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Elucidation of a Unique Pattern and the Role of Carbohydrate Binding Module of an Alginate Lyase.

ABSTRACT: Alginate oligosaccharides with different degrees of polymerization (DPs) possess diverse physiological activities. Therefore, in recent years, increasing attention has been drawn to the use of enzymes for the preparation of alginate oligosaccharides for food and industrial applications. Previously, we identified and characterized a novel bifunctional alginate lyase Aly7A, which can specifically release trisaccharide from three different substrate types with a unique degradation pattern. Herein, we investigated its degradation pattern by modular truncation and molecular docking. The results suggested that Aly7A adopted a unique action mode towards different substrates with the substrate chain sliding into the binding pocket of the catalytic domain to position the next trisaccharide for cleavage. Deletion of the Aly7A carbohydrate binding module (CBM) domain resulted in a complex distribution of degradation products and no preference for trisaccharide formation, indicating that the CBM may act as a "controller" during the trisaccharide release process. This study further testifies CBM as a regulator of product distribution and provides new insights into well-defined generation of alginate oligosaccharides with associated CBMs.

SUBMITTER: Hu F

PROVIDER: S-EPMC7024192 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Elucidation of a Unique Pattern and the Role of Carbohydrate Binding Module of an Alginate Lyase.

Hu Fu F Zhu Benwei B Li Qian Q Yin Heng H Sun Yun Y Yao Zhong Z Ming Dengming D

Marine drugs 20191230 1

Alginate oligosaccharides with different degrees of polymerization (DPs) possess diverse physiological activities. Therefore, in recent years, increasing attention has been drawn to the use of enzymes for the preparation of alginate oligosaccharides for food and industrial applications. Previously, we identified and characterized a novel bifunctional alginate lyase Aly7A, which can specifically release trisaccharide from three different substrate types with a unique degradation pattern. Herein, ...[more]

PMID: 31905894

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Project description:BackgroundHighly efficient enzymatic saccharification of pretreated lignocellulose is a key step in achieving lignocellulosic biorefinery. Cellobiohydrolase I (Cel7A) secreted by Trichoderma reesei is an industrially used cellulase that possesses carbohydrate-binding module 1 (TrCBM1) at the C-terminal domain. The nonproductive binding of TrCBM1 to lignin significantly decreases the enzymatic saccharification efficiency and increases the cost of biomass conversion because of the additionally required enzymes. Understanding the interaction mechanism between lignin and TrCBM1 is essential for realizing a cost-effective biofuel production; however, the binding sites in lignin have not been clearly elucidated.ResultsThree types of 13C-labeled ?-O-4 lignin oligomer models were synthesized and characterized. The 2D 1H-13C heteronuclear single-quantum correlation (HSQC) spectra of the 13C-labeled lignin models confirmed that the three types of the 13C labels were correctly incorporated in the (1) aromatic rings and ? positions, (2) ? positions, and (3) methoxy groups, respectively. The TrCBM1-binding sites in lignin were analyzed by observing NMR chemical shift perturbations (CSPs) using the synthetic 13C-labeled ?-O-4 lignin oligomer models. Obvious CSPs were observed in signals from the aromatic regions in oligomers bound to TrCBM1, whereas perturbations in the signals from aliphatic regions and methoxy groups were insignificant. These findings indicated that hydrophobic interactions and ?-? stacking were dominating factors in nonproductive binding. The synthetic lignin models have two configurations whose terminal units were differently aligned and donated C(I) and C(II). The C(I) ring showed remarkable perturbation compared with the C(II), which indicated that the binding of TrCBM1 was markedly affected by the configuration of the lignin models. The long-chain lignin models (degree of polymerization (DP) 4.16-4.70) clearly bound to TrCBM1. The interactions of TrCBM1 with the short-chain lignin models (DP 2.64-3.12) were insignificant, indicating that a DP greater than 4 was necessary for TrCBM1 binding.ConclusionThe CSP analysis using 13C-labeled ?-O-4 lignin oligomer models enabled the identification of the TrCBM1 binding sites in lignins at the atomic level. This specific interaction analysis will provide insights for new molecular designs of cellulase having a controlled affinity to cellulose and lignin for a cost-effective biorefinery process.

Dataset Information

Elucidation of a Unique Pattern and the Role of Carbohydrate Binding Module of an Alginate Lyase.

Publications

Elucidation of a Unique Pattern and the Role of Carbohydrate Binding Module of an Alginate Lyase.

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