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Alginate lyases, which degrade alginate into oligosaccharides, have broad applications in biorefinery, biomedical, and industrial fields. The Polysaccharide Lyase Family 7 (PL7) is particularly notable for its alginate lyase activity. In this study, two novel alginate lyases, PmAlg7A and PmAlg7B, from Pseudomonas mendocina E03 were cloned, heterologously expressed, and characterized. PmAlg7B exhibited limited activity toward alginate (0.10 U/mg-protein), while PmAlg7A demonstrated higher activity with a specific activity of 0.76 U/mg-protein. PmAlg7A was identified as an MG-specific alginate lyase, producing oligosaccharides with degrees of polymerization (Dp) ranging from 2 to 5. The enzyme exhibited optimal activity at a temperature of 30 °C and a pH of 8.0, with a K m of 7.94 ± 0.92 mg/ml and a k cat of 1.23 ± 0.06 s -1 . Structural comparisons and amino acid sequence alignments indicated a potential role for residue 55 in loop B in modulating the activity of PmAlg7B, which was supported by mutagenesis experiments and molecular dynamics simulations. These findings enhance our understanding of the critical role of loop B in regulating substrate binding in PL7 alginate lyases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In this study, a novel pectate lyase (ApPel1) was identified and characterized from Aspergillus parasiticus. The ApPel1 hydrolysed oligogalacturonides (OGs) effectively and produced 4,5-unsaturated OGs from low-methoxyl (LM) pectin, with DP 2 to DP 5 as the major products. Furthermore, the multiple sequence alignments, structure model and phylogenetic analyses of the ApPel1 indicated that its catalytic active sites were highly conserved with other pectin lyases (PLs) and the Ca 2+ binding amino acid residues are different compared with pectate lyases (Pels). N187D, N191D and N187D/N191D mutants were constructed to test for both Ca 2+ binding properties and the effects on catalytic ability. The three mutations sharply decreased the activity of ApPel1 and Ca 2+ tolerance, indicating that the Ca 2+ binding amino acid residues are different from the other Pels. Based on the sequence and structure comparison between PLs and Pels, and mutation analysis, the ApPel1 may be direct evolution from PLs. Thus, this enzyme has potential for use in producing unsaturated OGs for biological activity study, and contributes to an improved understanding of the evolutionary relationships between PLs and Pels., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Alginate is a family of industrially important linear polymers consisting of β-D-mannuronic acid (M) and its C-5 epimer α-L-guluronic acid (G). The function of alginate is closely related to the ratio of M/G. Mannuronan C-5 epimerase, which converts M to G, is a key enzyme involved in the biosynthesis of alginate. A new mannuronan C-5 epimerase isolated from Pseudomonas mendocina. sp. DICP-70 named PmC5A was characterized in this study. From the 1 H NMR analysis of the products, we have found that PmC5A possesses alginate lyase function in addition to mannuronan C-5-epimerase. The optimal pH and temperature of lyase and epimerase were found to be 8.0, 9.0 and 40 °C, 30 °C, respectively. PmC5A also shows lyase activity toward PolyMG and G-blocks., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Lytic polysaccharide monooxygenases (LPMOs) have attracted vast attention because of their unique mechanism of oxidative degradation of carbohydrate polymers and the potential application in biorefineries. This study characterized a novel LPMO from Myceliophthora thermophila, denoted MtLPMO9L. The structure model of the enzyme indicated that it belongs to the C1-oxidizing LPMO, which has neither an extra helix in the L3 loop nor extra loop region in the L2 loop. This was confirmed subsequently by the enzymatic assays since MtLPMO9L only acts on cellulose and generates C1-oxidized cello-oligosaccharides. Moreover, synergetic experiments showed that MtLPMO9L significantly improves the efficiency of cellobiohydrolase (CBH) II. In contrast, the inhibitory rather than synergetic effect was observed when combining used MtLPMO9L and CBHI. Changing the incubation time and concentration ratio of MtLPMO9L and CBHI could attenuate the inhibitory effects. This discovery suggests a different synergy detail between MtLPMO9L and two CBHs, which implies that the composition of cellulase cocktails may need reconsideration., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Enzymatic digestion of sodium alginate to produce specific oligosaccharides has attracted great attention. However, commercial enzymes that efficiently produce specific oligosaccharides are still unavailable. In the present study, a novel gene encoding an alginate lyase (designated alg7A) was cloned from the marine bacterium Vibrio sp. W13 and expressed in E. coli. The recombinant Alg7A shows high activities toward alginate, poly-α-l-guluronate (polyG), poly-β-d-mannuronate (polyM) and polyMG, and more preferred to polyMG. Moreover, the enzyme contains a highly conserved domain of the Polysaccharide Lyase (PL) 7 family (R*E*R, Q*H and Y*KAG*Y*Q), which indicates that it belongs to PL7. Furthermore, the thin layer chromatography and ESI-MS analysis showed that Alg7A mainly releases trisaccharides from alginate. These results demonstrated that Alg7A has a great potential to be used to produce oligosaccharides from alginate., (Copyright © 2019 Elsevier B.V. All rights reserved.)