Author Topic: Use of Peroxygenase for Epoxidation of C=C  (Read 1543 times)

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Use of Peroxygenase for Epoxidation of C=C
« on: December 21, 2002, 05:10:00 AM »

A method has been discovered for the epoxidation of a compound having at least one carbon-carbon double bond, the method involves reacting a compound having at least one carbon-carbon double bond, a solvent, an oxidant, and membrane bound peroxygenase. Also discovered is a method for preparing the membrane bound peroxygenase involving grinding seeds containing peroxygenase to produce ground seeds, homogenizing the ground seeds in a buffer to form a slurry, centrifuging the slurry to produce a first supernatant, centrifuging the first supernatant to produce a second supernatant, and filtering said second supernatant through a protein-binding membrane filter to produce membrane bound peroxygenase; optionally the second supernatant is filtered through a hydrophilic membrane filter prior to filtering the second supernatant through a protein-binding membrane filter.


Oat seeds (Avena sativa L.) were supplied by Equine Speciality Feed Co. (Ada, Minn.). Durapore (PVDF, hydrophilic) membranes and Fluoropore (PTFE, hydrophobic) membranes were from Millipore (Bedford, Mass.). Sodium oleate was purchased from Nu-Chek-Prep, Inc. (Elysian, Minn.). Heptane and hydrogen peroxide (30%) were purchased from Aldrich (Milwaukee, Wis.). Sigma (St. Louis, Mo.) was the source of t-butyl hydroperoxide (70%). Water was purified to a resistance of 18 m.OMEGA.-cm using a Bamstead (Dubuque, Iowa) NANO pure system. All other reagents were of the highest purity available. Preparation of 3 methyl 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate (Me-HPODE): Linoleic acid was enzymatically converted to HPODE using lipoxygenase as described previously (Piazza, G. J., et al., J. Am. Oil Chem. Soc., 74: 1385-1390 (1997)) and HPODE was methylated with CH.sub.2 N.sub.2 to give Me-HPODE.

Preparation of oat seed microsomes (membrane-bound peroxygenase): For small scale reactions, dry oat seeds (10 g) were ground in 5 g batches in a 37 mL Waring Blender (New Hartfod, Conn.) mini-jar for 30 s. The ground oat seeds were transferred to a 110 mL mini-jar containing 90 mL cold 0.1 M potassium phosphate buffer (pH 6.7) and blended for 90 s at high speed. The oat seed slurry was centrifuged at 9000.times.g for 10 min. The pellet was discarded and the supernatant was centrifuged for an additional 10 min at 9000.times.g. After the second centrifugation, the pellet was discarded and the supernatant was divided into four equal portions and each portion was subjected to vacuum infiltration with a Fluoropore membrane (0.2 .mu.m, 47 mm). The Fluoropore membrane was wetted with methanol before loading onto the membrane holder. After vacuum infiltration, the membrane was cut into four equal size pieces, and these pieces placed into a reaction flask.

For large scale reactions 100 g of oat were ground dry (30 s), and then the ground seeds were homogenized in 900 mL of cold 0.1 M potassium phosphate buffer (pH 6.7) using a Waring commercial blender (2 min). The slurry was centrifuged at 16,000.times.g for 15 min. The pellet was discarded and the supernatant was centrifuged for an additional 15 min at 16,000.times.g. The supernatant was passed through a hydrophilic Durapore membrane filter (0.65 .mu.m, 142 mm), and the filtrate was collected and divided into quarters and each was passed through a hydrophobic Fluoropore membrane (0.2 .mu.m, 142 mm).

Epoxidation reactions: The indicated amount of Me-HPODE, oleic acid, or elaidic acid dissolved in CH.sub.2 Cl.sub.2 was added to a 10 or 15 mL stoppered Erlenmeyer flask, and the solvent removed under a stream of nitrogen. Into each flask was added water-saturated heptane or 0.1 M potassium phosphate buffer (pH 6.7) containing 0.1% (w/v) Tween 20 and the membrane pieces. The reaction was initiated by adding t-butyl hydroperoxide. The suspension was agitated at C. for 2 h or as indicated. At the end of the incubation period, 3.5 mL methanol was added, and after removal of the membrane pieces, the contents were transferred to a 125 mL separatory funnel. The products were partitioned between 30 mL diethyl ether and 25 mL water. After separating the layers, the water layer was extracted with 25 mL diethyl ether. The ether fractions were combined, dried over sodium sulfate, and taken to dryness under a stream of nitrogen. The products were dissolved in 2 mL dichloromethane and stored at C. until analysis. When fatty acid was the substrate, the products were methylated with CH.sub.2 N.sub.2 before analysis.

Epoxidation of styrene and cyclohexene: Reaction mixtures contained 1.9 mg (18.2 .mu.mol) styrene or 1.5 mg (18.2 .mu.mol) cyclohexene, 7.0 mL 50 mM Hepes/0.1% (w/v) Tween 20, pH 7.5, and peroxygenase immobilized on a 47 mm, 0.2 .mu.m Fluoropore membrane. At 0, 1, 2, and 4 h, 3.34 .mu.mol t-butyl hydroperoxide was added; at 6 h, 20.0 .mu.mol t-butyl hydroperoxide was added. The reaction temperature was C. At 24 h a sample was removed using a solid phase microextractor with a polydimethyl siloxane filter (100 .mu.m thickness)(Supelco, Bellefonte, Pa.) and analyzed on an HP-MS 5890 GC/MS containing a 30 m HP-5MS column (Hewlett Packard, Palo Alto, Calif.). Epoxide yields shown below were obtained from separately prepared reaction:

                 Styrene               Cyclohexene
                    (Percent Yield of Epoxide)
                  60.0                    76.5
                  60.8                    72.8


Patent US6485949

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