title: Cloning, expression, and characterization of a highly active alkaline pectate lyase from alkaliphilic Bacillus sp. N16-5.
abstract: An alkaline pectate lyase, Bsp165PelA, was purified to homogeneity from the culture broth of alkaliphilic Bacillus sp. N16-5. The enzyme showed a specific activity as high as 1000 U/mg and had optimum activity at pH 11.5 and 50 degrees . It was composed of a single polypeptide chain with a molecular of 42 kDa deduced from SDS-PAGE, and its isoelectric point was around pH 6.0. It could efficiently depolymerize polygalacturonate and pectin. Characterization of product formation revealed unsaturated digalacturonate and trigalacturonate as the main product. The pectate lyase gene (pelA) contained an open reading frame (ORF) of 1089 bp, encoding a 36-amino acids signal peptide and a mature protein of 326 amino acids with a calculated molecular mass of 35.943 Da. The deduced amino acid sequence from the pelA ORF exhibited significant homology to those of known pectate lyases in polysaccharide lyase family 1. Some conserved active-site amino acids were found in the deduced amino acid sequence of Bsp165PelA. Ca2+ was not required for activity on pectic substrates.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Li
authors.forename: Lang
authors.initials: L
authors.lastname: Rao
authors.forename: Yanfen
authors.initials: Y
authors.lastname: Xue
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhou
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010368:Pectins / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011133:Polysaccharide-Lyases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; D010368:Pectins; D011133:Polysaccharide-Lyases; C023911:pectate lyase; C003181:polygalacturonic acid