modified biomolecule is placed in aqueous medium again then the imprinted active site will revert back to its native conformation and cease to demonstrate the acquired imprinted property. To restrict this flipping back of a modified active site to its native and thermodynamically favorable conformation we developed the combinatorial cross-linked imprinted protein approach (we termed it CLIP). The methodology is depicted in Fig. 2. Here, glucose oxidase (GO) is selected as an intricate model enzyme to modify its substrate selectivity (to see whether GO accepts galactose as its substrate in addition to its normal substrate, glucose) and thereby
to demonstrate the feasibility of the CLIP approach in inducing new catalytic properties. Further, the possibility of scaling-up the catalytic oxidation of galactose using CLIP-GO is described.
We previously demonstrated the enhanced stability of P-glucosidase using a two-step immobilization technique (9). In the present CLIP method a similar immobilization procedure is adopted, wherein the first step is derivatization using itaconic anhydride to introduce polymerizable vinyl groups into GO. It is important to optimize the percentage of derivatization in order to maintain or increase the specific activity of native enzyme. Then, after purification of the derivatized GO using size-exclusion chromatography, the resulting dried enzyme is used for imprinting. D-Galactose, a competitive inhibitor of GO, is used as a print molecule and imprinting is carried out in aqueous medium. The GO-galactose complex is precipitated using 1-propanol. Subsequently, the freeze-dried precipitate is suspended in dry anhydrous cyclohexane as a porogenic solvent and chemically cross-linked using excess of cross-linking agent. This excess amount is necessary to stabilize the imprinted conformation of the modified enzyme (10,11), and similar results were also found during synthesis of trypsin receptors prepared by affinity-imprinting procedure (12,13). After CLIP-GO is washed it is applied to oxidized galactose (an induced substrate), which yields galactono-1,4-lactone. This product cannot be synthesized using either native GO or galactose oxidase, the well known oxidase for galactose. The product of galactose oxidase is galactonohexodialdose. Thus, following this simple biochemical modification of crude GO on a mature protein level we not only broaden the substrate spectrum of the resulting enzyme in pure aqueous solution but yield a new product not possible to achieve by any of the oxidase enzymes so far reported in the literature.
All chemicals used in this work are of analytical grade.
2.1. Derivatization of Glucose Oxidase
1. GO from recombinant Aspergillus niger (specific activity 8500 nkat mg/protein (Roche, Mannheim, Germany).
2. Itaconic anhydride (Acros Organics; www.acros.be).
3. PD-10 desalting workmate columns (Amersham Pharmacia Biotech, Uppsala, Sweden).
4. 2,4,6-Trinitrobenzenesulfonic acid (TNBS) (Sigma Chemical Company, Steinheim, Germany).
5. Working buffer: 50 mMK-phosphate, pH 6.0.
2.2. Imprinting of Glucose Oxidase
1. Galactose/2 n-propanol (Fluka, Buchs, Germany).
2.3. Cross-Linking of Derivatized Imprinted Enzyme
1. Dry cyclohexane (Fluka, Buchs, Germany).
2. Glucose monohydrate (Fluka).
3. 30% Hydrogen peroxide (v/v) (Fluka, Buchs, Germany).
4. 2,2 Azobis(2-methyl-propionitrile) (AIBN; Acros Organics).
5. Ethyleneglycol dimethacrylate (EGDMA; Aldrich Chemical Company, Steinheim, Germany).
6. o-Dianisidine dihydrochloride (Sigma Chemical Company).
7. Horseradish peroxidase (specific activity 225 units mg/protein, one unit activity corresponds to production of 1 mg purpurogallin from pyrogallol in 20 s at pH 6.0 at 25°C) (Roche, Mannheim, Germany).
2.4. Bioconversion of Glucose or Galactose
1. Galactono-1,4-lactone (Fluka).
2. Glucono-1,5-lactone (Fluka).
3. Galactonohexodialdose (Fluka).
4. 2,6-Pyridinedicarboxylic acid (Fluka).
5. Cetyltrimethylammonium bromide (Fluka).
6. Basic anion buffer: in a 100-mL capacity stoppered conical glass flask 0.334 g 2,6-pyridinedicarboxylic acid and 10 mL cetyltrimethylammonium bromide (5 mM) are dissolved in 80 mL distilled water. The pH of this mixture is adjusted to 12.1 using 5 M NaOH and the final volume of this mixture is adjusted to 100 mL with distilled water.
1. The acylation of GO using various amounts of itaconic anhydride in working buffer is carried out. Here the procedure for 70% derivatization is explained.
2. In 25-mL capacity glass beaker placed on a magnetic stirrer add 60 mg of GO lyophilized powder and dissolve in 10 mL of working buffer. When the enzyme is completely soluble place this beaker in an ice bath at 4°C. A pH meter is dipped into this solution for monitoring pH during derivatization.
3. Then, slowly portion-wise, 420 mg itaconic anhydride is added with the help of a spatula (see Note 1) under constant stirring speed of 200 rpm. During this addition, pH of this solution is maintained at 6.0 using 3 MNaOH.
4. When all the itaconic anhydride has been added, the reaction mixture is stirred continually 30 min more in the same ice bath.
5. The unreacted monomer and other low-molecular-weight impurities (Mr< 1000) can be removed by gel filtration (PD 10 column) using deionized water as an eluting solvent (see Note 2).
6. All eluates are pulled together in a glass beaker at 4°C and derivatized enzyme is lyophilized to dryness and stored in a dry box at 4°C until further use (see Note 3).
1. In 10-mL capacity cellstar polypropylene tube with stopper, dissolve 30 mg of dry derivatized enzyme and 54 mg of galactose (as an imprint molecule) in 1 mLof 10 mM K-phosphate buffer, pH 5.0. Incubate at 25°C for 30 min.
2. GO-galactose complex is then precipitated by addition of 4 mL of supercooled (-20°C) ^-propanol in this mixture (see Note 4). The resulting precipitated enzyme is incubated on ice for 10 min more.
3. The precipitate is collected by centrifugation at 11,000 rpm for 15 min at 4°C (Eppendorf centrifuge 5804R with F-34-6-38 rotor).
4. The pellet is washed with 1 mL of supercooled (-20°C) n-propanol and dried over molecular vacuum pump (Alcatel, Drytel 31) for 12 h and kept under same vacuum until further use (see Note 5).
1. In 1.5-mL capacity Eppendorf tube 10 mg of dry derivatized GO is suspended in 1 mL of dry cyclohexane using an ultrasonication bath (Branson 2200; see Note 6). The bath is filled with cold water to absorb the heat generated during sonication. This procedure requires approx 30 min.
2. To this suspended enzyme 4 mg AIBN and 200 pL EGDMA are added. Check that AIBN is completely dissolved.
3. The free radical polymerization initiated under ultraviolet irradiation (Fluotest® Forte, Atlas Materials Testing, [email protected]) at l = 365 nm. The polymerization continued for 5 h at 25°C (see Note 7).
4. Keep the resulting white polymer refrigerated at 5°C for 12 h.
5. The polymer is then washed as follows: first wash with 2 mL cyclohexane to remove unreacted cross-linked, followed by three washes with 10 mL each of working buffer.
6. The protein and enzyme activity (see Note 8) are checked in aqueous washings to find out the leakage of the enzyme after cross-linking (see Note 9).
7. Cross-linked polymer is dried on a molecular vacuum pump. Similarly, a control polymer with nonimprinted, derivatized enzyme is also cross-linked in the same fashion (this is a classical immobilized enzyme).
3.4 Bioconversion of Glucose or Galactose
1. In a 2-mL capacity Eppendorf tube either 39.6 mg glucose monohydrate or 36.0 mg galactose are dissolved in 1 mL of working buffer.
2. To it either 50 Mg/mL native GO or immobilized/CLIP enzyme with equivalent amount of protein (~20 mg/ mL polymer) is added.
3. This reaction mixture is saturated with air and placed in thermomixer comfort (Eppendorf) kept at 25°C with stirring speed of 1000 rpm.
4. The enzymatic reaction is terminated by heat step (i.e., increasing the temperature to 70°C after 1 h).
5. The reaction mixture is centrifuged (Eppendorf centrifuge 5417 R) at 13,000 rpm for 10 min at 4°C.
6. The supernatant is analyzed by high-performance capillary electrophoresis (HPCE; see Note 10) and high-performance liquid chromatography (HPLC; see Note 11) separately. Structure of respective products are confirmed by !H or 13C NMR spectroscopy (see Note 12).
7 The bioconversion can be scaled up to a 50-mL reaction volume. For this, a stirred glass reactor (total capacity 200 mL) with continuous aeration is used. The corresponding amounts of enzyme and substrate equivalent to 50 mL reaction volume are taken in this reactor. The reaction is continued in a similar way as mentioned above except stirring at 1000 rpm is done with a mechanical propeller shaft and the temperature is maintained at 25°C by a thermostated water jacket. The products are analyzed by the same methods.
1. The portion-wise addition of itaconic anhydride and monitoring pH at 6.0 are the factors critical to achieving 70% derivatization.
2. The gel filtration should be carried out in a cold room.
3. The degree of derivatization is measured using TNBS assay. The detailed procedure is given in ref. 9. At 70% derivatization of GO an 8% increase in the specific activity is observed as compared with native enzyme.
4. The precipitant should be supercooled continuously at -20°C. The precipitation should be done abruptly in few seconds.
5. The precipitated imprinted complex is highly hygroscopic and therefore should be stored under dry conditions. The presence of traces of water in the precipitate will disrupt the imprinting efficiency.
6. Cyclohexane is dried by refluxing over metallic sodium for 12 h and then stored over molecular sieves (4 A, bead diameter 2 mm; Merck).
7. Proper lead shields should be used to work under ultraviolet radiation as the rays are mutagenic. The Eppendorf tube is placed in decline position and rotated intermittently so as to cover its maximum surface under the ultraviolet rays. Maintain the temperature of eppendorf tube strictly at 25°C during polymerization.
8. The protein is measured by Bradford method and enzyme activity is measured according to a procedure reported in ref. 14.
9. No enzyme leakage is found in washings after cross-linking procedure. A control polymer is synthesized for comparison with CLIP polymer. This whole process of CLIP preparation requires not more than 2.5 d.
10. HPCE is carried out using HP3D capillary electrophoresis system from Agilent Technologies GmbH (Waldbronn, Germany). The system comprises a CE unit with a built-in diode-array detector and an HP3D CE chemstation for system control, data collection, and analysis. Capillary electrophoresis is performed using a fused silica capillary (80.5-cm total length, 72-cm effective length, and internal diameter of 50 Mm). The sample is injected with a pressure of 50 mbar for 6 s. The applied voltage is set at -15 kV and the capillary temperature is maintained at 15°C. The elution is carried out using basic anion buffer and detection is carried out using diode-array detector. The signal wavelength is set at 350 nm with a reference at 275 nm. The calibration is done using pure external standards (15).
11. The conversion of respective monosaccharide is also analyzed using HPLC. The analysis is performed on Beckman HPLC system gold, which is composed of an injection module and an automatic sampling system (Bio-Rad AS-100 HRLC) connected to a refractive index (RI) detector (Erma RI ERC-7512). The column used for detection is carbohydrate Ca2+ column (capillary length 300 mm and thickness 8 mm). This column is pre-equilibrated at 85°C for 30 min. Then, 50 mL appropriately diluted reaction mixture is injected into the column and product eluted using deionized water at a flow rate of 0.6 mL/min. The detection is done at 50°C using a RI detector. The calibration is performed using pure external standards. The semipreparative HPLC method is applied to isolate product from scaled-up reaction volume of 50 mL. The LiChrospher®-NH2 column (250 x 25 mm, particle size 5 Mm) is employed. The undiluted reaction mixture (3-mL portions) is injected using BioCAD® sprint system. The elution is carried out with acetonitrile:water (80:20 v/v) mobile phase at a flow rate of 39 mL/min. The fractions are collected using autofractional collector (Advantec SF-2120) and lyophilized.
12. The structure of individual product is confirmed by 1H and 13C NMR analysis recorded at 300 MHz with a Varian, Unity Inova™ 300-MHz instrument. The
NMR spectral data is in accordance with the proposed structures of galactono-1,4-lactone, glucono-1,5-lactone and galactonohexodialdose, respectively (16).
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