Search Results (29)

Alginate lyases are critical enzymes in hydrolyzing alginate into alginate oligosaccharides (AOS), which are bioactive compounds known for their antioxidant properties and ability to lower serum glucose and lipid concentrations. However, elucidating catalytic mechanisms and discovering enzymes with enhanced catalytic efficiency remain long-term challenges. Here, we report AlgL2491, a novel bifunctional and cold-adapted alginate lyase from Pseudoalteromonas carrageenovora ASY5, belonging to the polysaccharide lyase family 18. This enzyme uniquely cleaves both polyguluronic (polyG) and polymannuronic (polyM), predominantly releasing disaccharides, trisaccharides, and tetrasaccharides after 12 h of hydrolysis. The enzyme achieves peak catalytic efficiency at 35 °C and pH 7.5, with activity increasing 5.5-fold in 0.5 M of NaCl. Molecular dynamics simulations demonstrate that salt ions enhance structural stability by minimizing conformational fluctuations and strengthening interdomain interactions, providing mechanistic insights into its salt-activated behavior. The alginate oligosaccharides (AOS) exhibit excellent free radical-scavenging activities of 86.79 ± 0.31%, 83.42 ± 0.18%, and 71.28 ± 2.27% toward hydroxyl, ABTS, and DPPH radicals, with IC50 values of 8.8, 6.74, and 9.71 mg/mL, respectively. These findings not only reveal the salt-activation mechanism of AlgL2491 and highlight the potential value of its hydrolysate in antioxidant activity but also provide a sustainable industrial solution in industrial-scale AOS production directly from marine biomass, eliminating the need for energy-intensive desalination of alginate, which may inform future biocatalyst design for marine polysaccharide valorization. Full article

This study investigates the biochemical properties of two xylanases, ZgXyn10A and CaXyn10B, which are members of the glycoside hydrolase family 10 (GH10) and originate from the marine Bacteroidetes species Zobellia galactanivorans and Cellulophaga algicola, respectively. Utilizing an auto-induction expression system in Escherichia coli, high-purity recombinant forms of these enzymes were successfully produced. Biochemical assays revealed that ZgXyn10A and CaXyn10B exhibit optimal activities at 40 °C and 30 °C, respectively, and demonstrate a high sensitivity to temperature fluctuations. Unlike conventional low-temperature enzymes, these xylanases retain only a fraction of their maximal activity at lower temperatures. To gain deeper insights into the structural and functional properties of these marine xylanases, two thermostable GH10 xylanases, TmxB and CoXyn10A, which share comparable amino acid sequence identity with ZgXyn10A and CaXyn10B, were selected for structural comparison. All four marine xylanases share a nearly similar three-dimensional structural topology. Molecular dynamics simulation indicated a striking difference in structural fluctuations between the low-temperature and thermostable xylanases, as evidenced by the distinct root mean square deviation values. Moreover, root mean square fluctuation analysis specifically identified the β3-α3 and β7-α7 loop regions within the substrate-binding cleft as crucial determinants of the temperature characteristics of these GH10 xylanases. Our findings establish loop dynamics as a key evolutionary driver in the thermal adaptation of GH10 xylanases and propose a loop engineering strategy for the development of industrial biocatalysts with tailored temperature responses, particularly for lignocellulosic biomass processing under moderate thermal conditions. Full article

Since the 2005 discovery of the first enzyme capable of depolymerizing polyethylene terephthalate (PET), an aromatic polyester once thought to be enzymatically inert, extensive research has been undertaken to identify and engineer new biocatalysts for plastic degradation. This effort was directed toward developing efficient enzymatic recycling technologies that could overcome the limitations of mechanical and chemical methods. These enzymes are versatile molecules obtained from microorganisms living in various environments, including soil, compost, surface seawater, and extreme habitats such as hot springs, hydrothermal vents, deep-sea regions, and Antarctic seawater. Among various plastics, PET and polylactic acid (PLA) have been the primary focus of enzymatic depolymerization research, greatly enhancing our knowledge of enzymes that degrade these specific polymers. They often display unique catalytic properties that reflect their particular ecological niches. This review explores recent advancements in marine-derived enzymes that can depolymerize synthetic plastic polymers, emphasizing their structural and functional features that influence the efficiency of these catalysts in biorecycling processes. Current status and future perspectives of enzymatic plastic depolymerization are also discussed, with a focus on the underexplored marine enzymatic resources. Full article

This study explores the reasons behind the variations in the enantioselectivity of the sulfoxidation of methyl phenyl sulfide by marine-derived vanadium-dependent haloperoxidases (VHPOs). Twelve new VHPOs of marine organisms were overexpressed, purified, and tested for their ability to oxidize sulfide. Most of these marine enzymes exhibited nonenantioselective behavior, underscoring the uniqueness of AnVBPO from the brown seaweed Ascophyllum nodosum and CpVBPO from the red seaweed Corallina pilulifera, which produce (R)- and (S)-sulfoxides, respectively. The enantioselective sulfoxidation pathway is likely due to direct oxygen transfer within the VHPO active site. This was demonstrated through molecular docking and molecular dynamics simulations, which revealed differences in the positioning of sulfide within AnVBPO and CpVBPO, thus explaining their distinct enantioselectivities. Nonenantioselective VHPOs probably follow a different oxidation pathway, initiating with sulfide oxidation to form a positively charged radical. Further insights were gained from studying the catalytic effect of VO₄³⁻ on H₂O₂-driven sulfoxidation. This research improves the understanding of VHPO-mediated sulfoxidation and aids in developing biocatalysts for sulfoxide synthesis. Full article

Kainoid synthases are key enzymes in the biosynthesis of kainoids. Kainoids, as represented by DA and KA, are a class of naturally occurring non-protein amino acids with strong neurotransmitter activity in the mammalian central nervous system. Marine algae kainoid synthases include PnDabC from diatoms, which synthesizes domoic acid (DA), and DsKabC and GfKabC from red algae, which synthesize kainic acid (KA). Elucidation of the catalytic mechanism of kainoid synthases is of great significance for the rational design of better biocatalysts to promote the industrial production of kainoids for use in new drugs. Through modeling, molecular docking, and molecular dynamics simulations, we investigated the conformational dynamics of kainoid synthases. We found that the kainoid synthase complexes showed different stability in the simulation, and the binding and catalytic processes showed significant conformational transformations of kainoid synthase. The residues involved in specific interactions with the substrate contributed to the binding energy throughout the simulation process. Binding energy, the relaxed active pocket, electrostatic potential energy of the active pocket, the number and rotation of aromatic residues interacting with substrates during catalysis, and the number and frequency of hydrogen bonds between the individual functional groups revealed the structure–activity relationships and affected the degree of promiscuity of kainoid synthases. Our research enriches the understanding of the conformational dynamics of kainoid synthases and has potential guiding significance for their rational design. Full article

Carbonic anhydrase (CA), an enzyme catalyzing the reversible hydration reaction of carbon dioxide (CO₂), is considered a promising biocatalyst for CO₂ reduction. The α-CA of Thermovibrio ammonificans (taCA) has emerged as a compelling candidate due to its high thermostability, a critical factor for industrial applications. However, the low-level expression and poor in vitro solubility have hampered further utilization of taCA. Recently, these limitations have been addressed through the fusion of the NEXT tag, a marine-derived, intrinsically disordered small peptide that enhances protein expression and solubility. In this study, the solubility and stability of NEXT-taCA were further investigated. When the linker length between the NEXT tag and the taCA was shortened, the expression level decreased without compromising solubility-enhancing performance. A comparison between the NEXT tag and the NT11 tag demonstrated the NEXT tag’s superiority in improving both the expression and solubility of taCA. While the thermostability of taCA was lower than that of the extensively engineered DvCA10, the NEXT-tagged taCA exhibited a 30% improvement in long-term thermostability compared to the untagged taCA, suggesting that enhanced solubility can contribute to enzyme thermostability. Furthermore, the bioprospecting of two intrinsically disordered peptides (Hcr and Hku tags) as novel solubility-enhancing fusion tags was explored, demonstrating their performance in improving the expression and solubility of taCA. These efforts will advance the practical application of taCA and provide tools and insights for enzyme biochemistry and bioengineering. Full article

The use of marine enzymes as catalysts for biotechnological applications is a topical subject. Marine enzymes usually display better operational properties than their animal, plant or bacterial counterparts, enlarging the range of possible biotechnological applications. Due to the fact that cytochrome P450 enzymes can degrade many different toxic environmental compounds, these enzymes have emerged as valuable tools in bioremediation processes. The present work describes the isolation, purification and biochemical characterization of a liver NADPH-dependent cytochrome P450 reductase (CPR) from the marine fish Liza klunzingeri (LkCPR). Experimental results revealed that LkCPR is a monomer of approximately 75 kDa that is active in a wide range of pH values (6–9) and temperatures (40–60 °C), showing the highest catalytic activity at pH 8 and 50 °C. The activation energy of the enzyme reaction was 16.3 kcal mol⁻¹ K⁻¹. The K_M values for cytochrome C and NADPH were 8.83 μM and 7.26 μM, and the k_cat values were 206.79 s⁻¹ and 202.93 s⁻¹, respectively. LkCPR displayed a specific activity versus cytochrome C of 402.07 µmol min⁻¹ mg¹, the highest activity value described for a CPR up to date (3.2–4.7 times higher than the most active reported CPRs) and showed the highest thermostability described for a CPR. Taking into account all these remarkable catalytic features, LkCPR offers great potential to be used as a suitable biocatalyst. Full article

Oceans possess tremendous diversity in microbial life. The enzymatic machinery that marine bacteria present is the result of extensive evolution to assist cell survival under the harsh and continuously changing conditions found in the marine environment. Several bacterial cells and enzymes are already used at an industrial scale, but novel biocatalysts are still needed for sustainable industrial applications, with benefits for both public health and the environment. Metagenomic techniques have enabled the discovery of novel biocatalysts, biosynthetic pathways, and microbial identification without their cultivation. However, a key stage for application of novel biocatalysts is the need for rapid evaluation of the feasibility of the bioprocess. Cultivation of not-yet-cultured bacteria is challenging and requires new methodologies to enable growth of the bacteria present in collected environmental samples, but, once a bacterium is isolated, its enzyme activities are easily measured. High-throughput screening techniques have also been used successfully, and innovative in vitro screening platforms to rapidly identify relevant enzymatic activities continue to improve. Small-scale approaches and process integration could improve the study and development of new bioprocesses to produce commercially interesting products. In this work, the latest studies related to (i) the growth of marine bacteria under laboratorial conditions, (ii) screening techniques for bioprospecting, and (iii) bioprocess development using microreactors and miniaturized systems are reviewed and discussed. Full article

Leucine dehydrogenase (LeuDH) has emerged as the most promising biocatalyst for L-tert-leucine (L-Tle) production via asymmetric reduction in trimethylpyruvate (TMP). In this study, a new LeuDH named PbLeuDH from marine Pseudomonas balearica was heterologously over-expressed in Escherichia coli, followed by purification and characterization. PbLeuDH possessed a broad substrate scope, displaying activities toward numerous L-amino acids and α-keto acids. Notably, compared with those reported LeuDHs, PbLeuDH exhibited excellent catalytic efficiency for TMP with a K_m value of 4.92 mM and a k_cat/K_m value of 24.49 s⁻¹ mM⁻¹. Subsequently, L-Tle efficient production was implemented from TMP by whole-cell biocatalysis using recombinant E. coli as a catalyst, which co-expressed PbLeuDH and glucose dehydrogenase (GDH). Ultimately, using a fed-batch feeding strategy, 273 mM (35.8 g L⁻¹) L-Tle was achieved with a 96.1% yield and 2.39 g L⁻¹ h⁻¹ productivity. In summary, our research provides a competitive biocatalyst for L-Tle green biosynthesis and lays a solid foundation for the realization of large-scale L-Tle industrial production. Full article

The ocean is an excellent source for new biocatalysts due to the tremendous genetic diversity of marine microorganisms, and it may contribute to the development of sustainable industrial processes. A marine bacterium was isolated and selected for the conversion of benzaldehyde to benzyl alcohol, which is an important chemical employed as a precursor for producing esters for cosmetics and other industries. Enzymatic production routes are of interest for sustainable processes. To overcome benzaldehyde low water solubility, DMSO was used as a biocompatible cosolvent up to a concentration of 10% (v/v). A two-phase system with n-hexane, n-heptane, or n-hexadecane as organic phase allowed at least a 44% higher relative conversion of benzaldehyde than the aqueous system, and allowed higher initial substrate concentrations. Cell performance decreased with increasing product concentration but immobilization of cells in alginate improved four-fold the robustness of the biocatalyst: free and immobilized cells were inhibited at concentrations of benzyl alcohol of 5 and 20 mM, respectively. Scaling up to a 100 mL stirred reactor, using a fed-batch approach, enabled a 1.5-fold increase in benzyl alcohol productivity when compared with batch mode. However, product accumulation in the reactor hindered the conversion. The use of a continuous flow reactor packed with immobilized cells enabled a 9.5-fold increase in productivity when compared with the fed-batch stirred reactor system. Full article

As an important enzyme involved in the marine carbon cycle, alginate lyase has received extensive attention because of its excellent degradation ability on brown algae, which is widely utilized for alginate oligosaccharide preparation or bioethanol production. In comparison with endo-type alginate lyases (PL-5, PL-7, and PL-18 families), limited studies have focused on PL-17 family alginate lyases, especially for those with special characteristics. In this study, a novel PL-17 family alginate lyase, Aly23, was identified and cloned from the marine bacterium Pseudoalteromonas carrageenovora ASY5. Aly23 exhibited maximum activity at 35 °C and retained 48.93% of its highest activity at 4 °C, representing an excellent cold-adaptation property. Comparative molecular dynamics analysis was implemented to explore the structural basis for the cold-adaptation property of Aly23. Aly23 had a high substrate preference for poly β-D-mannuronate and exhibited both endolytic and exolytic activities; its hydrolysis reaction mainly produced monosaccharides, disaccharides, and trisaccharides. Furthermore, the enzymatic hydrolyzed oligosaccharides displayed good antioxidant activities to reduce ferric and scavenge radicals, such as hydroxyl, ABTS⁺, and DPPH. Our work demonstrated that Aly23 is a promising cold-adapted biocatalyst for the preparation of natural antioxidants from brown algae. Full article

Cardiovascular diseases (CVDs) have emerged as a major threat to global health resulting in a decrease in life expectancy with respect to humans. Thrombosis is one of the foremost causes of CVDs, and it is characterized by the unwanted formation of fibrin clots. Recently, microbial fibrinolytic enzymes due to their specific features have gained much more attention than conventional thrombolytic agents for the treatment of thrombosis. Marine microorganisms including bacteria and microalgae have the significant ability to produce fibrinolytic enzymes with improved pharmacological properties and lesser side effects and, hence, are considered as prospective candidates for large scale production of these enzymes. There are no studies that have evaluated the fibrinolytic potential of marine fungal-derived enzymes. The current review presents an outline regarding isolation sources, production, features, and thrombolytic potential of fibrinolytic biocatalysts from marine microorganisms identified so far. Full article

Silicatein-α (Silα), a hydrolytic enzyme derived from siliceous marine sponges, is one of the few enzymes in nature capable of catalysing the metathesis of silicon–oxygen bonds. It is therefore of interest as a possible biocatalyst for the synthesis of organosiloxanes. To further investigate the substrate scope of this enzyme, a series of condensation reactions with a variety of phenols and aliphatic alcohols were carried out. In general, it was observed that Silα demonstrated a preference for phenols, though the conversions were relatively modest in most cases. In the two pairs of chiral alcohols that were investigated, it was found that the enzyme displayed a preference for the silylation of the S-enantiomers. Additionally, the enzyme’s tolerance to a range of solvents was tested. Silα had the highest level of substrate conversion in the nonpolar solvents n-octane and toluene, although the inclusion of up to 20% of 1,4-dioxane was tolerated. These results suggest that Silα is a potential candidate for directed evolution toward future application as a robust and selective biocatalyst for organosiloxane chemistry. Full article

The pivotal role played by ω-transaminases (ω-TAs) in the synthesis of chiral amines used as building blocks for drugs and pharmaceuticals is widely recognized. However, chiral bulky amines are challenging to produce. Herein, a ω-TA (TR₈) from a marine bacterium was used to synthesize a fluorine chiral amine from a bulky ketone. An analysis of the reaction conditions for process development showed that isopropylamine concentrations above 75 mM had an inhibitory effect on the enzyme. Five different organic solvents were investigated as co-solvents for the ketone (the amine acceptor), among which 25–30% (v/v) dimethyl sulfoxide (DMSO) produced the highest enzyme activity. The reaction reached equilibrium after 18 h at 30% of conversion. An in situ product removal (ISPR) approach using an aqueous organic two-phase system was tested to mitigate product inhibition. However, the enzyme activity initially decreased because the ketone substrate preferentially partitioned into the organic phase, n-hexadecane. Consequently, DMSO was added to the system to increase substrate mass transfer without affecting the ability of the organic phase to prevent inhibition of the enzyme activity by the product. Thus, the enzyme reaction was maintained, and the product amount was increased for a 62 h reaction time. The investigated ω-TA can be used in the bioconversion of bulky ketones to chiral amines for future bioprocess applications. Full article

The class EC 5.xx, a group of enzymes that interconvert optical, geometric, or positional isomers are interesting biocatalysts for the synthesis of pharmaceuticals and pharmaceutical intermediates. This class, named “isomerases,” can transform cheap biomolecules into expensive isomers with suitable stereochemistry useful in synthetic medicinal chemistry, and interesting cases of production of l-ribose, d-psicose, lactulose, and d-phenylalanine are known. However, in two published reports about potential biocatalysts of marine origin, isomerases are hardly mentioned. Therefore, it is of interest to deepen the knowledge of these biocatalysts from the marine environment with this specialized in-depth analysis conducted using a literature search without time limit constraints. In this review, the focus is dedicated mainly to example applications in biocatalysis that are not numerous confirming the general view previously reported. However, from this overall literature analysis, curiosity-driven scientific interest for marine isomerases seems to have been long-standing. However, the major fields in which application examples are framed are placed at the cutting edge of current biotechnological development. Since these enzymes can offer properties of industrial interest, this will act as a promoter for future studies of marine-originating isomerases in applied biocatalysis. Full article