Dissecting a two-domain alginate lyase of family PL6 reveals a mechanistic basis for substrate specificity and enzyme activity.

Enhancing the specific activity and thermostability of alginate lyase via semi-rational design

Microbial alginate lyases are essential biocatalysts for analyzing alginate structure and sustainably producing bioactive alginate oligosaccharides (AOS). In this study, we characterized Aly94, a novel alginate lyase from the polysaccharide lyase family 6 (PL6) family, identified from a marine sediment metagenomic library. Biochemical analyses showed Aly94 exhibits optimal activity at 40℃ in 50mM NaH₂PO₄-Na₂HPO₄ buffer (pH 7.0). Adding 20mM NaCl significantly increases its catalytic efficiency. The enzyme exhibits a strong preference for polyguluronate (polyG) over polymannuronate (polyM), with specific activities of 4.19U/mg (polyG), 0.25U/mg (polyM), and 2.45U/mg (alginate). When degrading substrates-particularly polyG-Aly94 primarily generates trisaccharides. Although Aly94 acts as an endolytic alginate lyase, it also could digest the monosaccharides from small oligosaccharide chains (∆G3, ∆G4). These catalytic properties, combined with its polyG-specific depolymerization, made Aly94 a promising candidate for biotechnological applications requiring controlled alginate saccharification and high-value AOS production.

Application of alginate lyase and advantages of its gene cloning: A review

The development of glycoscience has been impeded by the inherent structural complexity of carbohydrates, the lack of template encoding, and the limitations of current methodologies for their identification and quantification. Carbohydrate-binding modules (CBMs) and carbohydrate-active enzymes (CAZymes) are two protein families that have evolved to interact with distinct carbohydrates, offering the potential to function as specific carbohydrate recognition elements. Here, we present the first homogeneous assays for alginate as a representative polysaccharide, leveraging the unique properties of CBMs and CAZymes (e.g., polysaccharide lyases). In contrast to conventional antibody-antigen-antibody "sandwich" immunoassays, our approach takes advantage of the linear and repetitive nature of polysaccharides, allowing for simultaneous binding to multiple CBMs and lyases. We exploit this feature in our novel "Barbecue" assay design, utilizing the combination of CBMs/lyases that are orthogonally labeled with the Tb-based donor fluorophore CoraFluor-1 (CRF) and Cy5 as an acceptor to establish a time-resolved Förster resonance energy transfer (TR-FRET) detection system. The homogeneous single-step assay uses a straightforward protocol without wash steps, enabling the sensitive quantification of alginate. The lower limit of quantification was determined to be as low as 0.4 ng·mL-1 (0.01 ng) with CBMs and 1.6 ng·mL-1 (0.03 ng) with alginate lyases, representing a ≥100-fold improvement compared to that of conventional microarray-based assays. The broad applicability of the analytical method was rigorously validated across biomedical, food, and cosmetics samples. The "Barbecue" FRET concept is generalizable and can be applied to develop similar rapid and simple quantification assays for other polysaccharides beyond alginate.

Medium-chain fatty acids (MCFA) are scarcely produced when waste activated sludge (WAS) is used as the sole substrate, limiting their practical development. Production typically requires external electron donors following chemical or physical pretreatments. This study demonstrates that an enriched MCFA-producing consortium (MPC) can initiate MCFA production from WAS without any external electron donors. In the WAS group, the COD conversion ratios from WAS to SCFA (short-chain fatty acids) and MCFA were 6.0% and 0%, respectively. After dosing MPC in WAS, these ratios increased to 20.7% and 4.6%, respectively. These results were attributed to the higher hydrolytic activity in MPC compared to WAS; for instance, the relative activity of alginate lyase in WAS was only 2.8% of that in MPC. Bacteroides (33.1%) was identified as the key bacterium in MPC due to its role in secreting multihydrolases and intracellular enzymes to degrade uronic acids. Conversion of the hydrolysates to MCFA occurred predominantly via the fatty acid biosynthesis (FAB) pathway, as evidenced by the comprehensive identification of FAB-related genes and active enzymes via multiomics analysis. The initiating mechanism is thus attributed to the high activity of both multihydrolases and the FAB pathway without external electron donors. Overall, this study demonstrates the oriented microbial method, coupling the hydrolytic efficiency of FAB-related bacteria and the favorable thermodynamics of the reverse β-oxidation (RBO) pathway in the future, to support the recovery of MCFA from WAS.

Marine-derived enzymes often show distinct physiological properties and great potential for industrial use. Salt ions may improve the stability and expression efficiency of marine enzymes, which requires salt-resistant host based expression platform. Aspergillus oryzae of good protein expression and secretion was evaluated and explored for this purpose. Growth, sporulation, secreted extracellular proteins and transcriptome-based metabolic characteristics under artificial seawater condition were analyzed. Also, genetic manipulation system was developed and compared in various strains. Finally, A. oryzae AS 3.487 was recognized as a dominant chassis host. Efficient constitutive and artificial seawater-inducible promoters were screened from the transcriptomic data, which were further evaluated using marine alginate lyase AlgI as the reporter. Medium and condition optimizations improved enzyme production subsequently. Multicopy expression strategy further achieved a maximum enzyme production of 797 U/mL in flask culture. This study provides new references for the heterologous expression of marine-derived enzymes under high-salinity culture conditions.

Alginate lyases catalyze depolymerization of alginate, a major polysaccharide in brown algae, into bioactive oligosaccharides. Herein, the gene (CelPL7A) encoding a PL7 family alginate lyase from Cellulophaga lytica was cloned, heterologously expressed in Escherichia coli, and characterized. CelPL7A exhibited maximum activity at 30 °C and pH 8.0 but limited thermostability. To enhance its thermal stability, rational design was employed and yielded three single mutants (N99G, T331D, and G346S). All three mutants exhibited significantly enhanced thermal stability. Their specific activities were enhanced to 2.7-, 2.3-, and 3.2-fold, respectively, compared to the wild-type enzyme. Both CelPL7A and the G346S mutant efficiently degraded pretreated kelp powder, primarily yielding trisaccharides. Application tests demonstrated that alginate oligosaccharides (AOS) produced by CelPL7A significantly promoted the seed germination and seedling vigor of pak choi (Brassica rapa var. chinensis) under salt stress. These results suggest that engineered variants are promising biocatalysts for AOS production and agricultural biostimulant development.

Vibrio sp. is one of the main producers of alginate lyase; however, most strains have problems such as low and unstable enzyme production. In this study, the enzyme production conditions of V. sp. 32415, a marine bacterium capable of producing extracellular alginate lyase, were optimized through Response Surface Design. The optimized medium was as follows: NaCl 12 g/L, FeSO4·7H2O 0.067 g/L, NH4Cl 7 g/L, alginate 11 g/L, K2HPO4·3H2O 4 g/L, MgSO4·7H2O 1 g/L. Under 28 °C, 160 rpm, 30 mL/300 mL liquid volume, and an initial pH 5.5 culture condition, the extracellular enzyme activity was 51.06 U/mL, which was 2.8 times higher compared with the activity before optimization. The optimal temperature, pH, and NaCl concentration for the extracellular alginate lyase were 37 °C, 8.0, and 0.1 M, respectively. The enzyme remained more than 80% of its original activity at 30 °C for 4 h. 1 mM Fe3+, Ca2+, K+, Mg2+, and Na+ enhance enzyme activity, with a preference for polyG blocks. V. sp. 32415 has two circular chromosomes and one circular plasmid. Chromosome 2 has two polysaccharide utilization loci. It utilizes alginate through the Scatter pathway. The results of this study provide theoretical and data support for understanding the production of extracellular alginate lyase by marine Vibrio and their metabolism and utilization of alginate.

Alginate lyase is widely used in the preparation of alginate oligosaccharides, medicine production, energy conversion, and so on. In this study, we designed truncated mutants of a marine-derived alginate lyase Algl and identified a highly active mutant CD317 with less degradation when expressed in a yeast host. The enzyme activity of the secretory CD317 was 1.4-fold higher than that of the parent Algl. It degraded both polyM and polyG but had a stronger preference for polyM. The optimal temperature of Algl and CD317 was 40°C and 35°C respectively, for which CD317 showed better temperature tolerance. Additionally, Ca2+ highly improved the enzyme activity. Fermentation conditions for CD317 production were optimized as a culture time of 144h, an inoculum of an OD600nm of 0.5, and an inducer concentration of 2% (v/v), respectively. Bioreactor fermentation allowed the highest production of 19,500 U/mL, which was 4.6-fold higher than that in a well-plate. These results indicated that the truncated CD317 showed good potential for industrial use.

Biochemical characterization of a novel bifunctional cellulase-glucanase and its application for efficient degradation of kelp powder.

A review on the expanding biotechnological frontier of Pedobacter.

Alginate, a major polysaccharide in brown algae, is vital for the carbon cycling of the ocean ecosystem and holds promise for biotechnological applications. Marine Bacteroidetes, known for the ability to degrade complex polysaccharides, play an important role in the ocean carbon cycle; however, the detailed alginate degradation pattern remains to be further explored. In this study, an alginate utilization locus was identified in the genome of a new marine Bacteroidetes, Roseihalotalea indica gen. nov. sp. nov. TK19036T, and encodes two new alginate lyases, RiAlyPL6 and RiAlyPL17, which play potential roles in the degradation and utilization of alginate. RiAlyPL6 and RiAlyPL17 have distinct degradation products and substrate preferences, revealing the adaptation of the strain to utilize alginate with different M/G ratios. Based on the results in this paper, we have proposed a model for the degradation and utilization mechanism of alginate in Roseihalotalea indica gen. nov. sp. nov. TK19036T. All in all, our research provides a new insight into the alginate mechanisms within marine Roseihalotalea, and the two novel alginate lyases are excellent candidates for preparation and application.

A key loop in the catalytic pocket of the PL17 family of alginate lyases determines minimal substrate recognition.

The objective of this study was the evaluation of fungal solid-state fermentation (SSF) for the production of alginate lyase and extraction of uronic acids from Sargassum sp. For this purpose, the fungi Trichoderma asperellum, Aspergillus oryzae, and Rhizopus oryzae were applied (alone or combined) to Sargassum sp. biomass through SSF (107 spores gbiomass−1, 30 °C, and 7 days of treatment). In general, individual SSF with all three fungi degraded the biomass, achieving a marked synergy in the production of cellulase, laminarinase, and alginate lyase activities (especially for the last one). Trichoderma was the most efficient species in producing laminarinase, whereas Rhizophus was the best option for producing alginate lyase. However, when dual combinations were tested, the maximal values of alginate lyase activities were reached (13.4 ± 0.2 IU gbiomass−1 for Aspergillus oryzae and Rhizopus oryzae). Remarkably, uronic acids were the main monomeric units from algal biomass solubilization, achieving a maximum yield of 14.4 mguronic gbiomass−1, with the A + R condition being a feasible, eco-friendly alternative to chemical extraction of this monomer. Additionally, the application of all the fungal pretreatments drastically decreased the total phenolic content (TPC) in the biomass from 369 mg L−1 to values around 44–84 mg L−1, minimizing the inhibition for possible subsequent biological processes in which the residual solid can be used.

Acidic polysaccharides such as alginate, a key component of brown algae, have unique properties conferred by their carboxyl groups. Alginate is degraded by alginate lyases, a class of polysaccharide lyases (PLs) that cleave uronic acid glycoside bonds via β-elimination. These enzymes, which are classified into various PL families, differ in structure and substrate specificity but frequently share structural motifs including β-helices, β-jelly rolls, and (α/α)6 barrels coupled with antiparallel β-sheets. Moreover, marine bacteria from the genera Alteromonas, Pseudoalteromonas, and Vibrio produce alginate lyases that belong to several PL families that are associated with blue carbon cycling. Furthermore, some Bacteroides species in the human gut have acquired alginate-degrading genes via horizontal transfer from marine bacteria and/or other Bacteroides species. Alginate fermentation by gut microbes can produce short-chain fatty acids with potential prebiotic effects. This review explores alginate lyase diversity, ecological roles, and relevance in both marine and human microbial ecosystems.

A marine bacterial strain, Vibrio sp. E, capable of producing alginate lyases, was isolated from seawater. Three alginate lyase genes from this strain were cloned and expressed in Escherichia coli. The recombinant enzymes, designated AlyE1, AlyE2, and AlyE3, exhibited optimal activity at pH 9.0 and high pH stability, retaining over 80% of their initial activity across a pH range of 6.0 to 10.0. They retained more than 50% activity at 4 °C and over 30% at 100 °C. AlyE1 and AlyE3 demonstrated thermo-tolerance, recovering the majority of of their initial activity after heat treatment (>80 °C, <120 min) and cooling (>80 °C, >10 min). All three enzymes exhibited strong NaCl tolerance but were not NaCl-dependent. They were characterized as bifunctional and endolytic lyases, which was effective in kelp (Laminaria japonica) hydrolysis and in disrupting Pseudomonas aeruginosa biofilms.

The incredibly narrow protein fold bottleneck, which separates the billions of unique proteins on one side to deliver diverse biological functions on the other, arises from folds that tolerate mutations during evolution. One such fold, called the β-trefoil, is present in functionally diverse proteins including cytokines involved in the immune system such as interleukin-1. The unrecognizable sequence-level diversity, even among paralogs of interleukin-1 within the same chromosomal locus, suggests the resilience of this fold to mutational onslaught. Furthermore, β-trefoil domain containing-proteins are known to coexist with other domains to achieve functional diversity. In this study, we challenge the reach and limitations of function prediction using fold-fold comparison using β-trefoil fold as an example. We identified proteins containing β-trefoil fold belonging to thirty-two distinct functional classes based on diverse domain architecture and/or functional annotation by mining both the PDB and AlphaFold databases using fold-fold comparison. Among the proteins with novel domain architecture we find β-trefoil along with chitinase, lipase, β-glucosidase, protein kinase, peptidoglycan-binding + peptidase matrixin, glycosyl hydrolases family 3 + PA14 + fibronectin type- III, alpha galactosidase A, PhoD-like phosphatase, insecticidal crystal toxin, trypsin, alginate lyase and two novel structurally uncharacterized domains. We demonstrate that fold-fold comparison can extend function prediction beyond the reach of sequence-based approach and provides an opportunity to discover novel domain architecture associated with known folds. However, since extending fold similarity to functional similarity may be challenged by convergent fold evolution, we explore if β-trefoil may be a convergent evolution and share our hypothesis.

The incredibly narrow protein fold bottleneck, which separates the billions of unique proteins on one side to deliver diverse biological functions on the other, arises from folds that tolerate mutations during evolution. One such fold, called the β-trefoil, is present in functionally diverse proteins including cytokines involved in the immune system such as interleukin-1. The unrecognizable sequence-level diversity, even among paralogs of interleukin-1 within the same chromosomal locus, suggests the resilience of this fold to mutational onslaught. Furthermore, β-trefoil domain containing-proteins are known to coexist with other domains to achieve functional diversity. In this study, we challenge the reach and limitations of function prediction using fold-fold comparison using β-trefoil fold as an example. We identified proteins containing β-trefoil fold belonging to thirty-two distinct functional classes based on diverse domain architecture and/or functional annotation by mining both the PDB and AlphaFold databases using fold-fold comparison. Among the proteins with novel domain architecture we find β-trefoil along with chitinase, lipase, β-glucosidase, protein kinase, peptidoglycan-binding + peptidase matrixin, glycosyl hydrolases family 3 + PA14 + fibronectin type- III, alpha galactosidase A, PhoD-like phosphatase, insecticidal crystal toxin, trypsin, alginate lyase and two novel structurally uncharacterized domains. We demonstrate that fold-fold comparison can extend function prediction beyond the reach of sequence-based approach and provides an opportunity to discover novel domain architecture associated with known folds. However, since extending fold similarity to functional similarity may be challenged by convergent fold evolution, we explore if β-trefoil may be a convergent evolution and share our hypothesis.

Estuarine viral communities play a key role in microbial dynamics and ecosystem functioning. However, how viruses adapt to the highly dynamic estuarine environments remains largely underexplored. This study uses viromic sequencing to investigate the DNA viruses in estuarine water samples adjacent to the Shenzhen coast. Samples were divided into two major groups based on variations in quantified water parameters, corresponding to low-salinity and high-salinity waters. A total of 16,497 viral operational taxonomic units (vOTUs) were recovered, of which 85.59% were identified as novel viruses. β-diversity of viral communities supported the partition of samples based on salinity, and viral α-diversity differed significantly between low and high salinity. Taxonomically, Caudoviricetes dominated across all sites, with Myoviridae and Podoviridae more abundant in low salinity sites and Siphoviridae and Baculoviridae more abundant in high salinity sites. Gammaproteobacteria and Bacteroidota were the dominant host taxa, with distinct shifts in host abundance across the salinity gradient. Functional analysis revealed the abundant auxiliary metabolic genes involved in lipid, nucleotide, cofactor, and polysaccharide metabolisms. In particular, the alginate-degrading polysaccharide lyase family 6 was particularly abundant at high salinity sites. These results suggested that environmental factors, particularly salinity, shape the genomic diversity of estuarine viruses, which may further impact the biogeochemical processes in estuarine ecosystems.IMPORTANCEEstuaries are highly dynamic ecosystems with strong environmental fluctuations, particularly in salinity and nutrients. This study highlights how environmental factors shape viral diversity and function by examining viral populations across the salinity gradient, providing new insights into viral dynamics in these ecosystems. We identified novel viruses and viral-encoded auxiliary metabolic genes in estuarine samples, including the discovery of a previously unreported viral alginate lyase gene that was abundant at high salinity sites, which sheds light on the ecological role of viruses in nutrient cycling and ecosystem partition. In addition, the study provides valuable information on distinct viral populations and virus-host interactions across the salinity gradient, which are essential for predicting ecosystem responses to salinity changes. These findings provide important implications for a broader understanding of microbial and viral ecology in estuarine ecosystems.