Applied Microbiology and Biotechnology

, Volume 102, Issue 5, pp 2203–2212 | Cite as

Characterization of an α-agarase from Thalassomonas sp. LD5 and its hydrolysate

  • Weibin Zhang
  • Jingnan Xu
  • Dan Liu
  • Huan Liu
  • Xinzhi Lu
  • Wengong Yu
Biotechnologically relevant enzymes and proteins


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 1H-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.


α-Agarase Glycoside hydrolase family 96 Agarotetraose Alkaline-labile Odd-numbered oligosaccharide 


Funding information

This study was funded by the National Natural Science Foundation of China (41376144), NSFC-Shandong Joint Fund for Marine Science Research Centers (U1606403), Science and Technology Development Plan Project of Shandong Province (2014GGH215002), and the National High-tech R&D Program of China (2014AA093504).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethics approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_8762_MOESM1_ESM.pdf (403 kb)
ESM 1 (PDF 402 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.School of Medicine and Pharmacy, Key Laboratory of Marine Drugs, Chinese Ministry of Education, Key Laboratory of Glycoscience & Glycotechnology of Shandong ProvinceOcean University of ChinaQingdaoChina
  2. 2.Laboratory for Marine Drugs and BioproductsQingdao National Laboratory for Marine Science and TechnologyQingdaoChina

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