Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Preliminary characterization of a novel ?-agarase from Thalassospira profundimonas.

ABSTRACT: BACKGROUND:The objective of this study was to characterize the agarase from a newly isolated agarolytic bacterium Thalassospira profundimaris fst-13007. RESULTS:Agarase-fst was purified to homogeneity which apparent molecular weight was 66.2 kDa. Its activity was optimal at 45 °C and pH 8 and was stable at pH 5-9 or 30-50 °C. Agarase-fst required Mn(2+) for agarase activity and inhibition by Cu(2+), Fe(3+) and EDTA. Tests of hydrolysis pattern and substrate specificity, TLC analysis and mass spectrometry of the hydrolysis products revealed that it is an endo-type ?-agarase hydrolyzing agarose into neoagarobiose, neoagarotetraose and neoagarohexaose. Results of MALDI-TOF-TOF/MS indicate that it lack of homology to previously identified proteins and present conserved domain of ?-agarase. CONCLUSION:Agarase-fst from T. profundimaris fst-13007 was confirmed to be a novel endo-type ?-agarase.

SUBMITTER: Zeng C

PROVIDER: S-EPMC4947071 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Preliminary characterization of a novel β-agarase from Thalassospira profundimonas.

Zeng Cheng C Zhang Longtao L Miao Song S Zhang Yi Y Zeng Shaoxiao S Zheng Baodong B

SpringerPlus 20160715 1

<h4>Background</h4>The objective of this study was to characterize the agarase from a newly isolated agarolytic bacterium Thalassospira profundimaris fst-13007.<h4>Results</h4>Agarase-fst was purified to homogeneity which apparent molecular weight was 66.2 kDa. Its activity was optimal at 45 °C and pH 8 and was stable at pH 5-9 or 30-50 °C. Agarase-fst required Mn(2+) for agarase activity and inhibition by Cu(2+), Fe(3+) and EDTA. Tests of hydrolysis pattern and substrate specificity, TLC analys ...[more]

PMID: 27468386

Similar Datasets

Biochemical Characterization of a New β-Agarase from Cellulophaga Algicola.

Project description:Cellulophaga algicola DSM 14237, isolated from the Eastern Antarctic coastal zone, was found to be able to hydrolyze several types of polysaccharide materials. In this study, a predicted β-agarase (CaAga1) from C. algicola was heterologously expressed in Escherichia coli. The purified recombinant CaAga1 showed specific activities of 29.39, 20.20, 14.12, and 8.99 U/mg toward agarose, pure agar, and crude agars from Gracilaria lemaneiformis and Porphyra haitanensis, respectively. CaAga1 exhibited an optimal temperature and pH of 40 oC and 7, respectively. CaAga1 was stable over a wide pH range from 4 to 11. The recombinant enzyme showed an unusual thermostability, that is, it was stable at temperature below or equal to 40oC and around 70 oC, but was thermolabile at about 50 oC. With the agarose as the substrate, the Km and Vmax values for CaAga1 were 1.19 mg/mL and 36.21 U/mg, respectively. The reducing reagent (dithiothreitol) enhanced the activity of CaAga1 by more than one fold. In addition, CaAga1 was salt-tolerant given that it retained approximately 70% of the maximum activity in the presence of 2 M NaCl. The thin layer chromatography results indicated that CaAga1 is an endo-type β-agarase and efficiently hydrolyzed agarose into neoagarotetraose (NA4) and neoagarohexaose (NA6). A structural model of CaAga1 in complex with neoagarooctaose (NA8) was built by homology modeling and explained the hydrolysis pattern of CaAga1.

| S-EPMC6539560 | biostudies-literature

Overexpression and characterization of a novel thermostable β-agarase YM01-3, from marine bacterium Catenovulum agarivorans YM01(T).

Project description:Genome sequencing of Catenovulum agarivorans YM01T reveals 15 open-reading frames (ORFs) encoding various agarases. In this study, extracellular proteins of YM01T were precipitated by ammonium sulfate and separated by one-dimensional gel electrophoresis. The results of in-gel agarase activity assay and mass spectrometry analysis revealed that the protein, YM01-3, was an agarase with the most evident agarolytic activity. Agarase YM01-3, encoded by the YM01-3 gene, consisted of 420 amino acids with a calculated molecular mass of 46.9 kDa and contained a glycoside hydrolase family 16 β-agarase module followed by a RICIN superfamily in the C-terminal region. The YM01-3 gene was cloned and expressed in Escherichia coli. The recombinant agarase, YM01-3, showed optimum activity at pH 6.0 and 60 °C and had a K(m) of 3.78 mg mL⁻¹ for agarose and a Vmax of 1.14 × 10⁴ U mg⁻¹. YM01-3 hydrolyzed the β-1,4-glycosidic linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the main products. Notably, YM01-3 was stable below 50 °C and retained 13% activity after incubation at 80 °C for 1 h, characteristics much different from other agarases. The present study highlights a thermostable agarase with great potential application value in industrial production.

| S-EPMC4052312 | biostudies-literature

Purification and characterization of cold-adapted beta-agarase from an Antarctic psychrophilic strain.

Project description:An extracellular β-agarase was purified from Pseudoalteromonas sp. NJ21, a Psychrophilic agar-degrading bacterium isolated from Antarctic Prydz Bay sediments. The purified agarase (Aga21) revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of 80 kDa. The optimum pH and temperature of the agarase were 8.0 and 30 °C, respectively. However, it maintained as much as 85% of the maximum activities at 10 °C. Significant activation of the agarase was observed in the presence of Mg(2+), Mn(2+), K(+); Ca(2+), Na(+), Ba(2+), Zn(2+), Cu(2+), Co(2+), Fe(2+), Sr(2+) and EDTA inhibited the enzyme activity. The enzymatic hydrolyzed product of agar was characterized as neoagarobiose. Furthermore, this work is the first evidence of cold-adapted agarase in Antarctic psychrophilic bacteria and these results indicate the potential for the Antarctic agarase as a catalyst in medicine, food and cosmetic industries.

| S-EPMC4568857 | biostudies-literature

Cloning, expression, and characterization of a new beta-agarase from Vibrio sp. strain CN41.

Project description:A new agarase, AgaA(CN41), cloned from Vibrio sp. strain CN41, consists of 990 amino acids, with only 49% amino acid sequence identity with known ?-agarases. AgaA(CN41) belongs to the GH50 (glycoside hydrolase 50) family but yields neoagarotetraose as the end product. AgaA(CN41) was expressed and characterized.

| S-EPMC3187099 | biostudies-literature

Characterization of a novel β-agarase from Antarctic macroalgae-associated bacteria metagenomic library and anti-inflammatory activity of the enzymatic hydrolysates.

Project description:An agarase gene (aga1904) that codes a protein with 640 amino acids was obtained from the metagenomic library of macroalgae-associated bacteria collected from King George Island, Antarctica. Gene aga1904 was expressed in Escherichia coli BL21 (DE3) and recombinant Aga1904 was purified by His Bind Purification kit. The optimal temperature and pH for the activity of Aga1904 were 50°C and 6.0, respectively. Fe3+ and Cu2+ significantly inhibited the activity of Aga1904. The V max and K m values of recombinant Aga1904 were 108.70 mg/ml min and 6.51 mg/ml, respectively. The degradation products of Aga1904 against agarose substrate were mainly neoagarobiose, neoagarotetraose, and neoagarohexaose analyzed by thin layer chromatography. The cellular immunoassay of enzymatic hydrolysates was subsequently carried out, and the results showed that agaro-oligosaccharides dominated by neoagarobiose significantly inhibited key pro-inflammatory markers including, nitric oxide (NO), interleukins 6 (IL-6), and tumor necrosis factor α (TNF-α). This work provides a promising candidate for development recombinant industrial enzyme to prepare agaro-oligosaccharides, and paved up a new path for the exploitation of natural anti-inflammatory agent in the future.

| S-EPMC9478344 | biostudies-literature

Molecular cloning, expression, and functional characterization of the β-agarase AgaB-4 from Paenibacillus agarexedens.

Project description:In this study, a β-agarase gene, agaB-4, was isolated for the first time from the agar-degrading bacterium Paenibacillus agarexedens BCRC 17346 by using next-generation sequencing. agaB-4 consists of 2652 bp and encodes an 883-amino acid protein with an 18-amino acid signal peptide. agaB-4 without the signal peptide DNA was cloned and expressed in Escherichia coli BL21(DE3). His-tagged recombinant AgaB-4 (rAgaB-4) was purified from the soluble fraction of E. coli cell lysate through immobilized metal ion affinity chromatography. The optimal temperature and pH of rAgaB-4 were 55 °C and 6.0, respectively. The results of a substrate specificity test showed that rAgaB-4 could degrade agar, high-melting point agarose, and low-melting point agarose. The Vmax and Km of rAgaB-4 for low-melting point agarose were 183.45 U/mg and 3.60 mg/mL versus 874.61 U/mg and 9.29 mg/mL for high-melting point agarose, respectively. The main products of agar and agarose hydrolysis by rAgaB-4 were confirmed to be neoagarotetraose. Purified rAgaB-4 can be used in the recovery of DNA from agarose gels and has potential application in agar degradation for the production of neoagarotetraose.

| S-EPMC5874223 | biostudies-literature

Cloning, characterization, and molecular application of a beta-agarase gene from Vibrio sp. strain V134.

Project description:V134, a marine isolate of the Vibrio genus, was found to produce a new beta-agarase of the GH16 family. The relevant agarase gene agaV was cloned from V134 and conditionally expressed in Escherichia coli. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were around 40 degrees C and 7.0. AgaV was demonstrated to be useful in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at the genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for the selection of genes encoding secretion proteins from both gram-positive and gram-negative bacteria.

| S-EPMC1892855 | biostudies-literature

Expression and Characterization of a Novel Cold-Adapted and Stable β-Agarase Gene agaW1540 from the Deep-Sea Bacterium Shewanella sp. WPAGA9.

Project description:The neoagaro-oligosaccharides, degraded from agarose by agarases, are important natural substances with many bioactivities. In this study, a novel agarase gene, agaW1540, from the genome of a deep-sea bacterium Shewanella sp. WPAGA9, was expressed, and the recombinant AgaW1540 (rAgaW1540) displayed the maximum activity under the optimal pH and temperature of 7.0 and 35 °C, respectively. rAgaW1540 retained 85.4% of its maximum activity at 0 °C and retained more than 92% of its maximum activity at the temperature range of 20-40 °C and the pH range of 4.0-9.0, respectively, indicating its extensive working temperature and pH values. The activity of rAgaW1540 was dramatically suppressed by Cu2+ and Zn2+, whereas Fe2+ displayed an intensification of enzymatic activity. The Km and Vmax of rAgaW1540 for agarose degradation were 15.7 mg/mL and 23.4 U/mg, respectively. rAgaW1540 retained 94.7%, 97.9%, and 42.4% of its maximum activity after incubation at 20 °C, 25 °C, and 30 °C for 60 min, respectively. Thin-layer chromatography and ion chromatography analyses verified that rAgaW1540 is an endo-acting β-agarase that degrades agarose into neoagarotetraose and neoagarohexaose as the main products. The wide variety of working conditions and stable activity at room temperatures make rAgaW1540an appropriate bio-tool for further industrial production of neoagaro-oligosaccharides.

| S-EPMC8398281 | biostudies-literature

Biochemical characterization and key catalytic residue identification of a novel alpha-agarase with CBM2 domain.

Project description:Agarooligosaccharides have great potential in food industry because of their various bio-activities, while the limited availability and diversity of α-agarases hinder agarooligosaccharides' broader application. To overcome this limitation, a computer-assisted method was used to screen and identify novel agarases. Firstly, one novel α-agarase, AgaB, with an N-terminal CBM2 domain (the first report of this domain in agarases), was discovered. Purified agarases only exhibited activity against agarose, with optimum activity at 40℃ and pH 8.0. Analysis of hydrolysis products indicated that AgaB is an endo-type α-agarase, producing agarotetraose and agarohexaose. Secondly, AgaB truncated CBM2 showed increased Km values, suggesting that CBM2 aids in substrate binding. Thirdly, E468 and D333 are possibly catalytic amino acids, which was supported by molecular docking results and mutants. Biochemical characterization of first reported CBM2-containing agarase and catalytic mechanism study lay the foundation for the exploration and development of α-agarases in the future.

| S-EPMC10740060 | biostudies-literature

Cloning of the novel gene encoding beta-agarase C from a marine bacterium, Vibrio sp. strain PO-303, and characterization of the gene product.

Project description:The beta-agarase C gene (agaC) of a marine bacterium, Vibrio sp. strain PO-303, consisted of 1,437 bp encoding 478 amino acid residues. beta-Agarase C was identified as the first beta-agarase that cannot hydrolyze neoagarooctaose and smaller neoagarooligosaccharides and was assigned to a novel glycoside hydrolase family.

| S-EPMC1563652 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data