scholarly journals A Novel Neoagarotriose-Producing Agarase from the Marine Bacterium Gilvimarinus Agarilyticus JEA5

2020 ◽  
Author(s):  
Youngdeuk Lee ◽  
Eunyoung Jo ◽  
Yeon-Ju Lee ◽  
Hansol Choi ◽  
Tae-Yang Eom ◽  
...  
Keyword(s):  
Marine Bacterium ◽  
Industrial Applications ◽  
Biochemical Properties ◽  
Carbohydrate Binding ◽  
Ionization Mass ◽  
Coding Region ◽  
Protein Activity ◽  
Amino Acid Similarity ◽  
Chromatography Analysis ◽  
Layer Chromatography

Abstract Background The degradation of agar by bacterial agarases has many commercial and academic applications. We recently identified a novel neoagarotriose-producing β-agarase, Gaa16B, in the marine bacterium Gilvimarinus agarilyticus JEA5. This is the first report to describe neoagarotriose production from β-agarase.Results The Gaa16B agarase, which belongs to the glycoside hydrolase 16 (GH16) family of β-agarases, shows less than 70.9% amino acid similarity with previously characterized agarases. The coding region of Gaa16B is 1800 bp long, encoding 600 amino acids, and exhibits features typical of agarases belonging to the GH16 family. A recombinant Gaa16B lacking the carbohydrate binding region (rGaa16Bc) was overexpressed in Escherichia coli and purified as a maltose-binding protein (MBP) fusion protein. Activity assays revealed the optimal temperature and pH of rGaa16Bc to be 55 °C and pH 6–7, respectively, and the protein was highly stable at 55 °C for 90 min. Additionally, rGaa16Bc activity was strongly enhanced (2.3-fold) in the presence of 2.5 mM MnCl2. The Km and Vmax of rGaa16Bc for agarose were 6.4 mg/ml and 953 U/mg, respectively. Thin layer chromatography analysis revealed that rGaa16Bc can hydrolyze agarose into neoagarotetraose, neoagarotriose, and neoagarobiose, and the production of neoagarotriose by rGaa16Bc was successfully validated by high-resolution electrospray ionization mass spectrometry.Conclusion The biochemical properties of Gaa16B and the results of the hydrolytic pattern analysis suggest that Gaa16B could be useful to produce functional neoagaro-oligosaccharides for industrial applications.

scholarly journals A Novel Agarase, Gaa16B, Isolated from the Marine Bacterium Gilvimarinus agarilyticus JEA5, and the Moisturizing Effect of Its Partial Hydrolysis Products

Marine Drugs ◽  
2021 ◽  
Vol 20 (1) ◽  
pp. 2
Author(s):  
Youngdeuk Lee ◽  
Eunyoung Jo ◽  
Yeon-Ju Lee ◽  
Tae-Yang Eom ◽  
Yehui Gang ◽  
...  
Keyword(s):  
Marine Bacterium ◽  
Average Molecular Weight ◽  
Biochemical Properties ◽  
Partial Hydrolysis ◽  
Carbohydrate Binding ◽  
Thin Layer Chromatography Analysis ◽  
Amino Acid Similarity ◽  
Chromatography Analysis ◽  
Hydrolysis Products ◽  
Layer Chromatography

We recently identified a β-agarase, Gaa16B, in the marine bacterium Gilvimarinus agarilyticus JEA5. Gaa16B, belonging to the glycoside hydrolase 16 family of β-agarases, shows less than 70.9% amino acid similarity with previously characterized agarases. Recombinant Gaa16B lacking the carbohydrate-binding region (rGaa16Bc) was overexpressed in Escherichia coli and purified. Activity assays revealed the optimal temperature and pH of rGaa16Bc to be 55 ∘C and pH 6–7, respectively, and the protein was highly stable at 55 ∘C for 90 min. Additionally, rGaa16Bc activity was strongly enhanced (2.3-fold) in the presence of 2.5 mM MnCl2. The Km and Vmax of rGaa16Bc for agarose were 6.4 mg/mL and 953 U/mg, respectively. Thin-layer chromatography analysis revealed that rGaa16Bc can hydrolyze agarose into neoagarotetraose and neoagarobiose. Partial hydrolysis products (PHPs) of rGaa16Bc had an average molecular weight of 88–102 kDa and exhibited > 60% hyaluronidase inhibition activity at a concentration of 1 mg/mL, whereas the completely hydrolyzed product (CHP) showed no hyaluronidase at the same concentration. The biochemical properties of Gaa16B suggest that it could be useful for producing functional neoagaro-oligosaccharides. Additionally, the PHP of rGaa16Bc may be useful in promoting its utilization, which is limited due to the gel strength of agar.

scholarly journals Characterization of Properties and Transglycosylation Abilities of Recombinant α-Galactosidase from Cold-Adapted Marine Bacterium Pseudoalteromonas KMM 701 and Its C494N and D451A Mutants

Marine Drugs ◽  
2018 ◽  
Vol 16 (10) ◽  
pp. 349 ◽  
Author(s):  
Irina Bakunina ◽  
Lubov Slepchenko ◽  
Stanislav Anastyuk ◽  
Vladimir Isakov ◽  
Galina Likhatskaya ◽  
...  
Keyword(s):  
Active Center ◽  
Marine Bacterium ◽  
Three Dimensional ◽  
Resonance Spectroscopy ◽  
Ionization Mass ◽  
Amino Acid Residues ◽  
Cold Adapted ◽  
Important Amino Acid ◽  
Characterization Of Properties ◽  
Layer Chromatography

A novel wild-type recombinant cold-active α-d-galactosidase (α-PsGal) from the cold-adapted marine bacterium Pseudoalteromonas sp. KMM 701, and its mutants D451A and C494N, were studied in terms of their structural, physicochemical, and catalytic properties. Homology models of the three-dimensional α-PsGal structure, its active center, and complexes with D-galactose were constructed for identification of functionally important amino acid residues in the active site of the enzyme, using the crystal structure of the α-galactosidase from Lactobacillus acidophilus as a template. The circular dichroism spectra of the wild α-PsGal and mutant C494N were approximately identical. The C494N mutation decreased the efficiency of retaining the affinity of the enzyme to standard p-nitrophenyl-α-galactopiranoside (pNP-α-Gal). Thin-layer chromatography, matrix-assisted laser desorption/ionization mass spectrometry, and nuclear magnetic resonance spectroscopy methods were used to identify transglycosylation products in reaction mixtures. α-PsGal possessed a narrow acceptor specificity. Fructose, xylose, fucose, and glucose were inactive as acceptors in the transglycosylation reaction. α-PsGal synthesized -α(1→6)- and -α(1→4)-linked galactobiosides from melibiose as well as -α(1→6)- and -α(1→3)-linked p-nitrophenyl-digalactosides (Gal2-pNP) from pNP-α-Gal. The D451A mutation in the active center completely inactivated the enzyme. However, the substitution of C494N discontinued the Gal-α(1→3)-Gal-pNP synthesis and increased the Gal-α(1→4)-Gal yield compared to Gal-α(1→6)-Gal-pNP.

scholarly journals Characterization of a Hyaluronic Acid Utilization Locus and Identification of Two Hyaluronate Lyases in a Marine Bacterium Vibrio alginolyticus LWW-9

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiaoyi Wang ◽  
Ziwei Wei ◽  
Hao Wu ◽  
Yujiao Li ◽  
Feng Han ◽  
...  
Keyword(s):  
Hyaluronic Acid ◽  
Pathogenic Bacteria ◽  
Marine Bacterium ◽  
Opportunistic Pathogen ◽  
Bioinformatic Analysis ◽  
Industrial Applications ◽  
Biochemical Properties ◽  
Vibrio Alginolyticus ◽  
Host Cells ◽  
Marine Animals

Hyaluronic acid (HA) is a negatively charged and linear polysaccharide existing in the tissues and body fluids of all vertebrates. Some pathogenic bacteria target hyaluronic acid for adhesion and/or infection to host cells. Vibrio alginolyticus is an opportunistic pathogen related to infections of humans and marine animals, and the hyaluronic acid-degrading potential of Vibrio spp. has been well-demonstrated. However, little is known about how Vibrio spp. utilize hyaluronic acid. In this study, a marine bacterium V. alginolyticus LWW-9 capable of degrading hyaluronic acid has been isolated. Genetic and bioinformatic analysis showed that V. alginolyticus LWW-9 harbors a gene cluster involved in the degradation, transport, and metabolism of hyaluronic acid. Two novel PL8 family hyaluronate lyases, VaHly8A and VaHly8B, are the key enzymes for the degradation of hyaluronic acid. VaHly8A and VaHly8B have distinct biochemical properties, reflecting the adaptation of the strain to the changing parameters of the aquatic habitats and hosts. Based on genomic and functional analysis, we propose a model for the complete degradation of hyaluronic acid by V. alginolyticus LWW-9. Overall, our study expands our knowledge of the HA utilization paradigm within the Proteobacteria, and the two novel hyaluronate lyases are excellent candidates for industrial applications.

ANTI-CANCER COMPOUNDS ISOLATED FROM SOFT CORAL SINULARIA CRUCIATA - ALCYONIIDAE

2012 ◽  
pp. 84-89
Author(s):  
Quoc Hung Vo ◽  
Nguyen Phuong Nhi Doan ◽  
Dinh Quynh Phu Nguyen ◽  
Thi Dieu Tram Ho ◽  
Thi Hoai Nguyen
Keyword(s):  
Mass Spectrometry ◽  
Soft Coral ◽  
Normal Phase ◽  
Ionization Mass ◽  
Medical Field ◽  
Bioactive Substances ◽  
Anti Cancer ◽  
The World ◽  
Pure Compounds ◽  
Layer Chromatography

Objectives: Nowadays, bioactive substances isolated from marine organisms which are abundant and varied in Vietnamese sea attracted more and more the attention of scientists in the world and Vietnam as well. We have studied on soft coral Sinularia cruciata – Alcyoniidae, which has never been studied in Vietnam before, to find substances which are useful in medical field, especially in anti-cancer therapy. Materials and method: Specimens of soft coral Sinularia cruciata were collected from Con Co, Quang Tri province in May 2011. Pure compounds were isolated by using Thin Layer Chromatography; Column Chromatography normal phase and inverse phase; Shephadex LH 20. Structures of them were determined by spectral data of Nuclear Magnetic Resonance (NMR), Electrospray Ionization Mass Spectrometry (ESI-MS). Results & Conclusion: Structures of 4 compounds were identified: (1) 5.8-epidioxycholest-6-en-3-ol (2) Cholesterol (3) 1-O-hexadecyl-glycerol (Chimyl alcohol) (4) Glycerol 1-O-octadecyl ether (Batyl alcohol). The substance (1) was demonstrated to have strong anti-cancer effects in previous study. Key words Sinularia cruciata, Alcyoniidae, 5,8-epidioxycholest-6-en-3-ol, soft coral, cancer.

scholarly journals Abnormal physiological properties and altered cell wall composition in Streptococcus pneumoniae grown in the presence of clavulanic acid.

1997 ◽  
Vol 41 (3) ◽  
pp. 504-510 ◽  
Author(s):  
A Severin ◽  
E Severina ◽  
A Tomasz
Keyword(s):  
Streptococcus Pneumoniae ◽  
High Performance ◽  
Biochemical Properties ◽  
Beta Lactam ◽  
Chromatography Analysis ◽  
Peptide Composition ◽  
Increased Sensitivity ◽  
Altered Cell ◽  
Quantitative Changes

Subinhibitory concentrations of clavulanate caused premature induction of stationary-phase autolysis, sensitization to lysozyme, and reductions in the MICs of deoxycholate and penicillin for Streptococcus pneumoniae. In the range of clavulanate concentrations producing these effects, this beta-lactam compound was selectively bound to PBP 3. Cell walls isolated from pneumococci grown in the presence of clavulanate showed increased sensitivity to the hydrolytic action of purified pneumococcal autolysin in vitro. High-performance liquid chromatography analysis of the peptidoglycan isolated from the clavulanate-grown cells showed major qualitative and quantitative changes in stem peptide composition, the most striking feature of which was the accumulation of peptide species carrying intact D-alanyl-D-alanine residues at the carboxy termini. The altered biological and biochemical properties of the clavulanate-grown pneumococci appear to be the consequences of suppressed D,D-carboxypeptidase activity.

A High-Throughput Liposome Substrate Assay with Automated Lipid Extraction Process for PI 3-Kinase

2008 ◽  
Vol 13 (9) ◽  
pp. 906-911 ◽  
Author(s):  
Trupti Lingaraj ◽  
John Donovan ◽  
Zhi Li ◽  
Ping Li ◽  
Amanda Doucette ◽  
...  
Keyword(s):  
High Throughput ◽  
Lipid Extraction ◽  
Extraction Process ◽  
Enzyme Mechanism ◽  
Class I ◽  
Lipid Kinase ◽  
Chromatography Analysis ◽  
Regulate Cell Growth ◽  
Lipid Kinases ◽  
Layer Chromatography

The signaling pathways involving lipid kinase class I phosphatidylinositol 3-kinases (PI 3-kinases) regulate cell growth, proliferation, and survival. Class I PI 3-kinases catalyze the conversion of PI (4,5)P2 to PI (3,4,5)P3, which acts as a lipid second messenger to activate mitogenic signaling cascades. Recently, p110α, a class IA PI 3-kinase, was found to be mutated frequently in many human cancers. Therefore, it is increasingly studied as an anticancer drug target. Traditionally, PI 3-kinase activities have been studied using liposome substrates. This method, however, is hampered significantly by the labor-intensive manual lipid extraction followed by a low-throughput thin-layer chromatography analysis. The authors describe a high-throughput liposome substrate-based assay based on an automated lipid extraction method that allows them to study PI 3-kinase enzyme mechanism and quantitatively measure inhibitor activity using liposome substrates in a high-throughput mode. This improved assay format can easily be extended to study other classes of phosphoinositide lipid kinases. ( Journal of Biomolecular Screening 2008:906-911)

scholarly journals Identification and Quantification of an Adulterant in a Dietary Supplement Marketed for Sexual Enhancement

2018 ◽  
Vol 1 (4) ◽  
pp. 25-33 ◽  
Author(s):  
Flavia Redko ◽  
Sabrina Flor ◽  
Silvia Lucangioli ◽  
Jerónimo Ulloa ◽  
Rafael Ricco ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Performance ◽  
Spectroscopic Techniques ◽  
Easy Method ◽  
Isolation And Purification ◽  
Chromatography Analysis ◽  
Pharmaceutical Ingredients ◽  
Emerging Risk ◽  
The Mean ◽  
Layer Chromatography

In recent years, the consumption of dietary supplements (DS) has increased worldwide. In Argentina, approximately 14 million DS units were sold between 2015 and 2017. The adulteration of DS with active pharmaceutical ingredients or their analogues has been reported. This represents an alarming emerging risk to public health. The aim of this work was to detect the possible adulteration of a DS marketed in Argentina for the treatment of erectile dysfunction. Initially, thin layer chromatography analysis of the DS capsules content suggested the presence of a major compound. For the isolation and purification of this compound, an easy method consisted of a liquid-liquid extraction (water/CH2Cl2) followed by re-crystallisation from ethanol, is reported. Spectroscopic techniques such as mono- and bidimensional nuclear magnetic resonance, Fourier transform infrared spectroscopy and mass spectrometry allowed its identification as tadalafil. A rapid and reliable method was developed for the quantification of tadalafil in this DS by high performance liquid chromatography-mass spectrometry (HPLC-MS/MS). The mean content of tadalafil per capsule was 21.2 mg which represents a slightly higher value than that found in approved products in Argentina (5 or 20 mg per tablet). In addition, an undeclared alga was identified in the DS by microscopic techniques.

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