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.

Directed preparation, structure-activity relationship and applications of alginate oligosaccharides with specific structures: A systematic review.

The alginate oligosaccharides (AOS) possess versatile activities (such as antioxidant, anti-inflammatory, antitumor, and immune-regulatory activities) and have been the research topic in marine bioresource utilization fields. The degree of polymerization (DP) and the ß-D-mannuronic acid (M)/α-L-guluronic acid (G)-units ratio strongly affect the functionality of AOS. Therefore, directed preparation of AOS with specific structures is essential for expanding the applications of alginate polysaccharides and has been the research topic in the marine bioresource field. Alginate lyases could efficiently degrade alginate and specifically produce AOS with specific structures. Therefore, enzymatic preparation of AOS with specific structures has drawn increasing attention. Herein, we systematically summarized the current research progress on the structure-function relation of AOS and focuses on the application of the enzymatic properties of alginate lyase to the specific preparation of various types of AOS. At the same time, current challenges and opportunities for AOS applications are presented to guide and improve the preparation and application of AOS in the future.

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.

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.

Antitumor effect and molecular mechanism of fucoidan in NSCLC.

BACKGROUND: Fucoidan, a water-soluble polysaccharide, exerts anticoagulant and antiviral functions. It was recently reported that fucoidan also exerts an antitumor function. Lung cancer is one of the most common cancers in the world. The aim of this study was to investigate anti-tumor,apoptosis and anti-metastasis effects of fucoidan in both cell-based assays and mouse xenograft model, as well as to clarify possible role of m-TOR pathway in the protection. METHODS: In vitro: Different concentrations of fucoidan were given to act on non-small cell lung cancer (NSCLC) cell lines A549 and H1650. The effects of fucoidan on cell proliferation were observed by detecting cyclin expression levels, CCK8 and EDU experiments and cloning experiments. The apoptotic level was detected by flow cytometry and the apoptotic protein level was detected by Westernblot. By detecting the expression of adhesion molecules, the expression of matrix metalloproteinase (MMP) family, and Transwell cell invasion and migration experiment, the effect of fucoidan on cell adhesion, invasion and migration was observed. Meanwhile the effect of fucoidan on angiogenesis was observed by detecting the expression of vascular endothelial growth factor (VEGF). In vivo experiment: An animal model of NSCLC cell mouse subcutaneous xenograft tumor was established to analyze the correlation between the consumption of fucoidan and the size and volume of xenograft tumor through gross observation. Through immunohistochemical staining and immunofluorescence double staining, ki67 and cell adhesion molecules (E-cadherin, N-cadherin and CD31) and VEGF-A in the tumor were detected, and the correlation between the amount of fucoidan and the above indexes was analyzed. RESULTS: Fucoidan inhibited the proliferation and angiogenesis of NSCLC cells via the mTOR pathway and promoted their apoptosis by increasing the Bax/Bcl-2 ratio. Not only that, fucoidan inhibited NSCLC cell invasion via epithelial-mesenchymal transformation (EMT). The mice fed fucoidan exhibited significant reductions in tumor volumes and weights. These indicators (Ki67, VEGF-A,N-cadherin) were decreased and E-cadherin expression was up-regulated in A549 mice that treated with fucoidan. The results showed that fucoidan inhibited tumor proliferation in vivo by affecting the expression of related proteins. CONCLUSION: Fucoidan conveys antitumor effects and our results represent an ideal therapeutic agent for NSCLC.

Anti-inflammatory activity and structural identification of a sulfated polysaccharide CLGP4 from Caulerpa lentillifera.

In the present study, the in vitro anti-inflammatory activity of four purified polysaccharides (CLGP1, CLGP2, CLGP3 and CLGP4) extracted from edible green algae Caulerpa lentillifera was evaluated. As a result, CLGP4 exhibited more effectively inhibitory effect on LPS-induced HT29 cells, including reducing the production of IL-1ß, TNF-α, SIgA and mucin2, and decreasing the expression of IL-1ß and TNF-α. According to the results, CLGP4 showed a better anti-inflammatory effect, might highly related to the presence of sulfate groups. Furthermore, the structure of CLGP4 was analyzed by methylation analysis, GC-MS and NMR spectroscopy. It was found that CLGP4 was a novel xylogalactomanan consisting of ß-(1 â†’ 4)-Manp, →2,4)Manp(1→, ß-(1 â†’ 2)-Manp, ß-(1 â†’ 3)-Galp, ß-(1 â†’ 4)-Xylp, terminal ß-Galp and terminal ß-Xylp residues. Additionally, the sulfate groups were located on C-3 of â†’4)Xylp(1→, C-6 of â†’3)Galp(1→ and C-3 of â†’2)Manp(1→. These results could enlarge the potential application of CLGP4 as functional ingredient to attenuate inflammation.

Purification, structural features and immunostimulatory activity of novel polysaccharides from Caulerpa lentillifera.

In this study, four purified fractions (CLGP1, CLGP2, CLGP3 and CLGP4) were prepared from green seaweed Caulerpa lentillifera. They were identified to be a novel kind of xylogalactomanans, differed in molecular weight, monosaccharide composition, and the content of uronic acids and sulfate groups, leading to various ζ-potential, ultrastructure and immunostimulatory activity. Especially, CLGP4 was quite different from the others, as it was found to be a homogeneous heteropolysaccharide composed of Xyl, Man and Gal in a percentage ratio of 1.00:2.15:2.40 with 3877.8kDa. Moreover, CLGP4 contained minor uronic acids (2.37%±0.94%) and the highest sulfate content (21.26%±1.22%). These differences in structural features had an effect on the ζ-potential and ultrastructure of CLGP4, showing rod-, rubble- and ellipsoid-shaped particles with largest negatively charge. In vitro immunostimulatory activity evaluation revealed that all the four fractions significantly stimulated macrophages, but CLGP4 showed more potent immunostimulatory activity due to its stronger function on promoting proliferation of macrophages, enhancing phagocytosis, NO production and acid phosphatase activity in macrophages. Therefore, CLGP4 could be explored as a natural immunomodulator. These results would help a fully exploition of Caulerpa lentillifera polysaccharides recognized as health-improving ingredients in functional foods.

[Expression and characterization of a novel ω-transaminase from Burkholderia phytofirmans PsJN].

Production of chiral amines and unnatural amino-acid using ω-transaminase can be achieved by kinetic resolution and asymmetric synthesis, thus ω-transaminase is of great importance in the synthesis of pharmaceutical intermediates. By genomic data mining, a putative ω-transaminase gene hbp was found in Burkholderia phytofirmans PsJN. The gene was cloned and over-expressed in Escherichia coli BL21 (DE3). The recombinant enzyme (HBP) was purified by Ni-NTA column and its catalytic properties and substrate profile were studied. HBP showed high relative activity (33.80 U/mg) and enantioselectivity toward ß-phenylalanine (ß-Phe). The optimal reaction temperature and pH were 40 ℃ and 8.0-8.5, respectively. We also established a simpler and more effective method to detect the deamination reaction of ß-Phe by UV absorption method using microplate reader, and demonstrated the thermodynamic property of this reaction. The substrate profiling showed that HBP was specific to ß-Phe and its derivatives as the amino donor. HBP catalyzed the resolution of rac-ß-Phe and its derivatives, the products (R)-amino acids were obtained with about 50% conversions and 99% ee.

Characterization of (R)-selective amine transaminases identified by in silico motif sequence blast.

Compared to (S)-selective amine transaminase ((S)-AT), the (R)-selective counterpart ((R)-AT) has been less studied. As such, a simplified "Motif Sequence Blast" search (Höhne et al. Nat Chem Biol 6:807-813, 2010) was carried out to identify new (R)-ATs from the protein databases. The combined conserved sequence motifs of (R)-ATs based on the previous in silico method of predicting (R)-selective amine transaminase were used as the template sequence for BLASTP search at default settings in NCBI, and six candidate sequences were identified. These putative (R)-AT genes were synthesized and overexpressed in Escherichia coli. Among them, five new (R)-ATs were expressed as soluble protein and showed unusual substrate specificity and high stereoselectivity. Furthermore, several unnatural amino acids, such as D-alanine, D-2-aminobutyric acid, and D-norvaline, were synthesized via the (R)-AT-catalyzed amino transfer reaction to the corresponding keto acids. Optically pure (S)-amines were also obtained by kinetic resolution of racemic amines catalyzed with these new (R)-ATs. Therefore, the Motif Sequence Blast search offers a quick and effective method for in silico identification of new (R)-ATs, and the newly identified (R)-ATs are attractive additions to the toolbox of (R)-ATs for further study and industrial application.