Dobruchowska, J. | bioRxiv Science

bioRxiv2026

Characterization of a GH17 laminarinase, MlGH17A, from a laminarin polysaccharide utilization locus in the marine bacterium Muricauda lutaonensis

Heterotrophic marine microorganisms have the capability to degrade and metabolize laminarin, which is the most abundant source of energy and nutrients in the marine environment, via enzymes encoded by genes clustered in polysaccharide utilization loci (PULs). In this study, a PUL potentially responsible for laminarin utilization was identified in the genome of the marine bacterium Muricauda lutaonensis strain ISCAR-4703, with conserved synteny in the genus Muricauda. A GH17 laminarinase (MlGH17A) encoded in the newly identified PUL was cloned, produced, and characterized as an endo-acting laminarinase, exhibiting the ability to degrade laminarin and laminari-oligosaccharides with a degree of polymerization (DP) greater than four into laminaribiose, laminaritriose, and laminaritetraose, with laminaritriose as the main product making up >50% of the produced oligosaccharide products. The three-dimensional model of the enzyme revealed the presence of seven putative subsites, including four glycone subsites (-4 to -1) and three aglycone subsites (+1 to +3), with a wide cleft to accommodate branches at the -2 subsite, enabling it to act on {beta}-1,3 linked backbones in polysaccharides with {beta}-1,6 linked branches. This enzyme is, along with the recently characterized {beta}-1,3 glucanosyltransglycosylase (MlGH17B), conserved in several Muricauda species and is suggested to play a crucial role in the utilization of laminarin by these bacteria. ImportanceLaminarin, a {beta}-1,3-glucan with occasional {beta}-1,6 branching, is the most abundant source of energy and nutrients in the marine environment. In this study, polysaccharide utilization loci (PULs) for laminarin degradation were identified in various marine Muricauda species, encoding a range of glycoside hydrolases and transglycosylases. In Muricauda lutaonensis ISCAR-4703, the PUL included two GH17 enzymes, separated by a GH30 enzyme and a major facilitator superfamily (MFS) transporter, a feature observed in all corresponding Muricauda PULs. A novel endoacting laminarinase from the PUL, MlGH17A, was characterized and shown to hydrolyze laminarin into laminaribiose, laminaritriose, and laminaritetraose, with laminaritriose as main product. Bioinformatic analysis showed that the enzyme lacked the typical subdomain found in GH17 plant {beta}-glucanases, leading to a lower number of aglycone subsites (+1 to +3). Instead, MlGH17A possessed more glycone subsites (-1 to -4), attributed to the {beta}3-3 loop, which was longer than in GH17 plant {beta}-glucanases.

biochemistry↗

bioRxiv2025

Asymmetrical glycoengineering of monoclonal antibodies: new insights in a-gal immunogenicity

Glycosylation heterogeneity in therapeutic monoclonal antibodies (mAbs) necessitates precise glycoengineering to optimize effector functions, such as those mediated by Fc{gamma}-receptors (Fc{gamma}Rs). Current enzymatic remodeling methods are limited in scope, producing only a narrow spectrum of glycoforms and struggling to achieve complete control over the synthesis of defined asymmetrical structures, which are often critical for enhanced mAb efficacy. We report a multi-step, fully enzymatic platform for the solution-phase synthesis of a comprehensive library of biantennary mAb glycoforms with complete control over the 1,3- and 1,6-mannose arm architecture. The strategy relies on two key catalytic steps: the use of the single domain of the {beta}-N-acetylglucosaminidase StrH (GH20-2) to achieve the regioselective hydrolysis of the GlcNAc residue on the 1,3-mannose arm of the intact antibody, thereby introducing asymmetry; and exploiting the intrinsic, broad acceptor flexibility of GnT-I to reinstall the 1,3-mannose arm GlcNAc even after the 1,6-mannose arm has been selectively extended and terminally capped with Neu5Ac or the -gal epitope. This robust, two-stage enzymatic remodeling methodology, demonstrated on infliximab and cetuximab, unlocks the complete chemical space of biantennary Fc-glycans for comprehensive, functional studies. We employed these well-defined glycovariants to determine the minimum structural requirements for anti--gal IgE binding and systematically evaluate binding affinities toward Fc{gamma}Rs, providing essential insights for the rational design of next-generation immunotherapeutics.

biochemistry↗