Characterization of a New Biofunctional,Exolytic Alginate Lyase from Tamlana sp. s12 with High Catalytic Activity and Cold-Adapted Features

Alginate, a major acidic polysaccharide in brown algae, has attracted great attention as a promising carbon source for biorefinery systems. Alginate lyases, especially exo-type alginate lyase, play a critical role in the biorefinery process. Although a large number of alginate lyases have been characterized, few can efficiently degrade alginate comprised of mannuronate (M) and guluronate (G) at low temperatures by means of an exolytic mode. In this study, the gene of a new exo-alginate lyase—Alys1—with high activity (1350 U/mg) was cloned from a marine strain, Tamlana sp. s12. When sodium alginate was used as a substrate, the recombinant enzyme showed optimal activity at 35 °C and pH 7.0–8.0. Noticeably, recombinant Alys1 was unstable at temperatures above 30 °C and had a low melting temperature of 56.0 °C. SDS and EDTA significantly inhibit its activity. These data indicate that Alys1 is a cold-adapted enzyme. Moreover, the enzyme can depolymerize alginates polyM and polyG, and produce a monosaccharide as the minimal alginate oligosaccharide. Primary substrate preference tests and identification of the final oligosaccharide products demonstrated that Alys1 is a bifunctional alginate lyase and prefers M to G. These properties make Alys1 a valuable candidate in both basic research and industrial applications.     

Structure Characteristics,Biochemical Properties,and Pharmaceutical Applications of Alginate Lyases

Alginate, the most abundant polysaccharides of brown algae, consists of various proportions of uronic acid epimers α-L-guluronic acid (G) and β-D-mannuronic acid (M). Alginate oligosaccharides (AOs), the degradation products of alginates, exhibit excellent bioactivities and a great potential for broad applications in pharmaceutical fields. Alginate lyases can degrade alginate to functional AOs with unsaturated bonds or monosaccharides, which can facilitate the biorefinery of brown algae. On account of the increasing applications of AOs and biorefinery of brown algae, there is a scientific need to explore the important aspects of alginate lyase, such as catalytic mechanism, structure, and property. This review covers fundamental aspects and recent developments in basic information, structural characteristics, the structure–substrate specificity or catalytic efficiency relationship, property, molecular modification, and applications. To meet the needs of biorefinery systems of a broad array of biochemical products, alginate lyases with special properties, such as salt-activated, wide pH adaptation range, and cold adaptation are outlined. Withal, various challenges in alginate lyase research are traced out, and future directions, specifically on the molecular biology part of alginate lyases, are delineated to further widen the horizon of these exceptional alginate lyases.     

Cloning and Characterization of a Novel Alginate Lyase from Paenibacillus sp. LJ-23

As a low molecular weight alginate, alginate oligosaccharides (AOS) exhibit improved water solubility, better bioavailability, and comprehensive health benefits. In addition, their biocompatibility, biodegradability, non-toxicity, non-immunogenicity, and gelling capability make them an excellent biomaterial with a dual curative effect when applied in a drug delivery system. In this paper, a novel alginate lyase, Algpt, was cloned and characterized from a marine bacterium, Paenibacillus sp. LJ-23. The purified enzyme was composed of 387 amino acid residues, and had a molecular weight of 42.8 kDa. The optimal pH of Algpt was 7.0 and the optimal temperature was 45 °C. The analysis of the conserved domain and the prediction of the three-dimensional structure indicated that Algpt was a novel alginate lyase. The dominant degradation products of Algpt on alginate were AOS dimer to octamer, depending on the incubation time, which demonstrated that Algpt degraded alginate in an endolytic manner. In addition, Algpt was a salt-independent and thermo-tolerant alginate lyase. Its high stability and wide adaptability endow Algpt with great application potential for the efficient preparation of AOS with different sizes and AOS-based products.     

Biochemical Characterization and Cold-Adaption Mechanism of a PL-17 Family Alginate Lyase Aly23 from Marine Bacterium Pseudoalteromonas sp. ASY5 and Its Application for Oligosaccharides Production

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.     

Purification and Characterization of a Novel Alginate Lyase from a Marine Streptomyces Species Isolated from Seaweed

Alginate, a natural polysaccharide derived from brown seaweed, is finding multiple applications in biomedicine via its transformation through chemical, physical, and, increasingly, enzymatic processes. In this study a novel alginate lyase, AlyDS44, was purified and characterized from a marine actinobacterium, Streptomyces luridiscabiei, which was isolated from decomposing seaweed. The purified enzyme had a specific activity of 108.6 U/mg, with a molecular weight of 28.6 kDa, and was composed of 260 amino acid residues. AlyDS44 is a bifunctional alginate lyase, active on both polyguluronate and polymannuronate, though it preferentially degrades polyguluronate. The optimal pH of this enzyme is 8.5 and the optimal temperature is 45 °C. It is a salt-tolerant alginate lyase with an optimal activity at 0.6 M NaCl. Metal ions Mn²⁺, Co²⁺, and Fe²⁺ increased the alginate degrading activity, but it was inhibited in the presence of Zn²⁺ and Cu²⁺. The highly conserved regions of its amino acid sequences indicated that AlyDS44 belongs to the polysaccharide lyase family 7. The main breakdown products of the enzyme on alginate were disaccharides, trisaccharides, and tetrasaccharides, which demonstrated that this enzyme acted as an endo-type alginate lyase. AlyDS44 is a novel enzyme, with the potential for efficient production of alginate oligosaccharides with low degrees of polymerization.     

Expression and Characterization of a Cold-Adapted Alginate Lyase with Exo/Endo-Type Activity from a Novel Marine Bacterium Alteromonas portus HB161718T

The alginate lyases have unique advantages in the preparation of alginate oligosaccharides and processing of brown algae. Herein, a gene alg2951 encoding a PL7 family alginate lyase with exo/endo-type activity was cloned from a novel marine bacterium Alteromonas portus {"type":"entrez-nucleotide","attrs":{"text":"HB161718","term_id":"239323637"}}HB161718^T and then expressed in Escherichia coli. The recombinant Alg2951 in the culture supernatant reached the activity of 63.6 U/mL, with a molecular weight of approximate 60 kDa. Alg2951 exhibited the maximum activity at 25 °C and pH 8.0, was relatively stable at temperatures lower than 30 °C, and showed a special preference to poly-guluronic acid (polyG) as well. Both NaCl and KCl had the most promotion effect on the enzyme activity of Alg2951 at 0.2 M, increasing by 21.6 and 19.1 times, respectively. The TCL (Thin Layer Chromatography) and ESI-MS (Electrospray Ionization Mass Spectrometry) analyses suggested that Alg2951 could catalyze the hydrolysis of sodium alginate to produce monosaccharides and trisaccharides. Furthermore, the enzymatic hydrolysates displayed good antioxidant activity by assays of the scavenging abilities towards radicals (hydroxyl and ABTS+) and the reducing power. Due to its cold-adapted and dual exo/endo-type properties, Alg2951 can be a potential enzymatic tool for industrial production.     

Genome Analysis of a Novel Polysaccharide-Degrading Bacterium Paenibacillus algicola and Determination of Alginate Lyases

Carbohydrate-active enzymes (CAZymes) are an important characteristic of bacteria in marine systems. We herein describe the CAZymes of Paenibacillus algicola {"type":"entrez-nucleotide","attrs":{"text":"HB172198","term_id":"239334117"}}HB172198^T, a novel type species isolated from brown algae in Qishui Bay, Hainan, China. The genome of strain {"type":"entrez-nucleotide","attrs":{"text":"HB172198","term_id":"239334117"}}HB172198^T is a 4,475,055 bp circular chromosome with an average GC content of 51.2%. Analysis of the nucleotide sequences of the predicted genes shows that strain {"type":"entrez-nucleotide","attrs":{"text":"HB172198","term_id":"239334117"}}HB172198^T encodes 191 CAZymes. Abundant putative enzymes involved in the degradation of polysaccharides were identified, such as alginate lyase, agarase, carrageenase, xanthanase, xylanase, amylases, cellulase, chitinase, fucosidase and glucanase. Four of the putative polysaccharide lyases from families 7, 15 and 38 were involved in alginate degradation. The alginate lyases of strain {"type":"entrez-nucleotide","attrs":{"text":"HB172198","term_id":"239334117"}}HB172198^T exhibited the maximum activity 152 U/mL at 50 °C and pH 8.0, and were relatively stable at pH 7.0 and temperatures lower than 40 °C. The average degree of polymerization (DP) of the sodium alginate oligosaccharide (AOS) degraded by the partially purified alginate lyases remained around 14.2, and the thin layer chromatography (TCL) analysis indicated that it contained DP2-DP8 oligosaccharides. The complete genome sequence of P. algicola {"type":"entrez-nucleotide","attrs":{"text":"HB172198","term_id":"239334117"}}HB172198^T will enrich our knowledge of the mechanism of polysaccharide lyase production and provide insights into its potential applications in the degradation of polysaccharides such as alginate.     

Cloning and Characterization of a Novel Endo-Type Metal-Independent Alginate Lyase from the Marine Bacteria Vibrio sp. Ni1

The applications of alginate lyase are diverse, but efficient commercial enzymes are still unavailable. In this study, a novel alginate lyase with high activity was obtained from the marine bacteria Vibrio sp. Ni1. The ORF of the algB gene has 1824 bp, encoding 607 amino acids. Homology analysis shows that AlgB belongs to the PL7 family. There are two catalytic domains with the typical region of QIH found in AlgB. The purified recombinant enzyme of AlgB shows highest activity at 35 °C, pH 8.0, and 50 mmol/L Tris-HCl without any metal ions. Only K⁺ slightly enhances the activity, while Fe²⁺ and Cu²⁺ strongly inhibit the activity. The AlgB preferred polyM as substrate. The end products of enzymatic mixture are DP2 and DP3, without any metal ion to assist them. This enzyme has good industrial application prospects.     

Purification and characterization of a bifunctional alginate lyase from Pseudoalteromonas sp. SM0524

An alginate lyase-producing bacterial strain, Pseudoalteromonas sp. SM0524, was screened from marine rotten kelp. In an optimized condition, the production of alginate lyase from Pseudoalteromonas sp. SM0524 reached 62.6 U/mL, suggesting that strain SM0524 is a good producer of alginate lyases. The bifunctional alginate lyase aly-SJ02 secreted by strain SM0524 was purified. Aly-SJ02 had an apparent molecular mass of 32 kDa. The optimal temperature and pH of aly-SJ02 toward sodium alginate was 50 °C and 8.5, respectively. The half life period of aly-SJ02 was 41 min at 40 °C and 20 min at 50 °C. Aly-SJ02 was most stable at pH 8.0. N-terminal sequence analysis suggested that aly-SJ02 may be an alginate lyase of polysaccharide lyase family 18. Aly-SJ02 showed activities toward both polyG (α-l-guluronic acid) and polyM (β-D-mannuronic acid), indicating that it is a bifunctional alginate lyase. Aly-SJ02 had lower K(m) toward polyG than toward polyM and sodium alginate. Thin layer chromatography and ESI-MS analyses showed that aly-SJ02 mainly released dimers and trimers from polyM and alginate, and trimers and tetramers from polyG, which suggests that aly-SJ02 may be a good tool to produce dimers and trimers from alginate.     

Cultivable Alginate Lyase-Excreting Bacteria Associated with the Arctic Brown Alga Laminaria

Although some alginate lyases have been isolated from marine bacteria, alginate lyases-excreting bacteria from the Arctic alga have not yet been investigated. Here, the diversity of the bacteria associated with the brown alga Laminaria from the Arctic Ocean was investigated for the first time. Sixty five strains belonging to nine genera were recovered from six Laminaria samples, in which Psychrobacter (33/65), Psychromonas (10/65) and Polaribacter (8/65) were the predominant groups. Moreover, 21 alginate lyase-excreting strains were further screened from these Laminaria-associated bacteria. These alginate lyase-excreting strains belong to five genera. Psychromonas (8/21), Psedoalteromonas (6/21) and Polaribacter (4/21) are the predominant genera, and Psychrobacter, Winogradskyella, Psychromonas and Polaribacter were first found to produce alginate lyases. The optimal temperatures for the growth and algiante lyase production of many strains were as low as 10–20 °C, indicating that they are psychrophilic bacteria. The alginate lyases produced by 11 strains showed the highest activity at 20–30 °C, indicating that these enzymes are cold-adapted enzymes. Some strians showed high levels of extracellular alginate lyase activity around 200 U/mL. These results suggest that these algiante lyase-excreting bacteria from the Arctic alga are good materials for studying bacterial cold-adapted alginate lyases.     

Characterization of an Alginate Lyase,FlAlyA, from Flavobacterium sp. Strain UMI-01 and Its Expression in Escherichia coli

A major alginate lyase, FlAlyA, was purified from the periplasmic fraction of an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01. FlAlyA showed a single band of ~30 kDa on SDS-PAGE and exhibited the optimal temperature and pH at 55 °C and pH 7.7, respectively. Analyses for substrate preference and reaction products indicated that FlAlyA was an endolytic poly(mannuronate) lyase (EC 4.2.2.3). A gene fragment encoding the amino-acid sequence of 288 residues for FlAlyA was amplified by inverse PCR. The N-terminal region of 21 residues except for the initiation Met in the deduced sequence was predicted as the signal peptide and the following region of six residues was regarded as propeptide, while the C-terminal region of 260 residues was regarded as the polysaccharide-lyase-family-7-type catalytic domain. The entire coding region for FlAlyA was subjected to the pCold I—Escherichia coli BL21(DE3) expression system and ~eight times higher yield of recombinant FlAlyA (recFlAlyA) than that of native FlAlyA was achieved. The recFlAlyA recovered in the periplasmic fraction of E. coli had lost the signal peptide region along with the N-terminal 3 residues of propeptide region. This suggested that the signal peptide of FlAlyA could function in part in E. coli.     

Potential Food and Nutraceutical Applications of Alginate: A Review

Alginate is an acidic polysaccharide mainly extracted from kelp or sargassum, which comprises 40% of the dry weight of algae. It is a linear polymer consisting of β-D-mannuronic acid (M) and α-L-guluronic acid (G) with 1,4-glycosidic linkages, possessing various applications in the food and nutraceutical industries due to its unique physicochemical properties and health benefits. Additionally, alginate is able to form a gel matrix in the presence of Ca²⁺ ions. Alginate properties also affect its gelation, including its structure and experimental conditions such as pH, temperature, crosslinker concentration, residence time and ionic strength. These features of this polysaccharide have been widely used in the food industry, including in food gels, controlled-release systems and film packaging. This review comprehensively covers the analysis of alginate and discussed the potential applications of alginate in the food industry and nutraceuticals.     

Comparison of Biochemical Characteristics,Action Models,and Enzymatic Mechanisms of a Novel Exolytic and Two Endolytic Lyases with Mannuronate Preference

Recent explorations of tool-like alginate lyases have been focused on their oligosaccharide-yielding properties and corresponding mechanisms, whereas most were reported as endo-type with α-L-guluronate (G) preference. Less is known about the β-D-mannuronate (M) preference, whose commercial production and enzyme application is limited. In this study, we elucidated Aly6 of Flammeovirga sp. strain MY04 as a novel M-preferred exolytic bifunctional lyase and compared it with AlgLs of Pseudomonas aeruginosa (Pae-AlgL) and Azotobacter vinelandii (Avi-AlgL), two typical M-specific endolytic lyases. This study demonstrated that the AlgL and heparinase_II_III modules play indispensable roles in determining the characteristics of the recombinant exo-type enzyme rAly6, which is preferred to degrade M-enriched substrates by continuously cleaving various monosaccharide units from the nonreducing end, thus yielding various size-defined ΔG-terminated oligosaccharides as intermediate products. By contrast, the endolytic enzymes Pae-rAlgL and Avi-rAlgL varied their action modes specifically against M-enriched substrates and finally degraded associated substrate chains into various size-defined oligosaccharides with a succession rule, changing from ΔM to ΔG-terminus when the product size increased. Furthermore, site-directed mutations and further protein structure tests indicated that H¹⁹⁵NHSTW is an active, half-conserved, and essential enzyme motif. This study provided new insights into M-preferring lyases for novel resource discoveries, oligosaccharide preparations, and sequence determinations.     

Biochemical Properties of a New Polysaccharide Lyase Family 25 Ulvan Lyase TsUly25B from Marine Bacterium Thalassomonas sp. LD5

Marine macroalgae, contributing much to the bioeconomy, have inspired tremendous attention as sustainable raw materials. Ulvan, as one of the main structural components of green algae cell walls, can be degraded by ulvan lyase through the β-elimination mechanism to obtain oligosaccharides exhibiting several good physiological activities. Only a few ulvan lyases have been characterized until now. This thesis explores the properties of a new polysaccharide lyase family 25 ulvan lyase TsUly25B from the marine bacterium Thalassomonas sp. LD5. Its protein molecular weight was 54.54 KDa, and it was most active under the conditions of 60 °C and pH 9.0. The K_m and k_cat values were 1.01 ± 0.05 mg/mL and 10.52 ± 0.28 s⁻¹, respectively. TsUly25B was salt-tolerant and NaCl can significantly improve its thermal stability. Over 80% of activity can be preserved after being incubated at 30 °C for two days when the concentration of NaCl in the solution is above 1 M, while 60% can be preserved after incubation at 40 °C for 10 h with 2 M NaCl. TsUly25B adopted an endolytic manner to degrade ulvan polysaccharides, and the main end-products were unsaturated ulvan disaccharides and tetrasaccharides. In conclusion, our research enriches the ulvan lyase library and advances the utilization of ulvan lyases in further fundamental research as well as ulvan oligosaccharides production.     

Alginate Hydrogels Coated with Chitosan for Wound Dressing

In this work, a coating of chitosan onto alginate hydrogels was realized using the water-soluble hydrochloride form of chitosan (CH-Cl), with the dual purpose of imparting antibacterial activity and delaying the release of hydrophilic molecules from the alginate matrix. Alginate hydrogels with different calcium contents were prepared by the internal setting method and coated by immersion in a CH-Cl solution. Structural analysis by cryo-scanning electron microscopy was carried out to highlight morphological alterations due to the coating layer. Tests in vitro with human mesenchymal stromal cells (MSC) were assessed to check the absence of toxicity of CH-Cl. Swelling, stability in physiological solution and release characteristics using rhodamine B as the hydrophilic model drug were compared to those of relative uncoated hydrogels. Finally, antibacterial activity against Escherichia coli was tested. Results show that alginate hydrogels coated with chitosan hydrochloride described here can be proposed as a novel medicated dressing by associating intrinsic antimicrobial activity with improved sustained release characteristics.     

Advanced Strategies for 3D Bioprinting of Tissue and Organ Analogs Using Alginate Hydrogel Bioinks

Alginate is a natural polysaccharide that typically originates from various species of algae. Due to its low cost, good biocompatibility, and rapid ionic gelation, the alginate hydrogel has become a good option of bioink source for 3D bioprinting. However, the lack of cell adhesive moieties, erratic biodegradability, and poor printability are the critical limitations of alginate hydrogel bioink. This review discusses the pivotal properties of alginate hydrogel as a bioink for 3D bioprinting technologies. Afterward, a variety of advanced material formulations and biofabrication strategies that have recently been developed to overcome the drawbacks of alginate hydrogel bioink will be focused on. In addition, the applications of these advanced solutions for 3D bioprinting of tissue/organ mimicries such as regenerative implants and in vitro tissue models using alginate-based bioink will be systematically summarized.     

象耳豆根结线虫果胶酸裂解酶新基因Me-pel2的鉴定及发育表达分析

利用EST结合RACE方法从象耳豆根结线虫(Meloidogyne enterolobii)中克隆一个新的果胶酸裂解酶基因Me-pel2(Gen Bank登录号KP987180)。Me-pel2 c DNA开放阅读框全长834 bp,推导编码277个氨基酸残基的蛋白,属于多糖裂解酶第III家族新成员。Me-PEL2与南方根结线虫果胶酸裂解酶Mi-PEL3氨基酸具有较高的一致性,为54%。发育表达结果显示,Me-pel2在2龄幼虫和雄虫期高丰度表达,而在固着期寄生阶段转录水平急剧下降,推测Me-pel2主要在象耳豆根结线虫迁移性阶段起重要作用,通过降解寄主细胞壁果胶质成分,协助幼虫侵入寄主以及在寄主体内迁移。     

施用海藻酸复合肥料的双季稻产量和氮磷肥料效应

以华南大田双季稻为研究对象,设置5个处理:不施肥、普通复合肥、海藻复合肥、80%普通复合肥和80%海藻复合肥,研究海藻复合肥减施条件下对双季稻产量、肥效、养分吸收和土壤养分残留及土壤生物活性的影响。结果表明:海藻复合肥能够提高水稻有效穗和稻谷产量,晚稻海藻复合肥较普通复合肥有效穗增加14.5%,早稻稻谷增产7.6%,晚稻增产5.1%,均达显著水平;而80%海藻复合肥与普通复合肥相比稻谷产量无差异。海藻复合肥能够提高氮肥和磷肥偏生产力。与常量施肥相比,减量施氮肥和磷肥偏生产力分别提高6.1~8.5 kg/kg和15.2~21.4 kg/kg,氮和磷吸收效率分别提高11.3~19.7和12.0~19.8个百分点,均以80%海藻复合肥处理最高。减量施肥显著降低20~40 cm土壤硝态氮的含量。海藻复合肥可提高土壤微生物量碳（氮）和土壤蔗糖酶、脲酶及酸性磷酸酶活性。主成分分析结果表明,土壤肥力综合得分为80%海藻复合肥>海藻复合肥>普通复合肥>80%普通复合肥>CK。在华南双季稻种植条件下,与普通复合肥相比,海藻复合肥减肥20%具有较好的稳产效应,但是周年氮磷养分平衡表现为亏缺,建议根据土壤肥力状况及目标产量制订减肥措施,以保持土壤肥力的可持续性。     

Chitosan/Alginate Nanoparticles for the Enhanced Oral Antithrombotic Activity of Clam Heparinoid from the Clam Coelomactra antiquata

Chitosan/alginate nanoparticles (DG1-NPs and DG1/Cur-NPs) aiming to enhance the oral antithrombotic activity of clam heparinoid DG1 were prepared by ionotropic pre-gelation. The influence of parameters, such as the concentration of sodium alginate (SA), chitosan (CTS), CaCl₂, clam heparinoid DG1, and curcumin (Cur), on the characteristics of the nanoparticles, were investigated. Results indicate that chitosan and alginate can be used as polymer matrices to encapsulate DG1, and nanoparticle characteristics depend on the preparation parameters. Nano-particles should be prepared using 0.6 mg/mL SA, 0.33 mg/mL CaCl₂, 0.6 mg/mL CTS, 7.2 mg/mL DG1, and 0.24 mg/mL Cur under vigorous stirring to produce DG1-NPS and DG1/Cur-NPS with small size, high encapsulation efficiency, high loading capacity, and negative zeta potential from approximately −20 to 30 mV. Data from scanning electron microscopy, Fourier-transform infrared spectrometry, and differential scanning calorimetry analyses showed no chemical reaction between DG1, Cur, and the polymers; only physical mixing. Moreover, the drug was loaded in the amorphous phase within the nanoparticle matrix. In the acute pulmonary embolism murine model, DG1-NPs enhanced the oral antithrombotic activity of DG1, but DG1/Cur-NPs did not exhibit higher antithrombotic activity than DG1-NPs. Therefore, the chitosan/alginate nanoparticles enhanced the oral antithrombotic activity of DG1, but curcumin did not further enhance this effect.