scholarly journals Molecular Characterization of Novel Family IV and VIII Esterases from a Compost Metagenomic Library

2021 ◽  
Vol 9 (8) ◽  
pp. 1614
Author(s):  
Jong-Eun Park ◽  
Geum-Seok Jeong ◽  
Hyun-Woo Lee ◽  
Hoon Kim
Keyword(s):  
Metagenomic Library ◽  
Industrial Applications ◽  
Native Form ◽  
Metagenome Library ◽  
The Family ◽  
Alkaline Conditions ◽  
Molecular Masses ◽  
Novel Esterase ◽  
Family Iv

Two novel esterase genes, est8L and est13L, were isolated and identified from a compost metagenomic library. The encoded Est8L and Est13L had molecular masses of 33,181 and 44,913 Da consisting of 314 and 411 amino acids, respectively, without signal peptides. Est8L showed the highest identity (32.9%) to a hyper-thermophilic carboxylesterase AFEST from Archaeoglobus fulgidus compared to other esterases reported and was classified to be a novel member of family IV esterases with conserved regions such as HGGG, DY, GXSXG, DPL, and GXIH. Est13L showed the highest identity (98.5%) to the family VIII esterase Est7K from the metagenome library. Est8L and Est13L had the highest activities for p-nitrophenyl butyrate (C4) and p-nitrophenyl caproate (C6), respectively, and Est13L showed a broad substrate specificity for p-nitrophenyl substrates. Est8L and Est13L effectively hydrolyzed glyceryl tributyrate. The optimum temperatures for activities of Est8L and Est13L were identical (40 °C), and the optimum pH values were 9.0 and 10.0, respectively. Est13L showed higher thermostability than Est8L. Sephacryl S-200 HR chromatography showed that the native form of Est8L was a dimer. Interestingly, Est13L was found to be a tetramer, contrary to other family VIII esterases reported. Est8L was inhibited by 30% isopropanol, methanol, and acetonitrile; however, Est13L was activated to 182.9% and 356.1%, respectively, by 30% isopropanol and methanol. Est8L showed enantioselectivity for the S-form, but Est13L showed no enantioselectivity. These results show that intracellular Est8L and/or Est13L are oligomeric in terms of native forms and can be used for pharmaceutical and industrial applications with organic solvents under alkaline conditions.

scholarly journals Characterization of a Novel Family IV Esterase Containing a Predicted CzcO Domain and a Family V Esterase with Broad Substrate Specificity from an Oil-Polluted Mud Flat Metagenomic Library

2021 ◽  
Vol 11 (13) ◽  
pp. 5905
Author(s):  
Jong Eun Park ◽  
Geum Seok Jeong ◽  
Hyun Woo Lee ◽  
Sung Kyum Kim ◽  
Jungho Kim ◽  
...  
Keyword(s):  
Metagenomic Library ◽  
Signal Peptides ◽  
Mud Flat ◽  
Alkaline Conditions ◽  
Novel Esterase ◽  
Butyrylcholinesterase Activity ◽  
Triton X ◽  
Fusion Type ◽  
Family Iv

Two novel esterase genes, est2L and est4L, were identified from a previously constructed metagenomic library derived from an oil-polluted mud flat sample. The encoded Est2L and Est4L were composed of 839 and 267 amino acids, respectively, without signal peptides. Est2L was a unique fusion type of protein composed of two domains: a domain of the CzcO superfamily, associated with a cationic diffusion promoter with CzcD, and a domain of the acetylesterase superfamily, belonging to family IV with conserved motifs, such as HGG, GXSAG, and GXPP. Est2L was the first fused esterase with a CzcO domain. Est4L belonged to family V with GXS, GXSMGG, and PTL motifs. Native Est2L and Est4L were found to be in dimeric and tetrameric forms, respectively. Est2L and Est4L showed the highest activities at 60 °C and 50 °C, respectively, and at a pH of 10.0. Est2L preferred short length substrates, especially p-nitrophenyl (pNP)-acetate, with moderate butyrylcholinesterase activity, whereas Est4L showed the highest activity with pNP-decanoate and had broad specificity. Significant effects were not observed in Est2L from Co2+ and Zn2+, although Est2L contains the domain CzcD. Est2L and Est4L showed high stabilities in 30% methanol and 1% Triton X-100. These enzymes could be used for a variety of applications, such as detergent and mining processing under alkaline conditions.

scholarly journals Fucoidan Characterization: Determination of Purity and Physicochemical and Chemical Properties

Marine Drugs ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. 571
Author(s):  
Ahmed Zayed ◽  
Mona El-Aasr ◽  
Abdel-Rahim S. Ibrahim ◽  
Roland Ulber
Keyword(s):  
Structural Characteristics ◽  
Chemical Properties ◽  
Industrial Applications ◽  
Chemical Structures ◽  
Seasonal Factors ◽  
Molecular Masses ◽  
Current Article ◽  
Downstream Processes

Fucoidans are marine sulfated biopolysaccharides that have heterogenous and complicated chemical structures. Various sugar monomers, glycosidic linkages, molecular masses, branching sites, and sulfate ester pattern and content are involved within their backbones. Additionally, sources, downstream processes, and geographical and seasonal factors show potential effects on fucoidan structural characteristics. These characteristics are documented to be highly related to fucoidan potential activities. Therefore, numerous chemical qualitative and quantitative determinations and structural elucidation methods are conducted to characterize fucoidans regarding their physicochemical and chemical features. Characterization of fucoidan polymers is considered a bottleneck for further biological and industrial applications. Consequently, the obtained results may be related to different activities, which could be improved afterward by further functional modifications. The current article highlights the different spectrometric and nonspectrometric methods applied for the characterization of native fucoidans, including degree of purity, sugar monomeric composition, sulfation pattern and content, molecular mass, and glycosidic linkages.

scholarly journals Characterization of Fluorescent and Nonfluorescent Peptide Siderophores Produced by Pseudomonas syringaeStrains and Their Potential Use in Strain Identification

2001 ◽  
Vol 67 (4) ◽  
pp. 1718-1727 ◽  
Author(s):  
Alain Bultreys ◽  
Isabelle Gheysen ◽  
Henri Maraite ◽  
Edmond de Hoffmann
Keyword(s):  
Pseudomonas Syringae ◽  
Spectral Characteristics ◽  
Mass Spectrometry Data ◽  
Strain Identification ◽  
Virulent Strains ◽  
Electrophoretic Behavior ◽  
Fluorescent Peptide ◽  
Alkaline Conditions ◽  
Molecular Masses

ABSTRACT Nonfluorescent highly virulent strains of Pseudomonas syringae pv. aptata isolated in different European countries and in Uruguay produce a nonfluorescent peptide siderophore, the production of which is iron repressed and specific to these strains. The amino acid composition of this siderophore is identical to that of the dominant fluorescent peptide siderophore produced by fluorescentP. syringae strains, and the molecular masses of the respective Fe(III) chelates are 1,177 and 1,175 atomic mass units. The unchelated nonfluorescent siderophore is converted into the fluorescent siderophore at pH 10, and colors and spectral characteristics of the unchelated siderophores and of the Fe(III)-chelates in acidic conditions are similar to those of dihydropyoverdins and pyoverdins, respectively. The nonfluorescent siderophore is used by fluorescent and nonfluorescent P. syringae strains. These results and additional mass spectrometry data strongly suggest the presence of a pyoverdin chromophore in the fluorescent siderophore and a dihydropyoverdin chromophore in the nonfluorescent siderophore, which are both ligated to a succinamide residue. When chelated, the siderophores behave differently from typical pyoverdins and dihydropyoverdins in neutral and alkaline conditions, apparently because of the ionization occurring around pH 4.5 of carboxylic acids present in β-hydroxyaspartic acid residues of the peptide chains. These differences can be detected visually by pH-dependent changes of the chelate colors and spectrophotochemically. These characteristics and the electrophoretic behavior of the unchelated and chelated siderophores offer new tools to discriminate between saprophytic fluorescent Pseudomonas species and fluorescent P. syringae and P. viridiflava strains and to distinguish between the two siderovars in P. syringae pv. aptata.

scholarly journals Overproduction, purification, crystallization and preliminary X-ray characterization of the family 46 carbohydrate-binding module (CBM46) of endo-β-1,4-glucanase B (CelB) fromBacillus halodurans

2014 ◽  
Vol 70 (6) ◽  
pp. 754-757 ◽  
Author(s):  
Immacolata Venditto ◽  
Helena Santos ◽  
Luís M. A. Ferreira ◽  
Kazuo Sakka ◽  
Carlos M. G. A. Fontes ◽  
...  
Keyword(s):  
Metal Ion ◽  
Plant Cell Wall ◽  
Bacillus Halodurans ◽  
Carbohydrate Binding ◽  
Cell Wall Polysaccharides ◽  
Native Form ◽  
Carbohydrate Binding Modules ◽  
The Family ◽  
Carbon Recycling

Plant cell-wall polysaccharides offer an abundant energy source utilized by many microorganisms, thus playing a central role in carbon recycling. Aerobic microorganisms secrete carbohydrate-active enzymes (CAZymes) that catabolize this composite structure, comprising cellulose, hemicellulose and lignin, into simple compounds such as glucose. Carbohydrate-binding modules (CBMs) enhance the efficacy of associated CAZYmes. They are organized into families based on primary-sequence homology. CBM family 46 contains more than 40 different members, but has yet to be fully characterized. Here, a recombinant derivative of the C-terminal family 46 CBM module (BhCBM46) ofBacillus haloduransendo-β-1,4-glucanase B (CelB) was overexpressed inEscherichia coliand purified by immobilized metal-ion affinity chromatography. Preliminary structural characterization was carried out onBhCBM46 crystallized in different conditions. The crystals ofBhCBM46 belonged to the tetragonal space groupI4122. Data were collected for the native form and a selenomethionine derivative to 2.46 and 2.3 Å resolution, respectively. TheBhCBM46 structure was determined by a single-wavelength anomalous dispersion experiment usingAutoSolfrom thePHENIXsuite.

Purification and partial characterization of two extracellular alkaline proteases from Streptomyces corchorusii ST36

1995 ◽  
Vol 41 (1) ◽  
pp. 99-104 ◽  
Author(s):  
Abd El-Raheem R. El-Shanshoury ◽  
Mostafa A. El-Sayed ◽  
Reda H. Sammour ◽  
Wageeh A. El-Shouny
Keyword(s):  
Ethylenediaminetetraacetic Acid ◽  
Gel Filtration ◽  
Inhibitory Effect ◽  
Partial Characterization ◽  
Alkaline Proteases ◽  
Alkaline Conditions ◽  
Molecular Masses ◽  
Proteolytic Activities ◽  
Egyptian Soil

Two distinct alkaline proteases were detected in the culture medium of Streptomyces corchorusii ST36 isolated from an Egyptian soil sample. These enzymes were purified by precipitation with chilled ethanol and gel filtration on carboxymethyl Sepharose and on Sephadex G-120. The enzymes were purified 6 and 6.5 fold to homogeneity. The purified enzymes had final specific activities on substrate of 3.6 and 3.9 U/mg. Protease 1 had a molecular mass of 31 kDa and protease 2 was composed of two subunits, of molecular masses 18.6 and 17.4 kDa. The optimal pH and temperature for catalytic activity of protease 1 were pH 11 and 70 °C, respectively, and for protease 2 they were pH 10 and 70 °C, respectively. Protease 1 was more thermostable than protease 2. Both enzymes showed substrate specificity to casein, serum albumin, and ovalbumin. Calcium, copper, and cobalt stimulated protease 2 but did not significantly affect protease 1. Mercury was the strongest inhibitor for protease 2. The proteolytic activities of both proteases were inhibited by 10 mM phenanthroline and 50 mM ethylenediaminetetraacetic acid (EDTA). The inhibitory effect of EDTA on both enzymes was reversed by the addition of 50 mM of copper, calcium, or cobalt. Both enzymes were more stable at −20 °C under alkaline conditions than under neutral or acidic conditions.Key words: Streptomyces corchorusii, alkaline proteases, partial characterization.

Characterization of XtjR8: A novel esterase with phthalate-hydrolyzing activity from a metagenomic library of lotus pond sludge

2020 ◽  
Vol 164 ◽  
pp. 1510-1518
Author(s):  
Jiarong Qiu ◽  
Haiyan Yang ◽  
Zhenzhen Yan ◽  
Yaning Shi ◽  
Dandan Zou ◽  
...  
Keyword(s):  
Metagenomic Library ◽  
Novel Esterase

scholarly journals Mechanism and Structural Insights Into a Novel Esterase, E53, Isolated From Erythrobacter longus

2022 ◽  
Vol 12 ◽  
Author(s):  
Yi Ding ◽  
Laiyin Nie ◽  
Xiao-Chen Yang ◽  
Yang Li ◽  
Ying-Yi Huo ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Diffraction Method ◽  
Industrial Applications ◽  
X Ray Diffraction ◽  
Functional Studies ◽  
Detailed Structural Analysis ◽  
Alkaline Conditions ◽  
Novel Esterase ◽  
Reaction With Water ◽  
Mutagenesis Study

Esterases are a class of enzymes that split esters into an acid and an alcohol in a chemical reaction with water, having high potential in pharmaceutical, food and biofuel industrial applications. To advance the understanding of esterases, we have identified and characterized E53, an alkalophilic esterase from a marine bacterium Erythrobacter longus. The crystal structures of wild type E53 and three variants were solved successfully using the X-ray diffraction method. Phylogenetic analysis classified E53 as a member of the family IV esterase. The enzyme showed highest activity against p-nitrophenyl butyrate substrate at pH 8.5–9.5 and 40°C. Based on the structural feature, the catalytic pocket was defined as R1 (catalytic center), R2 (pocket entrance), and R3 (end area of pocket) regions. Nine variants were generated spanning R1–R3 and thorough functional studies were performed. Detailed structural analysis and the results obtained from the mutagenesis study revealed that mutations in the R1 region could regulate the catalytic reaction in both positive and negative directions; expanding the bottleneck in R2 region has improved the enzymatic activity; and R3 region was associated with the determination of the pH pattern of E53. N166A in R3 region showed reduced activity only under alkaline conditions, and structural analysis indicated the role of N166 in stabilizing the loop by forming a hydrogen bond with L193 and G233. In summary, the systematic studies on E53 performed in this work provide structural and functional insights into alkaliphilic esterases and further our knowledge of these enzymes.

Screening and characterization of a novel esterase from a metagenomic library

2006 ◽  
Vol 45 (2) ◽  
pp. 315-323 ◽  
Author(s):  
Yun-Jung Kim ◽  
Gi-Sub Choi ◽  
Seung-Bum Kim ◽  
Gee-Sun Yoon ◽  
Yong-Sung Kim ◽  
...  
Keyword(s):  
Metagenomic Library ◽  
Novel Esterase

scholarly journals Molecular Characterization of Four Alkaline Chitinases from Three Chitinolytic Bacteria Isolated from a Mudflat

2021 ◽  
Vol 22 (23) ◽  
pp. 12822
Author(s):  
Sung Kyum Kim ◽  
Jong Eun Park ◽  
Jong Min Oh ◽  
Hoon Kim
Keyword(s):  
Amino Acids ◽  
Main Product ◽  
Glycosyl Hydrolase ◽  
Glycosyl Hydrolase Family ◽  
Chitinolytic Bacteria ◽  
Binding Domains ◽  
Alkaline Conditions ◽  
Molecular Masses ◽  
Hydrolase Family

Four chitinases were cloned and characterized from three strains isolated from a mudflat: Aeromonas sp. SK10, Aeromonas sp. SK15, and Chitinibacter sp. SK16. In SK10, three genes, Chi18A, Pro2K, and Chi19B, were found as a cluster. Chi18A and Chi19B were chitinases, and Pro2K was a metalloprotease. With combinatorial amplification of the genes and analysis of the hydrolysis patterns of substrates, Chi18A and Chi19B were found to be an endochitinase and exochitinase, respectively. Chi18A and Chi19B belonged to the glycosyl hydrolase family 18 (GH18) and GH19, with 869 and 659 amino acids, respectively. Chi18C from SK15 belonged to GH18 with 864 amino acids, and Chi18D from SK16 belonged to GH18 with 664 amino acids. These four chitinases had signal peptides and high molecular masses with one or two chitin-binding domains and, interestingly, preferred alkaline conditions. In the activity staining, their sizes were determined to be 96, 74, 95, and 73 kDa, respectively, corresponding to their expected sizes. Purified Chi18C and Chi18D after pET expression produced N,N′-diacetylchitobiose as the main product in hydrolyzing chitooligosaccharides and colloidal chitin. These results suggest that Chi18A, Chi18C, and Chi18D are endochitinases, that Chi19B is an exochitinase, and that these chitinases can be effectively used for hydrolyzing natural chitinous sources.

Prothrombin Metz : Purification and Characterization of a Variant of Human Prothrombin

1979 ◽  
Author(s):  
M Ribieto ◽  
J Elion ◽  
D Labie ◽  
F Josso
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Xa ◽  
Polyacrylamide Gel ◽  
Cleavage Pattern ◽  
Purification And Characterization ◽  
Double Immunodiffusion ◽  
The Family

For the purification of the abnormal prothrombin (Pt Metz), advantage has been taken of the existence in the family of three siblings who, being double heterozygotes for Pt Metz and a hypoprothrombinemia, have no normal Pt. Purification procedures included barium citrate adsorption and chromatography on DEAE Sephadex as for normal Pt. As opposed to some other variants (Pt Barcelona and Madrid), Pt Metz elutes as a single symetrical peak. By SDS polyacrylamide gel electrophoresis, this material is homogeneous and appears to have the same molecular weight as normal Pt. Comigration of normal and abnormal Pt in the absence of SDS, shows a double band suggesting an abnormal charge for the variant. Pt Metz exhibits an identity reaction with the control by double immunodiffusion. Upon activation by factor Xa, Pt Metz can generate amydolytic activity on Bz-Phe-Val-Arg-pNa (S2160), but only a very low clotting activity. Clear abnormalities are observed in the cleavage pattern of Pt Metz when monitored by SDS gel electrophoresis. The main feature are the accumulation of prethrombin l (Pl) and the appearance of abnormal intermediates migrating faster than Pl.

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