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Screening for New Hydroxynitrilases from Plants

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Screening for New Hydroxynitrilases from Plants Biosci. Biotechnol. Biochem., 69 (12), 2349–2357, 2005 Screening for New Hydroxynitrilases from Plants y Yasuhisa ASANO,1; Ken’ichirou TAMURA,1 Nobutaka DOI,1 Techawaree UEATRONGCHIT,2 Aran H-KITTIKUN,2 and Tohru OHMIYA3 1Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan 2Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, 90112, Thailand 3Botanic Gardens of Toyama, 42 Fuchumachi-kamikutsuwada, Toyama 939-2713, Japan Received June 29, 2005; Accepted August 22, 2005 We established a simple HPLC method to determine pharmaceutical industries.4) Effenberger et al. demon- the activity and stereochemistry of the chiral mandelo- strated that (R)-methylalkylketone cyanohydrin from nitrile synthesized from benzaldehyde and cyanide, and methylalkylketone is obtained with good chemical yields applied it to screen for hydroxynitrile lyase (HNL) and in high optically purity using partially purified (R)- activity of plant origin. A total of 163 species of plants mandelonitrile lyase from Prunus dulcis (Miller) D. A. among 74 families were examined for (R)- and (S)-HNL Webb. (syn: Prunus amygdalus Batsch.) (almond).4,5) activities using the method. We discovered that homo- Griengl et al. have used (S)-specific HNL from Hevea genate of leaves of Baliospermum montanum shows (S)- brasiliensis (H.B.K.) Muell. Arg. (rubber) in the chiral HNL activity, while leaves and seeds from Passiflora synthesis of (S)-cyanohydrins.6) As far as we know, there edulis, and seeds from Eriobotrya japonica, Chaenomles has been almost no systematic screening for enzymatic sinensis, Sorbus aucuparia, Prunus mume, and Prunus activity of (R)- and (S)-mandelonitrile lyases in plants, persica show (R)-HNL activity. Partially purified (R)- although microbial screening is a standard and versatile HNLs from Passiflora edulis and Eriobotrya japonica method to obtain industrially important new biocata- acted not only on benzaldehyde but also on aliphatic lysts.7) We found that only a small portion of cyanogenic ketone. The enantiomeric excess of (R)-methylpropyl- plants show detectable HNL activity. Especially (S)- ketone cyanohydrin synthesized from 2-pentanone using mandelonitrile lyase has been found only among Hevea homogenate from leaves of Passiflora edulis was 87.0%, brasiliensis (rubber),8) Manihot esculenta Crantz (cassa- and that of (R)-mandelonitrile synthesized by homoge- va),9) Ximenia americana Linn.,10) Sorghum bicolor nate from seeds of Eriobotrya japonica was 85.0%. Moench,11) and Annona squamosa Linn.12) In this study, we examined a variety of plants for (R)- Key words: hydroxynitrile lyase; Prunus dulcis; Balio- and (S)-mandelonitrile lyase activities using our newly spermum montanum; Passiflora edulis; developed HPLC method to determine the activity and Eriobotrya japonica the stereochemistry of the chiral mandelonitrile synthe- sized from benzaldehyde and potassium cyanide. After Cyanogenesis is an ability of plants to release extensive screening of plants, we discovered producers hydrogen cyanide in the cells, and is considered a of (R)- and (S)-mandelonitrile lyases. They were used to mechanism to protect plants from attack by fungi and catalyze the synthesis of optically active mandelonitrile predators. Hydroxynitrile lyases (HNLs) are one of the and methylpropylketone cyanohydrin from benzalde- key enzymes in cyanogenesis and catalyze the final step hyde and 2-pentanone respectively. in the biodegration of cyanogenic glycosides.1) Cyano- genesis is believed to be distributed in over 2,650 Materials and Methods species of higher plants.2,3) Hydroxynitrile lyase (HNL) is a general term referring Materials. Seeds of Prunus dulcis (almond), kindly to four enzymes, mandelonitrile lyase (EC 4.1.2.10), donated by Lotte Co., Ltd., Tokyo, Japan, and Meiji p-hydroxymandelonitrile lyase (EC 4.1.2.11), acetone– Seika Kaisha Ltd., Tokyo, Japan, were used for cyanohydrin lyase (EC 4.1.2.37), and hydroxynitrilase extraction of (R)-mandelonitrile lyase. Bodies of various (EC 4.1.2.39). HNLs are used as biocatalysts for the plants (leaves, roots, shoots, spikes, and rhizomes) were synthesis of optically active cyanohydrins, which are mainly collected at the Botanic Gardens of Toyama and important building blocks in the fine chemical and used for extraction of mandelonitrile lyase. An HPLC y To whom correspondence should be addressed. Tel: +81-766-56-7500 ext 530; Fax: +81-766-56-2498; E-mail: [email protected] Abbreviation: HNL, hydroxynitrile lyase 2350 Y. ASANO et al. column, CHIRALCEL OJ-H, was purchased from 15 min at 4 C. The supernatant, which was adjusted to Daicel Chemical Industries Ltd., Osaka, Japan. 2- pH 6.4, was precipitated with ethanol to obtain partially Pentanone was purchased from Tokyo Kasei Kogyo purified enzyme. The precipitate was suspended in 10 ml Co., Ltd., Tokyo, Japan. (Trimethylsilyl) diazometane of 50 mM imidazole–HCl buffer (pH 6.0). The suspen- was obtained from Aldrich Chemical Co., Inc., Milwau- sion was dialyzed overnight against 2 liters of 10 mM kee, WI, U.S.A. Potassium cyanide was from Wako Pure imidazole–HCl buffer (pH 6.0), and the buffer was Chemical Industries, Ltd., Osaka, Japan. All other changed once. The dialyzed preparation was pooled and chemicals were from commercial sources and were (R)-mandelonitrile lyase activity was measured. used without further purification. 1H-NMR and 13C- NMR spectra were recorded with a JEOL LA-400 Typical procedure for extraction of HNL from plants. spectrophotometer (Tokyo, Japan). Seeds from plants were cracked with a hammer to release the soft kernels inside. The kernels were Enzyme assay. Enzyme activity was assayed by collected and crushed in process homogenizer at 4 C, measuring the production of optically active mandelo- with 10 mM KPB (potassium phosphate buffer, pH 6.0), nitrile synthesized from benzaldehyde and cyanide. The to give a milky suspension. The suspension was filtered standard assay solution contained 300 mmol citrate through four layers of cheese cloth to remove the buffer (pH 3.5–6.0), 50 mmol of benzaldehyde, 100 mmol insoluble part. The suspension was then centrifuged of potassium cyanide, and 100 ml of the enzyme in a final (18;000 Â g, 20 min at 4 C), and removal of the residue volume of 1.0 ml. The reaction was started by the gave a crude enzyme preparation, which was fractio- addition of 100 ml of the enzyme solution, which was nated with (NH4)2SO4. Proteins precipitating with 30% incubated at 25 C for 1 to 120 min. Aliquots (100 ml) saturation were collected by centrifugation (18;000 Â g, were withdrawn at various reaction times, and the 20 min at 4 C), dissolved in minimum volume of 10 mM reaction was stopped by the addition of 0.9 ml of organic KPB, pH 6.0, and dialyzed against the same buffer with solvent (hexane:isopropanol = 9:1 by volume), the three changes. After that the dialyzed solution was mandelonitrile formed was extracted, and the super- centrifuged and the supernatant was stored at 4 C and natant, obtained by centrifugation (15;000 Â g, 1.0 min assayed for HNL activity. at 4 C), was assayed by HPLC. The amounts of Leaves, roots, shoots, spikes, or rhizomes were placed benzaldehyde and (R)- and (S)-mandelonitrile were in four layers of cheese cloth and frozen by soaking in determined by HPLC with a CHIRALCEL OJ-H column liquid nitrogen. The frozen samples were cracked with a at 254 nm using a mobile phase of the solvent hammer to release small broken pieces of the samples (hexane:isopropanol = 9:1 volume) at a flow rate inside. They were made into finer pieces or powder by 1.0 ml/min. The retention times of benzaldehyde and grinding on mortar and pestle on ice. The finely ground (R)- and (S)-mandelonitrile were about 5.8, 16.9, and samples were suspended in 10 mM KPB, pH 7.0, and 21.8 min respectively. One unit of enzyme activity was extracted overnight with stirring at 4 C. The suspension defined as the amount of enzyme that produced 1 mmol was centrifuged (18;000 Â g, 20 min at 4 C), and of optically active mandelonitrile from benzaldehyde removal of the residue gave a crude enzyme preparation, per min under the standard assay condition. In the which was concentrated about 10 times by centrifuga- control experiment, boiled enzyme was used. The tion at 2;000 Â g and 4 C with a Centriprep YM-10 amount of racemic mandelonitrile formed non-enzymat- (Amicon, Beverly, MA, U.S.A.), and then used for ically in the reaction mixture was subtracted from the activity measurement. amount in the former reaction mixture. The reaction The homogenates of the following 156 species among progressed linearly in proportion to the amount of 74 families were inactive. L and S stand for the origin of enzyme in the first few minutes. the organ of the plant samples, leaves and seeds respectively. Other organs are specified in parentheses. Preparation of partially purified (R)-mandelonitrile Acanthaceae: Thunbergia grandiflora (Roxb. ex Rottl.) lyase from Prunus dulcis (almond). The surfaces of Roxb. (L); almonds were sterilized with 0.52% (w/v) sodium Aizoaceae: Mollugo pentaphylla Linn. (L); hypochlorite and then dried. All the purification steps Apocynaceae (2 species): Nerium indicum Mill. (L), were performed at 4 C. Almond (26.0 g) was homo- Trachelospermum jasminoides (Lindl.) Lem. (S); genized with 250 ml of 20 mM ammonia solution in a Aquifoliaceae: Ilex verticillata (Linn.) A. Gray (L); SMT PH91 process homogenizer (Tokyo, Japan). The Araceae (9 species): Alocasia macrorrhiza (Linn.) G. homogenate was filtered through four layers of cheese Don (L), Anthurium andreanum Linden ‘Elizabeth’ (L), cloth and centrifuged at 28;000 Â g for 15 min at 4 C. Anthurium andreanum Linden ‘Obake’ (L), Anthurium To remove unnecessary protein, the homogenate was andreanum Linden cv.
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