title: Characterization of a new (R)-hydroxynitrile lyase from the Japanese apricot Prunus mume and cDNA cloning and secretory expression of one of the isozymes in Pichia pastoris.
abstract: PmHNL, a hydroxynitrile lyase from Japanese apricot ume (Prunus mume) seed was purified to homogeneity by ammonium sulfate fractionation and chromatographic steps. The purified enzyme was a monomer with molecular mass of 58 kDa. It was a flavoprotein similar to other hydroxynitrile lyases of the Rosaceae family. It was active over a broad temperature, and pH range. The N-terminal amino acid sequence (20 amino acids) was identical with that of the enzyme from almond (Prunus dulcis). Based on the N-terminal sequence of the purified enzyme and the conserved amino acid sequences of the enzymes from Pr. dulcis, inverse PCR method was used for cloning of a putative PmHNL (PmHNL2) gene from a Pr. mume seedling. Then the cDNA for the enzyme was cloned. The deduced amino acid sequence was found to be highly similar (95%) to that of an enzyme from Pr. serotina, isozyme 2. The recombinant Pichia pastoris transformed with the PmHNL2 gene secreted an active enzyme in glycosylated form.
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, Toyama, Japan.
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Fukuta
authors.forename: Samik
authors.initials: S
authors.lastname: Nanda
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Kato
authors.forename: Hiroya
authors.initials: H
authors.lastname: Yurimoto
authors.forename: Yasuyoshi
authors.initials: Y
authors.lastname: Sakai
authors.forename: Hidenobu
authors.initials: H
authors.lastname: Komeda
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Asano
mesh_terms: D000446:Aldehyde-Lyases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D015870:Gene Expression; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D027861:Prunus / Q000201:enzymology* / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D018076:DNA, Complementary; D007527:Isoenzymes; D000446:Aldehyde-Lyases