PURIFICATION, CHARACTERIZATION, AND CLONING OF ALPHA-HYDROXYNITRILE LYASE FROM CASSAVA (MANIHOT-ESCULENTA CRANTZ)

alpha-Hydroxynitrile lyase (HNL, acetone cyanohydrin lyase, EC 4.1.2.3 7) was purified to homogeneity from young leaves of the cyanogenic tro pical crop plant cassava (Manihot esculenta Crantz). The purified prot ein is a homo-trimer with a subunit relative molecular mass of 28,500 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The acti ve protein is not glycosylated and does not contain a flavin group. HN L exhibits complex kinetics which vary according to substrate concentr ation and may be related to aggregation of the enzyme. HNL activity ag ainst two natural substrates, acetone cyanohydrin and 2-butanone cyano hydrin, and one nonphysiological substrate, 2-pentanone cyanohydrin, w as demonstrated. N-terminal and internal peptide sequences, obtained f rom HNL digested with the endoproteinase Glu-C, were used to design de generate oligonucleotide primers for polymerase chain reaction with si ngle-strand cDNA, using purified mRNA from cotyledons as template. The resulting DNA fragment was used to probe a cassava cotyledon cDNA lib rary. Four cDNA clones were isolated, sequenced, and shown to contain derived amino acid sequences identical to those obtained from the puri fied protein. (C) 1994 Academic Press, Inc.