Molecular Cloning and Characterization of a Novel β-Agarase, AgaB, from Marine Pseudoalteromonas sp. CY24

Abstract

Agarases are generally classified into glycoside hydrolase families 16, 50, and 86 and are found to degrade agarose to frequently generate neoagarobiose, neoagarotetraose, or neoagarohexaose as the main products. In this study we have cloned a novel endo-type ␤-agarase gene, agaB, from marine Pseudoalteromonas sp. CY24. The novel agarase encoded by agaB gene has no significant sequence similarity with any known proteins including all glycoside hydrolases. It degrades agarose to generate neoagarooctaose and neoagarodecaose as the main end products. Based on the analyses of enzymatic kinetics and degradation patterns of different oligosaccharides, the agarase AgaB appears to have a large substrate binding cleft that accommodates 12 sugar units, with 8 sugar units toward the reducing end spanning subsites ؉1 to ؉8 and 4 sugar units toward the non-reducing end spanning subsites ؊4 to ؊1, and enzymatic cleavage taking place between subsites ؊1 and ؉1. In addition, 1 H NMR analysis shows that this enzyme hydrolyzes the glycosidic bond with inversion of anomeric configuration, in contrast to other known agarases that are retaining. Altogether, AgaB is structurally and functionally different from other known agarases and appears to represent a new family of glycoside hydrolase.

Cloning, Characterization, and Molecular Application of a Beta-Agarase Gene fromVibriosp. Strain V134

“…In comparing AgaV with these two enzymes, we found that, like AgaB, AgaV was unable to degrade neoagarohexaose and smaller neoagarooligosaccharides, in contrast to AgaD, which can utilize DP6 as a substrate. However, similar to those of AgaD, the end hydrolysis products of AgaV were DP4 and DP6, while those of AgaB were DP8 and DP10 (4,17).…”

“…The AgaV presented in this report revealed no significant homology to the above-mentioned Vibrio AgaA, AgaB, and AgaC but was significantly similar to AgaD and the beta-agarase AgaB of the Pseudoalteromonas sp. strain CY24 (17), especially in the catalytic domain (83 to 84% identity). The AgaB from the Pseudoalteromonas sp.…”

Microorganisms living on macroalgae: diversity, interactions, and biotechnological applications

“…This GH118 enzyme proceeds according to a mechanism of inversion of the anomeric configuration (Ma et al 2007), in contrast to GH16 beta-agarases, which act via a retaining mechanism (Jam et al 2005). The families GH50 and GH86 are also predicted to encompass retaining enzymes (Pluvinage et al 2013).…”

An Extra Peptide within the Catalytic Module of a β-Agarase Affects the Agarose Degradation Pattern

“…Based on GH sequence similarity, the characterized ␣-agarases are classified as members of the GH96 family (12,13). Most of the reported ␤-agarases belong to the GH16 family in the CAZy (carbohydrate-active enzyme) database, but some ␤-agarases belong to the GH50, GH86, or GH118 family (5,14,15). Crystal structures of the GH modules (10,16,17) and their catalytic mechanisms have been well elucidated (18 -21).…”