Degradation of Cyanoglucosides by Brevibacterium Sp. Strain R 312 Jean Luc Legras, Mohamed Riad Kaakeh, Alain Arnaud, Pierre Galzy

Degradation of cyanoglucosides by Brevibacterium sp. strain R 312 Jean Luc Legras, Mohamed Riad Kaakeh, Alain Arnaud, Pierre Galzy

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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

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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 previously (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 method (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). It is noteworthy that on buffered media the nitrite hydratase activity stays constant during the whole growth period (7).

Degradation of cyanoglucosides by a supernatant S2 The degradation of cyanoglucosides involves the hydrolysis of the f3-glucosidic bond and of the nitrite function. As a consequence these compounds could theoretically be degraded in several ways: hydrolysis of the /3-glucosidic linkage and then of the a-hydroxynitrile by the nitrite hydratase, and of the amide subsequently produced by the amidase; or by the primary hydrolysis of the nitrite group, then of the /3-glucosidic bond and further of the amide. A third possibility is to hydrolyze the nitrite group then the amide group before the final hydrolysis of the /3-glycosidic bond. In each case the theoretical end products should be glucose and the organic acid corresponding to the aglycone.

Table 1. Comparison of the three enzymes participating in the degradation of cyanoglucosides by Brevibacterium sp. R 312. 1989 Degradation of Cyanoglucosides by Brevibacterium 455

Fig. 1. Biosynthesis of the different activities participating in the degradation of cyanoglucosides by Brevibacterium sp. R 312. (•): OD of culture. An (o): nitrile hydratase activity (pH 7, propionitrile 50 mM). Aa (A): amidase activity (pH 7, propionamide 50 mM). A~( x): /3-glucosidase activity (pH 7, pNPG 5 mM).o: pH. Specific activities measured at 30°C in U mg-~. Brevibacterium sp. R 312 was grown on the medium reported in MATERIALS AND METHODS supplemented with the amidase inductor N-methylacetamide (30 mM).

We investigated the degradation of two simple cyanoglucosides: prunassin and linamarin by a cellular extract (supernatant S2). The appearance of glucose, a-hydroxynitrile (and cyanide) and of the organic acids were monitored as well as the disappearance of the cyanoglucoside. But for technical reasons, we could not measure the amount of amides or the glucosides other than glucose and cyanoglucosides. Figure 2 presents the curves obtained during the kinetics with each cyanoglucoside. These curves are different depending on the cyanoglucoside used. The amount of glucose produced from prunassin was equal to the amount of cyanoglucoside hydrolyzed, but these amounts are different with linamarin. This difference was explained by the synthesis of the amidoglucoside 2-hydrox- yisobutyramide l-/3-D-glucoside from linamarin, while the corresponding amidoglucoside was not synthesized from prunassin. It is noteworthy that the concentrations of nitriles and of a-hydroxynitrile remained low during the whole 456 LEGRAS, KAAKEH, ARNAUD, and GALZY VOL. 35

Fig. 2. Degradation of two cyanoglucosides by a supernatant S2 at pH 6; 30°C (cyanoglucoside 4 mM; supernatant S2 0.6 ml; 0.1 M phosphate buffer pH 6, 2.4 ml). A: •, cyanoglucoside (linamarin); o, amidoglucoside + acidoglucoside; A, 2- hydroxyisobutyric acid; o, cyanide + 2-hydroxybutyronitrile; +, glucose. B: •, cyanoglucoside (prunassin); A, mandelic acid; o, cyanide + mandelonitrile; +, glucose.

experiment; this shows the high affinity of the nitrile hydratase for the free a-hydroxynitrile. To strengthen our hypothesis regarding the production of amidoglucoside, we prepared partially purified nitrite hydratase and f3-glucosidase extracts with only one activity.

Preparation of partially purified extracts The partially purified extracts were prepared as described above. The two 1989 Degradation of Cyanoglucosides by Brevibacterium 457

Table 2. Procedures for purifying /3-glucosidase and nitrile hydratase.

Fig. 3. Elution profile on Q-Sepharose fast-flow chromatography column. OD at 280 nm; +, nitrile hydratase; o, amidase; X, /3-glucosidase; •, NaCI concentration. enzyme purification ratios are given in Table 2. The peaks of activity were well separated and presented only one type of activity (Fig. 3).

Hydrolysis of nitrite by the nitrite hydratase preparation We determined the nitrile hydratase Vm and Km for linamarin and for the nitriles corresponding to the aglycones of the cyanoglucosides (Table 3). Propionitrile and lactonitrile were chosen as references. The values found were in good agreement with those given by Bui et al. (9). On the other hand, the value of 458 LEGRAS, KAAKEH, ARNAUD, and GALZY VOL. 35

Table 3. Km and Vm of the nitrile hydratase for various substrates. Kinetics at pH 7, 30°C.

the Km estimated for HIBN (9 mM) was much higher than that reported by Bui et al. for isobutyronitrile, even though both substrates are inhibited when in excess. We observed the direct hydrolysis of the nitrile function of linamarin by the nitrile hydratase. This reaction occurs very slowly, but in the presence of large amounts of the enzyme (3,000 U/ml of reaction media) and after 2 h of incubation, neither cyanoglucosides nor glucose appeared in the reaction media. Thin layer chromatography of the reaction products showed a new spot compared to the standards. It was adjacent to glucose but turned pink after treatment with ninhydrin. This supports the hypothesis that a new compound is formed, which must be the amidoglucoside corresponding to linamarin:

The high value of the Km and the low effect on linamarin indicate that the nitrile hydratase of Brevibacterium sp. R 312 has a low affinity for cyanoglucosides. We think that this low affinity results from the steric inhibition caused by the glucosidic bond in the a position of the nitrile group. Lower activities did not permit us to determine the Km for prunassin and amygdalin. As the nitrile hydratase showed less activity for mandelonitrile than for aromatic nitrile, apparently the inhibition due to the aromatic group and the glucose make the hydrolysis of prunassin or amygdalin very difficult.

Hydrolysis of cyanoglucosides by the f3-glucosidase of Brevibacterium sp. The f3-glucosidase of Brevibacterium sp. R 312 hydrolyzes mainly aryl-glycosides but also linamarin (23). Table 4 shows the values found for commercially available 1989 Degradation of Cyanoglucosides by Brevibacterium 459

Table 4. Vm and Km of the J3-glucosidase of Brevibacterium sp. R 312 for different cyanoglucosides. Kinetics at pH 6, 30°C.

Fig. 4. Pathways of degradation of the two cyanoglucosides linamarin and prunassin by Brevibacterium sp. R 312 strain. 65%: the ratio between the glucose produced and the linamarin introduced. 35%: the percentage of linamarin that disappeared without glucose formation. cyanoglucosides with pNPG given as a reference. We were unable to measure the degradation of the two amido-glucosides as we could not dispose from these products.

Degradation pathway of cyanoglucosides The degradation of the linamarin in fungi and almonds has been described by Lebeau et al. (22) and Haisman and Knight (16). In these two cases the mechanism was first hydrolysis of the fl-glucosidic linkage by a specific fl-glucosidase and then decomposition of the a-hydroxynitriles of linamarin into cyanide and of prunassin into acetone or benzaldehyde. Moreover, the fungi described by Lebeau et al., as well as other fungal pathogens of cyanogenic plants (14) hydrolyzed cyanide thanks to a cyanase. The mechanism that we deduced from our in vitro experiments (Fig. 4) 460 LEGRAS, KAAKEH, ARNAUD, and GALZY VOL. 35 is different in several ways from that previously reported . At pH 6, Brevibacterium sp. R 312 usually degraded cyanoglucosides by the hydrolysis of the f3-glucosidic bond by a /3-glucosidase, then hydrolysis of the a-hydroxynitrile by the nitrile hydratase and of the amide by the amidase. This seems to be the principal way of degradation for prunassin, but another way exists for linamarin in which the nitrile function is hydrolyzed first. This has not been reported before . This study shows the great potential of such a nitrile metabolizing agent as the Brevibacterium strain for detoxicating staple food containing cyanoglucosides such as cassava .

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