Cyanide degradation by Rhizopus oryzae G. PADMAJA AND C . BALAGOPAL Division of Post Harvest Technology, Central Tuber Crops Research Institute, Trivandrurn, India Accepted April 16, 1985

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PADMAJA, G., and C. BALAGOPAL. 1985. Cyanide degradation by Rhizopus oryzae. Can. J . Microbiol. 31: 663-669. Rhizopus oryzae, a mucoraceous fungus associated with the postharvest spoilage of cassava was found to effectively metabolize cyanide. Degradation of cyanogenic glycosides of cassava by R. oryzae was studied by growing the organism in potato dextrose broth with and without linamarin and potassium cyanide. The influence of adaptation of the organism to low and high cyanide concentrations on both growth and the release of extracellular rhodanese into cyanide-containing media was studied. Nonadapted cultures of R. oryzae grow poorly when compared with the cyanide-adapted cultures. However nonadapted R. oryzae cultures released large quantities of rhodanese when compared with the adapted ones. Potassium cyanide (1.0 mM) was found to be an efficient inducer of rhodanese whereas potassium cyanide (5.0 mM) repressed the release of rhodanese. A significant inductive effect was produced by thiosulphate and thiocyanate. Linamarin repressed the rhodanese activity of cultures during the growth phase. Rhizopus oryzae also elaborated extracellular linamarase during its growth in broth with and without linamarin. This study revealed the potential use of R. oryzae in detoxifying the cyanogenic glycosides in cassava feed and food preparations as well as in the effective disposal of cyanide in industrial wastes. G., et C. BALAGOPAL. 1985. Cyanide degradation by Rhizopus oryzae. Can. J . Microbiol. 31: 663-669. PADMAJA, Le Rhizopus oryzae, un champignon mucoracCen associC a I1altCrationdu manioc aprks la rkcolte, s'est avCrC capable de mCtaboliser le cyanure de faqon efficace. La dtgradation des glucosides cyanogknes du manioc par R. oryzae a CtC CtudiCe en faisant croitre I'organisme dans un bouillon de dextrose de pomme de terre, avec et sans linamarine et cyanure de potassium. L'influence de I'adaptation de I'organisme 21 de faibles concentrations de cyanure ou a des concentrations Clevees dans les deux milieux de croissance, ainsi que la libtration de rhodankse extracellulaire dans les milieux contenant du cyanure ont CtC CtudiCes. Les cultures de R. oryzae qui n'Ctaient pas adapttes au cyanure n'ont present6 qu'une faible croissance comparativement aux cultures qui Ctaient adaptCes. Le cyanure de potassium 2I 1,O mM s'est avCrC un inducteur efficace de rhodankse, tandis qu'une concentration de 5,O mM a rCprimC la liberation de rhodankse. Un effet inducteur significatif a CtC produit par du thiosulfate et du thiocyanate. La linamarine a rCprimC llactivitC rhodankse au cours de la phase de croissance des cultures. Le R. oryzae a aussi ClaborC une linamarase extracellulaire au cours de sa croissance dans les bouillons contenant ou non de la linamarine. Cette Ctude a rCvC1C I'emploi potentiel de R. oryzae pour dCtoxifier les glycosides cyanogknes du Manioc tant aux fins alimentaires directes que pour les prCparations d'aliments, ainsi que pour llClimination efficace du cyanure des eaux industrielles. [Traduit par le journal]

Introduction

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A number of microorganisms have been reported to possess the ability to produce, assimilate, or detoxify hydrogen cyanide (Castric 1981). Most of the fungal pathogens of cyanogenic plants that have been studied are tolerant to HCN (Fry and Millar 1971). Variations exist in the modes of assimilation of HCN by bacteria and fungi. Chromobacterium violaceum (Brysk et al. 1969), Bacillus megaterium (Castric and Strobe1 1969), and Escherichia coli (Dunnill and Forwden 1965) possess a P-cyanoalanine synthase activity which enables them to condense HCN with cysteine to form P-cyanoalanine. Another product of HCN assimilation in C . violaceum is y-cyanoa-aminobutyric acid (Brysk and Ressler 1970; Ressler et al. 1973). Some fungi incorporate HCN into the amino acids alanine, glutamine, and asparagine (Strobe1 1964; Akopyan 1975). Fusarium solani converts HCN into ammonia and presumably further to carbon dioxide (Shimizu and Taguchi 1969). Rhizoctonia solani converts HCN to propionaldehyde and then to a-aminobutyronitrile and a-aminobutyric acid (Mundy et al. 1973). Rhodanese, which is widely distributed in bacteria and fungi, is also used for cyanide assimilation (Allen and Strobel 1966; Hall and Berk 1968; Schook and Berk 1978; Sorbo 1975). Induction of HCN-insensitive respiration is an alternate device by which microorganisms can cope with HCN (Henry and N ~ n 1975; s and 1977). A new HCN assimilation has been reported in the fungi S t e m ~ h ~ l i uloti m and Gloeocercospora sorghi which attack the cyanogenic plants Lotus corniculatus and Sorghum vulgare, respectively

(Fry and Myers 1981). These fungi release an enzyme formamide hydrolyase (FHL) which condenses HCN with water to form formamide. The presence of FHL has also been reported in Fusarium solani and Helminthosporium maydis (Fry and Myers 1981). Although many microorganisms have been reported to be associated with the spoilage of cassava (Majumdar 1955; Affran 1968; Ekundayo and Daniel 1973; Burton 1970; Noon and Booth 1977), Rhizopus oryzae, a mucoraceous fungus, was found to be one of the predominant organisms associated with the decay of harvested cassava tubers in Kerala (Balagopal et al. 1981). Earlier studies have revealed that the fungus was capable of releasing polyphenol oxidase, peroxidase, and polysaccharide-degrading enzymes into the growth medium and hence a key role in cassava deterioration was attributed to this fungus (Padmaja and Balagopal 1985). The mode of cyanide degradation catalysed by the rhodanese of R . oryzae and the regulation of this enzyme have been studied in detail and are reported here. The ability of the organism to elaborate linamarase, an enzyme which hydrolyses the cyanogenic glycosides of cassava, was also investigated.

Materials and methods and growth conditions Rhizopus oryzae isolated from rotten cassava tubers (isolate No. 1

of the Central Tuber Crops Research Institute (CTCRI)) was used in the study. Spore suspensions of R. oryzae were prepared from I-week-old cultures grown at 30°C on Difco potato dextrose agar

CAN. J . MICROBIOL. VOL. 31, 1985

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TABLE1. Effect of KCN on the free thiocyanate levels (in milligrams per 100 mL culture filtrate) in R. oryzae culture filtrates

Time of incubation (h)

Concn. of KCN

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(mM)

24

48

Nonadapted cultures (with and without KCN) 0.0 0.40 0.80 1 .O 1.40 1.08 1 .oo 0.88 2.0 3.0 1 .OO 1.00 4.0 1.60 1.40 5.0 1.40 1.00 Low KCN (1.0 mM) adapted cultures 2.0 0.28 3.0 0.48 4.0 0.60 High KCN (5.0 mM) adapted cultures 2.0 1.80 3.0 1.80 4.0 2.00

72

2.80 0.92 0.80 0.72 1.00 0.60

96

1.60 0.40 0.20 0.20 0.40 0.20

120

Mycelial dry weight (mg)

0.40 ND* ND ND ND 0.20

*ND, thiocyanate not detected.

slants in 150-mL screw-cap bottles. Spores were harvested in sterile water and were filtered to remove mycelial fragments, washed, and diluted to the desired concentration (5 X lo7 spores/mL) after counting in a haemocytometer. The same concentration was used in all the subsequent experiments.

Adaptation of the organism to cyanide To study whether the adaptation of the organism to low and high cyanide levels has any effect on the growth of the fungus and also on the release of extracellular rhodanese, a spore suspension of the fungus was inoculated into 250-mL Erlenmeyer flasks containing 100 mL potato dextrose with low and high cyanide content (1.0 and 5.0 mM, respectively). After incubation for 120 h, spores produced by the cultures were harvested and washed twice with sterile distilled water. The washed spores were used for adaptability studies. Rhodanese activity studies using R. oryzae Spores from the low cyanide adapted (ICNa) and high cyanide adapted (hCNa) cultures of R. oryzae prepared as above were transferred to flasks containing potato dextrose (PD) broth with 2.0, 3.0, and 4.0 mM potassium cyanide (KCN). An equal number of spores (5 X lo7 spores/mL) was inoculated into each flask. The flasks were incubated at 28OC and duplicate samples were drawn from each flask every 24 h up to 120 h. Two replications were maintained for each concentration of cyanide as well as for the control. The samples were centrifuged at 10 000 X g for 10 min and the supernatants were used as the enzyme source. Control flasks inoculated with nonadapted (nCNa) spores were also simultaneously incubated. The rhodanese activity of the culture filtrates was assayed by the method of Sorbo (1955). The protein content of the culture filtrates was determined by Lowry's method (Lowry et al. 1951). In subsequent studies only spores from nonadapted cultures of R. oryzae were used as inoculum since the resulting cultures produced more rhodanese than those inoculated with sporei from cyanide-adapted cultures. Induction of rhodanese of R. oryzae by KCN was investigated by growing the organism in PD broth with KCN (I .O and 5.0 mM). The sampling and enzyme assay were done as described above. The regulatory effect of thiosulphate and thiocyanate on the rhodanese released by R. oryzae during its growth phase was investigated by growing the organism in PD broth containing varying levels of

thiosulphate and thiocyanate (2.0,4.0,6.0, and 8.0 mM). The enzyme sampling and assay were done as described previously. The free thiocyanate levels of the culture filtrates were also determined (Sorbo 1955) for all growth conditions (except in medium containing thiocyanate, in which case the thiocyanate vilues cannot be correlated with the thiocyanate produced as a result of rhodanese activity). The influence of linamarin, the cyanogenic glycoside of cassava (purchased from Calbiochem Ltd., La Jolla, CA), on rhodanese release by R. oryzae was also studied by growing the organism in PD broth containing varying levels of linamarin (0.2, 0.4, 0.6, and 0.8 mM). The enzyme sampling and assay were done as described previously.

Linamarase activity studies using R . oryzae The ability of R. oryzae to elaborate linamarase was assessed by withdrawing samples at 24-h intervals from the culture filtrates containing linamarin (0.2-0.8 mM). The effect of KCN on linamarase release was also studied by incorporating 1.0 mM KCN along with the various linamarin levels in a duplicate set of flasks. Control flasks without linamarin were also incubated simultaneously. Linamarase activity of the culture filtrates was tested by the following method: the assay system consisted of 0.5 mL linamarin (0.2 mM), 1.5 mL sodium phosphate buffer (0.1 M, pH 6.5), and 1.0 mL enzyme source. Incubation was carried out at 28OC for 1 h after which the reaction was terminated with 1.0 mL 0.2 N sodium hydroxide. The released cyanide was estimated using chloramine-T and barbituric acid - pyridine reagent (Asmus and Garschagen 1953). In the case of rhodanese and linamarase, the pH optima were confirmed and activities were measured at optimum pH values. Substrate blanks and enzyme blanks were used for each enzyme assay. The specific activities of the enzymes are expressed as follows: 1 rhodanese unit (RU) = 1.0 mg of thiocyanate liberated-h-' 100 mg protein-' and 1 linamarase unit (LU) = I .O p,g of cyanide liberated- h-' . 100 mg protein-'.

-

Growth performance of the organism The growth of the organism under the various growth conditions provided in the study was assessed from the mycelial dry weights taken after 120 h of growth. The mycelial mat was collected, washed twice with distilled water, and dried to a constant weight at 105OC.

Results and discussion Adaptation of R. oryzae to cyanide and release of rhodanese Exposure of R . oryzae to low and high KCN (1.0 and 5.0 mM, respectively) had a significant effect on the release of rhodanese by the organism (Figs. 1A- IC). When the KCN concentration was low (2.0 mM), the nonadapted (nCNa) cultures eFborated more rhodanese during the growth period up to 96 h. This was followed by a sharp decrease after growth was completed and sporulation had begun (120 h). For lCNa cultures, there was an initial induction of rhodanese activity (48 h); however, the lCNa cultures released only very low quantities of rhodanese and there was a reduction in the enzyme activity as the growth advanced (Fig. 1A). With a concentration of 3.0 mM, the nCNa cultures and lCNa cultures showed an initial induction of rhodanese and as growth advanced, the activity decreased (Fig. 1B). Similar repression in rhodanese aclivity (as with 2.0 mM KCN) was exhibited by hCNa cultures. When the KCN concentration was raised to 4.0 mM, the rate of rhodanese induction by nCNa and lCNa cultures was significantly reduced, while hCNa cultures exhibited a similar repression during the growth phase (Fig. 1C). The mycelial dry weights (Table 1) indicated that potassium cyanide was toxic to nCNa cultures of R. oryzae. The growth began only after appreciable detoxi-

PADMAJA AND BALAGOPAL

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FIG. 1. (A) Influence of adaptability of R. oryzae to low KCN (1 mM) and high KCN (5 mM) on the rhodanese activity of R. oryzae grown in PD media with KCN (2 mM). (B) Influence of adaptability of R. oryzae to low KCN (1 mM) and high KCN (5 mM) on the rhodanese activity of R. oryzae grown in PD media with KCN (3 mM). (C) Influence of adaptability of R. oryzae to low KCN (1 mM) and high KCN (5 mM) on the rhodanese activity of R. oryzae grown in PD media with KCN (4 mM).

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CAN. J . MICROBIOI VOL. 31, 1985

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FIG. 2. Induction of rhodanese activity of R. oryzae by KCN during the growth phase with I (O),5 (A), or 0 ( 0 )mM KCN.

fication of cyanide had taken place. Thus growth is poor in media containing 2.0, 3.0, and 4.0 mM KCN (mycelial dry weights of 115.0, 90.0, and 50.0 mg, respectively). Although adaptation to low and high KCN significantly reduced the ability of the organism to elaborate rhodanese, growth was better in adapted cultures (Table 1). However hCNa cultures had greater mycelial dry weights indicating that once the organism was adapted to high KCN levels, it could easily cope with lower cyanide levels in the growth medium. Tolerance to HCN was similarly induced by exposing the fungus, Stemphylium loti, to minute concentrations of HCN and once adapted, the spores were reported to be insensitive to even 25.0 mM HCN concentrations (Fry and Evans 1977). Unlike Gloeocercospora sorghi which elaborates large quantities of formamide hydrolyase when adapted to HCN (Fry and Myers 1981), R. oryzae elaborates more rhodanese when spores from nCNa cultures are used. This could be attributed to the lack of sensitivity of adapted cultures to KCN as shown by the high mycelial dry weights obtained for the cyanide-adapted cultures. Thus the need for rhodanese for detoxification was low in adapted cultures and, as a result, only small quantities of the enzyme were released during the growth phase. The free thiocyanate levels of R. oryzae culture filtrates (nCNa spores) indicated that there was a steady decline in the thiocyanate as the growth progressed (Table 1). Hence thiocyanate was not the end product of the assimilation of cyanide by R. oryzae which otherwise would have accumulated as in the case of formamide during the growth of G . sorghi (Fry and Myers 1981). Regulation of rhodanese KCN (1.0 mM) was found to be an efficient inducer of rhodanese production by R. oryzae (Fig. 2). Enhancement of rhodanese activity by the addition of HCN has also been reported in the bacteria Thiobacillus denitr~ficans(Bowen et al.

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1965) and Rhodopseudomonas palustris (Yoch and Lindstrom 1971). Rhizopus oryzae culture filtrates (without KCN) also possessed rhodanese activity and this indicated that the enzyme may also have vital functions other than the detoxification of cyanide as reported earlier (Westley 1981). The regulatory effect of thiosulphate and thiocyanate on the rhodanese produced by R. oryzae is presented in Figs. 3 and 4. Lower molar concentrations (2.0 and 4.0 mM) of thiosulphate produced an initial induction in rhodanese, while higher levels (6.0 and 8.0 mM) steadily repressed enzyme synthesis during the growth phase (Fig. 3). Thiocyanate (2.0-8.0 mM) significantly repressed the enzyme activity during the growth phase from 24 to 120 h (Fig. 4) and the enzyme activity at all levels of thiocyanate was significantly higher when compared with the activity of culture filtrates without thiocyanate. The free thiocyanate levels of thiosulphate-incorporated medium (with and without 1.0 mM KCN) indicated that there was a continuous utilization of thiocyanate up to 96 h of growth and thereafter thiocyanate accumulated in the medium (Table 2). Thiosulphate and thiocyanate promoted the growth of R. oryzae (Table 2 and Fig. 4). Linamarin at all levels repressed the release of rhodanese by R. oryzae cultures during growth phase (Fig. 5). However, there was significant initial induction in rhodanese activity (24 h) when linamarin was incorporated into the medium (Fig. 5). A low molar concentration of linamarin (0.2 mM) significantly induced rhodanese release in the presence of 1.0 mM KCN, while higher molar concentrations (0.4, 0.6, and 0.8

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mM) repressed it (Fig. 6). The free thiocyanate levels showed a steady decrease as growth advanced, with only trace amounts of thiocyanate being present when stationary growth was reached (120 h). Growth was poor in linamarin-incorporated media also, which supports the possibility of a defensive role for cyanogenic glycosides against invading pathogens (Seigler and Price 1976). Linamarase release by R. oryzae Rhizopus oryzae elaborated linamarase during its growth phase in the presence or absence of linamarin (Table 3). The activity of linamarase progressively decreased as growth advanced in the control media without linamarin or KCN. However, when linamarin was incorporated into the medium, there was significant induction of linamarase from 48 to 72 h of growth followed by a sharp decline thereafter. Release of linamarase into the broth without linamarin suggests the possibility that the enzyme may be a P-glycosidase with low aglycone specificity. Stevens and Strobe1 (1968) detected two P-glycosidases with differing substrate specificities in snow mold cultures which could hydrolyse the host plant cyanogenic glycosides. Studies with Klebsiella have indicated that the organism is able to effectively hydrolyse linamarin by releasing a P-glycosidase (Barrett et al. 1977). Owing to its ability to elaborate the P-glycosidase, R. oryzae seems to play a key role in the postharvest degradation of cyanogenic glycosides in cassava. The fungus has an efficient mechanism for detoxifying the cyanogenic glycosides and assimilating the cyanide in a utilizable form. Reduction in HCN content of tubers during postharvest storage of cassava has been reported earlier (Maini and Balagopal 1978). The survival of R. oryzae in an HCN-rich microenvironment is dependent on its ability to effectively assimilate HCN. Adaptation to cyanide seems to be a slow

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FIG.6. Specific activities of rhodanese released by R . oryzae in increasing concentrations of linamarin (with and without 1.0 mM

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process and once detoxification is completed, the fungus can grow rapidly. This explains the lag phase observed in the freshly harvested cassava tubers before the appearance of visual symptoms of fungal attack. This study indicates that R. oryzae has potential use in the processing of cassava feed and

CAN. J.

MICROBIOL. VOL. 3 1,

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TABLE2. Effect of thiosulphate or linamarin on the free thiocyanate levels (in milligrams per 100 mL of culture filtrate) in R . oryzae culture filtrates Time of incubation (h)

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Concn. of sz0:--linamarin

24

48

72

96

Mycelial dry weight (mg)

120

Sodium thiosulphate With KCN (I .O mM) 2.0 4.0 6.0 8.0 Without KCN (1.0 mM) 2.0 4.0 6.0 8.0 Linamarin With KCN (1.0 M ) 0.2 0.4 0.6 0.8 Without KCN (1.0 mM) 0.2 0.4 0.6 0.8 TABLE3. Linamarase activity of R. oryzae culture filtrates Time of incubation (h)

Concn. of linamarin ( M )

24

48

72

96

120

Without KCN (1.0 M ) 0.0 0.2 0.4 0.6 0.8 With KCN (1.0 M ) 0.2 0.4 0.6 0.8 NOTE:Linamarase observations.

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food preparations as well as in the effective disposal of industrial wastes which contain high levels of cyanide.

1977. Fate of orally dosed linamarin in the rats. Can. J. Physiol. Pharmacol. 55: 134- 136. BOWEN,T. J., P. J. BUTLER,and F. C. HAPPOLD.1965. Some proAFFRAN,D. K. 1968. Cassava and its economic importance. Ghana perties of the rhodanese system of Thiobacillus denitrificans. Biochem. J . 97: 651-657. Farmer, 12: 172-178. AKOPYAN, T. N., A. E. BRAUNSTEIN, and E. V. GORYACHENKOVA. BRYSK,M. M., W. A. CORPE,and L. V. HANKES.1969. P-Cyano1975. Beta-cyanoalanine synthase-purification and characterialanine formation by Chromobacterium violaceum. J. Bacterial. zation. Proc. Natl. Acad. Sci. U.S.A. 72: 1617-1621. 97: 322-327. ALLEN,J., and G. A. STROBEL. BRYSK,M. M., and C. RESSLER.1970. y-Cyano a-amino butyric 1966. The assimilation of HCN by a acid-a new product of cyanide fixation in Chromobacterium variety of fungi. Can. J. Microbial. 12: 414-41 6. violaceum. J. Biol. Chem. 245: 1156- 1 160. A s ~ u sE., , and H. GARSCHACEN. 1953. Use of barbituric acid for the photometric determination of cyanide and thiocyanate. Z. Anal. BURTON,C. C. 1970. Diseases of tropical vegetables in Chicago market. Trop. Agric. (Trinidad), 47: 303-313. Chem. 138: 414-416. BALACOPAL, C., S. B. MAINI,V. P. P o n y , and G. PADMAJA. 1981. CASTRIC,P. A. I98 1. The metabolism of hydrogen cyanide by bacMicrobial rotting of cassava roots. Proceedings of a Seminar on teria. In Cyanide in biology. Edited by B. Vennesland, E. E. Conn, Post Harvest Technology of Cassava, Trivandrum, India. February C. J. Knowles, J. Westley, and F. F. Wissing. Academic Press Inc., New York. pp. 233-261. 22-23, 1980. pp. 23-26. CASTRIC,P. A., and G. A. STROBEL.1969. Cyanide metabolism by BARRETT, M. D., D. C. HILL, J. C. ALEXANDER, and A. ZITNAK.

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