac ce p
mass transfer resistance due to their small size (Zaraki et al., 2015), and; 3) convenient surface functionalization is available for immobilization (Zhao et al., 2013a). Activity and enantioselectivity of immobilized CRL were evaluated and compared with free CRL under different pressures and water activities (a w), using the established model reaction of the asymmetric transesterification of 1-phenylpropan-2-ol (PP) with vinyl acetate (VA) (Herbst et al 2012, 2014).
2 Experimental 2.1 Materials
5
Page 5 of 19
Lipase from Candida rugosa in crude powder (ca. 2.5 % enzyme by protein mass) was a gift from Amano Enzyme Inc. Nagoya, Japan. All chemicals and solvents were of at least analytical grade or higher. Chemicals for particle preparation and immobilization including
ip t
tetraethyl orthosilicate (TEOS) and 3-glycidoxypropyltrimethoxysilane were purchased from Sigma-Aldrich Sydney, Australia. Chemicals for enzymatic reaction including vinyl acetate
cr
(VA), racemic 1-phenylethanol (PE), racemic 1-phenylpropan-2-ol (PP) and anhydrous hexane were obtained from Sigma-Aldrich Munich, Germany. Bradford assay reagent for
an
us
protein assay was obtained from Sigma-Aldrich Munich, Germany.
2.2 Preparation of SNP and lipase immobilization
M
SNP with diameters around 250 nm were synthesized using the Stoeber method (Stoeber et al., 1968). The synthesized SNP were functionalized with epoxy groups using 3-
d
glycidoxypropyltrimethoxysilane followed by immobilization of CRL using the method
te
reported previously (Zhao et al., 2013b). Then, the particles were dried in fume hood before
Ac ce p
controlling their water activity. (Detailed information is given in the Supporting Information).
2.3 CRL-catalyzed transesterification at ambient and high pressures In a typical experiment, 18.75 mL hexane and 6.25 mL VA with certain water activity (aw = 0.35 or 0.7, see control of water activity in the Supporting Information) were mixed with PP at a final concentration of 0.41 M. After stirring the reaction media for 5 min at 300 rpm to dissolve PP, 150 mg crude CRL powder or immobilized CRL with the equivalent CRL mass was added. For the reactions at atmospheric pressure the lipase-substrate-mixture was transferred into sealed glass vials which were kept in an oven to control the reaction temperature. For reactions at high-pressures the lipase-substrate-mixture was transferred into a leak-proof high pressure vessel using the set-up reported (Herbst et al., 2014) previously.
6
Page 6 of 19
Three pressure levels (50, 100 and 200 MPa) were applied for the experiments. The reaction solutions, both under atmospheric and high pressures, were continuously mixed with a stirrer bar at 300 rpm by a magnetic stirrer. The reaction was carried out for 48 hours at 35 °C
ip t
before samples were taken for analysis. All the experiments were conducted two times at least, with error bar which represent the standard deviation labelled on the data points of
us
cr
figures.
2.4 Analytical methods
an
The previous reported method was employed for the analysis of the (Herbst et al., 2014) product. Briefly, the product concentration of samples after the reaction were analyzed by gas
M
chromatography GC-FID (Clarus 500, Perkin, Elmer Rodgau, Germany) utilizing a chiral Hydrodex-ß-PM column (Macherey&Nagel, Dueren, Germany; column length 50 m; column
d
diameter 0.25 mm: condition: oven temperature from 80 °C (initial time 1min) to 120 °C for
te
17 min with a heating rate of 20 °C/min, split ratio 1:20, He as carrier gas) to determine the specific activity and the enantiomeric excess . The enantiomeric excess eeR [%] was
Ac ce p
calculated by
eeR
c R cS * 100 c R cS
Where c R is the concentration of (R)-1-phenylpropan-2-yl acetate ((R)-PPA) and cS is the concentration of (S)-1-phenylpropan-2-yl acetate ((S)-PPA). The enzymatic activity was calculated by the equation using previously reported method (Herbst et al., 2014) as below:
A
cp V t mE
Where cp is the concentration of the product, V is the reaction volume, t is the reaction time, and mE is mass of the enzyme
7
Page 7 of 19
3 Results and discussion 3.1 The enantioselectivity of immobilized CRL The amount of CRL immobilized onto SNP was estimated to be 19.2 mg g-1 silica, based on
ip t
the mass balance of CRL in the supernatant before and after immobilization. In our previous studies, aw of the reaction medium was shown to be an important parameter influencing the
cr
CRL performance (Herbst et al., 2014; Herbst et al., 2012). The functions of free and immobilized CRL are thus compared at two different water activities (aw = 0.35 and 0.7) in
an
us
this work.
Interestingly, the immobilization of CRL leads to a significant change of the CRL
M
enantioselectivity for transesterification of PP with VA (Figure 1A). Under atmospheric pressure, free CRL has only marginal enantioselectivity (Figure 1B and C). At the water
d
activity of 0.35, free CRL slightly favoured the (S)-PPA product (eeR=-4.3 %) (Figure 1B),
te
and the eeR value was 4.1 % at a water activity of 0.7 (Figure 1C). In contrast, the immobilized CRL (I-CRL) strongly favoured the S directed reaction at atmospheric pressure.
Ac ce p
The eeR values were -47.9 % (Figure 1B), and -69.6 % (Figure 1C) at water activities of 0.35, and 0.7, respectively. The possible reason for the change of enantiomeric selectivity by the CRL immobilization is that covalent binding onto the SNP introduces distortion of active site of the enzyme which may alter its catalytic properties, as discussed in a recent review by Mateo et al (2007).
At the water activity of 0.35, high pressures led to a shift of the enantiomeric excess towards (R)-PPA for both of free CRL, and immobilized CRL (Figure 1B). For free CRL, the eeR value was increased from 5.2 % at 50 MP to 13.2 % at 100 MPa, and finally to 21.1 % at 200 MPa, respectively. I-CRL provided little enantiomeric selectivity at 50 MPa (eeR = 0.18 %)
8
Page 8 of 19
and 100 MPa (eeR = 4.0 %), but had a higher eeR value of 13.6 % at 200 MPa. At water activity of 0.7, free CRL showed a similar trend of increased eeR at high pressures, and the value (eeR = 25.0 %) at 200 MPa was slightly higher than that (eeR = 21.4 %) at aw 0.35
ip t
(Figure 1C). However, at aw 0.7, the immobilized CRL exhibited little enantiomeric selectivity with marginal eeR values between -3.6 % and 2.6 % from 50 MPa to 200 MPa, in
cr
contrast to its strong preference for (S)-PPA (eeR = -69.6 %) at atmospheric pressure (Figure 1C). The results presented in Figure 1B and 1C have shown the complexity of the
us
enantiomeric selectivity of CRL, which is determined by the interplay of the state of the
an
enzyme (free or immobilized), pressures and water activities. Such complexity may be due to collective effects of these parameters on enzyme conformation (Boonyaratanakornkit et al.,
M
2002; Mateo et al., 2007; Mozhaev et al., 1996b).
d
3.2 High pressure enhances the activity of the immobilized CRL
te
The immobilization of CRL onto silica nanoparticles led to a significant decrease of CRL activities at atmospheric pressure. While free CRL had a specific activity of 95.9 U/g and
Ac ce p
25.6 U/g at the water activity of 0.7, and 0.35, respectively, immobilized CRL had a significantly lower specific activity of 2.37 U/g enzyme (aw = 0.7) and 1.56 U/g enzyme (aw = 0.35). Immobilization of CRL was realized by the reaction of the epoxy functional groups on SNP with primary amines (amine of lysine residues or amine at N-terminus) of the CRL. This immobilization condition is expected to result in multiple covalent bonds and rigid structure of enzymes that can decrease enzyme activity (Mateo et al., 2007). Moreover, exposing the CRL to air under room temperature in the process of immobilization also decreased the CRL activity. The application of high pressures, however, was efficient to increase the activities of the immobilized CRL as shown in Figure 2. At a water activity of 0.35, the relative activity of the immobilized CRL at 50 MPa was 560 % of that at atmospheric pressure (Figure 2A). The
9
Page 9 of 19
value decreased slightly with the increase of the pressure (460 % at 100 MPa and 330 % at 200 MPa), but was still significant higher than that at the atmospheric pressure. The relative activities of immobilized CRL at a water activity of 0.70 % shown a similar trend (Figure 2B)
ip t
although there was less improvement (440 % at 50 MPa) compared to those at 0.35 water activity. It is generally suggested that both, the changes of enzyme structure and alteration of
cr
the physical properties of the substrate and solvent by high pressures contribute to the improvement of the enzyme activities (Eisenmenger and Reyes-De-Corcuera, 2009b). Given
us
that immobilized CRL and free CRL responded differently to high pressures at the same
an
reaction conditions in this case, it was more likely that structural changes of enzymes played
M
a determined role in improving activities of immobilized CRL.
3.3 Particle stability under high pressures
d
Importantly, the SNP were highly stable under all tested high pressures. As shown in Figure
te
3, SNP remained their uniform spherical shapes after the reaction at high pressures. Given that the immobilized CRL has improved its activity at high pressures, the enzyme was likely
Ac ce p
to retain its intact structure as well. The SNP-based immobilization system was thus sufficiently robust for further exploring of advantages brought by high pressures in future.
4 Conclusions
In summary, this work demonstrated that high hydrostatic pressures could significantly improve activities as well as altering selectivities of the lipase immobilized onto silica nanoparticles. The asymmetric reaction catalyzed by CRL was significantly shifted by the immobilization of the CRL towards an excess of the (S)-enantiomer at atmospheric pressure towards the (R)-enantiomer of the product under high pressure. The enantiomeric excess value at high pressures depended on the state of enzyme (immobilized or free), water
10
Page 10 of 19
activities and the level of high pressures. This complexity of the enzyme’s enantiomeric selectivity may offer new opportunities to modulate enzyme functions through combination of high pressures and enzyme immobilization and warrants a systematic study of immobilized
ip t
enzymes at high pressures in future.
cr
Acknowledgement
The authors acknowledge travel support provide by Go8 - Germany Joint Research
us
Cooperation Scheme, a partnership between the Group of Eight and the German Academic
an
Exchange Service (DAAD). Z.Z. acknowledges New Horizons Joint PhD Scholarship Program provided by Monash University and Commonwealth Scientific and Industrial
M
Research Organisation (CSIRO). D.H. acknowledges the financial support by the Deutsche Forschungsgemeinschaft (DFG) for the joint project “Enzyme-catalyzed synthesis of chiral
d
substances under high pressures”. The authors thank Amano Enyzme Inc. Nagoya, Japan for
Ac ce p
Conflict of interest
te
free delivery of the CRL. The authors thank Oliver Wenzel for assistance in GC-analysis.
The authors declare no financial or commercial conflict of interest.
11
Page 11 of 19
5 References
Ac ce p
te
d
M
an
us
cr
ip t
Berheide, M., Peper, S., Kara, S., Long, W.S., Schenkel, S., Pohl, M., Niemeyer, B., Liese, A., 2010. Influence of the hydrostatic pressure and pH on the asymmetric 2hydroxyketone formation catalyzed by pseudomonas putida benzoylformate decarboxylase and variants thereof. Biotech Bioeng 106, 18-26. Boland, W., Frossl, C., Lorenz, M., 1991. Esterolytic and lipolytic enzymes in organic synthesis. Synthesis-Stuttgart, 1049-1072. Boonyaratanakornkit, B.B., Park, C.B., Clark, D.S., 2002. Pressure effects on intra- and intermolecular interactions within proteins. Bba-Protein Struct M 1595, 235-249. Bornscheuer, U.T., Huisman, G.W., Kazlauskas, R.J., Lutz, S., Moore, J.C., Robins, K., 2012. Engineering the third wave of biocatalysis. Nature 485, 185-194. Drauz, K., 1997. Chiral amino acids: A versatile tool in the synthesis of pharmaceuticals and fine chemicals. Chimia 51, 310-314. Eisenmenger, M.J., Reyes-De-Corcuera, J.I., 2009a. High hydrostatic pressure increased stability and activity of immobilized lipase in hexane. Enzyme Microb Tech 45, 118-125. Eisenmenger, M.J., Reyes-De-Corcuera, J.I., 2009b. High pressure enhancement of enzymes: A review. Enzyme Microb Tech 45, 331-347. Gardossi, L., Bianchi, D., Klibanov, A.M., 1991. Selective acylation of peptides catalyzed by lipases in organic solvents. J Am Chem Soc 113, 6328-6329. Herbst, D., Peper, S., Fernandez, J.F., Ruck, W., Niemeyer, B., 2014. Pressure effects on activity and selectivity of Candida rugosa lipase in organic solvents. J Mol Catal BEnzym 100, 104-110. Herbst, D., Peper, S., Niemeyer, B., 2012. Enzyme catalysis in organic solvents: influence of water content, solvent composition and temperature on Candida rugosa lipase catalyzed transesterification. J Biotechnol 162, 398-403. Heremans, K., 1982. High pressure effects on proteins and other biomolecules. Ann. Rev. Biophys. Bioeng 11, 1-21. Klibanov, A.M., 2001. Improving enzymes by using them in organic solvents. Nature 409, 241-246. Liu, B.K., Wu, Q., Lv, D.S., Lin, X.F., 2011. Modulating the synthetase activity of penicillin G acylase in organic media by addition of N-methylimidazole: Using vinyl acetate as activated acyl donor. J Biotechnol 153, 111-115. Mateo, C., Palomo, J.M., Fernandez-Lorente, G., Guisan, J.M., Fernandez-Lafuente, R., 2007. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb Tech 40, 1451-1463. Mozhaev, V.V., Heremans, K., Frank, J., Masson, P., Balny, C., 1996a. High pressure effects on protein structure and function. Proteins 24, 81-91. Mozhaev, V.V., Lange, R., Kudryashova, E.V., Balny, C., 1996b. Application of high hydrostatic pressure for increasing activity and stability of enzymes. Biotech Bioeng 52, 320-331. Schmid, A., Dordick, J.S., Hauer, B., Kiener, A., Wubbolts, M., Witholt, B., 2001. Industrial biocatalysis today and tomorrow. Nature 409, 258-268. Sicard, C., Brennan, J.D., 2013. Bioactive paper: Biomolecule immobilization methods and applications in environmental monitoring. Mrs Bull 38, 331-334. Stinson, S.C., 1998. Counting on chiral drugs. Chem Eng News 76, 83-104. Stoeber, W., Fink, A., Bohn, E., 1968. Controlled growth of monodisperse silica spheres in micron size range. J Colloid Interf Sci 26, 62-69.
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Ac ce p
te
d
M
an
us
cr
ip t
Volpato, G., Rodrigues, R.C., Fernandez-Lafuente, R., 2010. Use of enzymes in the production of semi-synthetic penicillins and cephalosporins: drawbacks and perspectives. Curr Med Chem 17, 3855-3873. Zhao, Z.Y., Liu, J., Hahn, M., Qiao, S.Z., Middelberg, A.P.J., He, L.Z., 2013a. Encapsulation of lipase in mesoporous silica yolk-shell spheres with enhanced enzyme stability. Rsc Adv 3, 22008-22013. Zhao, Z.Y., Tian, J.F., Wu, Z.X., Liu, J., Zhao, D.Y., Shen, W., He, L.Z., 2013b. Enhancing enzymatic stability of bioactive papers by implanting enzyme-immobilized mesoporous silica nanorods into paper. J Mater Chem B 1, 4719-4722. Klibanov, A.M., 2001. Improving enzymes by using them in organic solvents. Nature 409, 241-246. Zaraki, A., Ghalambaz, M., Chamkha, A.J., Ghalambaz, M., De Rossi, D., 2015. Theoretical analysis of natural convection boundary layer heat and mass transfer of nanofluids: Effects of size, shape and type of nanoparticles, type of base fluid and working temperature. Adv Powder Technol 26, 935-946.
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Page 13 of 19
Figure legends
ip t
Figure 1. A, Scheme of the transesterification reaction catalyzed by CRL or immobilized CRL (I-CRL). B, eeR values of reaction under different pressures at water activity of 0.35. C,
cr
eeR values of reaction under different pressures at water activity of 0.70.
us
Figure 2. Relative activity of CRL and I-CRL applying different pressures at: a water activity
an
of 0.35 (A), and at aw = 0.70 (B). Free CRL had a specific activity of 95.9 U/g and 25.6 U/g at a water activity of 0.7, and 0.35, respectively. I-CRL had a significantly lower specific
M
activity of 2.37 U/g enzyme (aw = 0.7), and 1.56 U/g enzyme (aw = 0.35), respectively. All
d
experiments were conducted at least twice, with standard deviation presented as error bars..
te
Figure 3. SEM imagines of SNP, onto which CRL was immobilized, after reactions under
Ac ce p
different pressures. The white label indicates the pressure that SNP was exposed.
14
Page 14 of 19
ip t cr us an M d te
Ac ce p
Figure 1
15
Page 15 of 19
ip t cr us an M d
Ac ce p
te
Figure 2
16
Page 16 of 19
ip t cr us an M d te
Ac ce p
Figure 3
17
Page 17 of 19
Highlights Lipase was successfully immobilized on solid silica nanospheres High hydrostatic pressures (50-200 MPa) improved activities of immobilized lipase by 4-6 folds.
ip t
The immobilization of lipase significantly change its enantiomeric selectivity
Ac ce p
te
d
M
an
us
cr
The level of pressures and the water activity determine enantiomeric excess at high pressures
18
Page 18 of 19
Ac
ce
pt
ed
M
an
us
cr
i
Graphical Abstract (for review)
Page 19 of 19
S0960-3085(15)00108-X http://dx.doi.org/doi:10.1016/j.fbp.2015.08.006 FBP 640
To appear in:
Food and Bioproducts Processing
Received date: Revised date: Accepted date:
10-6-2015 20-8-2015 21-8-2015
Please cite this article as: Zhao, Z., Herbst, D., Niemeyer, B., He, L.,High pressure enhances activity and selectivity of Candida rugosa lipase immobilized onto silica nanoparticles in organic solvent, Food and Bioproducts Processing (2015), http://dx.doi.org/10.1016/j.fbp.2015.08.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Short Communication
Title: High pressure enhances activity and selectivity of Candida rugosa lipase
ip t
immobilized onto silica nanoparticles in organic solvent
Zhengyang Zhao1, †
cr
Daniela Herbst2, †
us
Bernd Niemeyer2,* Lizhong He1,*
Department of Chemical Engineering, Monash University, Clayton, VIC3800, Australia
2
Institute of Thermodynamics, Helmut-Schmidt-University/University of Federal Armed
an
1
M
Forces Hamburg, Holstenhofweg 85, Hamburg, D-22043, Germany
* Correspondence: Bernd
Niemeyer,
Institute
of
Thermodynamics,
Helmut-Schmidt-
Ac ce p
Professor
d
These authors contributed equally to this work.
te
†
University/University of the Federal Armed Forces, Hamburg, Holstenhofweg 85, D-22043 Hamburg, Germany;
Email: [email protected] and Dr Lizhong He, Department of Chemical Engineering, Monash University, Clayton, VIC3800, Australia.
E-mail: [email protected]
Keywords: chiral access, enzyme immobilization, high pressure, lipase, silica nanoparticle
1
Page 1 of 19
Abbreviations:aw, water activity; CRL, Candida rugosa lipase; I-CRL, immobilized CRL; PP, 1-phenylpropan-2-ol; VA, vinyl acetate; SNP, silica nanoparticle; (R)-PPA, (R)-1phenylpropan-2-yl acetate; (S)-PPA, (S)-1-phenylpropan-2-yl acetate; TEOS, Tetraethyl
Ac ce p
te
d
M
an
us
cr
ip t
orthosilicate
2
Page 2 of 19
Abstract
High hydrostatic pressure has been increasingly utilized to improve functions of enzymes, and most of such studies are currently focused on free enzymes in aqueous solution or
ip t
organic solvent. In this work, Candida rugosa lipase (CRL) was immobilized onto silica nanoparticles and its activity and enantioselectivity in organic solvent were evaluated at high
cr
pressures under different water activities. The application of high hydrostatic pressures (50-
us
200 MPa) led to improved activities of immobilized CRL for transesterification of (R)-1phenylpropan-2-ol with vinyl acetate by 4-6 folds. Additionally the immobilization of CRL
an
resulted in a significant change of selectivities, shifting the of enantiomeric excess from the (R)- towards (S)-1-phenylpropan-2-yl acetate product at atmospheric pressure. The
M
application of high pressures led to either enantiomeric excess toward (R)-1-phenylpropan-2yl or no enantiomeric selectivity, depending on the water activities in the organic solvent and
d
the level of pressures. The interesting behaviour of immobilized CRL under high pressures
te
offers new opportunities to modulate enzyme functions through combination of high
Ac ce p
pressures and enzyme immobilization.
3
Page 3 of 19
1 Introduction Enzymatic synthesis has been increasingly applied in fine chemical industry to produce
ip t
enantiomeric pharmaceuticals (Stinson, 1998). Various chiral intermediates, including amino acids (Drauz, 1997), peptides (Gardossi et al., 1991; Sicard and Brennan, 2013), esters
cr
(Boland et al., 1991) and penicillin (Liu et al., 2011; Volpato et al., 2010) have been manufactured via enzymatic synthesis route. Sustainable application of enzymes for such
us
productions require enzymes with high stability and selectivity under industrial processing
an
conditions (Bornscheuer et al., 2012), and ideally, enzymes should be re-useable for a long period in order to keep the cost low (Mateo et al., 2007). Protein engineering and enzyme
M
immobilization are two classical approaches to engineer enzymes with these desired features (Bornscheuer et al., 2012; Schmid et al., 2001). Alternatively, the performance of enzymes
d
can also be enhanced through altering the reaction conditions such as utilizing organic
te
solvents as reaction medium (Klibanov, 2001), or employing high hydrostatic pressures
Ac ce p
(Eisenmenger and Reyes-De-Corcuera, 2009b).
Recently high hydrostatic pressure processes have shown potentials in enhancing performance of enzymes by increasing their stability and selectivity (Eisenmenger and Reyes-De-Corcuera, 2009b; Mozhaev et al., 1996b; Herbst et al., 2012; Herbst et al., 2014). Although it is well known many proteins denature upon exposure to high hydrostatic pressures (Heremans, 1982; Mozhaev et al., 1996a), more than 25 enzymes have been reported with improved performances under extreme pressures (Eisenmenger and Reyes-DeCorcuera, 2009b). Despite these progresses, applications of bio-catalysis under high pressures are still limited, in part, due to poor knowledge of enzymes at extreme conditions (Berheide et al., 2010). Particularly, there is little understanding about how high pressures affect the
4
Page 4 of 19
function of enzymes in their immobilized form that is important for re-use of enzymes in
ip t
industrial application (Eisenmenger and Reyes-De-Corcuera, 2009a).
Lipase catalysis in organic solvents is frequently used for enantiomeric products synthesis
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(Klibanov, 2001). Recently, high pressure has shown the ability to alter properties of free lipase in solvent including activity, stability and enantioselectivity. (Eisenmenger and Reyes-
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De-Corcuera, 2009a, b; Mozhaev et al., 1996b; Herbst et al., 2012; Herbst et al., 2014). The
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work by Herbst et al (2012, 2014) has evaluated high pressure effects on Candida rugosa lipase (CRL), one of the most used lipase for industrial application (e.g. transesterification
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reactions). However, the effect of high pressure on lipase under its immobilized form has not yet been studied. Thus, in this work, we aim to investigate the effect of high hydrostatic
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pressure on immobilized CRL in organic solvent at different water activities. Solid silica
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nanoparticle was selected as carrier for CRL immobilization, based on the following considerations: 1) solid silica nanoparticles are stable under high pressures; 2) they have low
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mass transfer resistance due to their small size (Zaraki et al., 2015), and; 3) convenient surface functionalization is available for immobilization (Zhao et al., 2013a). Activity and enantioselectivity of immobilized CRL were evaluated and compared with free CRL under different pressures and water activities (a w), using the established model reaction of the asymmetric transesterification of 1-phenylpropan-2-ol (PP) with vinyl acetate (VA) (Herbst et al 2012, 2014).
2 Experimental 2.1 Materials
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Lipase from Candida rugosa in crude powder (ca. 2.5 % enzyme by protein mass) was a gift from Amano Enzyme Inc. Nagoya, Japan. All chemicals and solvents were of at least analytical grade or higher. Chemicals for particle preparation and immobilization including
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tetraethyl orthosilicate (TEOS) and 3-glycidoxypropyltrimethoxysilane were purchased from Sigma-Aldrich Sydney, Australia. Chemicals for enzymatic reaction including vinyl acetate
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(VA), racemic 1-phenylethanol (PE), racemic 1-phenylpropan-2-ol (PP) and anhydrous hexane were obtained from Sigma-Aldrich Munich, Germany. Bradford assay reagent for
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protein assay was obtained from Sigma-Aldrich Munich, Germany.
2.2 Preparation of SNP and lipase immobilization
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SNP with diameters around 250 nm were synthesized using the Stoeber method (Stoeber et al., 1968). The synthesized SNP were functionalized with epoxy groups using 3-
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glycidoxypropyltrimethoxysilane followed by immobilization of CRL using the method
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reported previously (Zhao et al., 2013b). Then, the particles were dried in fume hood before
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controlling their water activity. (Detailed information is given in the Supporting Information).
2.3 CRL-catalyzed transesterification at ambient and high pressures In a typical experiment, 18.75 mL hexane and 6.25 mL VA with certain water activity (aw = 0.35 or 0.7, see control of water activity in the Supporting Information) were mixed with PP at a final concentration of 0.41 M. After stirring the reaction media for 5 min at 300 rpm to dissolve PP, 150 mg crude CRL powder or immobilized CRL with the equivalent CRL mass was added. For the reactions at atmospheric pressure the lipase-substrate-mixture was transferred into sealed glass vials which were kept in an oven to control the reaction temperature. For reactions at high-pressures the lipase-substrate-mixture was transferred into a leak-proof high pressure vessel using the set-up reported (Herbst et al., 2014) previously.
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Three pressure levels (50, 100 and 200 MPa) were applied for the experiments. The reaction solutions, both under atmospheric and high pressures, were continuously mixed with a stirrer bar at 300 rpm by a magnetic stirrer. The reaction was carried out for 48 hours at 35 °C
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before samples were taken for analysis. All the experiments were conducted two times at least, with error bar which represent the standard deviation labelled on the data points of
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figures.
2.4 Analytical methods
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The previous reported method was employed for the analysis of the (Herbst et al., 2014) product. Briefly, the product concentration of samples after the reaction were analyzed by gas
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chromatography GC-FID (Clarus 500, Perkin, Elmer Rodgau, Germany) utilizing a chiral Hydrodex-ß-PM column (Macherey&Nagel, Dueren, Germany; column length 50 m; column
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diameter 0.25 mm: condition: oven temperature from 80 °C (initial time 1min) to 120 °C for
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17 min with a heating rate of 20 °C/min, split ratio 1:20, He as carrier gas) to determine the specific activity and the enantiomeric excess . The enantiomeric excess eeR [%] was
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calculated by
eeR
c R cS * 100 c R cS
Where c R is the concentration of (R)-1-phenylpropan-2-yl acetate ((R)-PPA) and cS is the concentration of (S)-1-phenylpropan-2-yl acetate ((S)-PPA). The enzymatic activity was calculated by the equation using previously reported method (Herbst et al., 2014) as below:
A
cp V t mE
Where cp is the concentration of the product, V is the reaction volume, t is the reaction time, and mE is mass of the enzyme
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3 Results and discussion 3.1 The enantioselectivity of immobilized CRL The amount of CRL immobilized onto SNP was estimated to be 19.2 mg g-1 silica, based on
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the mass balance of CRL in the supernatant before and after immobilization. In our previous studies, aw of the reaction medium was shown to be an important parameter influencing the
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CRL performance (Herbst et al., 2014; Herbst et al., 2012). The functions of free and immobilized CRL are thus compared at two different water activities (aw = 0.35 and 0.7) in
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this work.
Interestingly, the immobilization of CRL leads to a significant change of the CRL
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enantioselectivity for transesterification of PP with VA (Figure 1A). Under atmospheric pressure, free CRL has only marginal enantioselectivity (Figure 1B and C). At the water
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activity of 0.35, free CRL slightly favoured the (S)-PPA product (eeR=-4.3 %) (Figure 1B),
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and the eeR value was 4.1 % at a water activity of 0.7 (Figure 1C). In contrast, the immobilized CRL (I-CRL) strongly favoured the S directed reaction at atmospheric pressure.
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The eeR values were -47.9 % (Figure 1B), and -69.6 % (Figure 1C) at water activities of 0.35, and 0.7, respectively. The possible reason for the change of enantiomeric selectivity by the CRL immobilization is that covalent binding onto the SNP introduces distortion of active site of the enzyme which may alter its catalytic properties, as discussed in a recent review by Mateo et al (2007).
At the water activity of 0.35, high pressures led to a shift of the enantiomeric excess towards (R)-PPA for both of free CRL, and immobilized CRL (Figure 1B). For free CRL, the eeR value was increased from 5.2 % at 50 MP to 13.2 % at 100 MPa, and finally to 21.1 % at 200 MPa, respectively. I-CRL provided little enantiomeric selectivity at 50 MPa (eeR = 0.18 %)
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and 100 MPa (eeR = 4.0 %), but had a higher eeR value of 13.6 % at 200 MPa. At water activity of 0.7, free CRL showed a similar trend of increased eeR at high pressures, and the value (eeR = 25.0 %) at 200 MPa was slightly higher than that (eeR = 21.4 %) at aw 0.35
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(Figure 1C). However, at aw 0.7, the immobilized CRL exhibited little enantiomeric selectivity with marginal eeR values between -3.6 % and 2.6 % from 50 MPa to 200 MPa, in
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contrast to its strong preference for (S)-PPA (eeR = -69.6 %) at atmospheric pressure (Figure 1C). The results presented in Figure 1B and 1C have shown the complexity of the
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enantiomeric selectivity of CRL, which is determined by the interplay of the state of the
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enzyme (free or immobilized), pressures and water activities. Such complexity may be due to collective effects of these parameters on enzyme conformation (Boonyaratanakornkit et al.,
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2002; Mateo et al., 2007; Mozhaev et al., 1996b).
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3.2 High pressure enhances the activity of the immobilized CRL
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The immobilization of CRL onto silica nanoparticles led to a significant decrease of CRL activities at atmospheric pressure. While free CRL had a specific activity of 95.9 U/g and
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25.6 U/g at the water activity of 0.7, and 0.35, respectively, immobilized CRL had a significantly lower specific activity of 2.37 U/g enzyme (aw = 0.7) and 1.56 U/g enzyme (aw = 0.35). Immobilization of CRL was realized by the reaction of the epoxy functional groups on SNP with primary amines (amine of lysine residues or amine at N-terminus) of the CRL. This immobilization condition is expected to result in multiple covalent bonds and rigid structure of enzymes that can decrease enzyme activity (Mateo et al., 2007). Moreover, exposing the CRL to air under room temperature in the process of immobilization also decreased the CRL activity. The application of high pressures, however, was efficient to increase the activities of the immobilized CRL as shown in Figure 2. At a water activity of 0.35, the relative activity of the immobilized CRL at 50 MPa was 560 % of that at atmospheric pressure (Figure 2A). The
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value decreased slightly with the increase of the pressure (460 % at 100 MPa and 330 % at 200 MPa), but was still significant higher than that at the atmospheric pressure. The relative activities of immobilized CRL at a water activity of 0.70 % shown a similar trend (Figure 2B)
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although there was less improvement (440 % at 50 MPa) compared to those at 0.35 water activity. It is generally suggested that both, the changes of enzyme structure and alteration of
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the physical properties of the substrate and solvent by high pressures contribute to the improvement of the enzyme activities (Eisenmenger and Reyes-De-Corcuera, 2009b). Given
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that immobilized CRL and free CRL responded differently to high pressures at the same
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reaction conditions in this case, it was more likely that structural changes of enzymes played
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a determined role in improving activities of immobilized CRL.
3.3 Particle stability under high pressures
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Importantly, the SNP were highly stable under all tested high pressures. As shown in Figure
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3, SNP remained their uniform spherical shapes after the reaction at high pressures. Given that the immobilized CRL has improved its activity at high pressures, the enzyme was likely
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to retain its intact structure as well. The SNP-based immobilization system was thus sufficiently robust for further exploring of advantages brought by high pressures in future.
4 Conclusions
In summary, this work demonstrated that high hydrostatic pressures could significantly improve activities as well as altering selectivities of the lipase immobilized onto silica nanoparticles. The asymmetric reaction catalyzed by CRL was significantly shifted by the immobilization of the CRL towards an excess of the (S)-enantiomer at atmospheric pressure towards the (R)-enantiomer of the product under high pressure. The enantiomeric excess value at high pressures depended on the state of enzyme (immobilized or free), water
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activities and the level of high pressures. This complexity of the enzyme’s enantiomeric selectivity may offer new opportunities to modulate enzyme functions through combination of high pressures and enzyme immobilization and warrants a systematic study of immobilized
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enzymes at high pressures in future.
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Acknowledgement
The authors acknowledge travel support provide by Go8 - Germany Joint Research
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Cooperation Scheme, a partnership between the Group of Eight and the German Academic
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Exchange Service (DAAD). Z.Z. acknowledges New Horizons Joint PhD Scholarship Program provided by Monash University and Commonwealth Scientific and Industrial
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Research Organisation (CSIRO). D.H. acknowledges the financial support by the Deutsche Forschungsgemeinschaft (DFG) for the joint project “Enzyme-catalyzed synthesis of chiral
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substances under high pressures”. The authors thank Amano Enyzme Inc. Nagoya, Japan for
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Conflict of interest
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free delivery of the CRL. The authors thank Oliver Wenzel for assistance in GC-analysis.
The authors declare no financial or commercial conflict of interest.
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5 References
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Berheide, M., Peper, S., Kara, S., Long, W.S., Schenkel, S., Pohl, M., Niemeyer, B., Liese, A., 2010. Influence of the hydrostatic pressure and pH on the asymmetric 2hydroxyketone formation catalyzed by pseudomonas putida benzoylformate decarboxylase and variants thereof. Biotech Bioeng 106, 18-26. Boland, W., Frossl, C., Lorenz, M., 1991. Esterolytic and lipolytic enzymes in organic synthesis. Synthesis-Stuttgart, 1049-1072. Boonyaratanakornkit, B.B., Park, C.B., Clark, D.S., 2002. Pressure effects on intra- and intermolecular interactions within proteins. Bba-Protein Struct M 1595, 235-249. Bornscheuer, U.T., Huisman, G.W., Kazlauskas, R.J., Lutz, S., Moore, J.C., Robins, K., 2012. Engineering the third wave of biocatalysis. Nature 485, 185-194. Drauz, K., 1997. Chiral amino acids: A versatile tool in the synthesis of pharmaceuticals and fine chemicals. Chimia 51, 310-314. Eisenmenger, M.J., Reyes-De-Corcuera, J.I., 2009a. High hydrostatic pressure increased stability and activity of immobilized lipase in hexane. Enzyme Microb Tech 45, 118-125. Eisenmenger, M.J., Reyes-De-Corcuera, J.I., 2009b. High pressure enhancement of enzymes: A review. Enzyme Microb Tech 45, 331-347. Gardossi, L., Bianchi, D., Klibanov, A.M., 1991. Selective acylation of peptides catalyzed by lipases in organic solvents. J Am Chem Soc 113, 6328-6329. Herbst, D., Peper, S., Fernandez, J.F., Ruck, W., Niemeyer, B., 2014. Pressure effects on activity and selectivity of Candida rugosa lipase in organic solvents. J Mol Catal BEnzym 100, 104-110. Herbst, D., Peper, S., Niemeyer, B., 2012. Enzyme catalysis in organic solvents: influence of water content, solvent composition and temperature on Candida rugosa lipase catalyzed transesterification. J Biotechnol 162, 398-403. Heremans, K., 1982. High pressure effects on proteins and other biomolecules. Ann. Rev. Biophys. Bioeng 11, 1-21. Klibanov, A.M., 2001. Improving enzymes by using them in organic solvents. Nature 409, 241-246. Liu, B.K., Wu, Q., Lv, D.S., Lin, X.F., 2011. Modulating the synthetase activity of penicillin G acylase in organic media by addition of N-methylimidazole: Using vinyl acetate as activated acyl donor. J Biotechnol 153, 111-115. Mateo, C., Palomo, J.M., Fernandez-Lorente, G., Guisan, J.M., Fernandez-Lafuente, R., 2007. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb Tech 40, 1451-1463. Mozhaev, V.V., Heremans, K., Frank, J., Masson, P., Balny, C., 1996a. High pressure effects on protein structure and function. Proteins 24, 81-91. Mozhaev, V.V., Lange, R., Kudryashova, E.V., Balny, C., 1996b. Application of high hydrostatic pressure for increasing activity and stability of enzymes. Biotech Bioeng 52, 320-331. Schmid, A., Dordick, J.S., Hauer, B., Kiener, A., Wubbolts, M., Witholt, B., 2001. Industrial biocatalysis today and tomorrow. Nature 409, 258-268. Sicard, C., Brennan, J.D., 2013. Bioactive paper: Biomolecule immobilization methods and applications in environmental monitoring. Mrs Bull 38, 331-334. Stinson, S.C., 1998. Counting on chiral drugs. Chem Eng News 76, 83-104. Stoeber, W., Fink, A., Bohn, E., 1968. Controlled growth of monodisperse silica spheres in micron size range. J Colloid Interf Sci 26, 62-69.
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Volpato, G., Rodrigues, R.C., Fernandez-Lafuente, R., 2010. Use of enzymes in the production of semi-synthetic penicillins and cephalosporins: drawbacks and perspectives. Curr Med Chem 17, 3855-3873. Zhao, Z.Y., Liu, J., Hahn, M., Qiao, S.Z., Middelberg, A.P.J., He, L.Z., 2013a. Encapsulation of lipase in mesoporous silica yolk-shell spheres with enhanced enzyme stability. Rsc Adv 3, 22008-22013. Zhao, Z.Y., Tian, J.F., Wu, Z.X., Liu, J., Zhao, D.Y., Shen, W., He, L.Z., 2013b. Enhancing enzymatic stability of bioactive papers by implanting enzyme-immobilized mesoporous silica nanorods into paper. J Mater Chem B 1, 4719-4722. Klibanov, A.M., 2001. Improving enzymes by using them in organic solvents. Nature 409, 241-246. Zaraki, A., Ghalambaz, M., Chamkha, A.J., Ghalambaz, M., De Rossi, D., 2015. Theoretical analysis of natural convection boundary layer heat and mass transfer of nanofluids: Effects of size, shape and type of nanoparticles, type of base fluid and working temperature. Adv Powder Technol 26, 935-946.
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Figure legends
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Figure 1. A, Scheme of the transesterification reaction catalyzed by CRL or immobilized CRL (I-CRL). B, eeR values of reaction under different pressures at water activity of 0.35. C,
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eeR values of reaction under different pressures at water activity of 0.70.
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Figure 2. Relative activity of CRL and I-CRL applying different pressures at: a water activity
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of 0.35 (A), and at aw = 0.70 (B). Free CRL had a specific activity of 95.9 U/g and 25.6 U/g at a water activity of 0.7, and 0.35, respectively. I-CRL had a significantly lower specific
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activity of 2.37 U/g enzyme (aw = 0.7), and 1.56 U/g enzyme (aw = 0.35), respectively. All
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experiments were conducted at least twice, with standard deviation presented as error bars..
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Figure 3. SEM imagines of SNP, onto which CRL was immobilized, after reactions under
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different pressures. The white label indicates the pressure that SNP was exposed.
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Figure 1
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Figure 2
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Figure 3
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Highlights Lipase was successfully immobilized on solid silica nanospheres High hydrostatic pressures (50-200 MPa) improved activities of immobilized lipase by 4-6 folds.
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The immobilization of lipase significantly change its enantiomeric selectivity
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The level of pressures and the water activity determine enantiomeric excess at high pressures
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Graphical Abstract (for review)
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