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Efficient resolution of 3-phenoxy-1,2-propanediol by immobilized lipase on amphiphilic comb polymer modified TiO2
Accepted Manuscript Title: Efficient resolution of 3-phenoxy-1,2-propanediol by immobilized lipase on amphiphilic comb polymer modified TiO2 Author: Bin Wang Wenhao Li Bin Wu Bingfang He PII: DOI: Reference:
S1381-1177(14)00141-6 http://dx.doi.org/doi:10.1016/j.molcatb.2014.04.019 MOLCAB 2945
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
11-12-2013 28-3-2014 29-4-2014
Please cite this article as: B. Wang, W. Li, B. Wu, B. He, Efficient resolution of 3-phenoxy-1,2-propanediol by immobilized lipase on amphiphilic comb polymer modified TiO2 , Journal of Molecular Catalysis B: Enzymatic (2014), http://dx.doi.org/10.1016/j.molcatb.2014.04.019 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.
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Efficient resolution of 3-phenoxy-1,2-propanediol by immobilized lipase on
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amphiphilic comb polymer modified TiO2
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Bin Wanga,b, Wenhao Lia, Bin Wua, Bingfang Hea,*
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College of Biotechnology and Pharmaceutical Engineering, Nanjing University of
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Technology, Nanjing211816, China
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School of Chemistry & Environmental Engineering, Jiangsu University of Technology,
Changzhou213001, China
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* Corresponding author: Bingfang He
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e-mail: [email protected]
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Phone: +86 25 58139902
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Fax: +86 25 58139902
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Abstract An amphiphilic microenvironment is a key element to the lipase which catalyzes
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substrates at the aqueous and hydrophobic interface. An amphiphilic comb copolymer,
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poly(sodium acrylate)-g-methoxy poly(ethylene oxide), was introduced for the
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post-functionalization of mesoporous TiO2. The lipase from Burkholderia ambifaria YCJ01
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was immobilized on the comb copolymer modified TiO2. The activity of immobilized lipase
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YCJ01 showed that comb polymer which side chain with a molecular weight of 1000 was the
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best material for the functionalization of TiO2. The amount of adsorbed lipase was 55.6 mg/g
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support and the immobilized lipase activity was 6076.1 U/g support. It was worth to note that
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the immobilized lipase achieved more than 200% of its origin activity in hydrophobic solvents
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and very stable in hydrophilic solvents. The immobilized lipase was used as a catalyst for
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resolution of 3-phenoxy-1,2-propanediol, and showed high enantioselectivity (eep≥99%) in
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all tested organic solvents. This might be explained that comb polymer with amphiphilic,
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multiple side chains features could well stabilize the favorite active conformation of lipase to
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the resolution substrate in all tested organic solvents. In diisopropyl ether reaction solvent,
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the yield of 49.8% and an enantiomeric excess of 99.2% for S-diacetate were achieved after
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one operation, and maintained relative yield of 72% for S-diacetate even after six reaction
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cycles with no significant decrease in the enantiomeric excess of product.
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Keywords Lipase, Comb polymer, Mesoporous TiO2, Immobilization, Biocatalysis
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1. Introduction
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Lipases have been widely utilized to desymmetrize prochiral molecules with high
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chemo-, regio- and stereo-selectivity in organic solvents [1-4]. However, their application is
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currently limited owing to shortage of reusability, stability and activity in organic solvents.
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Immobilization is a useful method to improve thermal and chemical stability of enzymes and
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ensure reusability of enzymes. Although, it is believed that the water layer on the molecular
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surface of enzymes determines their activity in organic media [5-6], an amphiphilic
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microenvironment not only preserves the structural water thus maintaining the protein in
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active conformation in organic media, but also is very important to the lipases which catalyze
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substrates at the aqueous and hydrophobic interface by removing the “lid” covering their
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active sites and making them more accessible to substrates [7]. So, amphiphilic polymers
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have been extensively utilized as supports for the immobilization of lipases. Crespo et al. [8]
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entrapped lipase from Candida rugosa in poly(ethylene oxide) for resolution of
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(R,S)-2-octanol, high activity was obtained in organic solvents. To further stabilize the lipase,
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Yasuda et al. [9] immobilized Rhizopus delemar lipase in amphiphilic polymer particles with
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grafted chains, and multipoint attachment to the support stabilized the active conformation of
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lipase in organic solvents. That meant that the amphiphilic polymers with grafted chains are
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potentially excellent materials for the immobilization of lipase. However, as the amphiphilic
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polymers have lower mechanical properties, their application in lipase immobilization is
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limited.
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The inorganic mesoporous materials have been found to be promising host matrixes for
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immobilization, for they have good mechanical properties, large surface areas, chemical
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inertia and biocompatibility with enzymes. Among the mesoporous materials, mesoporous
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TiO2, due to its unique pore structure and special physical and chemical properties, has
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many potential applications in catalytic supports, sensor materials and electrode materials
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[10-12]. For effective lipase immobilization, the surface of mesoporous materials was often
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modified with an amphiphilic layer due to the ‘‘interfacial activation” mechanism [7].
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Therefore, the mesoporous material with an amphiphilic layer is an effective support for the
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immobilization of lipase.
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In our previous report [4], the lipase from Burkholderia ambifaria YCJ01 showed thermal stability and distinct super-stability to the most tested solvents (25%, v/v) for 60 days and
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preferred to hydrolyze p-nitrophenyl esters with medium-long chain fatty acid. The resolution
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of mandelic acid by lipase YCJ01 with a theoretical conversion yield of 50%, eep of 99.9%
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and ees of 99.9%, showed its attractive potency in biocatalysis and the resolution of
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pharmaceutical intermediates.
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In this paper, we introduced amphiphilic poly(sodium acrylate)-g-methoxy poly(ethylene
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oxide) (herein after called PM), a comb copolymer, for the post-functionalization of
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mesoporous TiO2. The lipase from Burkholderia ambifaria YCJ01 was adsorbed into the
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pores of PM-modified TiO2. The immobilization exhibited the “interfacial activation” of lipase,
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and also significantly improved the stability of lipase in organic system and the
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enantioselectivity of lipase to the substrate. The immobilized lipase was used as a catalyst
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for the resolution of racemic 3-phenoxy-1,2-propanediol, an important intermediate for
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pharmaceuticals.
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2. Materials and methods
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2.1. Materials
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Lipase YCJ01 from Burkholderia ambifaria was characterized in our previous report, the
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amino acid sequence has been assigned GenBank Accession No. JQ733582. Burkholderia
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ambifaria YCJ01 was deposited in CCTCC (Wuhan, China) with an accession number of
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CCTCC M 2011058 [4]. p-Nitrophenyl palmitate (pNPP) was purchased from Sigma-Aldrich
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(St. Louis, MO, USA). 3-Phenoxy-1,2-propanediol, vinyl acetate, sodium acrylate (SAA),
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acrylic acid (AA), methoxy poly(ethylene oxide) (MPEO), dicyclohexyldimethylcarbodiimide
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(DCC), 4–dimethylaminopyridine (DMAP), Na2S2O8 and NaHSO3 were purchased from
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Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The mesoporous TiO2 used in
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this work was kindly supplied by Lu Group [10]. All the reagents were used directly without
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any further purification.
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2.2. Preparation of comb polymer
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The comb polymer poly(sodium acrylate)-g-methoxy poly(ethylene oxide) (PM) was
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synthesized by free radical copolymerization of SAA with methoxy poly(ethylene
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oxide)-acrylate (MPEOAA) according to the method established by [13]. MPEOAA
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macromonomer was first synthesized by a catalytic esterification reaction between acrylic
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acid (AA) and methoxy poly(ethylene oxide) (MPEO) (molar ratio of AA to MPEO was 2:1) in
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the presence of a DCC and DMAP system. The copolymerization reaction between SAA and
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MPEOAA was carried out in an aqueous medium at 95°C for 8h using Na2S2O8 as the
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initiator and NaHSO3 as the activator, under a nitrogen gas atmosphere. After the reaction,
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the PM was obtained. The 1H-NMR date of PM were shown in Fig. S1 and Table S1 (in
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supplementary material), and the average molecular mass of PM were shown in Fig. S2 and
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Table S2 (in supplementary material).
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2.3. Surface modification of TiO2
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TiO2 is amphoteric, rendering it to be an anion and cation exchanger at acidic and
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alkaline pH, respectively [14]. SEM patterns of TiO2 was shown in Fig. S3 (in supplementary
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material). Functional TiO2 was obtained as follows: 200 mg of PM was dissolved in 10 mL
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deionized water, 1 g of TiO2 was then added to the solution. The mixtures were stirred for 12
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h at 30°C and filtrated. The insoluble substance washed with water three times and
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successively dried in vacuum oven at 50°C for 8 h to yield the PM-modified TiO2. As the
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PM1000 has a high weight average molecular weight (Mw=31767 g/mol, Table S2 in
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supplementary material), the highest adsorbed amount of PM1000 on the TiO2 was about
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18mg/g TiO2 (Fig.S4 in supplementary material).
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2.4. Lipase Immobilization
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The lipase solution was prepared by adding crude lipase powder to phosphate buffer (pH=7.5, 0.05 M) and the insoluble was removed. The activity of lipase solution was 40~45
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U/mL and the protein concentration was 1.0 mg/mL. The adsorption experiment was carried
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out by mixing PM-TiO2 with lipase solution with the ratio of 10 mg PM-TiO2/mL enzyme
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solution, and the mixture was stirred for 12 h at 30°C. The PM-TiO2-lipase (immobilized
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lipase) was separated by filtration and washed with phosphate buffer. All portions of the
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filtration were retained for determination of protein concentration [15] and activity. The
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immobilized lipase was dried in vacuum and stored at 4°C.
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2.5. Enzyme activity assay
Activity of the free and immobilized lipase was assayed using pNPP as a substrate [4].
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The reaction mixture was composed of 240 μL of pNPP solution and 10 μL of lipase solution
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(for free lipase) or it was composed of 100 mL pNPP solution and 1 mg of immobilized lipase.
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The enzyme reaction was incubated at 40°C for 10 min. The liberated p–nitrophenol (pNP)
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was quantified spectrophotometrically at 410 nm. One unit of lipase activity was defined as
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the amount of enzyme that produced 1 μmol of pNP under the conditions mentioned above.
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2.6. Characterization of supports
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The textural properties were studied by N2 adsorption–desorption measurements
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(ASAP 2020 M) at the liquid N2 temperature of 77 K after the TiO2 samples were outgassed
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for 2 h at 200°C, and the PM–TiO2 and PM–TiO2 with adsorbed lipase were outgassed for 6
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h at 80°C. The Fourier transform infrared (FT–IR) analysis of the support was performed on
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a Nicolet 6700 FT–IR instrument with KBr pellets. Thermogravimetry measurement was
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determined by a thermogravimetric analyzer (TGA, NETZSCH TG 209 F3, Germany).
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Scanning electron microscopy (SEM) observations were performed on a Hitachi S-4800
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microscope operating at 20kV and a JEOL JSM 7401F microscope operating at 1.0kV. 1H
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NMR spectroscopy of sample was determined using BRUKER AVANCE DRX-500 with D2O
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as the solvent. Gel permeation chromatography (GPC) was carried out by an HPLC
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equipped with Styrage1 HR3, HR4 and HR5 columns, and Wyatt Optilab rEX was used as
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detector.
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2.7. Transesterification reaction
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After preliminary optimization, the enzymatic transesterification reaction of
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3-phenoxy-1,2-propanediol (0.2 mmol) and vinyl acetate (1.2 mmol) in various solvents (4
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mL) was carried out in 10 mL conical flasks with stoppers. The reaction was catalyzed in the
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presence of 40 mg of immobilized lipase or 6 mg of lipase powder (about the same lipase
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activity with 40mg of immobilized lipase) at 40°C with shaking at 180 rpm. Aliquots of
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samples were withdrawn from the reaction mixture at intervals and the samples were
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analyzed by an HPLC equipped with a Chiralcel ® OD–H column (Daicel, 4.6 mm×250 mm)
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and detected by UV at 270 nm, with a mobile phase composed of n–hexane/isopropanol
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(85/15, v/v) with a flow rate of 1 mL/min. The reaction was also catalyzed with deactivated
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catalyst as a control, and there was no product detected.
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3. Results
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3.1. FT-IR spectrum of PM1000, TiO2 and PM1000 modified TiO2
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An amphiphilic microenvironment is very important to the lipases which catalyze substrates at the aqueous and hydrophobic interface due to the ‘‘interfacial activation”
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mechanism [7], amphiphilic poly(sodium acrylate)-g-methoxy poly(ethylene oxide) (PM) with
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methoxy poly(ethylene oxide) group (MPEO) was designed for the modification of TiO2 as a
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support for the immobilization of lipase. Different MPEO with molecular weight of
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350,750,1000,1500 and 2000 were used to prepare the PM, signed as PM350, PM750,
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PM1000, PM1500 and PM2000 respectively.
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In Figs. 1a and 1b, the bands in the 2800–3000 cm−1 region correspond to the CH2 and
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CH3 groups of the polymer backbone. In Fig. 1b, the bands at 1247 and 1101 cm−1 are
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attributed to the vibration of the C–O–C bands. These results reveal the main structure
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characteristics of PM. In Figs. 1b and 1c, the wave band at 3419 and 3427 cm−1
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corresponding to –OH stretching vibrations shows light shift in the two spectra. This result is
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due to the TiO2–carboxyl interface interactions. As the 1721 cm−1 peak (COO-) appear in Fig.
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1b, which meant the PM attached to the TiO2 with electrostatic interactions. The region of
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bands at 500–800 cm−1 in Figs. 1b and 1c is tentatively assigned to the Ti–O–Ti groups.
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3.2. N2 adsorption–desorption isotherms of TiO2, PM1000-TiO2 and PM1000-TiO2-lipase
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The pore diameter distribution was calculated using the Barett–Joyner–Halenda (BJH)
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method based on the desorption isotherm, and the surface area was calculated using the
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Brunauer–Emmett–Teller (BET) method based on the adsorption isotherm.
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Adsorption–desorption isotherms and the corresponding pore size distribution of the TiO2
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and its derivatives are shown in Fig. 2. It is noted that the hysteresis loop occurs at a high
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relative pressure. This result suggests that these three materials were characterized by most
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mesopores. The observed changes of the specific surface area and pore volume as well as
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pore size are shown in Table 1. Compared to PM1000–TiO2, PM1000–TiO2-lipase exhibits a
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decrease in the specific surface area and pore volume, and this decrease is likely due to the
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loss of the adsorbed lipase and comb polymer. Sharp inflections occurring at around
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P/P0=0.85 are observed for all samples due to the capillary condensation and the
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evaporation in the uniform mesopores channels [16]. The average pore diameter decreased
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from 16.3 nm to 15.5 nm, the degree of reduction meant that the lipase were immobilized in
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a monolayer fashion on the support as explained by [17]. Monolayer adsorption may favor
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the contact of lipase with substrates.
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3.3. Immobilization of lipase and its interfacial activation
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The comb polymer PM for modification has dense COO- groups promoting significant interactions with Hδ+O on the surface of TiO2 by ionic bonds and yielding a strong anchoring
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block. It also has dense side chains composed of methoxy poly(ethylene oxide) (MPEO)
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which has the ability of adsorbing water and the lipase was adsorbed onto them (Scheme 1).
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The relative activity recovery of immobilized lipase on the PM modified TiO2 was much
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higher than that on the non-modified TiO2 with only 47.2% (Table 2). The significant
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activation was observed since the activity recovery of the immobilized lipase ranging from
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137.3% to 331.7% for all PM-modified TiO2 used as supports. The relative activity recovery
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was increased with the increasement of average length of side chain (MPEO) corresponding
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to the average molecular weight. As shown in Table 2, the amount of absorbed lipase
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increased with the increasement of average length of MPEO under 1000 of average
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molecular weight and then gradually decrease with the molecular weight increase further.
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The supports PM2000-TiO2 and PM1500-TiO2 with a higher average length of MPEO yielded
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a lower loading of lipase than that on the PM1000-TiO2, the higher steric hindrance effect of
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PM2000-TiO2 and PM1500-TiO2 could be conjectured [13]. Among the supports, Though
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PM2000-TiO2-lipase had the highest specific activity, PM1000-TiO2-lipase had the highest
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lipase activity of 6076.1 U/g support and the highest protein loading 55.6 mg/g support,
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which were used for the whole works.
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3.4. Tolerance of immobilized lipase in organic solvents
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The tolerance of immobilized lipase and the lipase powder in various organic solvents
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(100%, v/v) was compared in Table 3. The lipase was sensitive to organic solvents and only
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maintained 18.8% to 46.8% of its original activity in hydrophilic solvents (logP<1). The
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immobilized lipase in the same solvents showed much higher stability, even 71%~126% of
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its original activity was achieved in the tested hydrophilic solvents. Fortunately, the
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immobilized lipase was markedly activated with more than 200% of its original activity in
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hydrophobic solvents (logP>1).
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3.5. Efficient enzymatic resolution of racemic 3-phenoxy-1,2-propanediol
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Considering the solubility of substrate and activity of lipase YCJ01, four kinds of tested
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solvents were selected as reaction solvents for the resolution of racemic
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3-phenoxy-1,2-propanediol. The reaction was carried out in two steps, as shown in Scheme
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2. The RS-diol was prior transformed into corresponding primary monoacetate which was
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detected by HPLC after initial 2 hours reaction with high regioselectivity. Then, the primary
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monoacetate was continually converted enantioselectively into diacetates, a preference for
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the S compound was always observed. The significant effect of the reaction solvents on the
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enantioselectivity of lipase was observed when the lipase powder was used, and the least
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value of ee for S-diacetate was 36.8% in t-butanol (Table 4). Strikingly, the immobilized
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lipase showed the strict enantioselectivity with the values of ee for S-diacetate more than
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99% in all tested reaction solvents. Owing to the high activity in diisopropyl ether, the value
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of ee was 99.2% and the yield reached 49.8% (a close approximation to the theoretical yield
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of 50%) for S-diacetate were achieved by immobilized lipase.
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3.6. Temperature on the esterification activity and enantioselectivity of the immobilized lipase
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YCJ01 The temperature was varied in the range of 30–50°C. The highest esterification rate (2.7
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µmol/g/min) were observed at 45°C (Table 5). The E values were high (>276) for all the
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temperatures, and increased temperature leaded to a decrease in enantioselectivity.
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Considering the thermostability, esterification rate and enantioselectivity, the temperature
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40°C was selected as the reaction temperature.
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3.7. Reusability of immobilized lipase
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The reusability of immobilized enzyme is a key point for the practical catalysis. The
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relative yield of the immobilized lipase for S-diacetate was showed in Fig. 3, 72% of the
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relative yield of the lipase immobilized on PM1000-TiO2 was retained after six reaction
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cycles (108h of total operation), which was found to be much better than that of the lipase
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powder. Though about 30% of the relative yield of immobilized lipase was lost, the values of
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ee for S-diacetate fall slightly and the value of ee was 98.2% after six reaction cycles, which
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indicated that the long time of reaction had no significant influence on the enantioselectivity
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of PM1000-TiO2-lipase, the decrease of relative yield might be due to the detachment of
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adsorbed lipase on outer/inner surface of PM-TiO2. The detachment of adsorbed lipase may
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be remedied by connecting the lipase to activated poly(ethylene oxide) through a covalent
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bond in the further work.
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4. Discussion
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PM modification of TiO2 showed outstanding characteristics for immobilization of lipase
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YCJ01, tolerance of immobilized lipase to organic solvents of lipase YCJ01 was significantly
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improved. Jegannathan et al. [18] encapsulated Burkholderia cepacia lipase in
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k-carrageenan, 43.7%-93.5% residual activity of immobilized lipase was obtained after
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incubation in organic solvents for 1 h at 30°C. The Candida rugosa lipase was immobilized in
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kaolin, 90% residual activity of immobilized lipase was obtained after incubation in hexane
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for 1 days at 25°C [19]. In this paper, most of immobilized lipase YCJ01 activity was kept in
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hydrophilic solvents and more than 200% of its original activity was achieved in hydrophobic
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solvents (Table 3) after incubation in organic solvents for 3h at 40°C. It is well known that
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immobilization of enzymes within an aqueous microenvironment is essential to the activity of
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enzymes in the organic solvent. Wang et al. [20] immobilized enzymes in molecular hydrogel,
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superactivity and high stability was obtained in organic solvents, even in hydrophilic solvents.
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However, hydrophilic hydrogel would be not in favor of the immobilization of lipase due to the
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"interfacial activation" mechanism. In this paper, amphiphilic materials were used for the
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immobilization of lipase YCJ01, as shown in Scheme 1, PM modified TiO2 has the ability of
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adsorbing water and also supplies the aqueous and moderate hydrophobic long side chains
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(MPEO) for grasping lipase YCJ01 with multipoint attachment.
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The most advantages of TiO2 modified with comb polymer which supplied an aqueous
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and a moderate hydrophobic microenvironment was favorable to the lipase YCJ01 showing
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high activity as shown in Table 2 and 3. The opening of the “lid” of lipase YCJ01 might be
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speculated in amphiphilic microenvironment, and the activation of lipase by immobilization
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was obtained with all comb polymer modified TiO2 used as supports (Table 2). Strikingly,
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strict enantioselectivity of the lipase YCJ01 immobilized on the support of amphiphilic
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modified TiO2 was achieved to the substrate 3-phenoxy-1,2-propanediol, resulting more than
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99% of eep for S-diacetate in all tested reaction solvents (Table 4). While the ee of product
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S-diacetate by non-modified lipase was significantly affected by the reaction solvents. The
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appearance of strict enantioselectivity of immobilized lipase YCJ01 to substrate in all tested
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solvents was similar to the results obtained by few researchers[21-22]. The immobilized
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Pseudomonas cepacia lipase showed high enantioselectivity (E>150) in several solvents for
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the resolution of 1,2-diols [21]. The immobilized Pseudomonas sp. lipase exhibited strict
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enantioselectivity (eep>99%) in four solvents for the resolution of (R,S)-methyl
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mandelate[22]. The reasons for improvement of enantioselectivity and keeping the favorable
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active conformation of lipase YCJ01 in all tested solvents might be attributed to some
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protections on the protein conformation from the amphiphilic PM, greatly reducing the
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distortion of the lipase structures generated from the organic solvents.
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Efficient resolution of 3-phenoxy-1,2-propanediol with eep (S-diacetate) of 99.2% was
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achieved using lipase YCJ01 immobilized on amphiphilic comb polymer modified TiO2.
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S-3-phenoxy-1,2-propanediol is widely used as an important intermediate for the production
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of various pharmaceutical products [23-24]. The methods for the preparation of
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enantiomerically pure 3-phenoxy-1,2-propanediol include asymmetric catalysis and enzyme
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catalysis. Theil et al. [24] reported the transformation which was carried out in the presence
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of Amano lipase PS from Burkholderia cepacia, the yield of S-diacetate reached 49% and
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the ee for S-diacetate was 79%. Worthy et al. [23] applied a scaffolding catalyst for
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site-selective resolution of 3-phenoxy-1,2-propanediol, the yield of S-monoacetate reached
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32% and the ee was 96%. In this paper, high yield and enantioselectivity of S-diacetate was
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obtained with the immobilized lipase used as a catalyst, the yield reached 49.8% (a close
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approximation to the theoretical yield of 50%) and the value of ee was 99.2% (Table 4). Also,
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R-monoacetate was obtained with ee of 99.6% (Table 4), which meant that both of R product
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and S product could be obtained simultaneously with high optical purity.
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5. Conclusions
In this paper, the amphiphilic comb copolymer modified support not only markedly
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activated the activity of lipase YCJ01, more than 200% of its origin activity was achieved in
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hydrophobic solvents and it was very stable in hydrophilic solvents; but also significantly
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improved the enantioselectivity of lipase to the substrate (≥99% of ee for S-diacetate) in all
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tested solvents. The lipase YCJ01 which was immobilized in amphiphilic comb copolymer
347
modified TiO2 catalyzed 3-phenoxy-1,2-propanediol with high yield of 49.8% and excellent
348
ee value of 99.2% for S-diacetate after one operation, and retained up to 72% of its relative
349
yield after six reaction cycles with no significant decrease in the enantiomeric excess of
350
product. The strategy of amphiphilic comb copolymer modification will provide an available
351
approach to promoting the modification of various supports for lipase immobilization.
Ac ce p
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352
16
Page 16 of 29
353
Acknowledgments Financial supports for this research from the National Program on Key Basic Research Project
355
(2011CB710800) and the National High Technology Research and Development Key Program of China
356
(2012AA022205). We also acknowledge the support of the projects funded by PMSIRT (IRT1066) and PAPD.
ip t
354
cr
357 358
Reference
360
[1] U. T. Bornscheuer, Curr. Opin. Biotechnol. 13 (2002) 543–547.
361
[2] X. Tian, G.W. Zheng, C.X. Li, Z.L. Wang, J.H. Xu, J. Mol. Catal. B-Enzym. 73 (2011) 80−84.
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[3] G. Yang, J.P. Wu, G. Xu, L.R. Yang, Appl. Microbiol. Biotechnol. 81 (2009) 847−853.
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[4] C.J. Yao, Y. Cao, S.S. Wu, S. Li, B.F. He, J. Mol. Catal. B-Enzym. 85-86 (2012):105−110.
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[5] A. Ducret, M. Trani, R. Lortie, Enzyme. Microb. Technol. 22(1998) 212−216.
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[6] J.L. Schmitke, C.R. Wescott, A.M. Klibanov. J. Am. Chem. Soc. 118 (1996) 3360−3365.
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[7] C. Mateo, J.M. Palomo, G. Fernandez-Lorente, J.M. Guisan, R. Fernandez-Lafuente, Enzyme. Microb.
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Technol. 40 (2007) 1451–1463.
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[8] R. Dalla-Vecchia, D. Sebrão, M.G. Nascimento, V. Soldi, Process Biochem. 40 (2005) 2677–2682.
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[9] M. Yasuda, H. Nikaido, W.R. Glomm, H. Ogino, K. Ishimi, H. Ishikawa, Biochem. Eng. J. 48 (2009) 6−12.
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[10] M. He, X.H. Lu, P. Caimi, X. Feng, L. Yu, Z.H. Yang, Chem. Commun. 19 (2004) 2202−2203.
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[11] Y.J. Jiang, Q.Y. Sun, Z.Y. Jiang, L. Zhang, J. Li, L. Li, X.H. Sun, Mater. Sci.Eng. C 29 (2009) 328−334.
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[12] R.Y. Zhang, A.A. Elzatahry, S.S. Al-Deyab, D.Y. Zhao, Nano. Today 7 (2012) 344−366.
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[13] Q.P. Ran, P. Somasundaran, C.W. Miao, J.P. Liu, S.S. Wu, J. Shen, J. Colloid interf. Sci. 336 (2009)
374 375
Ac ce p
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d
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an
us
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624−633. [14] Y. Chen, Y.Y. Yi, J.D. Brennan, M.A. Brook, Chem. Mater. 18 (2006) 5326–5335.
17
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[15] M.M. Bradford, Anal. Biochem. 72 (1976) 248−254.
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[16] T.P.B. Nguyen, J.W. Lee, W.G. Shim, H. Moon,
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[17] B. Al-Duri, Y.P. Yong, Biochem. Eng. J. 4 (2000) 207−215.
379
[18] K.R. Jegannathan, E.-S. Chan, P. Ravindra, J. Mol. Catal. B-Enzym. 58 (2009) 78–83.
380
[19] M. B. A. Rahman, S. M. Tajudin, M. Z. Hussein, R. N. Z. R. A. Rahman, A. B. Salleh, M. Basri, Appl. Clay
381
Sci. 29 (2005) 111–116.
382
[20] Q.G. Wang, Z.M. Yang, L. Wang, M.L. Ma, B. Xu, Chem. Commun. 10 (2007) 1032−1034.
383
[21] A. Kamal, G. Chouhan, Tetrahedron Lett. 45 (2004) 8801-8805.
384
[22] N. Queiroz, M. G. Nascimento, Tetrahedron Lett. 43 (2002) 5225-5227.
385
[23] A.D. Worthy, X.X. Sun, K.L. Tan, J. Am. Chem. Soc.134 (2012) 7321−7324.
386
[24] F. Theil, J. Weidner, S. Ballschuh, A. Kunath, H. Schick,
390 391
ip t
cr us
an
M
te
FIGURE LEGENDS
Ac ce p
389
J. Org. Chem. 59 (1994) 388−393.
d
387
388
Micropor. Mesopor. Mat. 110 (2008) 560−569.
Fig.1 . FT-IR spectra of PM1000 (a), PM1000-TiO2 (b) and TiO2 (c)
392
Fig.2 . N2 adsorption–desorption isotherms and pore diameter distribution
393
curves (insert) of TiO2 and its derivatives. Symbols: (■)TiO2; (●)PM1000-TiO2;
394
(▲)PM1000-TiO2-lipase.
395 396
Fig.3. Reusability of lipase and immobilized lipase for transesterification. Relative yield= (the
397
yield of S-diacetate/the yield of S-diacetate for the first cycle)×100%. Operational conditions:
398
the first cycle ended with the yield of 49.8% for S-diacetate, the other cycles ended with the
399
same time of the first cycle. Symbols: (○) eep; (dense slant) lipase powders; (slant)
18
Page 18 of 29
400
PM1000-TiO2-lipase.
401 402
Scheme 1 Schematic representatives for the adsorption of lipase YCJ01 on the comb
404
polymer modified TiO2
ip t
403
405
Scheme 2 Enzymatic resolution of racemic 3-phenoxy-1,2-propanediol
cr
406
us
407 408
an
409
M
410 411
d
412
Tables
415 416
Table 1
Ac ce p
414
te
413
Textural properties of TiO2 and its derivatives Surface
Sample
2
area (m /g)
TiO2
Pore volume 3
(cm /g)
Average pore diameter (nm)
41.37
0.212
17.08
PM 1000–TiO2
38.02
0.194
16.25
PM1000–TiO2–lipase
34.76
0.176
15.51
417 418 19
Page 19 of 29
419 420
Table 2 Protein loading on supports and activity recovery of immobilized lipase Bound protein (mg/g
Lipase activity
Specific activity
Activity recovery
immobilized lipase) a
(U/g support)
(×105U/g protein)
(%)b
TiO2
37.6
738.3
2.0
47.2
PM350–TiO2
42.2
2410.3
5.7
PM750–TiO2
48.7
3752.0
7.7
PM1000–TiO2
55.6
6076.1
10.9
PM1500–TiO2
43.8
5695.8
PM2000–TiO2
34.5
4760.6
cr
ip t
Support
an
us
137.3 185.2 262.7 312.6
13.8
331.7
M
13.0
421
Data are means of at least three parallel reactions, where the average SD was 6.17% of the mean (range of 3.17–12.4%).
422
a
423
support weight after adsorption.
424
b
425
lipase)×100%
427 428
d
te
Activity recovery (%) = (activity of immobilized lipase/activity of free lipase with the same amount of adsorbed
Ac ce p
426
Bound protein= (amount of initial protein in the solution-remained amount of protein in the solution after adsorption/the
429 430 431 432
20
Page 20 of 29
433
Table 3 Stability of lipase and PM1000-TiO2-lipase in non–aqueous solvents Residual activity (%)a logP PM1000-TiO2-1ipase 246.3
Toluene
2.50
127.5
273.9
Diisopropyl ether
1.90
86.4
210.7
t-butanol
0.60
46.8
126.3
Tetrahydrofuran
0.49
40.6
89.5
Acetone
−0.23
32.7
71.3
1,4–Dioxane
−1.10
18.8
76.2
cr
111.0
us
3.50
an
Hexane
ip t
Lipase
M
Medium
The lipase powder and PM1000–TiO2–lipase were incubated in solvents (100%, v/v) at 40°C for 3 h. Data are means of at
435
least three parallel reactions, where the average SD was 4.63% of the mean (range of 1.46–8.7%).
436
a
437
lipase powder)×100%
439 440 441
te
Residual activity (%)= (total activity of immobilized lipase or lipase powder after 3h/total activity of immobilized lipase or
Ac ce p
438
d
434
442 443 444 445
21
Page 21 of 29
446
Resolution of racemic 3-phenoxy-1,2-propanediol in non–aqueous solvents
Table 4
S-diacetate Medium
cb
logP yield (%)
ees a
eep a
Ec
ip t
Catalyst
Lipase
Diisopropyl ether
1.9
45.7
92.0
99.1
0.481
YCJ01
t-butanol
0.6
24.9
11.5
36.8
0.238
powder
Tetrahydrofuran
0.49
1.1
0.75
81.8
0.009
Acetone
–0.23
3.1
6.9
93.6
0.069
32.1
Immobilized
Diisopropyl ether
1.9
49.8
99.6
99.2
0.501
>400
lipase
t-butanol
0.6
41.2
84.9
99.0
0.462
>400
YCJ01
Tetrahydrofuran
0.49
28.6
69.9
99.3
0.413
>400
Acetone
–0.23
35.5
77.4
99.2
0.438
>400
cr
us
an
2.4
10.8
d
M
>400
Data are means of at least three parallel reactions, where the average SD was 3.82% of the mean (range of 0.69–6.32%).
448
a
449
b
c=ees/(ees+eep).
450
c
E=ln[(1−c)(1−ees)]/ln[(1−c)(1+ees)].
452 453
ee of R-monoacetate (ees) and ee of S-diacetate (eep) were determined by HPLC.
Ac ce p
451
te
447
454 455 456 457
22
Page 22 of 29
458
Table 5 Effect of temperature on the esterification activity and enantioselectivity of the immobilized lipase
459
YCJ01 Temperature
Esterification activitya
(°C)
(µmol/g/min)
30
1.33
>400
35
1.96
>400
40
2.30
>400
45
2.70
389
50
2.62
276
an
us
cr
ip t
E
The reaction ended at the yield of S-diacetate reaching 49%. Data are means of at least three parallel reactions, where the
461
average SD was 2.57% of the mean (range of 1.28–4.82%).
462
a
465 466 467 468
d
te
464
Esterification activity=the amount of S-diacetate/( amount of immobilized lipase × reaction time)
Ac ce p
463
M
460
469 470 471 472
23
Page 23 of 29
te
d
Fig.1 .
Ac ce p
475 476 477 478 479 480 481 482 483
M
an
us
cr
ip t
473 474
24
Page 24 of 29
ip t cr us an te
d
Fig.2 .
Ac ce p
486 487 488 489 490 491 492 493 494
M
484 485
25
Page 25 of 29
ip t cr us an te
d
Fig.3.
Ac ce p
497 498 499 500 501 502 503 504 505
M
495 496
26
Page 26 of 29
ip t cr us an
506
te
d
Scheme 1
Ac ce p
510 511 512 513 514 515 516 517 518
M
507 508 509
519 520 521
Scheme 2
27
Page 27 of 29
521
Highlights:
522 523 524 525
The comb polymer modified TiO2 was first applied for immobilization of lipase. Greatly improved activity of immobilized lipase was obtained in all tested solvents. Strict enantioselectivity of the immobilized lipase was achieved in all tested solvents.
ip t
526
Ac ce p
te
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M
an
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cr
527
28
Page 28 of 29
ed
M
a
*Graphical Abstract (for review)
Ac
ce
pt
Solvent: Isopropyl ether
Amphiphilic comb-like polymer
=Lipase Applied to modifying mosoporous TiO2
yield of 49.8% ee of 99.2%
=Main line of polymer
H O
H O
H O
H O
H O
O Ti
O Ti
O Ti
O Ti
O Ti
O
O
O
O
O
H O O Ti O O
=Water Page 29 of 29
S1381-1177(14)00141-6 http://dx.doi.org/doi:10.1016/j.molcatb.2014.04.019 MOLCAB 2945
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
11-12-2013 28-3-2014 29-4-2014
Please cite this article as: B. Wang, W. Li, B. Wu, B. He, Efficient resolution of 3-phenoxy-1,2-propanediol by immobilized lipase on amphiphilic comb polymer modified TiO2 , Journal of Molecular Catalysis B: Enzymatic (2014), http://dx.doi.org/10.1016/j.molcatb.2014.04.019 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.
1
Efficient resolution of 3-phenoxy-1,2-propanediol by immobilized lipase on
2
amphiphilic comb polymer modified TiO2
3
Bin Wanga,b, Wenhao Lia, Bin Wua, Bingfang Hea,*
ip t
4
7
a
College of Biotechnology and Pharmaceutical Engineering, Nanjing University of
us
6
cr
5
Technology, Nanjing211816, China
10
b
School of Chemistry & Environmental Engineering, Jiangsu University of Technology,
Changzhou213001, China
M
9
an
8
11
* Corresponding author: Bingfang He
13
e-mail: [email protected]
14
Phone: +86 25 58139902
15
Fax: +86 25 58139902
17 18
te
Ac ce p
16
d
12
19 20 21 22
1
Page 1 of 29
Abstract An amphiphilic microenvironment is a key element to the lipase which catalyzes
24
substrates at the aqueous and hydrophobic interface. An amphiphilic comb copolymer,
25
poly(sodium acrylate)-g-methoxy poly(ethylene oxide), was introduced for the
26
post-functionalization of mesoporous TiO2. The lipase from Burkholderia ambifaria YCJ01
27
was immobilized on the comb copolymer modified TiO2. The activity of immobilized lipase
28
YCJ01 showed that comb polymer which side chain with a molecular weight of 1000 was the
29
best material for the functionalization of TiO2. The amount of adsorbed lipase was 55.6 mg/g
30
support and the immobilized lipase activity was 6076.1 U/g support. It was worth to note that
31
the immobilized lipase achieved more than 200% of its origin activity in hydrophobic solvents
32
and very stable in hydrophilic solvents. The immobilized lipase was used as a catalyst for
33
resolution of 3-phenoxy-1,2-propanediol, and showed high enantioselectivity (eep≥99%) in
34
all tested organic solvents. This might be explained that comb polymer with amphiphilic,
35
multiple side chains features could well stabilize the favorite active conformation of lipase to
36
the resolution substrate in all tested organic solvents. In diisopropyl ether reaction solvent,
37
the yield of 49.8% and an enantiomeric excess of 99.2% for S-diacetate were achieved after
38
one operation, and maintained relative yield of 72% for S-diacetate even after six reaction
39
cycles with no significant decrease in the enantiomeric excess of product.
40
Keywords Lipase, Comb polymer, Mesoporous TiO2, Immobilization, Biocatalysis
Ac ce p
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23
41 42 43 44
2
Page 2 of 29
45
1. Introduction
46
Lipases have been widely utilized to desymmetrize prochiral molecules with high
48
chemo-, regio- and stereo-selectivity in organic solvents [1-4]. However, their application is
49
currently limited owing to shortage of reusability, stability and activity in organic solvents.
50
Immobilization is a useful method to improve thermal and chemical stability of enzymes and
51
ensure reusability of enzymes. Although, it is believed that the water layer on the molecular
52
surface of enzymes determines their activity in organic media [5-6], an amphiphilic
53
microenvironment not only preserves the structural water thus maintaining the protein in
54
active conformation in organic media, but also is very important to the lipases which catalyze
55
substrates at the aqueous and hydrophobic interface by removing the “lid” covering their
56
active sites and making them more accessible to substrates [7]. So, amphiphilic polymers
57
have been extensively utilized as supports for the immobilization of lipases. Crespo et al. [8]
58
entrapped lipase from Candida rugosa in poly(ethylene oxide) for resolution of
59
(R,S)-2-octanol, high activity was obtained in organic solvents. To further stabilize the lipase,
60
Yasuda et al. [9] immobilized Rhizopus delemar lipase in amphiphilic polymer particles with
61
grafted chains, and multipoint attachment to the support stabilized the active conformation of
62
lipase in organic solvents. That meant that the amphiphilic polymers with grafted chains are
63
potentially excellent materials for the immobilization of lipase. However, as the amphiphilic
64
polymers have lower mechanical properties, their application in lipase immobilization is
65
limited.
66
Ac ce p
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M
an
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cr
ip t
47
The inorganic mesoporous materials have been found to be promising host matrixes for
3
Page 3 of 29
immobilization, for they have good mechanical properties, large surface areas, chemical
68
inertia and biocompatibility with enzymes. Among the mesoporous materials, mesoporous
69
TiO2, due to its unique pore structure and special physical and chemical properties, has
70
many potential applications in catalytic supports, sensor materials and electrode materials
71
[10-12]. For effective lipase immobilization, the surface of mesoporous materials was often
72
modified with an amphiphilic layer due to the ‘‘interfacial activation” mechanism [7].
73
Therefore, the mesoporous material with an amphiphilic layer is an effective support for the
74
immobilization of lipase.
cr
us
an
75
ip t
67
In our previous report [4], the lipase from Burkholderia ambifaria YCJ01 showed thermal stability and distinct super-stability to the most tested solvents (25%, v/v) for 60 days and
77
preferred to hydrolyze p-nitrophenyl esters with medium-long chain fatty acid. The resolution
78
of mandelic acid by lipase YCJ01 with a theoretical conversion yield of 50%, eep of 99.9%
79
and ees of 99.9%, showed its attractive potency in biocatalysis and the resolution of
80
pharmaceutical intermediates.
d
te
Ac ce p
81
M
76
In this paper, we introduced amphiphilic poly(sodium acrylate)-g-methoxy poly(ethylene
82
oxide) (herein after called PM), a comb copolymer, for the post-functionalization of
83
mesoporous TiO2. The lipase from Burkholderia ambifaria YCJ01 was adsorbed into the
84
pores of PM-modified TiO2. The immobilization exhibited the “interfacial activation” of lipase,
85
and also significantly improved the stability of lipase in organic system and the
86
enantioselectivity of lipase to the substrate. The immobilized lipase was used as a catalyst
87
for the resolution of racemic 3-phenoxy-1,2-propanediol, an important intermediate for
88
pharmaceuticals.
4
Page 4 of 29
89 90
2. Materials and methods
ip t
91 92
2.1. Materials
cr
93
us
94
Lipase YCJ01 from Burkholderia ambifaria was characterized in our previous report, the
96
amino acid sequence has been assigned GenBank Accession No. JQ733582. Burkholderia
97
ambifaria YCJ01 was deposited in CCTCC (Wuhan, China) with an accession number of
98
CCTCC M 2011058 [4]. p-Nitrophenyl palmitate (pNPP) was purchased from Sigma-Aldrich
99
(St. Louis, MO, USA). 3-Phenoxy-1,2-propanediol, vinyl acetate, sodium acrylate (SAA),
M
an
95
acrylic acid (AA), methoxy poly(ethylene oxide) (MPEO), dicyclohexyldimethylcarbodiimide
101
(DCC), 4–dimethylaminopyridine (DMAP), Na2S2O8 and NaHSO3 were purchased from
102
Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The mesoporous TiO2 used in
103
this work was kindly supplied by Lu Group [10]. All the reagents were used directly without
104
any further purification.
106
te
Ac ce p
105
d
100
2.2. Preparation of comb polymer
107 108
The comb polymer poly(sodium acrylate)-g-methoxy poly(ethylene oxide) (PM) was
109
synthesized by free radical copolymerization of SAA with methoxy poly(ethylene
110
oxide)-acrylate (MPEOAA) according to the method established by [13]. MPEOAA
5
Page 5 of 29
macromonomer was first synthesized by a catalytic esterification reaction between acrylic
112
acid (AA) and methoxy poly(ethylene oxide) (MPEO) (molar ratio of AA to MPEO was 2:1) in
113
the presence of a DCC and DMAP system. The copolymerization reaction between SAA and
114
MPEOAA was carried out in an aqueous medium at 95°C for 8h using Na2S2O8 as the
115
initiator and NaHSO3 as the activator, under a nitrogen gas atmosphere. After the reaction,
116
the PM was obtained. The 1H-NMR date of PM were shown in Fig. S1 and Table S1 (in
117
supplementary material), and the average molecular mass of PM were shown in Fig. S2 and
118
Table S2 (in supplementary material).
an
us
cr
ip t
111
119
d
122
2.3. Surface modification of TiO2
te
121
M
120
TiO2 is amphoteric, rendering it to be an anion and cation exchanger at acidic and
124
alkaline pH, respectively [14]. SEM patterns of TiO2 was shown in Fig. S3 (in supplementary
125
material). Functional TiO2 was obtained as follows: 200 mg of PM was dissolved in 10 mL
126
deionized water, 1 g of TiO2 was then added to the solution. The mixtures were stirred for 12
127
h at 30°C and filtrated. The insoluble substance washed with water three times and
128
successively dried in vacuum oven at 50°C for 8 h to yield the PM-modified TiO2. As the
129
PM1000 has a high weight average molecular weight (Mw=31767 g/mol, Table S2 in
130
supplementary material), the highest adsorbed amount of PM1000 on the TiO2 was about
131
18mg/g TiO2 (Fig.S4 in supplementary material).
Ac ce p
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132
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Page 6 of 29
133
2.4. Lipase Immobilization
134 135
The lipase solution was prepared by adding crude lipase powder to phosphate buffer (pH=7.5, 0.05 M) and the insoluble was removed. The activity of lipase solution was 40~45
137
U/mL and the protein concentration was 1.0 mg/mL. The adsorption experiment was carried
138
out by mixing PM-TiO2 with lipase solution with the ratio of 10 mg PM-TiO2/mL enzyme
139
solution, and the mixture was stirred for 12 h at 30°C. The PM-TiO2-lipase (immobilized
140
lipase) was separated by filtration and washed with phosphate buffer. All portions of the
141
filtration were retained for determination of protein concentration [15] and activity. The
142
immobilized lipase was dried in vacuum and stored at 4°C.
M
an
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ip t
136
143
146
d te
145
2.5. Enzyme activity assay
Activity of the free and immobilized lipase was assayed using pNPP as a substrate [4].
Ac ce p
144
147
The reaction mixture was composed of 240 μL of pNPP solution and 10 μL of lipase solution
148
(for free lipase) or it was composed of 100 mL pNPP solution and 1 mg of immobilized lipase.
149
The enzyme reaction was incubated at 40°C for 10 min. The liberated p–nitrophenol (pNP)
150
was quantified spectrophotometrically at 410 nm. One unit of lipase activity was defined as
151
the amount of enzyme that produced 1 μmol of pNP under the conditions mentioned above.
152 153
2.6. Characterization of supports
154
7
Page 7 of 29
The textural properties were studied by N2 adsorption–desorption measurements
156
(ASAP 2020 M) at the liquid N2 temperature of 77 K after the TiO2 samples were outgassed
157
for 2 h at 200°C, and the PM–TiO2 and PM–TiO2 with adsorbed lipase were outgassed for 6
158
h at 80°C. The Fourier transform infrared (FT–IR) analysis of the support was performed on
159
a Nicolet 6700 FT–IR instrument with KBr pellets. Thermogravimetry measurement was
160
determined by a thermogravimetric analyzer (TGA, NETZSCH TG 209 F3, Germany).
161
Scanning electron microscopy (SEM) observations were performed on a Hitachi S-4800
162
microscope operating at 20kV and a JEOL JSM 7401F microscope operating at 1.0kV. 1H
163
NMR spectroscopy of sample was determined using BRUKER AVANCE DRX-500 with D2O
164
as the solvent. Gel permeation chromatography (GPC) was carried out by an HPLC
165
equipped with Styrage1 HR3, HR4 and HR5 columns, and Wyatt Optilab rEX was used as
166
detector.
169 170
cr
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an
M
d te
168
2.7. Transesterification reaction
Ac ce p
167
ip t
155
After preliminary optimization, the enzymatic transesterification reaction of
171
3-phenoxy-1,2-propanediol (0.2 mmol) and vinyl acetate (1.2 mmol) in various solvents (4
172
mL) was carried out in 10 mL conical flasks with stoppers. The reaction was catalyzed in the
173
presence of 40 mg of immobilized lipase or 6 mg of lipase powder (about the same lipase
174
activity with 40mg of immobilized lipase) at 40°C with shaking at 180 rpm. Aliquots of
175
samples were withdrawn from the reaction mixture at intervals and the samples were
176
analyzed by an HPLC equipped with a Chiralcel ® OD–H column (Daicel, 4.6 mm×250 mm)
8
Page 8 of 29
and detected by UV at 270 nm, with a mobile phase composed of n–hexane/isopropanol
178
(85/15, v/v) with a flow rate of 1 mL/min. The reaction was also catalyzed with deactivated
179
catalyst as a control, and there was no product detected.
ip t
177
180
3. Results
cr
181
183
us
182
3.1. FT-IR spectrum of PM1000, TiO2 and PM1000 modified TiO2
185
an
184
An amphiphilic microenvironment is very important to the lipases which catalyze substrates at the aqueous and hydrophobic interface due to the ‘‘interfacial activation”
187
mechanism [7], amphiphilic poly(sodium acrylate)-g-methoxy poly(ethylene oxide) (PM) with
188
methoxy poly(ethylene oxide) group (MPEO) was designed for the modification of TiO2 as a
189
support for the immobilization of lipase. Different MPEO with molecular weight of
190
350,750,1000,1500 and 2000 were used to prepare the PM, signed as PM350, PM750,
191
PM1000, PM1500 and PM2000 respectively.
d
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Ac ce p
192
M
186
In Figs. 1a and 1b, the bands in the 2800–3000 cm−1 region correspond to the CH2 and
193
CH3 groups of the polymer backbone. In Fig. 1b, the bands at 1247 and 1101 cm−1 are
194
attributed to the vibration of the C–O–C bands. These results reveal the main structure
195
characteristics of PM. In Figs. 1b and 1c, the wave band at 3419 and 3427 cm−1
196
corresponding to –OH stretching vibrations shows light shift in the two spectra. This result is
197
due to the TiO2–carboxyl interface interactions. As the 1721 cm−1 peak (COO-) appear in Fig.
198
1b, which meant the PM attached to the TiO2 with electrostatic interactions. The region of
9
Page 9 of 29
199
bands at 500–800 cm−1 in Figs. 1b and 1c is tentatively assigned to the Ti–O–Ti groups.
200
3.2. N2 adsorption–desorption isotherms of TiO2, PM1000-TiO2 and PM1000-TiO2-lipase
ip t
201 202
The pore diameter distribution was calculated using the Barett–Joyner–Halenda (BJH)
cr
203
method based on the desorption isotherm, and the surface area was calculated using the
205
Brunauer–Emmett–Teller (BET) method based on the adsorption isotherm.
206
Adsorption–desorption isotherms and the corresponding pore size distribution of the TiO2
207
and its derivatives are shown in Fig. 2. It is noted that the hysteresis loop occurs at a high
208
relative pressure. This result suggests that these three materials were characterized by most
209
mesopores. The observed changes of the specific surface area and pore volume as well as
210
pore size are shown in Table 1. Compared to PM1000–TiO2, PM1000–TiO2-lipase exhibits a
211
decrease in the specific surface area and pore volume, and this decrease is likely due to the
212
loss of the adsorbed lipase and comb polymer. Sharp inflections occurring at around
213
P/P0=0.85 are observed for all samples due to the capillary condensation and the
214
evaporation in the uniform mesopores channels [16]. The average pore diameter decreased
215
from 16.3 nm to 15.5 nm, the degree of reduction meant that the lipase were immobilized in
216
a monolayer fashion on the support as explained by [17]. Monolayer adsorption may favor
217
the contact of lipase with substrates.
Ac ce p
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218 219
3.3. Immobilization of lipase and its interfacial activation
220
10
Page 10 of 29
221
The comb polymer PM for modification has dense COO- groups promoting significant interactions with Hδ+O on the surface of TiO2 by ionic bonds and yielding a strong anchoring
223
block. It also has dense side chains composed of methoxy poly(ethylene oxide) (MPEO)
224
which has the ability of adsorbing water and the lipase was adsorbed onto them (Scheme 1).
225
The relative activity recovery of immobilized lipase on the PM modified TiO2 was much
226
higher than that on the non-modified TiO2 with only 47.2% (Table 2). The significant
227
activation was observed since the activity recovery of the immobilized lipase ranging from
228
137.3% to 331.7% for all PM-modified TiO2 used as supports. The relative activity recovery
229
was increased with the increasement of average length of side chain (MPEO) corresponding
230
to the average molecular weight. As shown in Table 2, the amount of absorbed lipase
231
increased with the increasement of average length of MPEO under 1000 of average
232
molecular weight and then gradually decrease with the molecular weight increase further.
233
The supports PM2000-TiO2 and PM1500-TiO2 with a higher average length of MPEO yielded
234
a lower loading of lipase than that on the PM1000-TiO2, the higher steric hindrance effect of
235
PM2000-TiO2 and PM1500-TiO2 could be conjectured [13]. Among the supports, Though
236
PM2000-TiO2-lipase had the highest specific activity, PM1000-TiO2-lipase had the highest
237
lipase activity of 6076.1 U/g support and the highest protein loading 55.6 mg/g support,
238
which were used for the whole works.
Ac ce p
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222
239 240
3.4. Tolerance of immobilized lipase in organic solvents
241 242
The tolerance of immobilized lipase and the lipase powder in various organic solvents
11
Page 11 of 29
(100%, v/v) was compared in Table 3. The lipase was sensitive to organic solvents and only
244
maintained 18.8% to 46.8% of its original activity in hydrophilic solvents (logP<1). The
245
immobilized lipase in the same solvents showed much higher stability, even 71%~126% of
246
its original activity was achieved in the tested hydrophilic solvents. Fortunately, the
247
immobilized lipase was markedly activated with more than 200% of its original activity in
248
hydrophobic solvents (logP>1).
us
cr
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243
249
3.5. Efficient enzymatic resolution of racemic 3-phenoxy-1,2-propanediol
an
250 251
Considering the solubility of substrate and activity of lipase YCJ01, four kinds of tested
M
252
solvents were selected as reaction solvents for the resolution of racemic
254
3-phenoxy-1,2-propanediol. The reaction was carried out in two steps, as shown in Scheme
255
2. The RS-diol was prior transformed into corresponding primary monoacetate which was
256
detected by HPLC after initial 2 hours reaction with high regioselectivity. Then, the primary
257
monoacetate was continually converted enantioselectively into diacetates, a preference for
258
the S compound was always observed. The significant effect of the reaction solvents on the
259
enantioselectivity of lipase was observed when the lipase powder was used, and the least
260
value of ee for S-diacetate was 36.8% in t-butanol (Table 4). Strikingly, the immobilized
261
lipase showed the strict enantioselectivity with the values of ee for S-diacetate more than
262
99% in all tested reaction solvents. Owing to the high activity in diisopropyl ether, the value
263
of ee was 99.2% and the yield reached 49.8% (a close approximation to the theoretical yield
264
of 50%) for S-diacetate were achieved by immobilized lipase.
Ac ce p
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Page 12 of 29
265 266
3.6. Temperature on the esterification activity and enantioselectivity of the immobilized lipase
267
YCJ01 The temperature was varied in the range of 30–50°C. The highest esterification rate (2.7
ip t
268
µmol/g/min) were observed at 45°C (Table 5). The E values were high (>276) for all the
270
temperatures, and increased temperature leaded to a decrease in enantioselectivity.
271
Considering the thermostability, esterification rate and enantioselectivity, the temperature
272
40°C was selected as the reaction temperature.
an
3.7. Reusability of immobilized lipase
275
M
273 274
us
cr
269
The reusability of immobilized enzyme is a key point for the practical catalysis. The
277
relative yield of the immobilized lipase for S-diacetate was showed in Fig. 3, 72% of the
278
relative yield of the lipase immobilized on PM1000-TiO2 was retained after six reaction
279
cycles (108h of total operation), which was found to be much better than that of the lipase
280
powder. Though about 30% of the relative yield of immobilized lipase was lost, the values of
281
ee for S-diacetate fall slightly and the value of ee was 98.2% after six reaction cycles, which
282
indicated that the long time of reaction had no significant influence on the enantioselectivity
283
of PM1000-TiO2-lipase, the decrease of relative yield might be due to the detachment of
284
adsorbed lipase on outer/inner surface of PM-TiO2. The detachment of adsorbed lipase may
285
be remedied by connecting the lipase to activated poly(ethylene oxide) through a covalent
286
bond in the further work.
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287 288
4. Discussion
ip t
289 290
PM modification of TiO2 showed outstanding characteristics for immobilization of lipase
292
YCJ01, tolerance of immobilized lipase to organic solvents of lipase YCJ01 was significantly
293
improved. Jegannathan et al. [18] encapsulated Burkholderia cepacia lipase in
294
k-carrageenan, 43.7%-93.5% residual activity of immobilized lipase was obtained after
295
incubation in organic solvents for 1 h at 30°C. The Candida rugosa lipase was immobilized in
296
kaolin, 90% residual activity of immobilized lipase was obtained after incubation in hexane
297
for 1 days at 25°C [19]. In this paper, most of immobilized lipase YCJ01 activity was kept in
298
hydrophilic solvents and more than 200% of its original activity was achieved in hydrophobic
299
solvents (Table 3) after incubation in organic solvents for 3h at 40°C. It is well known that
300
immobilization of enzymes within an aqueous microenvironment is essential to the activity of
301
enzymes in the organic solvent. Wang et al. [20] immobilized enzymes in molecular hydrogel,
302
superactivity and high stability was obtained in organic solvents, even in hydrophilic solvents.
303
However, hydrophilic hydrogel would be not in favor of the immobilization of lipase due to the
304
"interfacial activation" mechanism. In this paper, amphiphilic materials were used for the
305
immobilization of lipase YCJ01, as shown in Scheme 1, PM modified TiO2 has the ability of
306
adsorbing water and also supplies the aqueous and moderate hydrophobic long side chains
307
(MPEO) for grasping lipase YCJ01 with multipoint attachment.
308
Ac ce p
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291
The most advantages of TiO2 modified with comb polymer which supplied an aqueous
14
Page 14 of 29
and a moderate hydrophobic microenvironment was favorable to the lipase YCJ01 showing
310
high activity as shown in Table 2 and 3. The opening of the “lid” of lipase YCJ01 might be
311
speculated in amphiphilic microenvironment, and the activation of lipase by immobilization
312
was obtained with all comb polymer modified TiO2 used as supports (Table 2). Strikingly,
313
strict enantioselectivity of the lipase YCJ01 immobilized on the support of amphiphilic
314
modified TiO2 was achieved to the substrate 3-phenoxy-1,2-propanediol, resulting more than
315
99% of eep for S-diacetate in all tested reaction solvents (Table 4). While the ee of product
316
S-diacetate by non-modified lipase was significantly affected by the reaction solvents. The
317
appearance of strict enantioselectivity of immobilized lipase YCJ01 to substrate in all tested
318
solvents was similar to the results obtained by few researchers[21-22]. The immobilized
319
Pseudomonas cepacia lipase showed high enantioselectivity (E>150) in several solvents for
320
the resolution of 1,2-diols [21]. The immobilized Pseudomonas sp. lipase exhibited strict
321
enantioselectivity (eep>99%) in four solvents for the resolution of (R,S)-methyl
322
mandelate[22]. The reasons for improvement of enantioselectivity and keeping the favorable
323
active conformation of lipase YCJ01 in all tested solvents might be attributed to some
324
protections on the protein conformation from the amphiphilic PM, greatly reducing the
325
distortion of the lipase structures generated from the organic solvents.
cr
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d
te
Ac ce p
326
ip t
309
Efficient resolution of 3-phenoxy-1,2-propanediol with eep (S-diacetate) of 99.2% was
327
achieved using lipase YCJ01 immobilized on amphiphilic comb polymer modified TiO2.
328
S-3-phenoxy-1,2-propanediol is widely used as an important intermediate for the production
329
of various pharmaceutical products [23-24]. The methods for the preparation of
330
enantiomerically pure 3-phenoxy-1,2-propanediol include asymmetric catalysis and enzyme
15
Page 15 of 29
catalysis. Theil et al. [24] reported the transformation which was carried out in the presence
332
of Amano lipase PS from Burkholderia cepacia, the yield of S-diacetate reached 49% and
333
the ee for S-diacetate was 79%. Worthy et al. [23] applied a scaffolding catalyst for
334
site-selective resolution of 3-phenoxy-1,2-propanediol, the yield of S-monoacetate reached
335
32% and the ee was 96%. In this paper, high yield and enantioselectivity of S-diacetate was
336
obtained with the immobilized lipase used as a catalyst, the yield reached 49.8% (a close
337
approximation to the theoretical yield of 50%) and the value of ee was 99.2% (Table 4). Also,
338
R-monoacetate was obtained with ee of 99.6% (Table 4), which meant that both of R product
339
and S product could be obtained simultaneously with high optical purity.
an
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331
341
M
340
5. Conclusions
In this paper, the amphiphilic comb copolymer modified support not only markedly
343
activated the activity of lipase YCJ01, more than 200% of its origin activity was achieved in
344
hydrophobic solvents and it was very stable in hydrophilic solvents; but also significantly
345
improved the enantioselectivity of lipase to the substrate (≥99% of ee for S-diacetate) in all
346
tested solvents. The lipase YCJ01 which was immobilized in amphiphilic comb copolymer
347
modified TiO2 catalyzed 3-phenoxy-1,2-propanediol with high yield of 49.8% and excellent
348
ee value of 99.2% for S-diacetate after one operation, and retained up to 72% of its relative
349
yield after six reaction cycles with no significant decrease in the enantiomeric excess of
350
product. The strategy of amphiphilic comb copolymer modification will provide an available
351
approach to promoting the modification of various supports for lipase immobilization.
Ac ce p
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352
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Page 16 of 29
353
Acknowledgments Financial supports for this research from the National Program on Key Basic Research Project
355
(2011CB710800) and the National High Technology Research and Development Key Program of China
356
(2012AA022205). We also acknowledge the support of the projects funded by PMSIRT (IRT1066) and PAPD.
ip t
354
cr
357 358
Reference
360
[1] U. T. Bornscheuer, Curr. Opin. Biotechnol. 13 (2002) 543–547.
361
[2] X. Tian, G.W. Zheng, C.X. Li, Z.L. Wang, J.H. Xu, J. Mol. Catal. B-Enzym. 73 (2011) 80−84.
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[3] G. Yang, J.P. Wu, G. Xu, L.R. Yang, Appl. Microbiol. Biotechnol. 81 (2009) 847−853.
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[4] C.J. Yao, Y. Cao, S.S. Wu, S. Li, B.F. He, J. Mol. Catal. B-Enzym. 85-86 (2012):105−110.
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[5] A. Ducret, M. Trani, R. Lortie, Enzyme. Microb. Technol. 22(1998) 212−216.
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[6] J.L. Schmitke, C.R. Wescott, A.M. Klibanov. J. Am. Chem. Soc. 118 (1996) 3360−3365.
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[7] C. Mateo, J.M. Palomo, G. Fernandez-Lorente, J.M. Guisan, R. Fernandez-Lafuente, Enzyme. Microb.
367
Technol. 40 (2007) 1451–1463.
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[8] R. Dalla-Vecchia, D. Sebrão, M.G. Nascimento, V. Soldi, Process Biochem. 40 (2005) 2677–2682.
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[9] M. Yasuda, H. Nikaido, W.R. Glomm, H. Ogino, K. Ishimi, H. Ishikawa, Biochem. Eng. J. 48 (2009) 6−12.
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[10] M. He, X.H. Lu, P. Caimi, X. Feng, L. Yu, Z.H. Yang, Chem. Commun. 19 (2004) 2202−2203.
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[11] Y.J. Jiang, Q.Y. Sun, Z.Y. Jiang, L. Zhang, J. Li, L. Li, X.H. Sun, Mater. Sci.Eng. C 29 (2009) 328−334.
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[12] R.Y. Zhang, A.A. Elzatahry, S.S. Al-Deyab, D.Y. Zhao, Nano. Today 7 (2012) 344−366.
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[13] Q.P. Ran, P. Somasundaran, C.W. Miao, J.P. Liu, S.S. Wu, J. Shen, J. Colloid interf. Sci. 336 (2009)
374 375
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624−633. [14] Y. Chen, Y.Y. Yi, J.D. Brennan, M.A. Brook, Chem. Mater. 18 (2006) 5326–5335.
17
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[15] M.M. Bradford, Anal. Biochem. 72 (1976) 248−254.
377
[16] T.P.B. Nguyen, J.W. Lee, W.G. Shim, H. Moon,
378
[17] B. Al-Duri, Y.P. Yong, Biochem. Eng. J. 4 (2000) 207−215.
379
[18] K.R. Jegannathan, E.-S. Chan, P. Ravindra, J. Mol. Catal. B-Enzym. 58 (2009) 78–83.
380
[19] M. B. A. Rahman, S. M. Tajudin, M. Z. Hussein, R. N. Z. R. A. Rahman, A. B. Salleh, M. Basri, Appl. Clay
381
Sci. 29 (2005) 111–116.
382
[20] Q.G. Wang, Z.M. Yang, L. Wang, M.L. Ma, B. Xu, Chem. Commun. 10 (2007) 1032−1034.
383
[21] A. Kamal, G. Chouhan, Tetrahedron Lett. 45 (2004) 8801-8805.
384
[22] N. Queiroz, M. G. Nascimento, Tetrahedron Lett. 43 (2002) 5225-5227.
385
[23] A.D. Worthy, X.X. Sun, K.L. Tan, J. Am. Chem. Soc.134 (2012) 7321−7324.
386
[24] F. Theil, J. Weidner, S. Ballschuh, A. Kunath, H. Schick,
390 391
ip t
cr us
an
M
te
FIGURE LEGENDS
Ac ce p
389
J. Org. Chem. 59 (1994) 388−393.
d
387
388
Micropor. Mesopor. Mat. 110 (2008) 560−569.
Fig.1 . FT-IR spectra of PM1000 (a), PM1000-TiO2 (b) and TiO2 (c)
392
Fig.2 . N2 adsorption–desorption isotherms and pore diameter distribution
393
curves (insert) of TiO2 and its derivatives. Symbols: (■)TiO2; (●)PM1000-TiO2;
394
(▲)PM1000-TiO2-lipase.
395 396
Fig.3. Reusability of lipase and immobilized lipase for transesterification. Relative yield= (the
397
yield of S-diacetate/the yield of S-diacetate for the first cycle)×100%. Operational conditions:
398
the first cycle ended with the yield of 49.8% for S-diacetate, the other cycles ended with the
399
same time of the first cycle. Symbols: (○) eep; (dense slant) lipase powders; (slant)
18
Page 18 of 29
400
PM1000-TiO2-lipase.
401 402
Scheme 1 Schematic representatives for the adsorption of lipase YCJ01 on the comb
404
polymer modified TiO2
ip t
403
405
Scheme 2 Enzymatic resolution of racemic 3-phenoxy-1,2-propanediol
cr
406
us
407 408
an
409
M
410 411
d
412
Tables
415 416
Table 1
Ac ce p
414
te
413
Textural properties of TiO2 and its derivatives Surface
Sample
2
area (m /g)
TiO2
Pore volume 3
(cm /g)
Average pore diameter (nm)
41.37
0.212
17.08
PM 1000–TiO2
38.02
0.194
16.25
PM1000–TiO2–lipase
34.76
0.176
15.51
417 418 19
Page 19 of 29
419 420
Table 2 Protein loading on supports and activity recovery of immobilized lipase Bound protein (mg/g
Lipase activity
Specific activity
Activity recovery
immobilized lipase) a
(U/g support)
(×105U/g protein)
(%)b
TiO2
37.6
738.3
2.0
47.2
PM350–TiO2
42.2
2410.3
5.7
PM750–TiO2
48.7
3752.0
7.7
PM1000–TiO2
55.6
6076.1
10.9
PM1500–TiO2
43.8
5695.8
PM2000–TiO2
34.5
4760.6
cr
ip t
Support
an
us
137.3 185.2 262.7 312.6
13.8
331.7
M
13.0
421
Data are means of at least three parallel reactions, where the average SD was 6.17% of the mean (range of 3.17–12.4%).
422
a
423
support weight after adsorption.
424
b
425
lipase)×100%
427 428
d
te
Activity recovery (%) = (activity of immobilized lipase/activity of free lipase with the same amount of adsorbed
Ac ce p
426
Bound protein= (amount of initial protein in the solution-remained amount of protein in the solution after adsorption/the
429 430 431 432
20
Page 20 of 29
433
Table 3 Stability of lipase and PM1000-TiO2-lipase in non–aqueous solvents Residual activity (%)a logP PM1000-TiO2-1ipase 246.3
Toluene
2.50
127.5
273.9
Diisopropyl ether
1.90
86.4
210.7
t-butanol
0.60
46.8
126.3
Tetrahydrofuran
0.49
40.6
89.5
Acetone
−0.23
32.7
71.3
1,4–Dioxane
−1.10
18.8
76.2
cr
111.0
us
3.50
an
Hexane
ip t
Lipase
M
Medium
The lipase powder and PM1000–TiO2–lipase were incubated in solvents (100%, v/v) at 40°C for 3 h. Data are means of at
435
least three parallel reactions, where the average SD was 4.63% of the mean (range of 1.46–8.7%).
436
a
437
lipase powder)×100%
439 440 441
te
Residual activity (%)= (total activity of immobilized lipase or lipase powder after 3h/total activity of immobilized lipase or
Ac ce p
438
d
434
442 443 444 445
21
Page 21 of 29
446
Resolution of racemic 3-phenoxy-1,2-propanediol in non–aqueous solvents
Table 4
S-diacetate Medium
cb
logP yield (%)
ees a
eep a
Ec
ip t
Catalyst
Lipase
Diisopropyl ether
1.9
45.7
92.0
99.1
0.481
YCJ01
t-butanol
0.6
24.9
11.5
36.8
0.238
powder
Tetrahydrofuran
0.49
1.1
0.75
81.8
0.009
Acetone
–0.23
3.1
6.9
93.6
0.069
32.1
Immobilized
Diisopropyl ether
1.9
49.8
99.6
99.2
0.501
>400
lipase
t-butanol
0.6
41.2
84.9
99.0
0.462
>400
YCJ01
Tetrahydrofuran
0.49
28.6
69.9
99.3
0.413
>400
Acetone
–0.23
35.5
77.4
99.2
0.438
>400
cr
us
an
2.4
10.8
d
M
>400
Data are means of at least three parallel reactions, where the average SD was 3.82% of the mean (range of 0.69–6.32%).
448
a
449
b
c=ees/(ees+eep).
450
c
E=ln[(1−c)(1−ees)]/ln[(1−c)(1+ees)].
452 453
ee of R-monoacetate (ees) and ee of S-diacetate (eep) were determined by HPLC.
Ac ce p
451
te
447
454 455 456 457
22
Page 22 of 29
458
Table 5 Effect of temperature on the esterification activity and enantioselectivity of the immobilized lipase
459
YCJ01 Temperature
Esterification activitya
(°C)
(µmol/g/min)
30
1.33
>400
35
1.96
>400
40
2.30
>400
45
2.70
389
50
2.62
276
an
us
cr
ip t
E
The reaction ended at the yield of S-diacetate reaching 49%. Data are means of at least three parallel reactions, where the
461
average SD was 2.57% of the mean (range of 1.28–4.82%).
462
a
465 466 467 468
d
te
464
Esterification activity=the amount of S-diacetate/( amount of immobilized lipase × reaction time)
Ac ce p
463
M
460
469 470 471 472
23
Page 23 of 29
te
d
Fig.1 .
Ac ce p
475 476 477 478 479 480 481 482 483
M
an
us
cr
ip t
473 474
24
Page 24 of 29
ip t cr us an te
d
Fig.2 .
Ac ce p
486 487 488 489 490 491 492 493 494
M
484 485
25
Page 25 of 29
ip t cr us an te
d
Fig.3.
Ac ce p
497 498 499 500 501 502 503 504 505
M
495 496
26
Page 26 of 29
ip t cr us an
506
te
d
Scheme 1
Ac ce p
510 511 512 513 514 515 516 517 518
M
507 508 509
519 520 521
Scheme 2
27
Page 27 of 29
521
Highlights:
522 523 524 525
The comb polymer modified TiO2 was first applied for immobilization of lipase. Greatly improved activity of immobilized lipase was obtained in all tested solvents. Strict enantioselectivity of the immobilized lipase was achieved in all tested solvents.
ip t
526
Ac ce p
te
d
M
an
us
cr
527
28
Page 28 of 29
ed
M
a
*Graphical Abstract (for review)
Ac
ce
pt
Solvent: Isopropyl ether
Amphiphilic comb-like polymer
=Lipase Applied to modifying mosoporous TiO2
yield of 49.8% ee of 99.2%
=Main line of polymer
H O
H O
H O
H O
H O
O Ti
O Ti
O Ti
O Ti
O Ti
O
O
O
O
O
H O O Ti O O
=Water Page 29 of 29