An agar-degrading bacterium, Catenovulum sp. X3, was isolated from the seawater of Shantou, China. A novel β-agarase gene agaXa was cloned from the strain Catenovulum sp. X3. The gene agaXa consists of 1,590 bp and encodes a protein of 529 amino acids, with only 40 % amino acid sequence identity with known agarases. AgaXa should belong to the glycoside hydrolase family GH118 based on the amino acid sequence similarity. The molecular mass of the recombinant AgaXa (rAgaXa) was estimated to be 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It had a maximal agarase activity at 52 °C and pH 7.4 and was stable over pH 5.0 ~ 9.0 and at temperatures below 42 °C. The K m and V max for agarose were 10.5 mg/ml and 588.2 U/mg, respectively. The purified rAgaXa showed endolytic activity on agarose degradation, yielding neoagarohexaose, neoagarooctaose, neoagarodecaose, and neoagarododecaose as the end products. The results showed that AgaXa has potential applications in agar degradation for the production of oligosaccharides with various bioactivities.

A novel yellow-pigmented, agarolytic bacterial strain, designated ZC1 T , was isolated from the surface of the marine red alga Porphyra haitanensis collected near Nan Ao Island, Guangdong province, China. The isolate was Gram-stain-negative, strictly aerobic and rod-shaped and displayed b-galactosidase, alkaline phosphatase, catalase and oxidase activities. The predominant cellular fatty acids were iso-C 15 : 0 , summed feature 3 (comprising C 16 : 1 v7c and/or iso-C 15 : 0 2-OH) and iso-C 17 : 0 3-OH. The major menaquinone was menaquinone 6 (MK-6). The DNA G+C content was 32.8 mol%. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain ZC1T was closely related to members of the genus Aquimarina in the family Flavobacteriaceae, phylum Bacteroidetes. Based on phylogenetic and phenotypic evidence, strain ZC1 T (5CCTCC AB 2010229 T 5NBRC 107695 T ) represents the type strain of a novel species in the genus Aquimarina, for which the name Aquimarina agarilytica sp. nov. is proposed.The genus Aquimarina (family Flavobacteriaceae, phylum In the course of a screening of marine environments for agardegrading bacteria, a yellow-pigmented bacterium, designated strain ZC1 T , was isolated from the surface of a red alga (Porphyra haitanensis) collected from shallow water near the coast of Nan Ao Island, located on the Tropic of Cancer, at 117 u E, near the city of Shantou in Guangdong province, in south-eastern China. The red alga was crushed in a mortar with sterile seawater and spread on plates of marine agar (MA) consisting of 1.5 % agar, 0.5 % tryptone (Oxoid) and 0.1 % yeast extract (Oxoid) in artificial seawater containing (w/v) 2.5 % NaCl, 0.63 % MgSO 4 . 7H 2 O, 0.46 % MgCl 2 . 6H 2 O, 0.1 % CaCl 2 and 0.07 % KCl. The plates were incubated at 25 u C for 3 days under aerobic conditions. Strain ZC1 T , which formed colonies that sank into the agar and were each surrounded by a clear halo of liquid, was purified by successive streaking on MA. The purified strain was preserved at 280 u C in marine broth (MB) containing 15 % (v/v) glycerol.For DNA extraction, strain ZC1T was cultivated aerobically in MB supplemented with 0

A β-agarase gene hz2 with 2,868 bp was cloned from the marine agarolytic bacterium Agarivorans sp. HZ105. It encoded a mature agarase HZ2 of 102,393 Da (920 amino acids). Based on the amino acid sequence similarity, agarase HZ2 was assigned to the glycoside hydrolase family 50. The β-agarase shared a gene sequence identity of 98.6% with the reported but much less characterized β-agarase agaB from Vibrio sp. JT0107. Its recombinant agarase rHZ2 was produced in E. coli cells and purified to homogeneity. The agarase rHZ2 degraded agarose and neoagarooligosaccharides with degrees of polymerization above four, to yield neoagarotetraose as the dominant product, which was different from β-agarase agaB of Vibrio sp. JT0107. The agarose hydrolysis pattern suggested that rHZ2 was an endo-type β-agarase. Beta-mercaptoethanol (90 mM) and dithiothreitol (9 mM) increased the agarase activity of rHZ2 by 72.9% and 17.3% respectively, while SDS (9 mM) inhibited the activity completely. The agarase activity was independent of Na(+), K(+), Mg(2+) and Ca(2+). The maximal enzyme activity was observed at 40°C and pH 7. The kinetic parameters K (m), V (max), K (cat), and K (cat)/K (m) values toward agarose of agarase rHZ2 were 5.9 mg ml(-1), 235 U mg(-1), 401 s(-1) and 6.8 × 10(5) M(-1) s(-1), respectively. Agarase rHZ2 could have a potential application in the production of bioactive neoagarotetraose.

A novel β-agarase gene aga672 was cloned from strain ZC1, the typical strain of agar-degrading Aquimarina agarilytica. Gene aga672 is composed of 2130 bp, and the encoded protein Aga672 showed an amino acid sequence identity of only 42% with reported agarases. Aga672 should belong to glycoside hydrolase family 16 according to the protein sequence similarity. The molecular mass of the recombinant Aga672 was estimated to be 98 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Aga672 decomposed agarose to produce neoagarotetraose, neoagarohexaose and neoagarooctaose as the main products. That is the main difference between Aga672 and other reported agarases of family GH16. The Km and Vmax for agarose degradation were 59.8 mg mL-1 and 154.3 U mg-1, respectively. The activity of Aga672 was stable at temperatures below 40°C and at pH 7.0-11.0 with the maximal agarase activity at 25°C and pH 7.0. The results showed that agarase Aga672 could be suitable to hydrolyze the gelated agarose. Thus, it has potential applications in the production of neoagarooligosaccharides directly from red alga.

The marine bacterium ZC1 is the type strain of the recently identified novel species Aquimarina agarilytica. It can produce multiple agarases. Here we report the draft genome sequence of strain ZC1 (4,253,672 bp, with a GC content of 32.8%) and major findings from its annotation. It is the first reported genome in the genus Aquimarina. The polysaccharides in the cell wall of both marine and terrestrial plants represent the most abundant reservoir of organic carbon in the biosphere. The microbial hydrolysis of these polymers is not only central to the carbon cycle but also of considerable industrial significance (3). Agar, which consists of agarose and agaropectin, is a component present in the cell walls of red algae. Agarase is a kind of enzyme that can degrade agarose and mainly comes from agarolytic bacteria. The agarase-producing bacterium ZC1 isolated from the surface of marine red alga is the type strain of the novel species Aquimarina agarilytica (7). The genome sequencing was performed to study the agar-degrading system of the novel type strain. Here, we present the draft genome sequence of strain ZC1. To our knowledge, this is the first genome report of the bacterial genus Aquimarina.The genome of ZC1 was sequenced by a whole-genome shotgun strategy using the Illumina HiSeq 2000 at the Beijing Genomics Institute (BGI; Shenzhen, China). Genome sequences were assembled in silico using SOAPdenovo (6), resulting in 155 contigs with an N 50 length of 56,586 bp. The protein-coding genes were predicted using Glimmer 3.02 (2). The functions of predicted protein-coding genes were then annotated through comparisons with the NCBI-NR, KEGG (4), and GO (1) databases. The annotation was done by using tRNAscan-SE 1.21 (8) to find tRNA and by using rRNAmmer 1.2 (5) to search rRNA.The draft genome includes 4,253,672 bases with a GC content of 32.8% and contains 3,594 predicted genes and 3,456 predicted coding sequences (CDSs). An estimated 87.0% of nucleotides are predicted genes. There are 36 tRNAs and single-copy genes predicted for 5S, 16S and 23S rRNA. The 1,728 CDSs annotated by GO can be classified into 20 GO categories, and 2,047 CDSs can be annotated in the KEGG orthology system. A total of 246 CDSs were related to amino acid metabolism, while 204 CDSs were related to carbohydrate metabolism in the KEGG orthology system. A total of 207 CDSs were annotated as poorly characterized in the KEGG orthology system, indicating the novelty of the strain and its genes.Forty CDSs were putative agarases in the KEGG orthology system. To our knowledge, this is the largest amount of predicted agarases in one bacterium. The lengths of these agarase genes ranged from 870 to 5,127 bp, and their protein sequence similarities ranged from 29% to 70% (with 78% of them below 50%) to the submitted sequences in GenBank database. The low similarity indicated the novelty of the strain and its agarases. Gene cloning and expression of all 40 putative agarase genes are ongoing to study their roles in agarose degradation.Nucleotide sequence a...