β-cyclodextrin as an additive to improve the thermostability of Yarrowia lipolytica Lipase 2: Experimental and simulation insights

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β -cyclodextrin as an additive to improve the thermostability of Yarrowia lipolytica Lipase 2: Experimental and simulation insights Hao Cao a, Meng Wang a, Kaili Nie a,b, Xin Zhang c, Ming Lei c, Li Deng a,b,∗, Fang Wang a, Tianwei Tan a a

Beijing Bioprocess Key Laboratory, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 1500029, PR China Amoy-BUCT Industrial of Bio-technovation Institute, Amoy 361026, PR China State Key Laboratory of Chemical Resource Engineering, Institute of Materia Medica, College of Science, Beijing University of Chemical Technology, Beijing 100029, PR China

b c

a r t i c l e

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Article history: Received 31 March 2015 Revised 14 October 2016 Accepted 20 October 2016 Available online xxx Keywords: Thermostability β -cyclodextrin Yarrowia lipolytica Lipase 2 Molecular dynamic simulation

a b s t r a c t The influence of β -cyclodextrin on the thermostability of Yarrowia lipolytica Lipase 2 (YLLIP2) has been simultaneously studied by in vitro experiments and molecular simulations. Both circular dichroism (CD) measurement and molecular dynamic (MD) simulation results verified that the content of the α -helix increased in the presence of β -cyclodextrin. Additionally, the reduction of the mean square fluctuation (RMSF) successfully demonstrated that β -cyclodextrin, as additive, restored the thermosensitive region of YLLIP2 (SER 90-VAL 125, SER 146-ASP 153 and GLN 282-GLY 287) over the optimum temperature (333 K) in MD simulations. Subsequently, further researches exhibited that the β -cyclodextrin molecule connected the separated region (SER 90-VAL 125 and LYS 176-210 ASN) of YLLIP2 through interactions with surface polar residues. The β -cyclodextrin molecule was like a bridge anchoring THR 117 and ASN 178, ASP 121 and LYS 203, THR 117 and GLU 209 at 313 K, 323 K and 333 K, respectively. The present study identities the effect of β -cyclodextrin on thermostability of YLLIP2 at molecular level and facilitates the extensive application of YLLIP2 in bio-catalysis. © 2016 Published by Elsevier B.V. on behalf of Taiwan Institute of Chemical Engineers.

1. Introduction Yarrowia lipolytica is studied extensively as the nonconventional yeast, and it processes 16 paralogs of genes coding for lipase. However, only three lipases (YLLIP2, YLLIP7, and YLLIP8) have been characterized so far [1,2]. In our previous work, Yarrowia lipolytica Lipase 2 (YLLIP2) was isolated from a mutant strain Candida sp. 99-125 (belonging to Yarrowia lipolytica) [3]. YLLIP2 has a broad range of applications in bio-energy, food chemistry, and pharmaceuticals through hydrolysis, esterification and transesterification reactions [4–6]. Nevertheless, as mesophilic enzymes, the optimum temperature (40 °C) of YLLIP2 has become a bottleneck considering the harsh reaction conditions in industrial processes [7]. Various technical methods have been employed to enhance the thermostability of lipase, including screening of strains, modification of genes or structure, fermentation engineering and others [8–10]. With regard to YLLIP2,

∗

Corresponding authors. E-mail addresses: [email protected] (K. Nie), [email protected], [email protected] (L. Deng).

Bordes et al. [11] and Wen et al. [12] reported the significative genetic engineering methods for improving the thermostability. From the structural view, YLLIP2 is based on the classical Rossmannfold [13]. The catalytic triad of YLLIP2 is formed by Ser 162, Asp 230, and His 289. And its oxyanion hole is composed by Arg 86 and Leu 163, which could act on substrate directly in the process of catalysis through an electrophilic environment [14]. According to our previous simulation results [15], the distance between Thr88Leu105 region and Ala277-Leu290 region extended with increasing temperature; And the interaction between His289 and Asp230, an essential part in proton transfer among catalytic triad based on the bi-bi-ping-pong mechanism, was affected by the high temperature (333 K) [16,17] (see supplementary material Fig. S1). Treatment of the reaction mixture with an additive has been a convenient way of improving the property of lipase because of its practical simplicity [18]. Furthermore, a growing interest toward the using of cyclodextrin or crown ethers as additives to enhance the catalytic characters of lipase has been reported [19,20]. Yurie et al. confirmed that methylated β -cyclodextrin enhanced the activity and enantioselectivity of Pseudomonas cepacia lipase (PCL) [21]. Fernández et al. investigated the thermostability of α chymotrypsin by enzymic modification with β -cyclodextrin derivatives. The modified enzyme was resistant to thermal inactivation

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at 50 °C and retained 70% of the original activity [22]. In our previous work, β -cyclodextrin, as an assistant, improved the catalytic properties of Candida sp. 99-125 for the synthesis of monoglyceride (MAG) and diglyceride (DAG) in solvent-free system [20]. The effect of the protein conformational changes in different timescale (from femtosecond to second) on enzymes’ features in catalytic reaction has been reviewed [23]. And classical molecular dynamics (MD) simulations have also offered logically explained and rationally predicted about enzyme characters (such as the thermal stability, solvent tolerance and ligand binding) concerning the physical basis of the structure of protein in the order of nanoseconds [24–26]. Therefore, in the present work, for exploring the effect of β -cyclodextrin on the thermostability of YLLIP2, experiments and simulations were simultaneously carried out. The results of molecular dynamics (MD) provided the structural basis to interpret the thermo-resistance of YLLIP2 in experiment. The potential of β -cyclodextrin for protecting YLLIP2 against thermodenaturation was demonstrated, and the mechanism of thermoresistance enhancement was clarified at molecular level. This study is significant for YLLIP2 in more extensive bio-catalytic applications. 2. Materials and methods 2.1. Experimental 2.1.1. Reagents β -cyclodextrin was analytical grade and purchased from Ziyi Chemical Reagent Co. (Shanghai, China). Olive oil and polyvinyl alcohol (PVA), obtained from Sanbo Biotech Co. (Beijing, China), were of chemical grade. The rest of the chemicals were analytical grade and purchased from Beijing Chemical Factory. 2.1.2. Enzyme purification The method of purification and deglycosylation was reported by Yu et al. [7]. Purified deglycosylated YLLIP2 was obtained from the crude extract of mutant strain Candida sp. 99-125 (belonging to Yarrowia lipolytica) via ion-exchange chromatography and hydrophobic interaction chromatography successively. 2.1.3. Lipase activity assay Lipase activity was measured by titrimetric assay according to an olive oil emulsion method [7,27]. The protein content was determined by the Bradford protein assay method using bovine serum albumin as standard [28]. 2.1.4. Thermostability assay 200 ml of purified YLLIP2 (0.1 mg/ml protein in 100 mM phosphate buffer, pH 7.0) was added and incubated at different temperatures (30 °C, 40 °C, and 50 °C) for 180 min with β -cyclodextrin (β -cyclodextrin:lipase = 1:1, w/w) in a water bath. Then, 1.5 ml reaction mixture was removed at 0 min, 10 min, 30 min, 60 min, 120 min and 180 min and was measured for lipase activity at 37 °C. The control experiments without β -cyclodextrin were performed and the lipase activity was also assayed. 2.1.5. Circular dichroism measurement The protein secondary structure of YLLIP2 was measured by circular dichroism spectrum (CD) between 180 nm and 280 nm at 25 °C. According to the data shown in supplementary material (Fig. S2), β -cyclodextrin lightly absorbs the signal of circular dichroism. Therefore, a baseline that was not added enzyme but added β cyclodextrin was used for the system with β -cyclodextrin. Another baseline that was not added enzyme and β -cyclodextrin was used for the system without β -cyclodextrin. The secondary structure element content was estimated using the DICHROWEB application

package based on the SELCON3 algorithm described by Sreerama et al. [29,30]. 2.2. Computational 2.2.1. Preparing molecules β -cyclodextrin’s geometric parameters were derived by ab initio calculation and optimization on the HF/6–31G∗ level using Gaussian 03 [31]. The partial charges were derived by fitting partial charges using the RESP program of AMBER 10 to the electrostatic potential [32]. The initial opened conformation of deglycosylated YLLIP2, based on the X-ray structure of YLLIP2 (PDB ID: 4JEI), was obtained in our previous study [15]. It was a semi-open conformation and was similar to the reported one by Bordes et al. [14]. The protonation states of catalytic triad (the protonation state of HIS 289 is HID state without the HE2 atom and with HD1 atom) and the crystal waters around surface were reserved in the molecular dynamics simulations. 2.2.2. Molecular dynamics simulations All MD simulations were performed with the GROMACS software package (version 4.5.4) using the AMBER99 force field [33]. The lipase was embedded in a cubic box with a 1.0 nm space left around the lipase for adding solvent molecules. 33 β -cyclodextrin molecules and 12,450 TIP3P water molecules were added in the cubic box. Ten Na+ ions were added by replacing the water molecules to neutralize the system charge. PME (Particle Mesh Ewald) algorithm was applied for the calculation of the electrostatic interactions setting the limit at 1.0 nm [34]. The cut-off for van der Waals interactions was 1.0 nm. Temperature and pressure controls were imposed using a Berendsen-type algorithm [35]. For energy minimization, the steepest descent method was used in the first 50 0 0 steps and a conjugate gradient method in the last 50 0 0 steps. After energy minimization, each system was heated gradually from 0 to final temperature (313 K, 323 K, or 333 K) during 200 ps and was equilibrated for 300 ps with constant volume and temperature coupling constant of 0.2 ps. After gradual heating, production simulations of the protein-solvent systems were performed at different temperatures (313 K, 323 K, and 333 K) and at atmospheric pressure in aqueous solution with β -cyclodextrin. Control experiments without β -cyclodextrin were also performed. Each MD run was performed for 10 ns using a time step of 2 fs. 2.2.3. Analysis The trajectories were analyzed by GROMACS tools; root mean square deviation (RMSD) values between structures following least-squares fitting to a referenced initial structure were computed using g_rms, the root mean square fluctuations (RMSF) of residues were computed to check the flexibility of each residue using g_rmsf, the radial distribution functions (RDF) to describe molecular distribution using g_rdf. The images of molecular graphics were generated and viewed using the Pymol package [36]. Secondary structure analysis was performed using the program DSSP [37]. 3. Results and discussion 3.1. Effect of β -cyclodextrin on YLLIP2 thermostability YLLIP2 thermostability studies were carried out at three chosen temperatures (30 °C, 40 °C and 50 °C) in the presence or absence of β -cyclodextrin (see Fig. 1). In the absence of β -cyclodextrin, a significant loss of activity was noticed at 50 °C: the specific activity decreased quickly to reach 30% of the initial activity at

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temperature in the control group. However, the content of β -sheet shows delicate fluctuations for all the systems. The β -sheet structure, which resides in the inner region of a protein, is more difficult to be influenced by temperature than α -helix. Circular dichroism analysis showed that the improvement of thermostability of YLLIP2 was correlated to the increase of its α -helix content. Therefore, there was an assumption that the conformation of YLLIP2 with β -cyclodextrin enhanced structural stability through increasing the content of α -helix at high temperature in comparison with the conformation of YLLIP2 without β -cyclodextrin. 3.3. Rearrangement of conformation for YLLIP2 with β -cyclodextrin

Fig. 1. The specific activity of YLLIP2 with β -cyclodextrin (—) and without β cyclodextrin (—) at 30 °C (), 40 °C (◦) and 50 °C () for 180 min.

Fig. 2. Quantitative estimation (%) of the secondary structure elements of YLLIP2 by circular dichroism at 30 °C, 40 °C and 50 °C after 180 min.

50 °C after 60 min. However, the specific activity in the presence of β -cyclodextrin remained at 70% of the initial activity at the same temperature and time. Moreover, at 180 min, YLLIP2 with β cyclodextrin still exhibited the catalytic activity (about 700 U/mg) at 50 °C. In contrast, YLLIP2 without β -cyclodextrin was inactivated at 50 °C after 180 min. As above shown, the results demonstrated that β -cyclodextrin, as a safe and inexpensive additive, protects YLLIP2 against thermo-inactivation when the temperature exceeded 40 °C, which is the optimal temperature of YLLIP2 [7]. Subsequently, the mechanism of β -cyclodextrin for enhancement of thermostability of YLLIP2 was investigated intensively. 3.2. Effect of β -cyclodextrin on YLLIP2 secondary structure Circular dichroism was conducted to analyze the secondary structure variation of YLLIP2. As shown in Fig. 2, the content of α -helix of YLLIP2 with β -cyclodextrin is always more than that of YLLIP2 without β -cyclodextrin at any temperature (30 °C, 40 °C and 50 °C). The content of α -helix is 32.3% of overall structure in the presence of β -cyclodextrin at 50 °C but is only 26.4% at same

Further, in order to interpret the correlation between thermostability enhancement and conformation change of YLLIP2 and to explore the functional mechanism of β -cyclodextrin, MD simulations were performed. The backbone RMSD values of YLLIP2 at 313 K, 323 K and 333 K were plotted versus simulation time with different colors, respectively (see Fig. 3). The patterns of backbone RMSD value exhibited that each MD run reached the plateau before 10.0 ns, and it indicated that the change to initial structure of YLLIP2 was less in the presence of β -cyclodextrin than that in absence of β -cyclodextrin at 313 K, 323 K and 333 K. Additionally, increasing the temperature up to 333 K, which is a relative inactivation temperature for YLLIP2 in MD simulation [15], backbone RMSD value of YLLIP2 with β -cyclodextrin exhibited was less than that of YLLIP2 without β -cyclodextrin. This reduced backbone RMSD value confirmed that the original structure of YLLIP2 was better maintained by β -cyclodextrin. Moreover, the reduced backbone RMSD value also has been regarded as an effective parameter for verification of more structural rigidity, which could enhance the thermostability of the enzyme [38]. For further investigation of the structural rearrangement of YLLIP2, the simulation snapshots were created at 10.0 ns at all temperatures. According to the simulation results, the effect of β cyclodextrin on the secondary structure of YLLIP2 is shown in Fig. 4. At 313 K, 323 K and 333 K, in the adding β -cyclodextrin guesthouse, secondary structure of YLLIP2 preserved 25.9%, 24.6%, and 23.9% of the α -helix of the overall structure, respectively. For the control group, the content of α -helix was preserved 23.3%, 22.3%, and 21.9% at 313 K, 323 K and 333 K, respectively. Additionally, the content of β -sheet was maintained at about 17.5% (±1.0%) at all temperatures (including control group). This simulation result strongly validated the above circularly dichroism analysis. The structural change of the catalytic triad (SER 162, ASP 230 and HIS 289) was investigated through comparing the average RMSD value of the catalytic triad in 10 ns MD simulation between with β -cyclodextrin and without β -cyclodextrin. As shown in Fig. 5, a severe structural rearrangement of the catalytic triad occurred at 333 K in the absence of β -cyclodextrin. In contrast, in the presence β -cyclodextrin, the average RMSD value of catalytic triad was nearly maintained; even though, this value was up to 0.27 nm at 333 K from 0.23 nm at 323 K. From the bi-bi-ping-pong mechanism view, the regular position of catalytic triad is essential to complete the catalytic process of lipase. In the present work, the average RMSD value of the catalytic triad in the simulation correlated well with the catalytic activity in experiments at all temperatures with and without β -cyclodextrin. From different aspect, the lid region of lipase could adopt the mobility to control substrates approaching the catalytic site. Hence, the Cα distance between ILE95 and VAL281 at 333 K was calculated to research the effect of β -cyclodextrin on the separated extent of YLLIP2’s lid. However, the opened extent of the lid was stable regardless of whether β cyclodextrin was added in system (see supplementary material Fig. S3). According to the analysis above, it could be confirmed that the improvement of thermostability of YLLIP2 attributed to the effect

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Fig. 3. The backbone root mean square deviations (RMSD) value of YLLIP2 with β -cyclodextrin and without β -cyclodextrin at 313 K, 323 K and 333 K for 10 ns MD simulation.

Fig. 5. Average RMSD of catalytic triad (Ser162, Asp230, and His289) of YLLIP2 with β -cyclodextrin (Cross lines) and without β -cyclodextrin (Parallel lines) at 313 K, 323 K and 333 K in 10 ns MD simulation. Fig. 4. Quantitative estimation (%) of the secondary structure elements of YLLIP2 by the program DSSP at 313 K, 323 K and 333 K after 10 ns MD simulation.

of β -cyclodextrin on the structural stability of the overall conformation and the catalytic triad of YLLIP2. 3.4. Modification for YLLIP2 with β -cyclodextrin RMSF value was employed to investigate the thermosensitive residues during the heat-denaturation process of YLLIP2. As shown in Fig. 6, SER 90-VAL 125, SER 146-ASP 153 and GLN 282-GLY 287 region of YLLIP2 show major fluctuation at 333 K in absence of β cyclodextrin. However, when β -cyclodextrin was added, the fluctuant range of RMSF value of SER 90-VAL 125, SER 146-ASP 153 and GLN 282-GLY 287 region was distinctly reduced. The RMSF value is commonly used to evaluate the flexibility of residues, with lower

RMSF values corresponding to a more rigid residue. In the present work, the reduction of the RMSF value using β -cyclodextrin at 333 K in MD simulation correlates well with the enhancement of thermostability by adding β -cyclodextrin at 50 °C in the experiments. Furthermore, the developed strategy restoring the RMSF value might help in selecting other valuable additives for the improvements of thermostability in further application of lipase. Another significant finding of the structural analysis was that YLLIP2 was surrounded by some β -cyclodextrin molecules. Furthermore, a similar region at all temperatures through weak interactions with YLLIP2, which was nearby the thermosensitive region (SER 90-VAL 125), was observed (see Fig. 7). Subsequently, the radial distribution functions (RDF) of β -cyclodextrin around the SER 90-VAL 125 region were computed. According to the data shown in Fig. 8, β -cyclodextrin molecules tended to gather around the region (SER 90-VAL 125) surface. This self-assembly

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Fig. 6. Root mean square fluctuations (RMSF) value of YLLIP2’s residues with β -cyclodextrin (—) and without β -cyclodextrin (- - -) at 333 K for 10 ns MD simulation.

Fig. 7. The modification of β -cyclodextrin with regular region of YLLIP2 at 313 K, 323 K and 333 K was amplified exhibited step by step. Blue denotes the YLLIP2 in ribbon; Gold denotes the β -cyclodextrin molecule in sphere and stick; Green denotes the polar residues of YLLIP2 in stick. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Acknowledgments This work was supported by National Natural Science Foundation of China (NSFC grant nos. 21406011 and 21203006), Amoy Industrial Biotechnology R&D and Pilot Conversion Platform (3502Z20121009), Small and mid-sized enterprise technology innovation project (14C26213511838), Transformation of Agricultural Science and Technology Achievements Project (2013GB2C410539) and Hong Kong, Macao and Taiwan science and technology cooperation projects (L2015TGA0018). Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.jtice.2016.10.035. References Fig. 8. Radial distribution functions (RDF) of β -cyclodextrin around the SER 90-VAL 125 at 313 K (Blue), 323 K (Cyan) and 333 K (Purple) in 10 ns MD simulation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

phenomenon directly facilitated the interactions between cylcodextrine and the thermosensitive region. As shown in Fig. 7 (the closed-up view), the THR 117 and ASN 178 (at 313 K), ASP 121 and LYS 203 (at 323 K) THR 117 and GLU 209 (at 333 K) were located in two original non-interaction secondary structures of YLLIP2. The β -cyclodextrin molecule was close to the above polar residues ˚ and might connect with secondary structures of (within 4.0 A) YLLIP2 through weak interactions (such as electrostatic, van der Waals interactions and hydrogen bonds) with two polar residues as a bridge at 313 K, 323 K and 333 K, respectively [39,40]. The new interaction between the β -cyclodextrin molecule and the polar residues provides a better anchoring of the flexible thermosensitive region of YLLIP2 to the more settled core elements of the lipase (such as catalytic triad), which can provide increased protein stability and aggregation resistance [41]. In a traditional view, β -cyclodextrin could interact with hydrophobic amino acids via its hydrophobic cage [42,43]. In contrast, β -cyclodextrin interaction with polar residues of YLLIP2 was observed in MD simulation due to the presence of hydroxyl groups at the outside of the β cyclodextrin molecule. Fernández et al. have proven the chemical interaction between β -cyclodextrin derivative and polar residues (ASP and GLU) of bovine pancreatic trypsin using reverse-phase chromatography-FABMS [44]. In the present work, β -cyclodextrin exhibited a significant assembly potential with the surface of lipase molecules in water, with improved thermostability firstly demonstrated. This finding will also be used in further work to enhance catalytic characters of YLLIP2 for biotechnological purposes. 4. Conclusion The thermostability of YLLIP2 was significantly increased by utilizing β -cyclodextrin as additive. According to experimental and simulation analysis for secondary structure, both the increased α helix content and stable catalytic triad revealed the improvement of thermostability of YLLIP2. Subsequently, the fluctuating RMSF values were restored in MD simulation through addition of β cyclodextrin, which means that the rigidity of the thermosensitive regions (SER 90-VAL 125, SER 146-ASP 153 and GLN 282-GLY 287) was successfully increased by β -cyclodextrin at 333 K. Finally, the modification of β -cyclodextrin with the thermosensitive region (SER 90-VAL 125) of YLLIP2 thorough interaction with two polar residues (THR 117 and GLU 209) was firstly observed so far at denatured temperatures (333 K) in MD simulation.

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Please cite this article as: H. Cao et al., β -cyclodextrin as an additive to improve the thermostability of Yarrowia lipolytica Lipase 2: Experimental and simulation insights, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2016.10.035