Year: 1967
Type: dissertation
Abstract: It has long been known that cyanide is evolved in basidiomycetes, however, the mechanism by which it is formed has not been established.Possible precursors and intermediates for cyanide were investigated by administering eight different carbon-14 labeled amino acids to cultures of a psychrophilic basidiomycete (strain W-2, J. B. Lebeau, Lethbridge, Alberta, Canada).Comparison of the dilution factors indicated that isoleucine and valine were the most effective sources of HC14N.Inasmuch as isoleucine and valine served as sources of HC14N, the cyanogenic glucosides lotaustralin and linamarin, were investigated as possible intermediates in cyanide formation in this organism.Both of these cyanogenic glucosides were isolated from the fungus and identified by paper chromatography and infrared spectrophotometry.Some evidence that the glucosides may serve as a source of cyanide was established by incubating mycelial mats with carbon-14 labeled valine and isoleucine and determining the specific activity of the glucosides formed at various times during the incubation period (3 to 48 hr.).Carbon-14 labeled lotaustralin was prepared by the administration of isoleucine-U-C14 to flax seedlings.When labeled lotaustraline was incubated with a crude enzyme preparation of the fungus, there was a decrease in the concentration of lotaustralin with a concomitant increase in HC14N as measured by liquid-scintillation counting.Heat-inactivated controls showed no activity.Inasmuch as the organism contained the necessary enzymes to release cyanide from the cyanogenic glucosides, it was desirable to purify and characterize these enzymes.A cell free extract was prepared from mycelial mats of the organism and subjected to precipitations by acetone and protamine sulfate, ammonium sulfate fractionation, dialysis and finally separation on a DEAE cellulose column.The elution pattern from the DEAE cellulose column showed two separate peaks having β-glucosidase activity against the chromogenic substrate p-nitrophenyl-β-D-glucoside.The peaks represented two different enzymes since their pH and temperature optima, energies or activation, Km's, and substrate specificities were different.The enzyme from one peak hydrolyzed linamarin to∝ -hydroxyisobutyronitrile and glucose, the other hydrolyzed lotaustralin to methyl-ethyl-ketone cyanohydrin and glucose.That the fungus produced an oxynitrilase capable of oxidizing acetone cyanohydrin and methyl-ethyl-ketone cyanohydrin to HCN and the corresponding ketone, was demonstrated by incubating the cyanohydrins with a crude enzyme preparation and then analyzing the products in a gas chromatograph.The oxynitrilase from the fungus oxidized acetone cyanohydrin, methyl-ethyl-ketone cyanohydrin and lactonitrile but had no activity on aromatic cyanohydrins In summary, the release of cyanide by the psychrophilic basidiomycete first involves synthesis of cyanogenic glucosides from amino acid precursors followed by a two step enzymic breakdown of the cyanogenic glucosides by β β-glucosidase and a crude preparation showing oxynitrilase activity.
Author: Dennis L. Stevens
Language: English
Topic: Cassava research and cyanide
Subfield: Plant Science
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W1922490332