| CAS No.: | 9083-24-3 |
|---|---|
| Resource: | Natural |
| Transport Package: | Paper |
| Specification: | large |
| Trademark: | china |
| Origin: | China |
| Samples: |
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Audited Supplier Funoran, agarose and porphyran all belong to agaran, and share the similar skeleton. Although the glycoside hydrolase for agarose and porphyran, i.e. agarase and porphyranase, have been extensively studied, the enzyme hydrolyzing funoran has not been reported hitherto. The crystal structure of a previously characterized GH86 β-agarase Aga86A_Wa showed a large cavity at subsite −1, which implied its ability to accommodate sulfate ester group. By using glycomics and NMR analysis, the activity of Aga86A_Wa on the characteristic structure of funoran was validated, which signified the first discovery of funoran hydrolase, i.e. funoranase. Aga86A_Wa hydrolyzed the β-1,4 glycosidic bond between β-d-galactopyranose-6-sulfate (G6S) and 3,6-anhydro-α-l-galactopyranose (LA) unit of funoran, and released disaccharide LA-G6S as the predominant end product. Considering the hydrolysis pattern, we proposed to name the activity represented by Aga86A_Wa on funoran as "β-funoranase" and suggested to assign it an EC number.
Agarans are the predominant constituents of the cell wall of agarophyte, which represent a family of water soluble sulfated galactans (Pomin & Mourao, 2008; Prado, Ciancia, & Matulewicz, 2008). It has been reported as chemically heterogeneous polysaccharides with the common skeleton consisting of alternating 3-linked β-d-galactopyranose (G) and 4-linked α-l-galactopyranose (L) units (G-L)n, while containing subtle difference between different polysaccharides (Pomin & Mourao, 2008). Currently, three types of agarans-agarose, funoran and porphyran-were extensively studied (Correc, Hehemann, Czjzek, & Helbert, 2011; Hu et al., 2012; Lee et al., 2017).
Funoran derived from edible red algae of genus Gloiopeltis builds up of β-d-galactopyranose-6-sulfate (G6S) and cyclized L (3,6-anhydro-α-l-galactopyranose, LA) (G6S-LA)n (Hu et al., 2012; Takano, Hayashi, Hara, & Hirase, 1995; Tuvikene et al., 2015). It exhibits a similar repetition moiety with agarose (G-LA)n, while distinctively differed by the occurrence of ester sulfate groups in G unit. Funoran has already been used commercially due to its fine properties, similar to agarose and porphyran (composed of G and α-l-galactopyranose-6-sulfate (L6S)). It has been widely applied as adhesive in pottery and textiles industries (Takano, Iwane-Sakata, Hayashi, Hara, & Hirase, 1998), and safe thickener in food industries (Yu et al., 2010). Besides, it has also been confirmed with various biological activities, including anti-inflammatory (Keukenmeester, Slot, Putt, & Van der Weijden, 2014), anti-tumor (Bae & Choi, 2007) and antibacterial (Kurihara, Goto, Aida, Hosokawa, & Takahashi, 1999), etc., which highlighted its potential as a functional polysaccharide as other marine sulfated polysaccharides.
Enzymatic degradation is a specific and mild method for the depolymerization of polysaccharides and production of oligosaccharides (Long et al., 2022). The previously characterized glycoside hydrolases targeting agaran were classified as agarase (Park, Lee, & Hong, 2020) and porphyranase (Hehemann et al., 2010). According to the mode of action, the reported agaran glycoside hydrolases were divided into three groups, β-agarase, β-porphyranase and α-agarase. β-agarase (EC 3.2.1.81) and β-porphyranase (EC 3.2.1.178) catalyzed the hydrolysis of the internal β-1,4 glycosidic linkage in agarose and porphyran respectively, while α-agarase (EC 3.2.1.158) cleaved the α-1,3 glycosidic bond in agarose (Chi, Chang, & Hong, 2012). Nevertheless, the enzyme for hydrolyzing funoran has not been clarified hitherto, and the particular EC number was not assigned.
In our previous study, a β-agarase Aga86A_Wa belonging to glycoside hydrolase (GH) family 86 from Wenyingzhuangia aestuarii OF219 was characterized to have tolerating capacity to methyl-galactose (GMe) besides G unit at subsite −1 (Cao, Shen, Zhang, Chang, & Xue, 2020). The crystal structure of Aga86A_Wa combined the molecular docking result revealed that the tolerance capacity to methyl-galactose could be attributed to the accommodation pocket at subsite −1 of Aga86A_Wa (Zhang et al., 2023). According to this discovery, we further observed that the accommodation pocket was much larger than the space required for methyl groups. Considering the characteristics of the chemical structure of agaran, we therefore speculated that whether the accommodation pocket could accept larger groups, such as sulfate ester group. Our preliminary molecular docking result showed that the canonical tetrasaccharide of funoran could adapt to the active cleft of Aga86A_Wa, and the G6S could be accommodate by the subsite −1. Enlightened by the structure, we aimed to verify the tolerance capacity to G6S of the subsite −1 of Aga86A_Wa, thereby elucidating its ability to act on the β-1,4 glycosidic bond of funoran backbone.
The subsite −1 of Aga86A_Wa (PDB 8H97) is a relatively large cavity. For investigating the methyl-galactose tolerance capacity, the tetrasaccharide ligand (LA-G6Me)2 was docked into the catalytic cleft of the protein structure of Aga86A_Wa (Zhang et al., 2023). The docking result showed that the G6Me unit occupied the inside of the cavity, while still leaving a relatively huge space. Based on the discovery, we supposed that the cavity might be able to accommodate bulky groups bigger than methyl
To the best of our knowledge, Aga86A_Wa is the first and recombinant funoran hydrolase reported so far. It should be mentioned that a wild-type porphyran degrading enzyme from Zobellia uliginosa was reported with a secondary funoran degradation activity (Howlader, Niroda, Bai, Premarathna, & Tuvikene, 2022), while the type of the catalytic reaction was undefined. Herein, our results elucidated that Aga86A_Wa hydrolyzed the characteristic structure of funoran and catalyzed the breakdown of the
The potential activity of Aga86A_Wa on the characteristic structure of funoran was validated in this work. Aga86A_Wa acted on funoran and agarose, while had no activity on porphyran. It hydrolyzed the β-1,4 glycosidic linkage in G6Sβ1 → 4LA of funoran in a random endo-acting manner, and released the disaccharide LA-G6S as the predominant component of the end products. Our results confirmed the funoran hydrolase activity for the first time, and the activity against funoran represented by
Dried G. furcate was purchased from a market (Guangzhou, China) and pulverized before subsequent procedures. Crude polysaccharides were extracted from the powder using hot water. The insoluble seaweed residues were centrifuged at 4102g for 20 min and removed. The supernatant was subsequently mixed with 3-fold volume of ethanol (95 % v/v) and centrifuged at 4102g for 20 min to recover precipitated polysaccharides. The obtained polysaccharide was then purified on ÄKTA Prime Plus system (GE
This work was supported by the Fundamental Research Funds for the Central Universities (202012020). We thank the Shanghai Synchrotron Radiation Facility (SSRF) for providing the platform of collecting diffraction data.
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