Article pubs.acs.org/IECR
Dramatically Improved Performance of an Esterase for Cilastatin Synthesis by Cap Domain Engineering Zheng-Jiao Luan,†,§ Hui-Lei Yu,†,§ Bao-Di Ma,‡ Yi-Ke Qi,† Qi Chen,† and Jian-He Xu*,† †
State Key Laboratory of Bioreactor Engineering and Shanghai Collaborative Innovation Centre for Biomanufacturing, East China University of Science and Technology, Shanghai 200237, China ‡ School of Chemical and Environmental Engineering, Shanghai Institute of Technology, 100 Haiquan Road, Shanghai 201418, China S Supporting Information *
ABSTRACT: Whole-protein random mutation and substrate tunnel evolution have recently been applied to the pharmaceutically relevant esterase RhEst1 for the synthesis of a cilastatin precursor. The mutant RhEst1 M1 (=RhEst1A147I/V148F/G254A) was identified from a large library consisting of 1.5 × 104 variants. Though the activity of this mutant was improved 5-fold, the enantioselectivity for biohydrolysis decreased at the same time. Herein a smart library (3.0 × 103) focused on the cap domain of RhEst1 was constructed to improve its catalytic performance comprehensively. As a result, a variant designated as RhEst1M2 (=RhEst1M1‑A143T), showed a 6-fold increase in specific activity compared with the wild type. Meanwhile, the decreased enantioselectivity for enzymatic resolution was recovered to the native enzyme level. The melting temperature of RhEst1M2 was nearly 11 °C higher than that of the wild type. This work provides detailed insight into the vital role of α/β hydrolase cap domains in influencing all aspects of enzyme characteristics. Furthermore, the commercial resin ESR-1 with free amino groups was used for enzyme immobilization to enhance the operational performance of RhEst1M2. No obvious activity loss was observed when the immobilized enzyme was incubated at 30 °C for 200 h. The immobilized enzyme could be repeatedly used for up to 20 batches, and the total turnover number (TTN) reached up to 8.0 × 105.
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INTRODUCTION The α/β-fold family is one group of the most frequently reported enzymes in nature, including lipases, carboxylic esterases, peroxidases, dehalogenases, epoxide hydrolases, and proteases.1,2 Though their sequence identities with each other are low, the three-dimensional structures of the α/β core domain share a surprisingly high similarity. The α/β domain is composed of a central parallel or mixed β-sheet surrounded by an α-helix. Meanwhile, the catalytic residues always constitute a highly conserved triad: a nucleophile, an acidic residue, and an absolutely conserved histidine residue. In order to accommodate different substrates or reactions, loops and helices are inserted into the center of the structure during natural evolution. These structures may be composed of only a few residues or large enough to form a complete domain, such as the movable lid domain or the cap domain.3,4 The lid domain can be triggered to open by formation of the lipid−water interface, followed by an activity improvement of the lipases. © XXXX American Chemical Society
The movable lid domain is a distinguishing character for lipases, known as “interfacial activation”.5 Different from lipases, some carboxylic esterases have an α-helical cap domain, which completely covers the catalytic cavity. Though no interfacial activation is observed, the cap domain may influence the enzyme stability, substrate binding, turnover frequency, and product release. To date, the cap domain engineering approach has been developed to study the hydrolase catalysis mechanism and to promote catalytic efficiencies.6,7 The cap domain fragment of BsteE was incorporated into the homologous gene encoding for BsubE, and the melting temperature (Tm) of the hybrid was increased by 4 °C compared with BsubE.8 Molecular dynamics Received: Revised: Accepted: Published: A
June 25, 2016 November 1, 2016 November 9, 2016 November 9, 2016 DOI: 10.1021/acs.iecr.6b02440 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
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Industrial & Engineering Chemistry Research (MD) simulations of haloalkane dehalogenase DhlA showed high flexibility in the cap domain, involving residues 184−211. A disulfide cross-link was engineered between residue 201 of the cap domain and residue 16 of the main domain. The oxidized form of the mutant enzyme showed an increase in the apparent transition temperature from 47.5 to 52.5 °C.9 A range of mutation experiments has emphasized the importance of the cap domain in haloalkane dehalogenases.10 Phe172 is located in a helix−loop−helix cap domain that covers the active-site cavity of the haloalkane dehalogenase. The mutant Phe172Trp showed 10-fold higher kcat/Km toward 1-chlorohexane than the wild type. The X-ray structure of the Phe172Trp enzyme showed a local conformational change in the cap domain and allowed the large substrate to bind more easily in the active-site cavity.11 MD trajectories of three haloalkane dehalogenases (DhlA, LinB, and DhaA) showed that the difference of water exchange among the three dehalogenases might be associated with the flexibility of their cap domains.12 These facts all underline the significance of the cap region for enzyme stability and catalytic efficiency. RhEst1, a pharmaceutically relevant carboxylic esterase discovered in our laboratory from Rhodococcus sp. ECU1013, exhibited excellent enantioselectivity toward the asymmetric hydrolysis of ethyl 2,2-dimethylcyclopropanecarboxylate (DmCpCe) for preparing (S)-2,2-dimethylcyclopropane carboxylic acid [(S)-DmCpCa].13,14 (S)-DmCpCa is the key chiral building block for the synthesis of cilastatin, which is used to prevent the breakdown of imipenem, a carbapenem antibiotic drug.15−18 We previously performed random mutagenesis (with a library of 104 mutants) and site-directed saturation mutagenesis around the substrate tunnel (5.0 × 103 mutants) to improve the catalytic efficiency of RhEst1.19 The resulting mutant RhEst1M1 (=RhEst1A147I/V148F/G254A) showed a 5-fold increase in specific activity compared with the wild type as well as a 4-fold increase in protein solubility. However, the eep value dropped to 90% eep at around 42% conversion. The requirements for both activity improvement and enantioselectivity maintenance were not satisfied simultaneously. On the basis of the first-generation mutant enzyme RhEst1M1, a new round of evolution focusing on the cap domain was carried out in the present work to further improve the catalytic performance of the biocatalyst (Figure 1). Moreover, enzyme immobilization was also tried in order to enhance the operational stability of the best mutant and to facilitate the repeated utilization of the pharmaceutically important biocatalyst.
Figure 1. (A) Biocatalytic resolution of (±)-DmCpCe for the synthesis of a cilastatin precursor mediated by esterase RhEst1. (B) Domain composition of the RhEst1 monomer. The core α/β-fold domain is shown in gray, and the cap domain is shown in blue. The catalytic triad residues S101, D225, and H253 are shown as sticks. The topology diagram of RhEst1 shows the insertion of the cap domain (blue) between β6 and α9.
(25% v/v) was purchased from Lingfeng Chemical Reagent Co., Ltd. (Shanghai, China). Construction of the Local Mutation Library Focused on the Cap Domain. Random mutagenesis was performed on the cap domain (amino acids 125−207) of RhEst1M1 (Table S1 and Figure S1). For the cap domain of the target gene (fragment 1, base pairs 375−621), 200 μM Mn2+ was used to obtain the desired mutagenesis rate. Primers F1 and R1 were designed for error-prone polymerase chain reaction (PCR) of fragment 1, and F2/R2 and F3/R3 were used for the highfidelity amplification of the other two parts of the gene (fragments 2 and 3). The three parts were amplified by overlap extension PCR with high fidelity. The following steps for random library construction and screening were performed as described previously.19 Site-Directed Evolution of the α4- and α5-Helices. Site-directed saturation mutagenesis of the α4- and α5-helices, belonging to the cap domain, was performed using RhEst1M1 as the parent gene. Primers for mutations are shown in Table S2. The process for site-directed mutagenesis and library screening was described previously.19 Kinetic Parameter Determination toward (S)-DmCpCe or (R)-DmCpCe. The kinetic parameters of the purified variants toward the substrate (S)-DmCpCe or (R)-DmCpCe were determined by measuring the activity at varied substrate concentrations (0.1−10 mM). The Michaelis−Menten constant (Km) and the maximal reaction rate (Vmax) of the enzyme were calculated from Lineweaver−Burk plots. Circular Dichroism Spectroscopy. The melting temperature was measured by circular dichroism (CD) spectroscopy. The Tm values of RhEst1 and its variants were measured on a Chirascan CD spectrometer (Applied Photophysics Ltd., Leatherhead, UK) equipped with a TC125 temperature-control system (Quantum Northwest, Liberty Lake, WA, USA). The unfolding curves were measured from 200 to 280 nm at temperatures of 30−80 °C using the temperature scan mode with a gradient of 1 °C/min. The purified enzyme was diluted
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MATERIALS AND METHODS Materials. Racemic ester (RS)-DmCpCe was prepared from the acid (RS)-DmCpCa and ethanol by chemical esterification in our laboratory. The acid (RS)-DmCpCa was obtained commercially with an analytical grade (Zhejiang Hisoar Pharmaceutical Co., Ltd.). Tryptone and yeast extract were obtained from Oxoid (Shanghai, China). rTaq polymerase, restriction endonucleases (DpnI, EcoRI, HindIII), T4 DNA ligase, and PrimeSTARHS were all purchased from Takara Biotechnology Co., Ltd. (Dalian, China) and stored at −20 °C. Primers for RhEst1 cap domain engineering were synthesized by Generay Biotech Co., Ltd. (Shanghai, China) (Tables S1 and S2). The plasmid RhEst1M1 constructed was used as the parent for cap domain evolution. The resins used for immobilization of the enzyme were kindly donated by Nankai Hecheng S. & T. Co., Ltd. (Tianjin, China). Glutaraldehyde B
DOI: 10.1021/acs.iecr.6b02440 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
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Figure 2. Structural comparison of RhEst1 with other reported α/β hydrolases from Pseudomonas fluorescens. The enzymes show differences in active-site accessibility. The catalytic serine residue is presented as sticks. The substrate tunnel is shown in the transverse figures. (A) RhEst1 (PDB ID 4RNC). (B) Chloroperoxidase CPO-F (PDB ID 1A8S). (C) Carboxylesterase PFE (PDB ID 1VA4). (D) Carboxylesterase (PDB ID 1AUO).
activated resin was added to the enzyme solution, and the following steps were similar to the procedures described above. Repeated Reaction of Immobilized Enzyme. The 10 mL reaction was performed at 30 °C and 200 rpm. The reaction mixture contained 0.2 M phosphate buffer (pH 8.0), 0.3 g of immobilized enzyme, and 100 mM (RS)-DmCpCe (with 0.5% w/v Tween-80 to help with dispersion). Samples were taken periodically to measure the conversions and eep values. The immobilized enzyme was isolated when the conversion reached 45% and then washed with buffer for reuse in the next round of reaction.
to 0.1 mg of protein/mL in 10 mM potassium phosphate buffer (pH 7.0). Preparation of (S)-DmCpCa Using RhEst1 Variants. Enzymatic resolution of (±)-DmCpCe by the variants and the product detection method were the same as described previously.19 For preparative resolution, (±)-DmCpCe (50 mmol) was dissolved in 100 mL of potassium phosphate buffer (100 mM, pH 8.0). Lyophilized cell-free extracts (0.5 g) were added to initiate the reaction. The pH was automatically maintained around 8.0 by titration with 1 M K2CO3. After the reaction was terminated, the reaction mixture was extracted with dichloromethane under alkaline conditions to remove the residual substrate; the aqueous phase was then acidified to pH < 2.0. The resultant suspension was extracted with 100 mL of dichloromethane three times. The organic layers were combined, dried over anhydrous sodium sulfate, and evaporated under vacuum. Immobilization of Mutant Esterase RhEst1M2. The mutant RhEst1M2 was immobilized on amino group resins (ESR1, ESR2, and ESR3) according the following procedures.20 First, the resins were activated by glutaraldehyde for enzyme immobilization. Each of the resins (0.5 g) and 1.6 mL of glutaraldehyde solution (25% w/v) were added into 6.4 mL of phosphate buffer (0.2 M, pH 7.8), and the resultant mixtures were then shaken at 170 rpm and 25 °C for 2.5 h. The resins were collected by centrifugation and then rinsed five times with distilled water to remove the free glutaraldehyde. Second, each activated resin (0.2 g) were added to a 5 mL solution of the enzyme containing 1.5 mg of protein (in 0.2 M phosphate buffer, pH 7.8). The resulting mixtures were shaken at 20 °C and 170 rpm for 6 h. The immobilized enzyme was then collected and washed with phosphate buffer until no free protein could be detected in the wash buffer. The steps for enzyme immobilization on the epoxy group resins (ES1, ES101, and ES103) were very simple: 0.5 g of each resin was washed with phosphate buffer (0.2 M, pH 7.8) three times. Then the
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RESULTS AND DISCUSSION Cap Domain Engineering of Esterase RhEst1. The crystal structure of RhEst1 (PDB ID 4RNC) comprises two domains: the α/β core domain and the V-shaped cap domain. The active-site Ser101 is located in the center of RhEst1. The whole structure results in an unusually long and narrow activesite pocket with topological features to restrict binding to small substrates. The cap domain consisting of four helices lies on the top of the main domain, and the two parts are connected by loops. The main domain is rigid and connected to the core properties of the enzyme, whereas the cap domain shows considerably higher mobility and might function in adapting its structure to a specific substrate. Homologous sequence alignment indicated a high diversity for the positions between amino acids 140 and 200 in the central part of RhEst1, which was predicted as the cap domain.14 Meanwhile, comparison of the crystal structure of RhEst1 with other reported α/β hydrolases showed that the cap domain has a great influence on the characteristics of the substrate tunnel. Figure 2 displays the high similarity of the core domain fold for different types of α/ β hydrolases, while differences in the cap domains resulted in diversity of the substrate tunnel, including its shape, size, and hydrophobicity. These differences greatly influence the catalytic properties of the enzyme toward a specific substrate. C
DOI: 10.1021/acs.iecr.6b02440 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
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lectivity toward the hydrolysis of (S)-DmCpCe. The eep value of this mutant was improved to 96% when the conversion reached 47%, whereas for RhEst1M1‑R145E and RhEst1M1‑A143F there was no significant change in the enantioselectivity compared with RhEst1M1. To confirm the feasibility of the biotransformation process on a preparative scale (100 mL), (RS)-DmCpCe was resolved with the variant RhEst1M2. The reaction was conducted at 30 °C, and the pH of reaction system was controlled around 8.0. The catalyst (5 g/L, lyophilized cell-free extract) was loaded for the bioresolution of 500 mM (RS)-DmCpCe. After 6.5 h, the reaction was terminated with 45% conversion, affording 2.3 g of (S)-DmCpCa with 96% eep. Enantioselectivity Analysis of RhEst1WT and Its Variants. Kinetic parameters of the wild type and mutants toward (S)/(R)-DmCpCe were determined to illustrate the improvement in hydrolysis enantioselectivity (Table 2). Compared with those for the parent RhEst1M1, kcat of RhEst1M2 toward (S)-DmCpCe was further enhanced while that toward (R)-DmCpCe was decreased. This phenomenon illustrates that the RhEst1M2 mutant might more easily form the tetrahedral intermediate of (S)-DmCpCe, which promotes the enantioselectivity of hydrolysis. Therefore, RhEst1M2 was chosen for further research. Thermostabilities of the Native Enzyme and Its Variants. The melting temperature (Tm) of RhEst1M2 was 1.7 °C higher than that of its parent RhEst1M1 and nearly 11 °C higher than that of the wild type (Table 3). All-atom MD
On the basis of the crystal structure analysis and our previous research, the present work was focused on engineering of the cap domain. An enriched random library was constructed for the positions between amino acid residues 125 and 207 in the cap domain using RhEst1M1 as the parent. The loop region connecting the cap domain and the core structure was also included, considering that this loop region might influence the entrance of substrate. The mutant RhEst1M2 (=RhEst1M1‑A143T) was identified from 2000 mutants showing a higher enantioselectivity of DmCpCe hydrolysis than RhEst1M1. Together with our previous research, the sensitive mutation sites for activity and selectivity, including residues 143, 147, and 148, were mainly located on the α4- and α5-helices of the cap domain. Therefore, the site-directed saturation mutagenesis was then focused on the α4- and α5-helices (the positions between 142 and 152) with RhEst1M1 as the parent. Library screening (1000 mutants) and sequence analysis showed that the variants RhEst1 M 2 (=RhEst1 M 1 ‑ A 1 4 3 T ), RhEst1 M 1 ‑ A 1 4 3 F , and RhEst1M1‑R145E had higher specific activity toward DmCpCe (Table 1). The mutant RhEst1M2 was screened out during this Table 1. Enzymatic Resolution of (RS)-DmCpCe by WildType RhEst1 and Its Variants enzyme WT M1a M2b M1-A143F M1-R145E M1-A143T-R145E
specific activity (unit/mg of protein)
time (h)
conv. (%)
eep (%)
± ± ± ± ± ±
15 4.0 2.0 2.5 5.5 4.0
9.4 41 47 47 46 48
97 92 96 91 91 95
0.17 0.78 1.08 0.99 1.43 0.89
0.01 0.02 0.02 0.01 0.01 0.01
Table 3. Tm Determination for RhEst1 and Its Variants
a
M1 refers to the mutant RhEst1A147I/V148F/G254A. bM2 refers to the mutant RhEst1M1‑A143T.
round of evolution for its recovered 96% enantioselectivity compared with the parent RhEst1M1 and also for the nearly 1.5fold higher activity compared with RhEst1M1 and the 6-fold activity improvement relative to the wild type (WT) enzyme (RhEst1). The activities of the mutant RhEst1M1‑R145E and other A143 mutants were also increased to different degrees, but the enantioselectivity was not changed much compared with RhEst1M1. Moreover, no further activity improvement was obtained by combining the two mutation sites A143T and R145E together. Enzymatic Resolution of (RS)-DmCpCe Using RhEst1WT or Its Variants. For further enzymatic resolution, the mutant RhEst1M2 (=RhEst1M1‑A143T) showed the best performance. Under the same conditions, the reaction catalyzed by RhEst1M2 achieved 47% conversion after 2.0 h. In contrast, the wild type reached just 9.4% conversion after 15 h. More importantly, the mutant RhEst1M2 showed excellent enantiose-
mutant
Tm (°C)
RhEst1 RhEst1M1 RhEst1M2
48.3 ± 0.1 57.5 ± 0.2 59.2 ± 0.1
simulations indicated that α-helices and loops in the cap domain exhibit high fluctuations, showing relatively high Bfactors.21 Mutations of these sites might greatly improve the enzyme thermostability and simultaneously decrease the thermal motion and positional disorder of these sites.22 The improvement of thermostability for this enzyme has provided a significant clue that indicates the relationship between cap domain engineering and enzyme thermostability. Meanwhile, the mutant with higher stability might be helpful for the enzymatic resolution and enzyme immobilization. The Cap Domain Significantly Influences the Catalytic Properties of RhEst1. The evolutionary pathway of RhEst1 reflects that the cap domain plays a vital role in the protein engineering. All of the mutation sites related to either enzyme activity or enantioselectivity were focused on the cap domain
Table 2. Kinetic Constants of RhEst1 and Its Variants toward (R)-DmCpCe and (S)-DmCpCe mutant RhEst1 RhEst1M1 RhEst1M2
a
DmCpCe
Km (mM)
kcat (min−1)
kcat/Km (min−1mM−1)
E valuea
(S) (R) (S) (R) (S) (R)
0.26 ± 0.08 1.7 ± 0.5 0.18 ± 0.05 1.4 ± 0.1 0.71 ± 0.13 3.6 ± 0.7
3.2 ± 0.3 0.23 ± 0.03 13 ± 0.8 2.2 ± 0.1 27 ± 2 1.4 ± 0.2
12 0.14 72 1.6 38 0.39
86 45 97
E value= [kcat/Km for (S)-DmCpCa]/[kcat/Km for (R)-DmCpCa]. D
DOI: 10.1021/acs.iecr.6b02440 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
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Industrial & Engineering Chemistry Research step by step. Figure 3 presents B-factor variation during protein engineering. The whole evolution process of RhEst1 decreased
Figure 4. Reusability of RhEst1M2@ESR1 for the kinetic resolution of 100 mM (RS)-DmCpCe. Reactions were conducted at 30 °C in 200 mM phosphate buffer (pH 8.0). After each reaction run, the immobilized enzymes were collected, washed with buffer, and reused in the next round of reaction.
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CONCLUSIONS On the basis of the protein’s structure and our former research, error-prone PCR and site-directed saturation mutagenesis approaches were performed focusing on the cap domain. Screened from the smart library, RhEst1M2 showed a 6-fold increase in its specific activity compared with the native enzyme. Kinetic analysis and structure prediction revealed that mutation of these sites greatly influences the substrate binding and enzyme activity for the two opposite enantiomers of the substrate. Determination of the melting temprature showed that the thermostability of the RhEst1M2 mutant was greatly improved. This is consistent with the flexibility of the cap domain, which has a high B-factor in the whole structure. This work suggests that the cap domain of α/β hydrolase appears to be an important element for fine-tuning the catalytic performance of the enzyme. Enzyme immobilization on a resin with amino groups allowed the biocatalyst to be recycled for up to 20 runs. The TTN of the hydrolysis was enhanced to 8.0 × 105, and the productivity of the immobilized RhEst1M2 was improved to 2.5 g of product/g of catalyst after immobilization.
Figure 3. Evolutionary pathway of RhEst1. B-factors of RhEst1 and its mutant RhEst1M2 are shown. The B-factor of the mutant RhEst1M2 was predicted using the I-TASSER suite.23−25
the fluctuation frequency of the cap domain, which might facilitate binding of the substrate to the active site of the enzyme. The space-time yield (STY) of the mutant RhEst1M2 reached 53 g L−1 day−1, which is 50 times higher than that of the wild type. Also, the thermostability of the mutant was improved greatly. Meanwhile, the loops connecting the cap domain and the α/ β-fold domain should also be considered because these loops have a direct impact on the shape of substrate channel entrance. For example, Val140 is situated directly at the entrance of the substrate channel. In our former research, mutations of this site could improve the enzyme specific activity toward DmCpCe, although the enantioselectivity for substrate hydrolysis decreased obviously. The evolution of the RhEst1 cap domain could skillfully balance the enzyme activity, enantioselectivity, and thermostability, which are difficult to coordinate with each other at the same time. Repeated Batch Reaction of the Immobilized Enzyme. Enzyme immobilization can enhance the operational stability of biocatalysts. Meanwhile, it facilitates the recovery of enzymes in the downstream process, making the enzyme recyclable and decreasing the cost of the reaction.26−30 Herein, a carrierbinding immobilization strategy was adopted to optimize the operational performance of the mutant RhEst1M2 in batch reactions. The resins with amino groups showed a higher activity recovery, and the specific activity of the immobilized enzyme for RhEst1M2@ESR1 was 6.0 units/g (Figure S2 and Table S3). Enzyme immobilization further improved the thermostability of the mutant. There was no activity loss when the immobilized enzyme was incubated at 30 °C for 200 h (Figure S3). Figure 4 shows that RhEst1M2@ESR1 exhibited excellent operational stability. The immobilized enzyme could be repeatedly used for up to 20 batches with nearly no activity loss. A high total turnover number (TTN) of 8.0 × 105 was obtained using the immobilized enzyme. The product-tocatalyst ratio (P/C) of the immobilized enzyme was improved from 0.4 to 2.5 g/g.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.iecr.6b02440. Additional experimental results, including primers for cap domain engineering, parameters for enzyme immobilization on resins, thermostability of the mutant, and other data (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Fax: +86-21-6425-0840. Author Contributions §
Z.-J.L. and H.-L.Y. contributed equally to this paper.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (21276082, 21536004, and E
DOI: 10.1021/acs.iecr.6b02440 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
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operation of a continuous packed bed reactor. Biochem. Eng. J. 2016, 107, 45−51. (21) Chen, Q.; Luan, Z. J.; Yu, H. L.; Cheng, X.; Xu, J. H. Rational design of a carboxylic esterase RhEst1 based on computational analysis of substrate binding. J. Mol. Graphics Modell. 2015, 62, 319−324. (22) Reetz, M. T.; Carballeira, J. D.; Vogel, A. Iterative saturation mutagenesis on the basis of B factors as a strategy for increasing protein thermostability. Angew. Chem., Int. Ed. 2006, 45, 7745−7751. (23) Yang, J.; Yan, R.; Roy, A.; Xu, D.; Poisson, J.; Zhang, Y. The ITASSER suite: protein structure and function prediction. Nat. Methods 2015, 12, 7−8. (24) Roy, A.; Kucukural, A.; Zhang, Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nat. Protoc. 2010, 5, 725−738. (25) Zhang, Y. I-TASSER server for protein 3D structure prediction. BMC Bioinf. 2008, 9, 40−48. (26) Mateo, C.; Palomo, J. M.; Fernandez-Lorente, G.; Guisan, J. M.; Fernandez-Lafuente, R. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb. Technol. 2007, 40, 1451−1463. (27) Sheldon, R. A. Enzyme immobilization: The quest for optimum performance. Adv. Synth. Catal. 2007, 349, 1289−1307. (28) Tran, D. N.; Balkus, K. J. Perspective of recent progress in immobilization of enzymes. ACS Catal. 2011, 1, 956−968. (29) Barbosa, O.; Torres, R.; Ortiz, C.; Berenguer-Murcia, A.; Rodrigues, R. C.; Fernandez-Lafuente, R. Heterofunctional supports in enzyme immobilization: from traditional immobilization protocols to opportunities in tuning enzyme properties. Biomacromolecules 2013, 14, 2433−2462. (30) Pan, J.; Dang, N. D.; Zheng, G. W.; Cheng, B.; Ye, Q.; Xu, J. H. Efficient production of L-menthol in a two-phase system with SDS using an immobilized Bacillus subtilis esterase. Bioresour. Bioprocess. 2014, 1, 12−18.
31500592) and the Shanghai Science and Technology Program (15JC1400403).
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DOI: 10.1021/acs.iecr.6b02440 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
