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31762100113701.Pdf (3.427Mb) The origin of cyanide in a psychrophilic basidiomycete by Dennis Leroy Stevens A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Microbiology Montana State University © Copyright by Dennis Leroy Stevens (1967) 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. THE ORIGIN OF CYANIDE IN A PSYCHROPHILIC BASIDIOMYCETE by DENNIS LEROY STEVENS A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Microbiology Approved: Head, Major Department MONTANA STATE UNIVERSITY Bozeman, Montana December, 1967 iii ACKNOWLEDGMENTS I should like to take this opportunity to express my sincere appreciation and gratitude to Dr. Gary A. Strobel for his enthusiasm and interest in my research and for his guidance and support through­ out my graduate career. Many thanks are extended to Dr„ Larry Jackson for his help in analyzing samples in the gas chromatograph. I would also like to thank Dr. Strobel, Dr. McBee, Dr. Jutila, Dr0 Nelson, Dr. Temple and Dr. Julian for their help in the prepa­ ration of this manuscript, I am grateful to Darlene Harpster who typed this manuscript and to my wife, Carolyn, who prepared the thesis illustrations. •iv TABLE OF CONTENTS Page VITA ........ ............................................. ii ACKNOWLEDGMENT.............. .i, iii TABLE OF CONTENTS. ............................................ ■ iv- LIST OF TABLES ......................................... .... vii LIST OF FIGURES ....................................................viii ABSTRACT .............................................. .. , ix. INTRODUCTION ................ I MATERIALS AND METHODS. ............................ 8 Culturing . .......................... 8 M a t e r i a l s .......................................... 8 General Methods . ........................... 9 Analytical Methods ............................ .9 Radioactivity Determinations ....................... 9 Chromatography ............... 10 Isolation and Characterization of Cyanogenic Compounds , , 10 C14 Feeding Experiments ................................. .11 Feeding U-C^4 Amino Acids to the Fungus to Determine Dilution Factors . .................... 11 -Glucosida.se Studies.............................. 12 Assay of ^ -Glucosidase .......... .. , 12 Energy of Activation ........................ .... 12 Purification of y$-Glucosidase .13 V Page Oxynxtrilase Studies .......................... .......... ,14 Preparation of Oxynitrilase from the PsychrophiIi,c Basidiomycete . .............. 14 Assay of Oxynitrilase Activity .................... 14 Determination of Aglycone Hydrolysis Products .... 20 EXPERIMENTAL RESULTS .................................... .. 21 Utilization of Amino Acids as Precursors to Cyanide Formation by the Psychrophilic Basidiomycete . 21 Isolation and Characterization of Linamarin and Lotaustralin ................................ 21 Evidence for Isoleucine Incorporation into Lotaustralin ............ ................ 24 Enzyme Studies .......................................... 24 Purification of ^-Glucosidase from the Psychrophilic Basidiomycete . .....................'........... 27 Properties of the ^-Glucosidases............ - . 27 a. Effect of pH on Activity . ................ 27 b. Effect of Substrate Concentration on Enzyme Activity ........ .......... 27 c . Substrate Specificity ...................... 30 d. Energy of Activation ....................... 30 e „ Stability .................................. 30 f. Effect of In h i b i t o r s..................... 36 g. Effect of Cellobiose as an Inducer of Enzyme S y n t h e s i s ...................... .. 36 Oxynitrilase Studies .... ........................ 40 a. Products of, Aglycone Oxidation ....... 40 vi Page b„ Substrate Specificity . ................... 43 DISCUSSION ..................................................... 45 . S U M M A R Y .................................... •................ 56 LITERATURE CITED ................. 57 vii LIST OF TABLES Page Table I . Purification of Glucosidase from the Psychrophilic Basidiomycete .................... 15 Table II. Conversion of Amino Acid-U-C^ Precursors to HC14N ..................................... 22 Table III. Comparison of Rf Values of Unknown Compounds to Those of Linamarin and Lotaustralin in Three Different Solvent Systems.......................... 23 Table IV. Isoleucine-U-C^ as a Precursor of Lotaustralin and HCN During a 48 hr. S t u d y .................. 25 Table V. Evidence that the Liberation of Cyanide from Lotaustralin was an Enzymic Process ............ 26 Table VI. Relative Reaction Rates of the 2 /?-Glucosidases on Several Substrates...................... ' . , 33 Table VII. The Effect of Competitive Inhibitors on the Reaction Rate of -Glucosidase.............. 39 Table VIII. /2 -Glucosidase Levels after Growth in Various Carbohydrate Media ............................ 41 Table IX. Comparison of Retention Times of Standards with Aglycone Breakdown Products .............. 42 Table X. Substrate Specificity of Oxynitrilase from the Psychrophilic Basidiomycete .................... 44 Table XI. Summary of the Characteristics of Fraction.I and Fraction 2 /3 -Glucosidases........... .. 48 viii LIST OF FIGURES Page Figure I. Two proposed mechanisms of cyanide production in the psychrophilic basidiomycete........ 7 Figure 2. The elution pattern of -glucosidase on a DEAE cellulose colu m n............ ............. .. 17 Figure 3. The reaction vessel used for determining oxynitrilase activity ...................... .. 19 Figure 4. The effect of pH on the activity of fraction I and fraction 2 ^J-glucosidases ................... 29 Figure 5. The effect of substrate concentration on the activity of fraction I arid fraction 2 -glucosidases 32 Figure 6. The effect of temperature on the rate of hydrolysis . (Kg) of p-nitrophenyl- jQ -D-glucoside by fraction I and fraction 2 /6 -glucosidases.................... 35 Figure 7. The effect of several competitive inhibitors on the reaction rate of /$ -glucosidase . .. , 38 Figure 8. Present proposed mechanism of linamarin biosynthesis ^ 50 ix . 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
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