Purification, Characterization, and Cloning of α-Hydroxynitrile Lyase from Cassava (Manihot esculenta Crantz)

University of California, Davis, Davis, California, United States
Archives of Biochemistry and Biophysics (Impact Factor: 3.04). 07/1994; 311(2):496-502. DOI: 10.1006/abbi.1994.1267
Source: PubMed

ABSTRACT alpha-Hydroxynitrile lyase (HNL, acetone cyanohydrin lyase, EC was purified to homogeneity from young leaves of the cyanogenic tropical crop plant cassava (Manihot esculenta Crantz). The purified protein is a homo-trimer with a subunit relative molecular mass of 28,500 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The active protein is not glycosylated and does not contain a flavin group. HNL exhibits complex kinetics which vary according to substrate concentration and may be related to aggregation of the enzyme. HNL activity against two natural substrates, acetone cyanohydrin and 2-butanone cyanohydrin, and one nonphysiological substrate, 2-pentanone cyanohydrin, was demonstrated. N-terminal and internal peptide sequences, obtained from HNL digested with the endoproteinase Glu-C, were used to design degenerate oligonucleotide primers for polymerase chain reaction with single-strand cDNA, using purified mRNA from cotyledons as template. The resulting DNA fragment was used to probe a cassava cotyledon cDNA library. Four cDNA clones were isolated, sequenced, and shown to contain derived amino acid sequences identical to those obtained from the purified protein.

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