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.

An agarolytic bacterium was isolated from the sea coast of Shantou in China and identified as Vibrio sp. LA1. The agarase gene agaA was cloned from Vibrio sp. LA1 by using degenerate oligonucleotide-primed PCR and genome walking technique. Gene agaA consists of a 2913 bp open reading frame encoding 970 amino acids; and the predicted molecular mass and isoelectric point were 108 kDa and 4.46, respectively. Based on the amino acid sequence similarity, the encoded protein (AgaA) of gene agaA should be an agarase of glycoside hydrolase family GH50 with a catalytic domain of glycoside hydrolase family GH42. Soluble expression of AgaA in Escherichia coli was obtained and investigated. The optimal temperature and pH for the activity of the purified recombinant agarase were 35°C and pH 6, respectively. The reducing reagent β-mercaptoethanol could increase the activity of agarase AgaA by more than 80%. AgaA showed exo-lytic activity on agarose degradation. It decomposed agarose to yield neoagarobiose as the sole product, which was different from the agarase Aga41A from Vibrio sp. CN41 that showed 95% identities of amino acid sequence to agarase AgaA. With single end product, purification procedure is easier than that with multi-products. Therefore, agarase AgaA could be a useful tool for producing the bioactive neoagarobiose.