Micro-Biocatalyst: Soybean Epoxide Hydrolase

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Novel Nano/micro-Biocatalyst: Soybean Epoxide Hydrolase Immobilized on UiO-66-NH2 MOF for Efficient Biosynthesis of Enantipure (R)-1, 2-Octanediol in Deep Eutectic Solvents Shi-Lin Cao, Dong-Mei Yue, Xuehui Li, Thomas John Smith, Ning Li, Min-Hua Zong, Hong Wu, Yongzheng Ma, and Wen-Yong Lou ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b00777 • Publication Date (Web): 18 May 2016 Downloaded from http://pubs.acs.org on May 19, 2016

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ACS Sustainable Chemistry & Engineering

Novel Nano/micro-Biocatalyst: Soybean Epoxide Hydrolase Immobilized on UiO-66-NH2 MOF for Efficient Biosynthesis of Enantipure (R)-1, 2-Octanediol in Deep Eutectic Solvents Shi-Lin Cao 1,2,3,4, Dong-Mei Yue 1,3, Xue-Hui Li 2, Thomas J Smith 5, Ning Li 3, Min-Hua Zong 2,3, Hong Wu 3, Yong-Zheng Ma 4,Wen-Yong Lou 1,3,*, 1

Lab of Applied Biocatalysis, School of Food Science and Engineering, South China

University of Technology, No. 13 Building, 381th Wushan Road, Guangzhou 510640, China. 2

School of Chemistry and Chemical Engineering, South China University of

Technology, No. 16 Building, 381th Wushan Road, Guangzhou 510640, China. 3

State Key Laboratory of Pulp and Paper Engineering, South China University of

Technology, Zaozhi Building, 381th Wushan Road, Guangzhou 510640, China. 4

Department of Applied Biology and Chemical Technology, The Hong Kong

Polytechnic University, Block Y, 1st Yuk Choi Rd, Kowloon, Hong Kong. 5

Biomolecular Sciences Research Centre, Sheffield Hallam University, Owen

Building, Howard Street, Sheffield, S1 1WB, UK *Corresponding author. Tel.: +86-20-22236669; fax: +86-20-22236669. E-mail address: [email protected] (Prof. W. Y. Lou).

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Abstract: The nano/micro-scale UiO-66-NH2 metal-organic framework (MOF) materials were successfully prepared with a uniform size of about 350-400 nm and structurally characterized.

Soybean epoxide hydrolase (SEH), a useful hydrolase for

synthesis of valuable vicinal diols, was for the first time efficiently immobilized onto the prepared UiO-66-NH2 MOF. The resulting novel nano/microbiocatalyst SEH@UiO-66-NH2 manifested high SEH loading (87.3 mg/g) and enzyme activity recovery (88.0%). The novel SEH@UiO-66-NH2 greatly surpassed the free SEH with resepct

to

pH-,

thermo

stabilities

and

tolerance

to

organic

solvents.

SEH@UiO-66-NH2 retained more than 17.6 U activity after 2 h incubation at 45oC, whereas free SEH maintained around 10.1 U activity under the same condition. After storage at 4 oC for 4 weeks, the prepared SEH@UiO-66-NH2 still retained around 97.5% of its initial activity. The significant enhancements resulted from the increase of structure rigidity of SEH@UiO-66-NH2, which was demonstrated by the secondary structure analysis of the enzyme. The optimun pH and tempearture of SEH@UiO-66-NH2 were significantly superior to the corresponding levels of its free counterpart.

Also,

SEH@UiO-66-NH2

manifested

markedly

enhanced

enzyme-substrate affinity and catalytic efficiency compared to free SEH, as supported by

a lower apparent Km value (6.5 vs. 19.2 mM) and a increased Vmax/ Km value

(8.0×10-3 vs.

5.8×10-3

min-1),

respectively.

Furthermore,

the

as-prepared

SEH@UiO-66-NH2, for the first time, was successfully applied as an efficient biocatalyst for the asymmetric hydrolysis of 1, 2-epoxyoctane to (R)-1,2-octanediol in novel deep eutectic solvent (DES) with a yield of around 41.4% and a product e.e. value of 81.2%. Remarkably, the nano/micro-scale UiO-66-NH2 MOFs as novel

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enzyme support materials are promising for enzyme immobilization and the prepared SEH@UiO-66-NH2 exhibited great potential for efficient biosynthesis of enantipure (R)-1,2-octanediol.

Keywords: Metal Organic Framework, Epoxide Hydrolase, Dipiptide, Deep eutectic solvent, Optically Active Vicinal Diols, Green Chemistry, Sustainable Chemistry.

INTRODUCTION Metal organic frameworks (MOFs), a class of hybrid material composed of metal ions and organic ligands (linkers) connected via coordinate bond to form two- or three-dimensional reticular crystalline structure1-3, have attracted increasing attention because of their unique diverse topologies, porosity and other favorable physical characteristics4. Presently, MOFs are mainly used in gas storage5-7, and homogeneous and

heterogeneous

catalysis8-10.

As

described

in

previous

reports11-13,

microperoxidase-11, laccase, trypsin and lipase have been successfully immobilized at Tb-meso MOF, MOF-199, MIL-88B and MIL-53(Al), respectively. The main immobilization methods used to date have been encapsulation14, and physical adsorption11, 15-17. When the enzymes was trapped into MOF mesopores, the reported immobilization process was time-consuming and in some cases 50 hours was necessary for the adsorption11. Also, the enzyme loading capacity of the MOF needed to be improved 16, 18. In view of the above limitations, chemical cross-linking has been used to attach enzyme covalently to the MOF, as well as complex post-synthesis



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modification of MOF was utilized19. However, the post-synthesis modification only gave the carrier the limited active sites, which could influence the protein loading and relative activity recovery. As an alternative to the micro-scale MOFs described in the above reports, nano/micro-scale MOFs can be produced with uniform sizes and morphologies so that they can be well dispersed not only in aqueous media but also in other solvents20. Our recent study21 showed that the enzyme papain could be immobilized using formaldehyde onto a magnetic cellulose nano-material had enhanced activity, stability, catalytic efficiency and enzyme-substrate affinity22. This stimulated our interest to explore a relatively simple and chemically milder ways to prepare amino-functionalized MOFs with good solvent stability that could be used as the enzyme-carrier without further modification. Moreover, our previous literature indicated that both papain22 and pseudomonas cepacia lipase23 can be easily immobilized onto the surface of the nano-carrier via the precipitation-crosslinking method.

In order to explore the universality of this method, in the work described

here, we firstly immobilized enzyme onto the surface of the nano/micro-scale UiO-66-NH2 MOF via the precipitation-crosslinking method. The enzyme loading amount was improved and the immobilization time was shortened compared to previous studies. Epoxide hydrolase (EH) was chosen as the enzyme to test the as-prepared new nano/micro-material. Optically active vicinal diols and epoxides, which can be obtained from EH-mediated asymmetric hydrolysis of racemic epoxides, are important building blocks in the production of many bioactive compounds such as leukotriene, insect pheromones, steroides, β-blockers, adrenaline, nerve protectants, 3 ACS Paragon Plus Environment

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and HIV protease inhibitors24, and the starting materials of ferroelectric liquid crystals25, as well as optically pure carboxylic acids, esters and other high-added-value

chemicals26.

Here,

hydrolysis

(R)-1,2-octanediol was used as the test reaction.

of

1,2-epoxyoctane

to

This product is used for treating

head louse infestations and as a starting material for production of ferroelectric liquid crystals. Compared with EHs from microorganisms, EHs from plants have many advantages such as a wide range of potential sources, low cost and facile preparation 27-31

. The observed product e.e. of epoxide hydrolysis catalyzed by EHs from

microorganisms is relatively low in some instances32-34. In contrast, it has been found that mung bean EHs can enantioselectively catalyze the hydrolysis of epoxides to produce enantiopure vicinal diols such as (R)-1-phenyl-1, 2-ethanediol, and (R)-p-nitrophenylglycol35-38. Nowadays, many researchers have focused on the enzymatic catalysis in deep eutectic solvent (DES)-containing systems. DESs are a kind of sustainable solvents, which are more biodegradable and less toxic than many traditional organic solvents, and can be conveniently obtained by mixing hydrogen bond donors and ammonium salt39. DESs have been efficiently used for several enzymatic processes such as lipase40, potato epoxide hydrolase41, protease22, and the result showed that the enzyme exhibited higher activity and stability in the presence of DESs. Widersten et.al

41

firstly reported the efficient use of DESs as co-solvents for

the hydrolysis of (1,2)-trans-2-methylstyrene oxide catalyzed by potato epoxide hydrolase with significantly increased solubility of substrate and regioselectivity of product. Therefore, it is of great interest to explore the catalytic performances of SEH in DES-containing co-solvent systems.



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In the present study, we firstly carried out a comparative study on catalytic efficiency of EHs from five different plants for the asymmetric hydrolysis of 1, 2-epoxyoctane to (R)-1, 2-octanediol. Soybean epoxide hydrolase (SEH), a dimer and non-glycosylated protein with a molecular mass of about 31 kDa40, was found the most suitable enzyme for the reaction.

SEH was effectively immobilized onto the

surface of nano/micro-scale UiO-66-NH2 MOF support by using the precipitation and cross-linking method, and a comparative study was performed of the enzymatic hydrolysis of 1, 2-epoxyoctane catalyzed by the immobilized SEH (SEH@ UiO-66-NH2) and the free SEH. The obtained results show that SEH@ UiO-66-NH2 is an efficient biocatalyst for the asymmetric hydrolysis of 1,2-epoxyoctane to (R)-1,2-octanediol and that deep eutectic solvent (DES)-containing system can be used to further improve the product yield and the e.e. value. To the best of our knowledge, this is the first report of the asymmetric hydrolysis of 1, 2-epoxyoctane catalyzed by EHs from a plant source in DES-containing system.

EXPERIMENTAL SECTION Preparation of the UiO-66-NH2 metal-organic frameworks UiO-66-NH2 (all samples of UiO-66-NH2 were prepared by stirring unless stated otherwise): the synthesis of UiO-66-NH2 was performed by dissolving 1.4914g ZrCl4 (6.4mmol) and 1.1464g H2N-H2BDC in DMF (about 100 mL) in a beaker

42

. The

beaker was sealed with plastic wrap and placed in a preheated 80 oC oil-bath pan on a heating magnetic stirrer and stirred for 2h. The reaction was then heated to 100 oC for 4h. The reaction was cooled in air to room temperature and filtered. After washing 5 ACS Paragon Plus Environment

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three times with ethanol, the product was dried at 70 oC overnight in a drying oven. The resulting pale yellow powder was the metal-organic frameworks UiO-66-NH2. Preparation of the immobilized SEH (SEH@UiO-66-NH2) SEH immobilized onto UiO-66-NH2 (SEH@UiO-66-NH2) was prepared as follows. The as-prepared UiO-66-NH2 MOF (50 mg) was mixed with crude SEH preparation (5 mg of crude protein containing 0.625 mg of SEH) in PBS (phosphate buffer saline, 2ml, 50 mmol/L, pH 7.5), followed by adding 100% saturated ammonium sulfate solution (to give 80% final saturation) to precipitate the enzyme under magnetic stirring for 30 min at 4 oC. Then an appropriate amount of 25% glutaraldehyde (GA) was injected into the above mixture and stirred to cross-link the enzyme to the UiO-66-NH2 MOF. After cross-linking, the SEH@UiO-66-NH2 was then washed at least 3 times with PBS (50 mmol/L, pH 7.5) containing 10% glucose (m/v) and stored at 4 oC for subsequent use. The conditions to immobilize the enzyme onto the UiO-66-NH2 support, including GA concentration, cross-linking time and mass ratio of SEH to UiO-66-NH2 were studied. Assay of SEH activity The initial activity of the free SEH and the SEH@UiO-66-NH2 was determined as follows: a given amount of crude SEH protein or immobilized SEH was dispersed in 4 mL PBS buffer (50mmol/L, pH 6.75) containing 1,2-epoxyoctane (10mmol/L). The reaction was conducted for 10 min at 200r/min and 37.5 oC. Then, a sample (50µL) was taken out and the product was extracted from the sample with equivalent



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ethyl acetate. The product concentration was analyzed by GC described below. One unit (U) of enzyme activity (initial reaction rate) was defined as the amount of enzyme required to catalyze the release of 1 µM 1, 2-octanediol per min under the given conditions. For the immobilized SEH, the relative activity recovery was calculated as below:

Relative activity recovery % =

ℎ ℎ ! ℎ ℎ "

Enzymatic properties of free SEH and SEH@UiO-66-NH2 Optimal pH and temperature: the activities were measured according to the method described above over a pH range from 6.0 to 8.5 and a temperature range from 25 to 50 oC. Thermo-stability: 10.0 mg of crude SEH protein containing 1.25 mg of SEH or 125 mg of SEH@UiO-66-NH2 (containing 10 mg of crude SEH protein) were incubated in PBS (50 mmol/L, pH 6.7) at a range of temperatures (20–45oC) for 2 h, and the residual activity was determined as above. pH-stability: 10.0 mg of crude SEH protein containing 1.25 mg of SEH or 125 mg of SEH@UiO-66-NH2 (containing 10 mg of crude SEH protein) with various pH values (6.0 – 8.0) for 2 h at 37 oC, and the residual activity was determined as above. Tolerance to organic solvent: 10.0 mg of crude SEH protein containing 1.25 mg of SEH or 125 mg of SEH@UiO-66-NH2 (containing 10 mg of crude SEH protein) were incubated in 4 mL of various hydrophobic organic solvents (ethyl acetate, 7 ACS Paragon Plus Environment

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n-octyl alcohol, isopropyl ether, cyclohexane, n-hexane) at 37.5 oC for 2 h, and the residual activity of the enzyme was assayed as above. The initial activity was defined as 100%. Storage-stability: the immobilized enzyme SEH@UiO-66-NH2 was stored at 4 oC for a range time periods, and then the residual activity of the enzyme was assayed as above. The activity of newly prepared immobilized was defined as 100%. Apparent kinetics parameters: the kinetic parameters of free SEH and SEH@UiO-66-NH2 were determined according to the previous study36 with varied concentrations of 1,2-epoxyoctane from 1 to 25 mmol/L in PBS (50 mmol/L, pH 6.7) at 37.5 oC. 8 mg of SEH was used to start the reaction in each case. The maximum reaction rate (Vmax) and the Michaelis-Menten constant (Km) were calculated from Hanes-Woolf plots. Biocatalytic asymmetric hydrolysis of 1,2-epoxyoctane with SEH@UiO-66-NH2 in aqueous and DES-containing systems Aqueous buffer system: 4 mL of PBS (50 mmol/L, pH 6.7) containing 2% (v/v) DMSO and 5 mmol/L 1, 2-epoxyoctane was added into a 25-mL Erlenmeyer flask capped with a septum. After the mixture was incubated in a water-bath shaker for 5 min at 37.5 oC, the reaction was initiated by adding 15.0 mg of crude SEH protein containing 1.875 mg of SEH or 187.5 mg of SEH@UiO-66-NH2 (containing 15.0 mg of crude SEH protein) to the system. Samples (60µL) were taken at specified time intervals. The product was extracted with an equal volume of ethyl acetate



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containing 4’-chloroacetophenone as the internal standard and subsequently was analyzed by GC-2010. DESs containing system: 4 mL of the system consisting of PBS (50mmol/L, pH 7.0), and the four DESs above (15%, v/v) was contained in a 25-mLErlenmeyer flask capped with a septum. The reaction was started by adding 187.5 mg of SEH@UiO-66-NH2 (containing 15 mg of crude SEH protein) into the flask after incubated for 5 min at 40 oC. The samples were handled as those taken from the aqueous buffer system.

RESULTS AND DISSCUSSION Characterization of UiO-66-NH2 MOFs The UiO-66-NH2 MOFs were prepared and the resulting powders were yellow (Figure 1A). The morphologies of the MOFs were characterized via scanning electron microscopy (SEM) (Figure 1B). The size of the UiO-66-NH2 was shown via SEM to be uniform at about 350-400 nm. Then the materials were characterized by FTIR and XRD. The FTIR spectrum (Figure 2) was recorded to confirm the chemical composition of the nano/micro-composites. The peaks at 3375 and 3460 cm-1 were identified as the asymmetric and symmetric N-H stretching of the MOF (Figure 2). The vibration frequency at approximately 1656 cm-1 was assigned to the N-H bending vibration. The observed bending signals at 1388 cm-1 are typical frequencies of strong C-N stretching absorption peaks of aromatic amines (Figure 2). The assignments of 9 ACS Paragon Plus Environment

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these spectroscopic features were further confirmed by FTIR spectrum of the model compound NH2-H2BDC and the results agreed well with the above bands42-43. The Raman spectrum of UiO-66-NH2 (Figure S1) illustrated the fluorescence of the as-prepared MOF, which was also consistent with a previous report in the literature 42. The XRD pattern of nano/micro-scale UiO-66-NH2 (Figure 5A1) was in accordance with a previous report42 and our simulated XRD pattern (FigureS2). These results indicated the phase purity of our as-synthesized UiO-66-NH2 support. Preparation and Characterization of SEH@ UiO-66-NH2 Five EHs from different plant sources were investigated as biocatalysts for the asymmetric hydrolysis of 1, 2-epoxyoctane. As shown in Table S1, in spite of relatively lower activity towards 1, 2-epoxyoctane than EHs from mung bean, jumbie bean or cow gram (23.6 U vs. 29.5-47.3 U), EH from soybean (SEH) exhibited the highest enantioselectivity and afforded the highest e.e. value of the product (R)-1, 2-octanediol (84.9% vs. 30.6-76.1%) among all the examined EHs. Therefore, SEH was selected for further study. Additionally, the electrophoresis analysis showed that the content of pure SEH in crude SEH protein and the molecular mass of SEH were about 12.5% and 31 kDa, respectively, which was similar with the previous report44. The immobilization of SEH onto UiO-66-NH2 is illustratedin Scheme 1. Glutaraldehyde (GA) was used as cross-linker because it is known from previous studies that GA can easily access reactive groups within the protein and cross-link them to amino groups as found on the support45. As can be seen in Figure S3, the optimal cross-linker concentration was approximately 130 10 ACS Paragon Plus Environment


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mmol/L, with a relative activity recovery of 82.9%. The optimal cross-linking time was about 120 min (Figure S4). As indicated by Figure S5, the mass ratio of UiO-66-NH2 to SEH which was optimal for SEH immobilization was 10:1 (with relative activity recovery being 88.0% and the amount of crude SEH protein loaded onto the MOF being about 87.3 mg crude SEH protein /g UiO-66-NH2). Also, the SEM analysis showed that the SEH@UiO-66-NH2 had a relatively regular spherical shape of about 500 nm with smooth surface (Figure 3). The size of the immobilized SEH (SEH@UiO-66-NH2) relative to the support UiO-66-NH2 was increased from about 400 nm to 500 nm, due to the loading of SEH onto the surface of the MOF support. The average size of the SEH@UiO-66-NH2, measured by dynamic light scattering (DLS) at 25 oC, was around 465.2 nm (Figure S6A). The prepared SEH@UiO-66-NH2 well dispersed in the solution of DES and no significantly amorphous aggregates was observed (Figure S6B). FTIR, XRD were also used to characterize the SEH@ UiO-66-NH2. The N-H bending vibration peak at 1656 cm-1 of UiO-66-NH2 (Figure 2) shifted to 1652 cm-1 (Figure 4) after immobilization, indicating that SEH had bonded onto the surface of MOFs by cross-linking. The carrier of the immobilized enzymes (Figure 5B1) retained the crystal integrity of MOFs based on XRD (Figure 5A1 and Figure S2). It is well known that the degradation of many MOFs generally occurs in the presence of water as well as the hydrophilic solvents, possibly resulting from the relatively weak coordination bond strength of MOFs46. Recently, deep eutectic 11 ACS Paragon Plus Environment

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solvents (DESs), relatively hydrophilic solvents, have gained much attraction as green media for biocatalytic processes22. Moreover, the SEH@UiO-66-NH2-catalyzed asymmetric hydrolysis of 1, 2-epoxyoctane will be conducted in DES-containing reaction systems in subsequent investigation. Therefore, it is necessary to study the stability of the support MOF and the immobilized enzyme (SEH@UiO-66-NH2 MOF) in various DESs. To examine the structural stability of the MOF, the XRD analysis for the support materials was performed after being exposed to the reaction media (PBS and DESs) for 2 h at 40 oC (Figure 5). The XRD diffraction peaks depicted in Figure 5A2 and 5B2 were slightly broaden, mainly because of the coordination ability of phosphate to the Zr-clusters47. However, the prepared UiO-66-NH2 MOF still maintained the structural stability, possibly due to the low amount of phosphate (50 mM) in the

solvent system47. No significant change of XRD spectra was observed

after being treated by four kinds of DES(ChCl (Choline Chloride) :Uera; ChCl : Glycerol; ChCl : Xylitol; ChCl : Ethylene glycol; Figure 5A2- Figure 5A6 and Figure 5B2- Figure 5B6). These results showed that both UiO-66-NH2 and SEH@ UiO-66-NH2 displayed structural stability in the four kinds of DES examined. Catalytic Performance of SEH@UiO-66-NH2 The effects of pH on the activity of free and immobilized enzymes were depicted in Figure 6, the optimal pH value for free SEH was 6.5 (18.9 U/mg SEH), while that of SEH@UiO-66-NH2 was 7.0 (16.6 U/mg SEH). The reason for this may be that22 the cross-linking between GA and the basic residues of enzyme changed the ionization state of protein functional groups in the microenvironment around the catalytic site of



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the enzyme or the surface –NH2 groups of enzyme carriers, resulting in the alkaline shift of the optimal pH value of the immobilized enzyme46. Besides a higher relative activity (72.3% vs 52.4%), the as-prepared SEH@ UiO-66-NH2 exhibited a higher absolute activity of enzyme than free SEH (12.0 U/mg SEH vs 9.9 U/mg SEH) in PBS (50 mM, pH 8.0), which was consistent with the observation that the immobilization usually stabilizes enzymes against deactivation 48. The effects of assay temperature on the activity of free and immobilized enzymes were shown in Figure 7. The optimum temperature for the hydrolysis activity was 35oC for free SEH (17.6 U/mg SEH) and shifted to 40 oC for SEH@UiO-66-NH2 (16.6 U/mg SEH). Also, the SEH@UiO-66-NH2 retained more than 83.2% of relative activity (13.8 U/mg SEH) at 50 oC, while the free SEH maintained only about 58.1% of relative activity (11.0 U/mg SEH). The change in optimal temperature may have been due to a decline in the enzyme conformational flexibility and an improvement in SEH heat resistance after covalent cross-linking of the enzyme by GA during the immobilization process49-50. The kinetic behaviour of free SEH and SEH@ UiO-66-NH2 were compared (Table S2). It was found that the SEH-catalyzed hydrolysis of 1, 2-epoxyoctane followed the Michaelis-Menten model. The apparent Vmax of the SEH@ UiO-66-NH2 was lower than that of free SEH (5.2 × 10-2

vs. 11.2

×10-2 mM min-1) and the apparent kinetic parameter Km of SEH@ UiO-66-NH2 was also lower than that of free SEH (6.5 vs. 19.2 mM), demonstrating an


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increase in apparent enzyme-substrate affinity of the immobilized SEH49. One possible explanation for this observation would be a change of the three-dimensional structure of SEH upon immobilization making it easier for the substrate to access the enzyme’s active site. In addition, the catalytic efficiency Vmax/ Km value of SEH@ UiO-66-NH2 was higher than that of free enzyme (8.0 × 10-3 vs 5.8 × 10-3 min-1). It was of interest to investigate the internal mass transfer of SEH@UiO-66-NH2. The effectiveness factor #$ of SEH@UiO-66-NH2 was calculated to be about 0.954. The Thiele modulus (%) was determined to be around

0.285.

The

results

showed

that

the

rate

of

the

SEH@UiO-66-NH2-catalyzed reaction was limited by internal mass transfer to a moderate extent. SEH@ UiO-66-NH2 exhibited a higher pH-stability over a wider pH range (from 6.0 to 8.0) than free SEH Figure 8, most likely due to stabilization of the enzyme by cross-linking. When the SEH@UiO-66-NH2 was incubated at pH 7 for 2 h, it retained more than 95.1% of the relative activity (15.4 U/mg SEH) of its activity, whereas less than 82.9% of relative activity (14.1 U/mg SEH) remained with free enzyme under the same conditions. As can be seen in Figure 9, SEH@UiO-66-NH2 exhibited higher thermal stability than its free counterpart in the temperature range investigated. When the SEH@UiO-66-NH2 was incubated at 45oC for 2 h, it retained more than 84.6% of the relative activity (14.1 U/mg SEH) of its activity, whereas less than 42.8% of relative activity (8.1 U/mg SEH) remained with free enzyme under the



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same conditions. Among the five hydrophobic organic solvents (Figure S7) examined, n-hexane showed the least toxicity to both free and immobilized enzyme. The residual activity of SEH@ UiO-66-NH2 was 7.7% higher than that of the free SEH in n-hexane. Previous research in our group showed that organic solvents with higher polarity can more significantly damage the native structure of the enzyme and result in rapid deactivation of the enzyme51.In general, SEH@MOF showed somewhat better solvent tolerance to all these five solvents compared with its free counterpart. This higher organic solvent tolerance after immobilization may have been mainly due to the increased structural rigidity of the enzyme after being immobilized onto the UiO-66-NH2 support. Finally, after storing at 4 oC for 4 weeks, the immobilized enzyme still had 97.5% relative activity (20.3U) (Table S3). FTIR spectroscopy is a useful tool for the study of the secondary structure (conformation)- catalytic performance relationships of enzyme52-53. From the amide I band (1600-1700 cm-1) in FTIR spectrum, it is possible to estimate the secondary structure content of the free and immobilized enzymes. From Figure 10, it is apparent that the secondary structure of the enzyme changed significantly after immobilization. The comparison of the secondary structure content of SEH and SEH@UiO-66-NH2 shows that the amount of α-helixes sharply increased (34.4% vs 19.4%) upon immobilization, and this is consistent with the increase in the rigidity of the enzymes’ structure that was inferred from the observed increased stability of the immobilized enzyme.The α-helix content of the protein may reflect the structural rigidity of


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enzyme, which may play an important role in the stability of the enzyme to pH, temperature and organic solvents54-56. In the present study, FTIR results indicated that the enhancement in the stability of SEH@UiO-66-NH2 could be largely attributed to the increase in the structural rigidity of the enzyme after immobilization. Biocatalytic asymmetric hydrolysis of 1,2-epoxyoctane with SEH@UiO-66-NH2 in aqueous and DES-containing systems. As showed in Scheme 2, SEH preferentially catalyzed the hydrolysis of the (R)-1,2-epoxyoctane to produce (R)-1, 2-octanediol. When the concentration of (R)-isomer is low, the SEH could also hydrolyse the (S)-isomer to produce the opposite configuration of the product. Hence the e.e. of the product decreased with decreasing concentration of (R)-1,2-epoxyoctane substrate. As a result, we chose conditions of high substrate concentration that gave a relatively high yield and e.e. as the optimal reaction. Figure 11A displays the time course of (R)-1, 2-octanediol with free and immobilized SEH in phosphate buffer. Compared with the yield and product e.e. of free SEH (yield 41.5%, e.e. 78.7), the immobilized SEH has higher enantioselectivity (yield 41.4%, e.e. 81.2%). This may be due to the structural changes of SEH during the immobilization process 57. Then we studied this hydrolysis reaction in four DES-containing systems. It is apparent from Figure 11 that ChCl: Urea was the optimal DESs for asymmetric hydrolysis of 1, 2-epoxyoctane. Compared with phosphate buffer solution, ChCl: Uera system gave similar yield (41.2% vs 41.4%) but somewhat higher product e.e. (84.7% vs 81.2%), which was in accordance with our previous experience of whole-cell 16 ACS Paragon Plus Environment


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catalysed reduction reactions in similar solvent systems. Although as a hydrogen bond donor the alkaline urea solution may deactivate the enzymes, this effect may be mitigated since the ChCl and urea in the DES are expected to bind to one another via a hydrogen-bonded network58. Furthermore, in the DES (ChCl: Urea) the product e.e. value increased and this solvent system may have additional advantages such as increasing the solubility of the substrate.

Conclusion In summary, the main achievements of this work are as follows: (1) epoxide hydrolase was for the first time immobilized onto the surface of the nano/microscale amino-functionalized MOFs rather than internal to the MOFs; (2) immobilized soybean epoxide hydrolase (SEH) was prepared for the first time and its enzymatic properties were studied; (3) SEH@ UiO-66-NH2 was prepared and used as an efficient

biocatalyst

for

asymmetric

hydrolysis

of

1,2-epoxyoctane

to

(R)-1,2-octanediol in DESs-containing system to further improve the yield and the product e.e.; (4) the stability of UiO-66-NH2 MOF in four DESs was investigated and the immobilised biocatalyst was found to have high catalytic efficiency and stability in the asymmetric hydrolysis of 1, 2-epoxyoctane to (R)-1, 2-octanediol in DES-containing media with high product yield and product e.e. value (yield 41.4%, e.e. 81.2%).

Supporting Information The method and materials: preparation of epoxy hydrolases (EHs) crude protein; 17 ACS Paragon Plus Environment

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screening of EHs from different plant sources for enzymatic asymmetric hydrolysis of 1,2-epoxyoctane; analytic methods; The structural stability of MOFs and SEH@UiO-66-NH2 in aqueous buffer and DES-containing systems; determination of internal mass transfer parameters; preparation of the immobilized SEH for in absence of UiO-66-NH2; Supplementary Table: Results of the asymmetric hydrolysis of 1,2-epoxyoctane catalyzed by epoxide hydrolases from different sources (Table S1). Apparent kinetic parameters of free enzyme and immobilized enzyme (Table S2). The storage stability of the immobilized enzyme (Table S3). Supplementary Figure: Raman spectrum of UiO-66-NH2 (Figure S1). XRD of the simulated UiO-66-NH2 (Figure S2). The effect of glutaraldehyde concentration on relative activity recovery (Figure S3). The effect of cross-linking time on relative activity recovery (Figure S4). The effect of MOF and SEH mass ratio on relative activity recovery (Figure S5). The DLS analysis of SEH@UiO-66-NH2 (Figure S6A). SEH@UiO-66-NH2 dispersed in ChCl: Urea DES solution (Figure S6B). Organic solvent tolerance of free and immobilized enzymes (Figure S7).This information is available free of charge via the Internet at http: //pubs.acs.org.

ASSOCIATED CONTENT Corresponding Author *Prof. W.-Y. Lou. Tel.: +86-20-22236669. Fax: +86-20-22236669. E-mail: [email protected].



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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources The Program of State Key Laboratory of Pulp and Paper Engineering (2015C04), the National Natural Science Foundation of China (21336002; 21222606; 21376096), the Key Program of Guangdong Natural Science Foundation (S2013020013049), the Fundamental Research Funds for the Chinese Universities (2015PT002; 2015ZP009), and the Open Funding Project of the State Key Laboratory of Bioreactor Engineering, SCUT Doctoral Student Short-Term Overseas Visiting Study Funding Project. Note The authors declare no competing financial interest.

ACKNOWLEDGEMENT We wish to thank the Program of State Key Laboratory of Pulp and Paper Engineering (2015C04), the National Natural Science Foundation of China (21336002; 21222606; 21376096), the Key Program of Guangdong Natural Science Foundation (S2013020013049), the Fundamental Research Funds for the Chinese Universities (2015PT002; 2015ZP009), and the Open Funding Project of the State Key Laboratory of Bioreactor Engineering for partially funding this work. We also thank SCUT


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Doctoral Student Short-Term Overseas Visiting Study Funding Project.



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Table of Contents Novel nano/micro-biocatalyst: soybean epoxide hydrolase immobilized on UiO-66-NH2 MOF Shi-Lin Cao, Dong-Mei Yue, Xue-Hui Li, Thomas J Smith, Ning Li, Hong Wu, Yong-Zheng Ma, Wen-Yong Lou*

Min-Hua Zong,

Preparation of the UiO-66-NH2 MOF and its efficient use for biocatalysis.


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Figure Captions Scheme 1 Schematic illustration of the synthesis of the immobilization of SEH onto UiO-66-NH2. Scheme

Epoxide

2

hydrolase-catalysed

asymmetric

hydrolysis

of

UiO-66-NH2UiO-66-NH2

and

(R/S)-1,2-epoxyocatane to chiral (R)-1,2-octanediol. Figure

1

A,

macroscopic

appearance

of

SEH@UiO-66-NH2; B, SEM spectrum of SEH@UiO-66-NH2. Figure 2 FTIR analysis of UiO-66-NH2 MOFs, NH2-H2BDC, the N-H stretching of UiO-66-NH2 and the N-H stretching of H2N-H2BDC. Figure 3 The SEM graphs with high magnification of SEH@UiO-66-NH2 Figure 4 FTIR spectra of SEH and SEH@UiO-66-NH2. Figure 5 XRD of the UiO-66-NH2 (A1), the UiO-66-NH2 after treatment with PBS (A2), the UiO-66-NH2 after treatment with ChCl: Ethylene glycol (A3), the UiO-66-NH2 after treatment with ChCl: Xylitol (A4), the UiO-66-NH2 after treatment with ChCl: Glycerol (A5), the UiO-66-NH2 after treatment with ChCl: Urea (A6); XRD of the SEH@UiO-66-NH2 (B1), the SEH@UiO-66-NH2 after treatment with PBS (B2), the SEH@UiO-66-NH2 after treatment with ChCl: Ethylene glycol (B3), the

SEH@UiO-66-NH2

after

treatment

with

ChCl:

Xylitol

(B4),

the

SEH@UiO-66-NH2 after treatment with ChCl: Glycerol (B5), the SEH@UiO-66-NH2 after treatment with ChCl: Urea (B6). 30 ACS Paragon Plus Environment


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Figure 6 The effects of pH on the activity of free and immobilized enzymes. Figure 7 The effects of assay temperature on the activity of free and immobilized enzymes. Figure 8

pH stability of free and immobilized enzymes.

Figure 9 Thermal stability of free and immobilized enzymes. Figure 10 The amide I fitting results of free SEH and immobilized SEH via FTIR analysis. Figure 11 Time course of (R)-1, 2-octanediol with free and immobilized SEH in phosphate buffer (A) and immobilized SEH in various DESs containing system: ChCl: Urea(B), ChCl:Glycerol(C), ChCl:Ethanediol(D), ChCl:Xylitol(E).


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 1

Scheme 2



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


N-H stretching of H2N-H2BDC

N-H stretching of UiO-66-NH2

v(NH2)sym

v(NH2)asym

v(NH2)sym

v(NH2)asym 3350

3400

3650 3600 3550 3500 3450 3400 3350 3300 3250

3450 -1

Wavenumber/cm-1

Wavenumber/cm

H2N-H2BDC

1656

1388

UiO-66-NH2

1800

1600

1400

1200

1000

800 -1

Wavenumber/cm Figure 2


600

400


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


SEH@UiO-66-NH2

SEH

1800

1600

1400

1200

1000

800 -1

Wavenumber/cm Figure 4


600

400

10

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UiO-66-NH2 treated with ChCl:Urea

A6

UiO-66-NH2 treated with ChCl:Glycerol

A5

UiO-66-NH2 treated with ChCl : Xylitol

A4

UiO-66-NH2 treated with ChCl:Ethylene glycol

A3

UiO-66-NH2 treated with PBS

A2

UiO-66-NH2

A1

20

30

40

50

60

o

2 θ(

Relative Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Relative Intensity


)

SEH@UiO-66-NH2 treated with ChCl:Urea

B6

SEH@UiO-66-NH2 treated with ChCl:Glycerol

B5

SEH@UiO-66-NH2 treated with ChCl : Xylitol

B4

SEH@UiO-66-NH2 treated with ChCl:Ethylene glycol

B3

SEH@UiO-66-NH2 treated with PBS

B2

SEH@UiO-66-NH2 10

20

B1 30

40

o

2 θ(

)

Figure 5 37

ACS Paragon Plus Environment

50

60

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20

Enzyme specific activity (U/mg SEH)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


18 16 14 12 10 8

SEH@UiO66-NH2 Free SEH

6 6.0

6.5

7.0

7.5

8.0

pH Figure 6


8.5


20

Enzyme specific activity (U/ mg SEH)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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15

10 SEH@UIO-66NH2 Free SEH 5 25

30

35

40

45

Temperature/°C

Figure 7


50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



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16 SEH@UiO-66-NH2 Free SEH

14

12

10

6.0

6.5

7.0

7.5

pH Figure 8


8.0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



18

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SEH@UiO-66NH2 Free SEH

16 14 12 10 8 6

20

25

30

35

40 o

Tenperature/ C Figure 9


45

100

0.25

90

0.20

80

0.15

60 50 40

SEH@UiO-66-NH2 α-Helix

0.10

β-Sheet

β-Turn

0.10

Intensity

70

0.15

Free SEH α-Helix

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Secondary structure contents (%)

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β-Turn β-Sheet

0.05

0.05 0.00 1600

0.00

1620

1640 1660 -1 Wavenumber cm

1680

1700

1600

1620

1640 1660 -1 Wavenumber cm

Free SEH SEH@UiO-66-NH2

30 20 10 0 α-helix

β-sheet

β-turn

Assignment

Figure 10


1680

1700


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 11


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