Degradation of Cyanoglucosides by Brevibacterium Sp. Strain R 312 Jean Luc Legras, Mohamed Riad Kaakeh, Alain Arnaud, Pierre Galzy
Total Page：16
File Type：pdf, Size：1020Kb
Degradation of cyanoglucosides by Brevibacterium sp. strain R 312 Jean Luc Legras, Mohamed Riad Kaakeh, Alain Arnaud, Pierre Galzy To cite this version: Jean Luc Legras, Mohamed Riad Kaakeh, Alain Arnaud, Pierre Galzy. Degradation of cyanoglucosides by Brevibacterium sp. strain R 312. Journal of general and applied microbiology, The Microbiology Research Foundation, 1989, 35, pp.451-461. hal-02727589 HAL Id: hal-02727589 https://hal.inrae.fr/hal-02727589 Submitted on 2 Jun 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. J. Gen. Appl. Microbial., 35, 451-461 (1989) DEGRADATION OF CYANOGLUCOSIDES BY BREVIBACTERIUM SP. R 312 STRAIN JEAN-LUC LEGRAS, MOHAMED RIAD KAAKEH, ALAIN ARNAUD,* ANDPIERRE GALZY Laboratoire de Microbiologie Industrielle et de Genetique des Microorganismes, Ecole Nationale Superieure Agronomique, Place Viala, 34060 Montpellier Cedex, France (Received October 2, 1989) We report here the action of the f3-glucosidase, nitrite hydratase and amidase of Brevibacterium sp. R 312 during the in vitro degradation of some cyanoglucosides. The degradation pathway found for prunassin begins with the action of the f3-glucosidase followed by the action of the nitrite hydratase and of the amidase, but linamarin can also be attacked first by the nitrite hydratase. The Vm and Km of the nitrite hydratase for the main nitrite compounds and of the /3-glucosidase for cyanoglucosides were determined with partially purified extracts. Tissues of many plants and of some insects produce cyanoglucosides among which amygdalin, prunassin and linamarin are the most widespread. Their degradation was mostly described for fungi involving a fl-glucosidase, a cyanase, and in most cases a hydroxynitrilelyase (14). Brevibacterium sp. R. 312 was isolated by Arnaud et al. (1,2) and selected for its ability to hydrolyze water soluble nitrites. This bacterium has a constitutive nitrite hydratase (4, 9,10, 30, 31) and an inducible amidase (17, 19,25,28,29). These two enzymes have a wide spectrum allowing them to degrade many nitrite compounds and amides (3, 9, 20, 25) which makes them very attractive for industrial applications (6, 8, 20, 26, 32). In addition to these two enzymes, a fl-glucosidase was recently found, purified and characterized by Legras et al. (23). The /3-glucosidase hydrolyzes the two cyanoglucosides linamarin and prunassin, and releases a-hydroxynitrites that can be further degraded by the nitrite hydratase. Here we report a study of the enzymatic systems concerned with the degradation of cyanoglucosides by Brevibacterium sp. R 312. * Address reprint requests to: Dr . A. Arnaud, Laboratoire de Microbiologie Industrielle et de Genetique des Microorganismes, Ecole Nationale Superieure Agronomique, Place Viala, 34060 Montpellier Cedex, France. 451 452 LEGRAS, KAAKEH, ARNAUD, and GALZY VOL. 35 MATERIALS AND METHODS Strains. The bacterial strain that we used throughout this study was Brevibacterium sp. R 312, isolated from soil and described by Arnaud et al. (1, 2). We also used Candida molischiana (Zikes) Meyer and Yarrow CBS 136 to produce /3-glucosidase (15). Culture conditions. The cultures were incubated at 28°C and shaken (80 oscillations per min, 9 cm amplitude) in flasks filled to one tenth of their volume. The basal medium used for Brevibacterium sp. R 312 had the following composition: 10 mM phosphate buffer (KH2P04, Na2HP04) pH 7; CaClz, 10 mg; Mg504.7H20, 50 mg, FeS04, 10 mg; MnSO4, 1 mg; glucose, 10 g; (NH4)2S04, 5 g; thiamine hydrochloride, 2 mg. Growth was measured by monitoring the absorbance at 600 nm. Chemicals and enzymes. The following nitrile compounds were used as substrates: 2-hydroxyisobutyronitrile (HIBN), 2-hydroxyisobutyric acid (HIBAc) and mandelic acid (Aldrich-Chimie, Strasbourg, France). Mandelonitrile, prunassin and linamarin were obtained from Sigma-Chimie (L`Isle d`Abeau Chesnes, France). The almond f3-glucosidase (emulsin) was purchased from Fluka-Chemie AG (Buchs, Switzerland) and the f3-glucosidase of Candida molischiana was prepared as previously described by Gonde et al. (15). Preparation of cell-free extracts. Cells were sonicated as described pre- viously (23). The resulting suspension was centrifuged at 18,000 x g for 15 min, and at 180,000 x g for 90 min. The supernatant (S2) was saved and used for enzymatic studies. Preparation of partially purified enzymatic extracts. After salting out the /3-glucosidase and the nitrile hydratase activity at 30-70% saturation of ammonium sulfate followed by dissolution and dialysis overnight against the equilibration buffer (50 mM Tris-HCI, pH 7.5), the protein solution was applied to an ion exchange column (2.4 x 40 cm) filled with Q-Sepharose Fast-Flow (Pharmacia, Uppsala, Sweden). The elution began with a 200 ml wash with the equilibration buffer followed by a 600-ml linear gradient of NaCI (0 to 0.3 M) in the same buffer. After a short plateau (100 ml) at 0.3 M of NaCI a second gradient (500 ml, 0.3 to 0.6 of NaCI) was applied to the column. The eluate was collected in 10-m1fractions at a rate of 100 ml/h. Nitrile assay methods and determination of nitrile hydratase activity. Propioni- trile and propionamide were assayed by gas chromatography as described by Jallageas et al. (18) with an Intersmat IGC 121 DFL chromatograph equipped with an FID. a-Hydroxynitriles were assayed colorimetrically by a procedure developed by Jallageas et ai. (21). Nitrile hydratase activity was assayed at pH 7, 30°C, with propionitrile 50 mM substrate. For a-hydroxynitrile, an acidic primary solution was prepared with HCl 1 mM and the kinetics were performed quickly (1 or 2 min) to avoid the spontaneous decomposition of the a-hydroxynitrile at neutral or basic pH to cyanide and acetone or benzaldehyde (13). 1989 Degradation of Cyanoglucosides by Brevibacterium 453 Cyanoglucosides assay procedure. Cyanoglucosides (amygdalin, prunassin and linamarin) were assayed after the release of the aglycone in acidic media by a specific f3-glucosidase (emulsin for amygdalin and prunassin, and of Candida molischiana for linamarin). A primary solution of emulsin was prepared at a concentration of 10 mg/ml in 0.2 M citrate phosphate buffer, pH 4.5, and of the /3-glucosidase of Candida molischiana: 25 U/ml (on p-nitrophenyl-l,4-f3-D-glucopyranoside: pNPG 5 mM, at pH 4 and 30°C) in 0.2 M citrate phosphate, pH 4. To 400µl of 0.2 M citrate phosphate buffer, pH 4.5 was added 100µl of the amygdalin or prunassin solution and 50 µl of the emulsin solution. After mixing and 30 min of incubation, the free a-hydroxynitrile was measured colorimetrically. For linamarin assays the buffer was replaced by 0.2 M citrate phosphate, pH 4 and the f3-glucosidase from Candida molischiana. At these pHs, the nitrile hydratase, the amidase and the f3-glucosidase of Brevibacterium sp. R 312 were all inhibited and this stopped the degradation of cyanoglucosides by the enzymatic extracts of this bacteria. Acids determination. Two organic acids: mandelic acid and HIBAc were assayed by gas chromatography on a nickel column (0.3 x 50 cm) of Chromosorb 101 (60-80 mesh) at a colmn temperature of 170°C for HIBAc and 200'C for mandelic acid. The sample was acidified before injection with HCl 0.1 N to avoid ghost peaks as described by Dupreez and Lategan (12). Cyanide determination. Cyanide concentration was determined according to the method of Jallageas et al. (21). Glucose determination and f3-glucosidase activity. For Brevibacterium sp. R 312 /3-glucosidase activity was assayed using 5 mM pNPG at pH 6 and 30°C (23) or by glucose determination as described by Bergmeyer (5). During the study of degradation of cyanoglucosides by a supernatant S2, Bergmeyer's method could not be used because of high endogenous interference, however Somogyi's meth- od (27) was found convenient. Thin layer chromatography (TLC). We used the TLC method described by Brimer et al. (11) with silica gel precoated plates (Merck Art 5721, 20 x 20 cm). The solvent system was composed of ethyl acetate, pyridine, acetone, methanol and water (40: 30:12 :10 : 8). Two detection reagents were used: A mixture of 4 ml aniline, 4 g diphenylamine, 100 ml acetone, and 20 ml phosphoric acid 85% was used as a standard sugar detection reagent and the Merck ninhydrin spray was used for amidoglucoside. For both reagents the plates were heated at 100°C for 15 min after spraying. Protein determination. Protein was assayed according to the method of Lowry et al. (24) with bovine serum albumin standard. Enzymatic units. One unit of each enzyme was defined as the amount of enzyme that hydrolyzed 1 µmol of substrate per min under the above conditions. 454 LEGRAS, KAAKEH, ARNAUD, and GALZY VOL. 35 RESULTS AND DISCUSSION General properties of the enzymatic systems Table 1 summarizes the properties of the enzymes taking part in the degradation of cyanoglucosides by Brevibacterium sp. R 312: /3-glucosidase, nitrite hydratase and amidase. These enzymes are endocetlular, active at pH 6 to 7, and inhibited by thiol-reacting agents. However, the biosynthesis rates were very different (Fig. 1). Nitrite hydratase is constitutive (4, 9,10, 30, 31) and produced in large amounts, especially in the early log phase; the amidase activity is inducible (17, 25, 28, 29) and produced at the same period of growth while the /3-glucosidase is synthesized at a lower level in the late log phase (23).