It has been a long time since the first α-agarase was discovered. However, only two α-agarases have been cloned and partially characterized so far and the study of α-agarases has lagged far behind that of β-agarases. Here, we report an α-agarase, AgaD, cloned from marine bacterium Thalassomonas sp. LD5. Its cDNA consists of 4401 bp, encoding a protein of 1466 amino acids. Based on amino acid similarity, AgaD is classified into glycoside hydrolase (GH) family GH96. The recombinant enzyme gave a molecular weight of about 180 kDa on SDS-PAGE and 360 kDa on Native-PAGE indicating it acted as a dimer. However, the recombinant enzyme is labile and easy to be fractured into series of small active fragments, of which the smallest one is about 70 kDa, matching the size of catalytic module. The enzyme has maximal activity at 35 °C and pH 7.4, and shows a strong dependence on the presence of calcium ions. AgaD degrades agarose to yield agarotetraose as the predominate end product. However, the hydrolysates are rapidly degraded to odd-numbered oligosaccharides under strong alkaline condition. The spectra of ESI-MS and H-NMR proved that the main hydrolysate agarotetraose is degraded into neoagarotriose, bearing the sequence of G-A-G (G, D-galactose; A, 3,6-anhydro-α-L-galactose). Unlike the alkaline condition, the hydrolysates are further hydrolyzed into smaller degree polymerization (DP) of agaro-oligosaccharides (AOS) in dilute strong acid. Therefore, this study provides more insights into the properties for both the α-agarases and the AOS.

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The interaction of tumor cells with the microenvironment is like a relationship between the “seeds” and “soil,” which is a hotspot in recent cancer research. Targeting at tumor microenvironment as well as tumor cells has become a new strategy for cancer treatment. Conventional cancer treatments mostly focused on single targets or single mechanism (the seeds or part of the soil); few researches intervened in the whole tumor microenvironment and achieved ideal therapeutic effect as expected. Traditional Chinese medicine displays a broad range of biological effects, and increasing evidence has shown that it may relate with synergistic effect on regulating tumor microenvironment and cancer cells. Based on literature review and our previous studies, we summarize the synergistic effect and the molecular mechanisms of traditional Chinese medicine on regulating tumor microenvironment and cancer cells.