Biochemical characterization of a new ulvan lyase and its applicability in utilization of ulvan and preparation of ulva oligosaccharides.

Ulva is globally distributed specie and has a high economic value. Ulvan is one of the main active substances in Ulva, which has a variety of biological properties. Ulvan lyase degrades ulvan through a ß-elimination mechanism which cleaves the ß-glycosidic bond between Rha3S and GlcA or IdoA. The complex monosaccharide composition of ulvan makes it promising for use in food and pharmaceutical applications. This thesis explores a putative ulvan lyase from Alteromonas sp. KUL_42. We expressed and purified the protein, performed a series of characterizations and signal peptide had been removed. The results showed that the protein molecular weight of ULA-2 was 53.97 kDa, and it had the highest catalytic activity at 45 °C and pH 8.0 in Tris-HCl buffer. The Km and Vmax values were 2.24 mg · mL-1 and 2.048 µmol · min-1 · mL-1, respectively. The activity of ULA-2 was able to maintain more than 80% at 20 ~ 30 °C. ESI-MS analysis showed that the primary end-products were mainly disaccharides to tetrasaccharides. The study of ULA-2 enriches the ulvan lyase library, promotes the development and high-value utilization of Ulva resources, and facilitates further research applications of ulvan lyase in ulva oligosaccharides.

A novel alginate lyase and its domain functions for the preparation of unsaturated monosaccharides.

Brown algae are considered promising crops for the production of sustainable biofuels. However, the commercial application has been limited by lack of efficient methods for converting alginate into fermentable sugars. Herein, we cloned and characterized a novel alginate lyase AlyPL17 from Pedobacter hainanensis NJ-02. It possessed outstanding catalytic efficiency toward polymannuronic acid (polyM), polyguluronic acid (polyG), and alginate sodium, with kcat of 39.42 ± 1.9 s-1, 32.53 ± 0.88 s-1, and 38.30 ± 2.12 s-1, respectively. AlyPL17 showed maximum activity at 45 °C and pH 9.0. The domain truncation did not change the optimal temperature and optimal pH, but greatly reduced the activity. In addition, AlyPL17 degrades alginate through the cooperative action of two structural domains in an exolytic mode. The minimal degradation substrate of AlyPL17 is a disaccharide. Furthermore, AlyPL17 and AlyPL6 can synergistically degrade alginate to prepare unsaturated monosaccharides that can be converted to 4-deoxy-L-erythron-5-hexoseuloseuronate acid (DEH). DEH is reduced to KDG by DEH reductase (Sdr), which enters the Entner-Doudoroff (ED) pathway as a common metabolite and is converted to bioethanol. KEY POINTS: • Biochemical characterization of alginate lyase from Pedobacter hainanensis NJ-02 and its truncated form. • Degradation patterns of AlyPL17 and the role of its domains in product distribution and mode of action. • Potential of synergistic degradation system for efficient preparation of unsaturated monosaccharides.

Efficient Degradation of Alginate and Preparation of Alginate Oligosaccharides by a Novel Biofunctional Alginate Lyase with High Activity and Excellent Thermophilic Features.

The enzymatic degradation of seaweed polysaccharides is gaining interest for its potential in the production of functional oligosaccharides and fermentable sugars. Herein, a novel alginate lyase, AlyRm3, was cloned from a marine strain, Rhodothermus marinus DSM 4252. The AlyRm3 showed optimal activity (37,315.08 U/mg) at 70 °C and pH 8.0, with the sodium alginate used as a substrate. Noticeably, AlyRm3 was stable at 65 °C and also exhibited 30% of maximal activity at 90 °C. These results indicated that AlyRm3 is a thermophilic alginate lyase that efficiently degrades alginate at high industrial temperatures (>60 °C). The FPLC and ESI-MS analyses suggested that AlyRm3 primarily released disaccharides and trisaccharides from the alginate, polyM, and polyG in an endolytic manner. In the saccharification process of sodium alginate (0.5%, w/v), the AlyRm3 yielded numerous reducing sugars (1.73 g/L) after 2 h of reaction. These results indicated that AlyRm3 has a high enzymatic capacity for saccharifying the alginate, and could be used to saccharify the alginate biomass before the main fermentation process for biofuels. These properties make AlyRm3 a valuable candidate for both fundamental research and industrial applications.

Recent advances in the production, properties and applications of alginate oligosaccharides - a mini review.

Alginate oligosaccharides (AOS) made from the degradation of alginate, to some extent, makes up for the poor solubility and bioavailability of alginate as a macromolecular substance and possess several beneficial biological activities that are absent in alginate. These properties include prebiotic, glycolipid regulatory, immunomodulatory, antimicrobial, antioxidant, anti-tumor, promoting plant growth and other activities. Consequently, AOS has significant potential for use in the agricultural, biomedical, and food industries, and has been the focus of research in the field of marine biological resources. This review comprehensively covers methods (physical, chemical, and enzymatic methods) for the production of AOS from alginate. More importantly, this paper reviews recent advances in the biological activity and potentially industrial and therapeutic applications of AOS, providing a reference for future research and applications of AOS.

Biochemical characterization and elucidation of the action mode of a GH16 family κ-carrageenase for efficient preparation of carrageenan oligosaccharides.

κ-Carrageenan oligosaccharides have a variety of biological activities. Degradation of κ-carrageenan by κ-carrageenase leads to degradation products with different degrees of polymerization (DPs). A novel gene (CecgkA) encoding a new κ-carrageenase was cloned from Colwellia echini and heterologously expressed in Escherichia coli BL21 (DE3). The enzyme is 1104 bp in length, encodes 367 amino acid residues and has a molecular weight of 41.30 kDa. Multiple alignment analysis showed that CeCgkA belongs to the glycoside hydrolase (GH16) family and has the highest homology with the κ-carrageenase of Rhodopirellula maiorica SM1, with 58% homology. The CeCgkA showed maximum activity (453.15 U/mg) at pH 8.0 and 35 °C. Determination of biochemical properties showed that CeCgkA was a thermal recovery enzyme, and 51.6% of the initial enzyme activity was recovered by immediately placing the sample at 35 °C for 60 min after enzymatic inactivation by boiling for 10 min. K+, Na+, and EDTA had an activating effect on the enzyme activity, while Ni2+, Cu2+, and Zn2+ inhibited the activity of the enzyme. In addition, TLC and ESI-MS analysis showed that the maximum recognition unit of CecgkA was decasaccharide and that the main degradation products were disaccharides, tetrasaccharides and hexasaccharides, indicating that the enzyme is an endo-type carrageenase.

Purification and biochemical characterization of a novel chitosanase cloned from the gene of Kitasatospora setae KM-6054 and its application in the production of chitooligosaccharides.

Chitosanase could degrade chitosan efficiently under mild conditions to prepare chitosan oligosaccharides (COSs). COS possesses versatile physiological activities and has wide application prospects in food, pharmaceutical and cosmetic fields. Herein, a new glycoside hydrolase (GH) family 46 chitosanase (CscB) was cloned from Kitasatospora setae KM-6054 and heterologously expressed in Escherichia coli. The recombinant chitosanase CscB was purified by Ni-charged magnetic beads and showed a relative molecular weight of 29.19 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). CscB showed the maximal activity (1094.21 U/mg) at pH 6.0 and 30 °C. It was revealed that CscB is a cold-adapted enzyme. CscB was determined to be an endo-type chitosanase with a polymerization degree of the final product mainly in the range of 2-4. This new cold-adapted chitosanase provides an efficient enzyme tool for clean production of COSs.

Ulva (Enteromorpha) Polysaccharides and Oligosaccharides: A Potential Functional Food Source from Green-Tide-Forming Macroalgae.

The high-valued utilization of Ulva (previously known as Enteromorpha) bioresources has drawn increasing attention due to the periodic blooms of world-wide green tide. The polysaccharide is the main functional component of Ulva and exhibits various physiological activities. The Ulva oligosaccharide as the degradation product of polysaccharide not only possesses some obvious activities, but also possesses excellent solubility and bioavailability. Both Ulva polysaccharides and oligosaccharides hold promising potential in the food industry as new functional foods or food additives. Studies on Ulva polysaccharides and oligosaccharides are increasing and have been the focus of the marine bioresources field. However, the comprehensive review of this topic is still rare and do not cover the recent advances of the structure, isolation, preparation, activity and applications of Ulva polysaccharides and oligosaccharides. This review systematically summarizes and discusses the recent advances of chemical composition, extraction, purification, structure, and activity of Ulva polysaccharides as well as oligosaccharides. In addition, the potential applications as new functional food and food additives have also been considered, and these will definitely expand the applications of Ulva oligosaccharides in the food and medical fields.

Biochemical Characterization and Elucidation of the Hybrid Action Mode of a New Psychrophilic and Cold-Tolerant Alginate Lyase for Efficient Preparation of Alginate Oligosaccharides.

Alginate lyases with unique biochemical properties have irreplaceable value in food and biotechnology industries. Herein, the first new hybrid action mode Thalassotalea algicola-derived alginate lyase gene (TAPL7A) with both psychrophilic and cold-tolerance was cloned and expressed heterologously in E. coli. With the highest sequence identity (43%) to the exolytic alginate lyase AlyA5 obtained from Zobellia galactanivorans, TAPL7A was identified as a new polysaccharide lyases family 7 (PL7) alginate lyase. TAPL7A has broad substrate tolerance with specific activities of 4186.1 U/mg, 2494.8 U/mg, 2314.9 U/mg for polyM, polyG, and sodium alginate, respectively. Biochemical characterization of TAPL7A showed optimal activity at 15 °C, pH 8.0. Interestingly, TAPL7A exhibits both extreme psychrophilic and cold tolerance, which other cold-adapted alginate lyase do not possess. In a wide range of 5-30 °C, the activity can reach 80-100%, and the residual activity of more than 70% can still be maintained after 1 h of incubation. Product analysis showed that TAPL7A adopts a hybrid endo/exo-mode on all three substrates. FPLC and ESI-MS confirmed that the final products of TAPL7A are oligosaccharides with degrees of polymerization (Dps) of 1-2. This study provides excellent alginate lyase candidates for low-temperature environmental applications in food, agriculture, medicine and other industries.

Alginate degrading enzymes: an updated comprehensive review of the structure, catalytic mechanism, modification method and applications of alginate lyases.

Alginate, a kind of linear acidic polysaccharide, consists of α-L-guluronate (G) and ß-D-mannuronate (M). Both alginate and its degradation products (alginate oligosaccharides) possess abundant biological activities such as antioxidant activity, antitumor activity, and antimicrobial activity. Therefore, alginate and alginate oligosaccharides have great value in food, pharmaceutical, and agricultural fields. Alginate lyase can degrade alginate into alginate oligosaccharides via the ß-elimination reaction. It plays an important role in marine carbon recycling and the deep utilization of brown algae. Elucidating the structural features of alginate lyase can improve our knowledge of its catalytic mechanisms. With the development of structural analysis techniques, increasing numbers of alginate lyases have been characterized at the structural level. Hence, it is essential and helpful to summarize and discuss the up-to-date findings. In this review, we have summarized progress on the structural features and the catalytic mechanisms of alginate lyases. Furthermore, the molecular modification strategies and the applications of alginate lyases have also been discussed. This comprehensive information should be helpful to expand the applications of alginate lyases.

Marine oligosaccharides originated from seaweeds: Source, preparation, structure, physiological activity and applications.

Marine polysaccharides originated from seaweeds, including agar, alginate, carrageenan, and fucoidan, possess various kinds of physiological activities and have been widely used in food, agricultural and medical areas. However, the application has been greatly limited by their poor solubility and low bioavailability. Thus marine oligosaccharides, as the degradation products of those polysaccharides, have drawn increasing attentions due to their obvious biological activities, good solubility and excellent bioavailability. This review will summarize the recent advances on the source, molecular structure and physiological activity of marine oligosaccharides, emphasizing their application as functional food additives. Furthermore, the relationship between the structure and the physiological activity of marine oligosaccharides is also elucidated and highlighted. The review concludes with an outlook toward potential applications for preparing the functional oligosaccharides in food biotechnology and agriculture fields.

Effect of surface charge conditions of carriers on the immobilization of ß-d-glucosidase.

In this study, a series of acidic or alkaline polypeptide chains were designed and grafted onto DEG-AM resin using Fmoc solid-phase synthesis to study the relationship between enzyme conformation and carrier surface charge. ß-d-glucosidase (ßGase) was then immobilized onto these modified carriers by adsorption. Each form of immobilized ßGase showed decreasing specific activity compared to that of the free. It could be attributed to both the changes in the enzyme conformation and the decrease in mass transfer efficiency. The optimum temperature of free ßGase, DEG@B3-ßGase is 55 °C, which of DEG@A3-ßGase is 65 °C and they all have the highest activity at pH 5. The Ea values ​​of free ßGase, DEG@A3-ßGase, and DEG@B3-ßGase are 0.546 kJ/mol, 0.224 kJ/mol, and 0.446 kJ/mol, and the Km values were 1.30 mmol/L, 1.44 mmol/L and 2.63 mmol/L, respectively. It shows that free ßGase and DEG@A3-ßGase are more similar. Meanwhile, the free ßGase (1.0 g/L, pH 5.0) stored at 4 °C has a shorter half-life (t1/2), which is only 9 days. However, the half-life of DEG@B3-ßGase and DEG@A3-ßGase is 20 days and over 60 days, indicating that the negative charged surface was conducive to maintenance of the structure and catalytic property of ßGase.

Insights into ulvan lyase: review of source, biochemical characteristics, structure and catalytic mechanism.

Ulvan, a kind of polyanionic heteropolysaccharide consisting of 3-sulfated rhamnose, uronic acids (iduronic acid and glucuronic acid) and xylose, has been widely applied in food and cosmetic industries. In addition, ulvan can be converted into fermentable monosaccharides through the cascade system of carbohydrate-active enzymes. Ulvan lyases can degrade ulvan into ulvan oligosaccharides, which is the first step in the fully degradation of ulvan. Various ulvan lyases have been cloned and characterized from marine bacteria and grouped into five polysaccharide lyase (PL) families, namely: PL24, PL25, PL28, PL37 and PL40 families. The elucidation of the biochemical characterization, action pattern and catalytic mechanism of ulvan lyase would definitely enhance our understanding of the deep utilization of marine bioresource and marine carbon cycling. In this review, we summarized the recent progresses about the source and biochemical characteristics of ulvan lyase. Additionally, the structural characteristics and catalytic mechanisms have been introduced in detail. This comprehensive information should be helpful regarding the application of ulvan lyases.

Effects of module truncation on biochemical characteristics and products distribution of a new alginate lyase with two catalytic modules.

In this work, we investigated the functions of structural modules within alginate lyase by truncating an endo-type alginate lyase into two successive catalytic modules. The effects of module deletion on biochemical characteristics and product distributions were further investigated. The N-terminal module (Aly7B-CDI) exhibited no activity toward alginate, polyM or polyG, but the C-terminal module (Aly7B-CDII) retained its activity. The full-length enzyme (Aly7B) and its truncated counterpart (Aly7B-CDII) had similar substrate specificities, but Aly7B-CDII had lower activity. Moreover, the activity of Aly7B was much higher than Aly7B-CDII at 30°C. Aly7B-CDII, however, possessed higher optimal pH and better pH stability than the full-length enzyme. The final degradation products for Aly7B were unsaturated di-, tri- and tetra-oligosaccharides, and those for Aly7B-CDII were unsaturated mono-, di-, tri-, tetra- and penta-oligosaccharides. Therefore, the potential impact of the noncatalytic module Aly7B-CDI on the catalytic module Aly7B-CDII was further elucidated by characterizing Aly7B and its truncations. These data contribute to the functional understanding of these differing modules.

Biochemical Characterization and Elucidation of Action Pattern of a Novel Polysaccharide Lyase 6 Family Alginate Lyase from Marine Bacterium Flammeovirga sp. NJ-04.

Alginate lyases have been widely used to prepare alginate oligosaccharides in food, agricultural, and medical industries. Therefore, discovering and characterizing novel alginate lyases with excellent properties has drawn increasing attention. Herein, a novel alginate lyase FsAlyPL6 of Polysaccharide Lyase (PL) 6 family is identified and biochemically characterized from Flammeovirga sp. NJ-04. It shows highest activity at 45 °C and could retain 50% of activity after being incubated at 45 °C for 1 h. The Thin-Layer Chromatography (TLC) and Electrospray Ionization Mass Spectrometry (ESI-MS) analysis indicates that FsAlyPL6 endolytically degrades alginate polysaccharide into oligosaccharides ranging from monosaccharides to pentasaccharides. In addition, the action pattern of the enzyme is also elucidated and the result suggests that FsAlyPL6 could recognize tetrasaccharide as the minimal substrate and cleave the glycosidic bonds between the subsites of -1 and +3. The research provides extended insights into the substrate recognition and degradation pattern of PL6 alginate lyases, which may further expand the application of alginate lyases.

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

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.

Insight into carrageenases: major review of sources, category, property, purification method, structure, and applications.

Carrageenan, a kind of linear sulfated polysaccharides consisting of D-galactose with alternating α-1,3 and ß-1,4 linkages, has been widely applied in the food and cosmetic industries as thickening and gelling agents due to excellent properties, such as gel-forming ability and chemical stability. It can be degraded by carrageenases to produce a series of even-numbered carrageenan oligosaccharides, which exhibit various fascinating functions, such as anti-inflammation, anti-coagulation, anti-tumor, and anti-thrombosis effects. Numerous carrageenases have been isolated and identified from various sources. The enzymes are grouped into three categories, namely κ-carrageenase, ι-carrageenase, and λ-carrageenase based on their substrate specificities and primary sequences, respectively. Elucidating the paradigm of the enzyme at every aspect would definitely enhance our understanding of the marine carbon cycling and natural evolution of glycoside hydrolases (GHs). The structural features of these enzymes have been fully illustrated, which will improve our knowledge of its catalytic mechanisms. In this review, we have summarized the recent progresses of major sources, category, and the enzyme's biochemical characteristics. Additionally, structural characteristics and catalytic mechanisms have been introduced in detail. We conclude with a brief discussion of the potential of the carrageenases in possible future applications in preparing functional oligosaccharides with versatile activities. This comprehensive information should be helpful regarding the application of carrageenases.

Biochemical Characterization and Degradation Pattern of a Unique pH-Stable PolyM-Specific Alginate Lyase from Newly Isolated Serratia marcescens NJ-07.

Enzymatic preparation of alginate oligosaccharides with versatile bioactivities by alginate lyases has attracted increasing attention due to its featured characteristics, such as wild condition and specific products. In this study, AlgNJ-07, a novel polyM-specific alginate lyase with high specific activity and pH stability, has been purified from the newly isolated marine bacterium Serratia marcescens NJ-07. It has a molecular weight of approximately 25 kDa and exhibits the maximal activity of 2742.5 U/mg towards sodium alginate under 40 °C at pH 9.0. Additionally, AlgNJ-07 could retain more than 95% of its activity at pH range of 8.0-10.0, indicating it possesses excellent pH-stability. Moreover, it shows high activity and affinity towards polyM block and no activity to polyG block, which suggests that it is a strict polyM-specific alginate lyase. The degradation pattern of AlgNJ-07 has also been explored. The activity of AlgNJ-07 could be activated by NaCl with a low concentration (100-300 mM). It can be observed that AlgNJ-07 can recognize the trisaccharide as the minimal substrate and hydrolyze the trisaccharide into monosaccharide and disaccharide. The TLC and ESI-MS analysis indicate that it can hydrolyze substrates in a unique endolytic manner, producing not only oligosaccharides with Dp of 2-5 but also a large fraction of monosaccharide. Therefore, it may be a potent tool to produce alginate oligosaccharides with lower Dps (degree of polymerization).

Biochemical Characterization and Degradation Pattern of a Novel Endo-Type Bifunctional Alginate Lyase AlyA from Marine Bacterium Isoptericola halotolerans.

Alginate lyases are important tools to prepare oligosaccharides with various physiological activities by degrading alginate. Particularly, the bifunctional alginate lyase can efficiently hydrolyze the polysaccharide into oligosaccharides. Herein, we cloned and identified a novel bifunctional alginate lyase, AlyA, with a high activity and broad substrate specificity from bacterium Isoptericola halotolerans NJ-05 for oligosaccharides preparation. For further applications in industry, the enzyme has been characterized and its action mode has been also elucidated. It exhibited the highest activity (7984.82 U/mg) at pH 7.5 and 55 °C. Additionally, it possessed a broad substrate specificity, showing high activities towards not only polyM (polyß-d-mannuronate) (7658.63 U/mg), but also polyG (poly α-l-guluronate) (8643.29 U/mg). Furthermore, the Km value of AlyA towards polyG (3.2 mM) was lower than that towards sodium alginate (5.6 mM) and polyM (6.7 mM). TLC (Thin Layer Chromatography) and ESI-MS (Electrospray Ionization Mass Spectrometry) were used to study the action mode of the enzyme, showing that it can hydrolyze the substrates in an endolytic manner to release a series of oligosaccharides such as disaccharide, trisaccharide, and tetrasaccharide. This study provided extended insights into the substrate recognition and degrading pattern of the alginate lyases, with a broad substrate specificity.

Expression and characterization of a new heat-stable endo-type alginate lyase from deep-sea bacterium Flammeovirga sp. NJ-04.

Alginate lyases play an essential role in the production of oligosaccharides by degrading alginate polysaccharide. Although many alginate lyases from various microorganisms have been characterized, reports on alginate lyases with special characteristics and commercial potential are still rather rare. In this study, a new alginate lyase, FsAlgA, was cloned from the deep-sea marine bacterium Flammeovirga sp. NJ-04. The recombinant enzyme was purified on Ni-NTA sepharose and then characterized in detail. It exhibited the highest activity (3343.7 U/mg) at pH 7.0 and 50 °C. Notably, the FsAlgA retained more than 80% of its maximum activity after incubation at 50 °C for 30 min, suggesting that FsAlgA was a heat-stable alginate lyase. Additionally, FsAlgA possessed broad substrate specificity, showing high activities toward both poly ß-D-mannuronate (polyM) and poly α-L-guluronate (polyG). Furthermore, the K m values of FsAlgA toward sodium alginate (0.48 mM) and polyG (0.94 mM) were lower than that toward polyM (1.42 mM). The TLC and ESI-MS analyses indicated that FsAlgA endolytically degraded alginate polysaccharide and released oligosaccharides with degree of polymerization (DP) of 2-5. Therefore, it may be a potent tool to produce alginate oligosaccharides with low DPs.

Enzymatic Hydrolysis of Alginate to Produce Oligosaccharides by a New Purified Endo-Type Alginate Lyase.

Enzymatic hydrolysis of sodium alginate to produce alginate oligosaccharides has drawn increasing attention due to its advantages of containing a wild reaction condition, excellent gel properties and specific products easy for purification. However, the efficient commercial enzyme tools are rarely available. A new alginate lyase with high activity (24,038 U/mg) has been purified from a newly isolated marine strain, Cellulophaga sp. NJ-1. The enzyme was most active at 50 °C and pH 8.0 and maintained stability at a broad pH range (6.0-10.0) and temperature below 40 °C. It had broad substrate specificity toward sodium alginate, heteropolymeric MG blocks (polyMG), homopolymeric M blocks (polyM) and homopolymeric G blocks (polyG), and possessed higher affinity toward polyG (15.63 mM) as well as polyMG (23.90 mM) than polyM (53.61 mM) and sodium alginate (27.21 mM). The TLC and MS spectroscopy analysis of degradation products suggested that it completely hydrolyzed sodium alginate into oligosaccharides of low degrees of polymerization (DPs). The excellent properties would make it a promising tool for full use of sodium alginate to produce oligosaccharides.