Cloning and sequencing of alginate lyase genes from deep-sea strains of Vibrio and Agarivorans and characterization of a new Vibrio enzyme.

Institute of Biogeoscience, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima, Yokosuka, Japan.
Marine Biotechnology (Impact Factor: 3.15). 11/2009; 12(5):526-33. DOI: 10.1007/s10126-009-9237-7
Source: PubMed

ABSTRACT Four alginate lyase genes were cloned and sequenced from the genomic DNAs of deep-sea bacteria, namely members of Vibrio and Agarivorans. Three of them were from Vibrio sp. JAM-A9m, which encoded alginate lyases, A9mT, A9mC, and A9mL. A9mT was composed of 286 amino acids and 57% homologous to AlxM of Photobacterium sp. A9mC (221 amino acids) and A9mL (522 amino acids) had the highest degree of similarity to two individual alginate lyases of Vibrio splendidus with 74% and 84% identity, respectively. The other gene for alginate lyase, A1mU, was shotgun cloned from Agarivorans sp. JAM-A1m. A1mU (286 amino acids) showed the highest homology to AlyVOA of Vibrio sp. with 76% identity. All alginate lyases belong to polysaccharide lyase family 7, although, they do not show significant similarity to one another with 14% to 58% identity. Among the above lyases, the recombinant A9mT was purified to homogeneity and characterized. The molecular mass of A9mT was around 28 kDa. The enzyme was remarkably salt activated and showed the highest thermal stability in the presence of NaCl. A9mT favorably degraded mannuronate polymer in alginate. We discussed substrate specificities of family 7 alginate lyases based on their conserved amino acid sequences.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Previously we isolated the major alginate lyase isozyme LbAly35 from a marine snail Littorina brevicula and showed that this enzyme was significantly heat stable in a broad pH range compared with other molluscan alginate lyases (Hata et al., Fish Sci 75:755–763, 2009). LbAly35 showed practically no similarity to other molluscan alginate lyases in the N-terminal amino-acid sequence of 20 residues and no cross-reactivity with anti-abalone alginate lyase antiserum. These led us to consider that the primary structure of LbAly35 is considerably deviated from other molluscan enzymes. Thus, in the present study, we first compared the thermal stability of LbAly35 with an abalone alginate lyase, HdAly, and found that the first order inactivation rate constants for LbAly35 at 40 and 45 °C were 1/20 and 1/45 of those for HdAly, respectively. Then, we cloned cDNAs encoding LbAly35 and characterized its deduced amino-acid sequence comparing with those of other molluscan alginate lyases. The cDNAs were amplified by polymerase chain reaction (PCR) and 5′- and 3′-RACE PCRs from the L. brevicula hepatopancreas cDNA using degenerated primers synthesized on the basis of partial amino-acid sequences of LbAly35. The cDNA covering the entire translational region of LbAly35 comprised 1,093 bp and encoded an amino-acid sequence of 296 residues. The amino-acid sequence consisted of an initiation methionine, a putative signal peptide for secretion (22 residues), a propeptide-like region (10 residues), and a mature LbAly35 domain of 263 residues. Although the N-terminal region of LbAly35 was significantly deviated from those of other molluscan alginate lyases, the catalytic domain of LbAly35 showed ~45 % identity to other molluscan enzymes which had been classified under polysaccharide-lyase-family-14 (PL-14). In addition, the amino-acid residues crucially important for the catalytic actions of PL-14 enzymes were also conserved in LbAly35. Accordingly, LbAly35 was regarded as a member of PL-14 as other molluscan alginate lyases despite the significant deviation of its N-terminal region.
    Fisheries Science 78(4). · 0.90 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The aim of this research is to identify the variables that express the perception of the students towards biology laboratory class environment. The biology laboratory environment inventory, developed by Fraser, Gidding & McRobbie (1992), Learning style inventory of Kolb (1985) and biology self-efficacy inventory of Ekici (2009) are used in this research. The significant results of the research; the perception of the students towards biology laboratory environment has a positive and meaningful relation with their sex, biology self-efficacy perception level, learning style and overall academic success, but any relation is not defined with class variable. The results of regression analysis introduces that the perception of the students towards biology laboratory environment has a positive and meaningful relation with their sex, biology self-efficacy perception level, learning style and overall academic success, but the classroom variable has a negative and meaningful effect on it. These interpretative variables signify % 41.9 of total variance in the perception towards biology laboratory class environment.
    Procedia - Social and Behavioral Sciences 01/2011; 15:1901-1905.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: To reduce the volume of seaweed wastes and extract polysaccharides, seaweed-degrading bacteria were isolated from drifting macroalgae harvested along the coast of Toyama Bay, Japan. Sixty-four bacterial isolates were capable of degrading "Wakame" (Undaria pinnatifida) thallus fragments into single cell detritus (SCD) particles. Amongst these, strain 6532A was the most active degrader of thallus fragments, and was capable of degrading thallus fragments to SCD particles within a day. Although the sequence similarity of the 16S rRNA gene of strain 6532A was 100% similar to that of Microbulbifer elongatus JAMB-A7, several distinct differences were observed between strains, including motility, morphology, and utilization of D: -arabinose and gelatin. Consequently, strain 6532A was classified as a new Microbulbifer strain, and was designated Microbulbifer sp. 6532A. Strain 6532A was capable of degrading both alginate and cellulose in the culture medium, zymogram analysis of which revealed the presence of multiple alginate lyases and cellulases. To the best of our knowledge, this is the first study to directly demonstrate the existence of these enzymes in Microbulbifer species. Shotgun cloning and sequencing of the alginate lyase gene in 6532A revealed a 1,074-bp open reading frame, which was designated algMsp. The reading frame encoded a PL family seven enzyme composed of 358 amino acids (38,181 Da). With a similarity of 74.2%, the deduced amino acid sequence was most similar to a Saccharophagus enzyme (alg 7C). These findings suggest that algMsp in strain 6532A is a novel alginate lyase gene.
    Biodegradation 06/2011; 23(1):93-105. · 2.17 Impact Factor


Available from
May 30, 2014