title: Cloning, heterologous expression, and characterization of novel protease-resistant α-galactosidase from new Sphingomonas strain.
abstract: The α-galactosidase-coding gene agaAJB13 was cloned from Sphingomonas sp. JB13 showing 16S rDNA (1,343 bp) identities of < or =97.2% with other identified Sphingomonas strains. agaAJB13 (2,217 bp; 64.9% GC content) encodes a 738-residue polypeptide (AgaAJB13) with a calculated mass of 82.3 kDa. AgaAJB13 showed the highest identity of 61.4% with the putative glycosyl hydrolase family 36 α-galactosidase from Granulicella mallensis MP5ACTX8 (EFI56085). AgaAJB13 also showed <37% identities with reported protease-resistant or Sphingomonas alpha-galactosidases. A sequence analysis revealed different catalytic motifs between reported Sphingomonas α-galactosidases (KXD and RXXXD) and AgaAJB13 (KWD and SDXXDXXXR). Recombinant AgaAJB13 (rAgaAJB13) was expressed in Escherichia coli BL21 (DE3). The purified rAgaAJB13 was characterized using p-nitrophenyl-α-D-galactopyranoside as the substrate and showed an apparent optimum at pH 5.0 and 60 degrees C and strong resistance to trypsin and proteinase K digestion. Compared with reported proteaseresistant alpha-galactosidases showing thermolability at 50 degrees C or 60°C and specific activities of <71 U/mg with or without protease treatments, rAgaAJB13 exhibited a better thermal stability (half-life of >60 min at 60°C) and higher specific activities (225.0-256.5 U/mg). These sequence and enzymatic properties suggest AgaAJB13 is the first identified and characterized Sphingomonas α-galactosidase, and shows novel protease resistance with a potential value for basic research and industrial applications.
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming 650500, People's Republic of China.
authors.forename: Junpei
authors.initials: J
authors.lastname: Zhou
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Dong
authors.forename: Junjun
authors.initials: J
authors.lastname: Li
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.forename: Xianghua
authors.initials: X
authors.lastname: Tang
authors.forename: Yuelin
authors.initials: Y
authors.lastname: Mu
authors.forename: Bo
authors.initials: B
authors.lastname: Xu
authors.forename: Qian
authors.initials: Q
authors.lastname: Wu
authors.forename: Zunxi
authors.initials: Z
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D019015:Geologic Sediments / Q000382:microbiology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D020578:Sphingomonas / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity; D000519:alpha-Galactosidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D000519:alpha-Galactosidase