scholarly journals Functional Characterization of a New GH107 Endo-α-(1,4)-Fucoidanase from the Marine Bacterium Formosa haliotis

Marine Drugs ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. 562
Author(s):  
Marlene Vuillemin ◽  
Artem S. Silchenko ◽  
Hang Thi Thuy Cao ◽  
Maxim S. Kokoulin ◽  
Vo Thi Dieu Trang ◽  
...  
Keyword(s):  
Marine Bacterium ◽  
Divalent Cations ◽  
Recombinant Expression ◽  
Biological Activities ◽  
Functional Characterization ◽  
Maximal Activity ◽  
Brown Macroalgae ◽  
Pharmaceutical Uses ◽  
Enzymatic Depolymerization

Fucoidans from brown macroalgae are sulfated fucose-rich polysaccharides, that have several beneficial biological activities, including anti-inflammatory and anti-tumor effects. Controlled enzymatic depolymerization of the fucoidan backbone can help produce homogeneous, defined fucoidan products for structure-function research and pharmaceutical uses. However, only a few endo-fucoidanases have been described. This article reports the genome-based discovery, recombinant expression in Escherichia coli, stabilization, and functional characterization of a new bacterial endo-α-(1,4)-fucoidanase, Fhf1, from Formosa haliotis. Fhf1 catalyzes the cleavage of α-(1,4)-glycosidic linkages in fucoidans built of alternating α-(1,3)-/α-(1,4)-linked l-fucopyranosyl sulfated at C2. The native Fhf1 is 1120 amino acids long and belongs to glycoside hydrolase (GH) family 107. Deletion of the signal peptide and a 470 amino acid long C-terminal stretch led to the recombinant expression of a robust, minimized enzyme, Fhf1Δ470 (71 kDa). Fhf1Δ470 has optimal activity at pH 8, 37–40 °C, can tolerate up to 500 mM NaCl, and requires the presence of divalent cations, either Ca2+, Mn2+, Zn2+ or Ni2+, for maximal activity. This new enzyme has the potential to serve the need for controlled enzymatic fucoidan depolymerization to produce bioactive sulfated fucoidan oligomers.

scholarly journals Purification and Characterization of A New Cold-Adapted and Thermo-Tolerant Chitosanase from Marine Bacterium Pseudoalteromonas sp. SY39

Molecules ◽  
2019 ◽  
Vol 24 (1) ◽  
pp. 183 ◽  
Author(s):  
Yu Zhou ◽  
Xuehong Chen ◽  
Xiao Li ◽  
Yantao Han ◽  
Yanan Wang ◽  
...  
Keyword(s):  
Enzymatic Degradation ◽  
Marine Bacterium ◽  
Degradation Products ◽  
Biological Activities ◽  
Apparent Molecular Weight ◽  
Maximal Activity ◽  
Purification And Characterization ◽  
Cold Adapted ◽  
Total Product

Chitosanases play an important role in chitosan degradation, forming enzymatic degradation products with several biological activities. Although many chitosanases have been discovered and studied, the enzymes with special characteristics are still rather rare. In this study, a new chitosanase, CsnM, with an apparent molecular weight of 28 kDa was purified from the marine bacterium Pseudoalteromonas sp. SY39. CsnM is a cold-adapted enzyme, which shows highest activity at 40 °C and exhibits 30.6% and 49.4% of its maximal activity at 10 and 15 °C, respectively. CsnM is also a thermo-tolerant enzyme that recovers 95.2%, 89.1% and 88.1% of its initial activity after boiling for 5, 10 and 20 min, respectively. Additionally, CsnM is an endo-type chitosanase that yields chitodisaccharide as the main product (69.9% of the total product). It’s cold-adaptation, thermo-tolerance and high chitodisaccharide yield make CsnM a superior candidate for biotechnological application to produce chitooligosaccharides.

scholarly journals Characterization of a Cytotoxic Factor in Culture Filtrates of Serratia marcescens

2002 ◽  
Vol 70 (3) ◽  
pp. 1121-1128 ◽  
Author(s):  
Kent B. Marty ◽  
Christopher L. Williams ◽  
Linda J. Guynn ◽  
Michael J. Benedik ◽  
Steven R. Blanke
Keyword(s):  
Cytotoxic Activity ◽  
Serratia Marcescens ◽  
Mammalian Cells ◽  
Divalent Cations ◽  
Recombinant Expression ◽  
Cytotoxic Factor ◽  
Culture Filtrates ◽  
Type Strains

ABSTRACT Serratia marcescens culture filtrates have been reported to be cytotoxic to mammalian cells. Using biochemical and genetic approaches, we have identified a major source of this cytotoxic activity. Both heat and protease treatments abrogated the cytotoxicity of S. marcescens culture filtrates towards HeLa cells, suggesting the involvement of one or more protein factors. A screen for in vitro cytotoxic activity revealed that S. marcescens mutant strains that are deficient in production of a 56-kDa metalloprotease are significantly less cytotoxic to mammalian cells. Cytotoxicity was significantly reduced when culture filtrates prepared from wild-type strains were pretreated with either EDTA or 1,10-phenanthroline, which are potent inhibitors of the 56-kDa metalloprotease. Furthermore, cytotoxic activity was restored when the same culture filtrates were incubated with zinc divalent cations, which are essential for enzymatic activity of the 56-kDa metalloprotease. Finally, recombinant expression of the S. marcescens 56-kDa metalloprotease conferred a cytotoxic phenotype on the culture filtrates of a nonpathogenic Escherichia coli strain. Collectively, these data suggest that the 56-kDa metalloprotease contributes significantly to the in vitro cytotoxic activity commonly observed in S. marcescens culture filtrates.

Characterization of transport mechanisms and determinants critical for Na+-dependent Pisymport of the PiT family paralogs human PiT1 and PiT2

2006 ◽  
Vol 291 (6) ◽  
pp. C1377-C1387 ◽  
Author(s):  
Pernille Bøttger ◽  
Susanne E. Hede ◽  
Morten Grunnet ◽  
Boy Høyer ◽  
Dan A. Klærke ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Divalent Cations ◽  
Maximal Activity ◽  
Xenopus Laevis Oocytes ◽  
Transport Function ◽  
Knockout Mutant ◽  
Uptake Medium ◽  
The Impact ◽  
First Time

The general phosphate need in mammalian cells is accommodated by members of the Pitransport (PiT) family ( SLC20), which use either Na+or H+to mediate inorganic phosphate (Pi) symport. The mammalian PiT paralogs PiT1 and PiT2 are Na+-dependent Pi(NaPi) transporters and are exploited by a group of retroviruses for cell entry. Human PiT1 and PiT2 were characterized by expression in Xenopus laevis oocytes with32Pias a traceable Pisource. For PiT1, the Michaelis-Menten constant for Piwas determined as 322.5 ± 124.5 μM. PiT2 was analyzed for the first time and showed positive cooperativity in Piuptake with a half-maximal activity constant for Piof 163.5 ± 39.8 μM. PiT1- and PiT2-mediated Na+-dependent Piuptake functions were not significantly affected by acidic and alkaline pH and displayed similar Na+dependency patterns. However, only PiT2 was capable of Na+-independent Pitransport at acidic pH. Study of the impact of divalent cations Ca2+and Mg2+revealed that Ca2+was important, but not critical, for NaPitransport function of PiT proteins. To gain insight into the NaPicotransport function, we analyzed PiT2 and a PiT2 Pitransport knockout mutant using22Na+as a traceable Na+source. Na+was transported by PiT2 even without Piin the uptake medium and also when Pitransport function was knocked out. This is the first time decoupling of Pifrom Na+transport has been demonstrated for a PiT family member. Moreover, the results imply that putative transmembrane amino acids E55and E575are responsible for linking Piimport to Na+transport in PiT2.

scholarly journals The Quest for Phenolic Compounds from Macroalgae: A Review of Extraction and Identification Methodologies

Biomolecules ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 847 ◽  
Author(s):  
Sónia A. O. Santos ◽  
Rafael Félix ◽  
Adriana C. S. Pais ◽  
Sílvia M. Rocha ◽  
Armando J. D. Silvestre
Keyword(s):  
Phenolic Compounds ◽  
Biological Activities ◽  
Mass Spectrometry Data ◽  
Brown Macroalgae ◽  
Isolation And Characterization ◽  
Analytical Strategies ◽  
Critical Revision ◽  
Identification Techniques ◽  
First Time

The current interest of the scientific community for the exploitation of high-value compounds from macroalgae is related to the increasing knowledge of their biological activities and health benefits. Macroalgae phenolic compounds, particularly phlorotannins, have gained particular attention due to their specific bioactivities, including antioxidant, antiproliferative, or antidiabetic. Notwithstanding, the characterization of macroalgae phenolic compounds is a multi-step task, with high challenges associated with their isolation and characterization, due to the highly complex and polysaccharide-rich matrix of macroalgae. Therefore, this fraction is far from being fully explored. In fact, a critical revision of the extraction and characterization methodologies already used in the analysis of phenolic compounds from macroalgae is lacking in the literature, and it is of uttermost importance to compile validated methodologies and discourage misleading practices. The aim of this review is to discuss the state-of-the-art of phenolic compounds already identified in green, red, and brown macroalgae, reviewing their structural classification, as well as critically discussing extraction methodologies, chromatographic separation techniques, and the analytical strategies for their characterization, including information about structural identification techniques and key spectroscopic profiles. For the first time, mass spectrometry data of phlorotannins, a chemical family quite exclusive of macroalgae, is compiled and discussed.

Zebrafish tyrosylprotein sulfotransferase: molecular cloning, expression, and functional characterization

2004 ◽  
Vol 82 (2) ◽  
pp. 295-303 ◽  
Author(s):  
Emi Mishiro ◽  
Ming-Yih Liu ◽  
Yoichi Sakakibara ◽  
Masahito Suiko ◽  
Ming-Cheh Liu
Keyword(s):  
Molecular Cloning ◽  
Divalent Cations ◽  
Functional Characterization ◽  
The Other ◽  
Inhibitory Effects ◽  
Amino Acid Sequence Level ◽  
Ph Optimum ◽  
Rapid Amplification ◽  
High Degree

By employing the reverse transcriptase – polymerase chain reaction technique in conjunction with 3' rapid amplification of cDNA ends, a full-length cDNA encoding a zebrafish (Danio rerio) tyrosylprotein sulfotransferase (TPST) was cloned and sequenced. Sequence analysis revealed that this zebrafish TPST is, at the amino acid sequence level, 66% and 60% identical to the human and mouse TPST-1 and TPST-2, respectively. The recombinant form of the zebrafish TPST, expressed in COS-7 cells, exhibited a pH optimum at 5.75. Manganese appeared to exert a stimulatory effect on the zebrafish TPST. The activity of the enzyme determined in the presence of 20 mM MnCl2 was more than 2.5 times that determined in the absence of MnCl2. Of the other nine divalent metal cations tested at a 10 mM concentration, Co2+ also showed a considerable stimulatory effect, while Ca2+, Pb2+, and Cd2+ exerted some inhibitory effects. The other four divalent cations, Fe2+, Cu2+, Zn2+, and Hg2+, inhibited completely the sulfating activity of the zebrafish TPST. Using the wild-type and mutated P-selectin glycoprotein ligand-1 N-terminal peptides as substrates, the zebrafish TPST was shown to exhibit a high degree of substrate specificity for the tyrosine residue on the C-terminal side of the peptide. These results constitute a first study on the cloning, expression, and characterization of a zebrafish cytosolic TPST.Key words: zebra fish, tyrosylprotein sulfotransferase, molecular cloning.

scholarly journals Isolation and Functional Characterization of Beneficial Bacteria Associated with Roots of Thysanolaena Maxima and Rhizospheric Soil Enzymatic Activities in Jhum Agriculture

2019 ◽  
Vol 7 (2) ◽  
pp. 189-200
Author(s):  
Juri Deka ◽  
Dwipendra Thakuria ◽  
Alarisa Khyllep ◽  
Giasuddin Ahmed
Keyword(s):  
Endophytic Bacteria ◽  
Organic Phosphorus ◽  
Biological Activities ◽  
Functional Characterization ◽  
Inorganic Phosphorus ◽  
Rhizospheric Soil ◽  
Pgp Traits ◽  
Plant Growth Promoting ◽  
Fallow Phase

The activity of amylase (AMY), arylsulphatase (ASA), β-glucosidase (GSA), dehydrogenase (DHA), acid-phosphomonoestarase (PHA) and protease (PRO) enzymes were analyzed in rhizospheric soils of broom grass, Thysanolaena maxima (TM) collected from fallow phases of 5 and 20 years Jhum cycles (F5 and F20, respectively) and their corresponding bulk soils. The activities of soil enzymes from rhizospheric soil of TMF5 were significantly higher relative to that of bulk soils and the rhizospheric soils from TMF20. The counts of rhizobacteria [0.74 ± 0.056 x 107 colony forming unit (cfu) g-1 soil] and root endophytic bacteria (0.083 ± 0.004 x 104 cfu g-1 roots) of TM from F20 fallow phase were higher compared to the counts of rhizobacteria and endophytic bacteria (0.27 ± 0.029 x 107cfu g-1 soil and 0.05 ± 0.008 x 104 cfu g-1 roots, respectively) of TM from F5 fallow phase. Altogether 63 isolates associated with TM were screened for multifaceted plant growth promoting (PGP) traits viz. production of pectinase and cellulase, IAA like substances, 1-aminocyclopropane-1-carboxylate deaminase (ACCD), N2-fixation, solubilisation of inorganic phosphorus (iP) from Ca3(PO4)2, AlPO4 and FePO4 and mineralization of organic phosphorus (Na-phytate). The PGP screening results indicated that the percent incidence of rhizobacteria and root endophytic bacteria for PGP traits was higher in F5 fallow phase as compared to F20 fallow phase. These results provided clear indication that TM plants play an important role in rejuvenating the biological activities (in terms of higher activities of enzymes in rhizospheric soils and greater population of beneficial rhizobacteria and root endophytes) in frequently burnt soils under shorter Jhum cycles.

scholarly journals Cloning and Characterization of a New Chitosanase From a Deep-Sea Bacterium Serratia sp. QD07

2021 ◽  
Vol 12 ◽  
Author(s):  
Qiuling Zheng ◽  
Xiangjun Meng ◽  
Mingyang Cheng ◽  
Yanfeng Li ◽  
Yuanpeng Liu ◽  
...  
Keyword(s):  
Deep Sea ◽  
Biological Activities ◽  
Maximal Activity ◽  
Industrial Applications ◽  
Potential Candidate ◽  
Reaction Products ◽  
Industrial Manufacture ◽  
Encoding Gene ◽  
Ph Range

Chitosanase is a significant chitosan-degrading enzyme involved in industrial applications, which forms chitooligosaccharides (COS) as reaction products that are known to have various biological activities. In this study, the gene csnS was cloned from a deep-sea bacterium Serratia sp. QD07, as well as over-expressed in Escherichia coli, which is a new chitosanase encoding gene. The recombinant strain was cultured in a 5 L fermenter, which yielded 324 U/mL chitosanases. After purification, CsnS is a cold-adapted enzyme with the highest activity at 60°C, showing 37.5% of the maximal activity at 0°C and 42.6% of the maximal activity at 10°C. It exhibited optimum activity at pH 5.8 and was stable at a pH range of 3.4–8.8. Additionally, CsnS exhibited an endo-type cleavage pattern and hydrolyzed chitosan polymers to yield disaccharides and trisaccharides as the primary reaction products. These results make CsnS a potential candidate for the industrial manufacture of COS.

scholarly journals Functional Characterization of Carbohydrate-Binding Modules in a New Alginate Lyase, TsAly7B, from Thalassomonas sp. LD5

Marine Drugs ◽  
2019 ◽  
Vol 18 (1) ◽  
pp. 25 ◽  
Author(s):  
Zhelun Zhang ◽  
Luyao Tang ◽  
Mengmeng Bao ◽  
Zhigang Liu ◽  
Wengong Yu ◽  
...  
Keyword(s):  
Specific Activity ◽  
Biological Activities ◽  
Functional Characterization ◽  
Alginate Lyase ◽  
Carbohydrate Binding ◽  
Enzyme Degradation ◽  
Carbohydrate Binding Modules ◽  
Catalytic Module ◽  
Alginate Lyases

Alginate lyases degrade alginate into oligosaccharides, of which the biological activities have vital roles in various fields. Some alginate lyases contain one or more carbohydrate-binding modules (CBMs), which assist the function of the catalytic modules. However, the precise function of CBMs in alginate lyases has yet to be fully elucidated. We have identified a new multi-domain alginate lyase, TsAly7B, in the marine bacterium Thalassomonas sp. LD5. This novel lyase contains an N-terminal CBM9, an internal CBM32, and a C-terminal polysaccharide lyase family 7 (PL7) catalytic module. To investigate the specific function of each of these CBMs, we expressed and characterized the full-length TsAly7B and three truncated mutants: TM1 (CBM32-PL7), TM2 (CBM9-PL7), and TM3 (PL7 catalytic module). CBM9 and CBM32 could enhance the degradation of alginate. Notably, the specific activity of TM2 was 7.6-fold higher than that of TM3. CBM32 enhanced the resistance of the catalytic module to high temperatures. In addition, a combination of CBM9 and CBM32 showed enhanced thermostability when incubated at 80 °C for 1 h. This is the first report that finds CBM9 can significantly improve the ability of enzyme degradation. Our findings provide new insight into the interrelationships of tandem CBMs and alginate lyases and other polysaccharide-degrading enzymes, which may inspire CBM fusion strategies.

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