A metagenomic approach to discover a novel β-glucosidase from bovine rumens

2017 ◽  
Vol 89 (7) ◽  
pp. 941-950 ◽  
Author(s):  
Eukote Suwan ◽  
Siriphan Arthornthurasuk ◽  
Prachumporn T. Kongsaeree
Keyword(s):  
Environmental Samples ◽  
Amino Acid Residues ◽  
Cultivation Conditions ◽  
Rumen Microorganisms ◽  
Isolation And Characterization ◽  
Screening Approach ◽  
Metagenomic Sample ◽  
Ruminant Animals ◽  
Metagenomic Approach

Abstractβ-Glucosidases play an important role in biomass degradation as they hydrolyze cellobiose to glucose in a final step of cellulolysis. In particular, ruminant animals rely onβ-glucosidases from rumen microorganisms for conversion of plant cellulosic materials into glucose. In this study, we are interested in characterization of a novelβ-glucosidase from rumen microorganisms. However, most rumen microorganisms are obligate anaerobes, which require special cultivation conditions. Presently, the metagenomic techniques, which enable isolation and characterization of microbial genes directly from environmental samples, have been applied to overcome these problems. In this study, the sequence-based screening approach was successfully applied to identify a novelβ-glucosidase gene,Br2, from a bovine rumen metagenomic sample. A 1338-bp complete coding sequence ofBr2encodes a 51-kDa GH1β-glucosidase of 445 amino acid residues with 59% sequence identity to aβ-glucosidase fromCellulosilyticum ruminicolaJCM 14822. The recombinantly expressed Br2 exhibited an optimal activity at pH 6.5 and 40°C, reflecting its rumen bacterial origin, and relatively higher catalytic efficiencies toward glucoside and fucoside substrates than other glycosides, similar to many previously reported bacterialβ-glucosidases. Our sequence-based screening approach can be applied to identify other genes of interest from environmental samples.

scholarly journals Isolation and characterization of Anoxygenic phototrophs from shrimp ponds in Thailand

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Xelimar Ramirez ◽  
Imeleta Luamanu ◽  
Ruben Michael Ceballos ◽  
Elizabeth Padilla Crespo
Keyword(s):  
Environmental Samples ◽  
Production Systems ◽  
Phenotypic Diversity ◽  
Purple Bacteria ◽  
Shrimp Farming ◽  
Post Inoculation ◽  
Shrimp Ponds ◽  
Anoxygenic Phototrophs ◽  
Isolation And Characterization

Anoxygenic phototrophic purple bacteria are ubiquitous in aquatic and terrestrial environments and demonstrate broad phenotypic diversity. Purple bacteriaderive energy from light under anaerobic conditions via anoxygenic photosynthesis, a process in which water is not the electron donor. It has been suggested that these bacteria are useful for a variety of applications, including: wastewater treatment; heavy metal remediation; nitrogen fixation; and, control of CH4 emissions. In this study, the goal was to isolate and characterize PNSB from shrimp ponds in Thailand. Surface water and sediment were collected. Enrichment cultures were prepared using Pfenning’s mineral media. As indicated by development of reddish color and turbidity, anoxygenic phototrophic growth was observed within two days of incubation. Cultures in liquid media and on solid plates exhibited a deep red or purple color ten weeks post-inoculation. Under light microscopy, enrichments consist of communities dominated by thin, elongated gram-negative cells with granules of elemental sulfur, which are characteristic of purple bacteria. Molecular methods confirm the presence of pufLM, a genetic biomarker for purple bacteria (e.g., Thiohalocapsa marina, Allochromatium vinosum, Roseovarius tolerans). Initial sequencing of key genes (i.e., pufLM) indicate that these environmental samples contain novel isolates or “geographic variants” that have not been previously described. We have developed a few pure cultures of multiple species from these environmental samples. Since shrimp farming is a key industry in southern Thailand, the characterization of the microbial communities in these ecosystems, including anoxygenic phototrophs, will provide insights into how to maintain water quality in these food production systems.

Isolation and characterization of microcystins from laboratory cultures and environmental samples ofMicrocystis aeruginosa and from an associated animal toxicosis

1995 ◽  
Vol 3 (1) ◽  
pp. 50-57 ◽  
Author(s):  
Linda A. Lawton ◽  
Christine Edwards ◽  
Kenneth A. Beattie ◽  
Stephen Pleasance ◽  
Gordon J. Dear ◽  
...  
Keyword(s):  
Environmental Samples ◽  
Isolation And Characterization ◽  
Laboratory Cultures

scholarly journals Isolation and characterization of cyanogen bromide peptides from the collagen of bovine articular cartilage

1973 ◽  
Vol 133 (4) ◽  
pp. 615-622 ◽  
Author(s):  
Kalindi Deshmukh ◽  
Marcel E. Nimni
Keyword(s):  
Articular Cartilage ◽  
Total Amino Acid ◽  
Cross Linking ◽  
Amino Acid Residues ◽  
Bovine Articular Cartilage ◽  
Isolation And Characterization ◽  
Peptide Pattern ◽  
Intact Cartilage ◽  
Native Collagen

Significant amounts of native collagen can be extracted from bovine articular cartilage after removal of the acid mucopolysaccharides by controlled proteolysis. The fraction thus solubilized upon denaturation gives rise to three identical α chains. Cleavage of these chains with CNBr generated nine peptides, all of which contain glycine as one-third of their total amino acid residues. Two of the smaller peptides CB-1 and CB-2 contain partially hydroxylated proline. A similar CNBr digest of intact cartilage also gives a series of peptides identical with those obtained from the soluble cartilage collagen. The absence of cross-linking peptides, the fact that only few β components are seen in articular cartilage collagen and the similarity in peptide pattern between the two collagen fractions investigated, suggests that this collagen is stabilized by a different cross-linking mechanism, possibly involving an association with the tissue proteoglycans.

The Isolation and Characterization of the ATPase Inhibitory Protein (TN-I) from Bovine Cardiac Muscle

1975 ◽  
Vol 53 (11) ◽  
pp. 1207-1213 ◽  
Author(s):  
L. D. Burtnick ◽  
W. D. McCubbin ◽  
C. M. Kay
Keyword(s):  
Molecular Weight ◽  
Cardiac Muscle ◽  
Calcium Binding ◽  
Sodium Dodecyl ◽  
Basic Amino Acid ◽  
Sedimentation Equilibrium ◽  
Amino Acid Residues ◽  
Inhibitory Protein ◽  
Isolation And Characterization

The inhibitory component of the troponin complex (TN-I) was purified from bovine cardiac muscle, using a combination of ion exchange and molecular exclusion chromatographies in the presence of urea. It has the ability to inhibit the Mg2+-activated ATPase (EC 3.6.1.3) of a synthetic cardiac actomyosin preparation and this inhibition is reversed by the addition of cardiac calcium binding component of troponin (TN-C). Conventional sedimentation equilibrium experiments suggest a molecular weight for cardiac TN-I of 22 900 ± 500. However, sodium dodecyl sulfate (SDS) gels indicate a molecular weight of 27 000 ± 1000. The mobility of TN-I on SDS gels may be anomalous due to the high proportion of basic amino acid residues in the protein. Cardiac TN-I and TN-C interact to form a tight complex, even in the presence of 6 M urea. The results of this study invite direct comparison with results published for rabbit skeletal TN-I.

scholarly journals The isolation and characterization of corticotropin from the pituitary gland of the ostrich Struthio camelus

1977 ◽  
Vol 165 (3) ◽  
pp. 519-523 ◽  
Author(s):  
R J Naudé ◽  
W Oelofsen
Keyword(s):  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Amino Acid Residues ◽  
Struthio Camelus ◽  
Isolation And Characterization ◽  
Isoelectric Points ◽  
Pituitary Glands ◽  
Amide Content

1. Avian corticotropin (ACTH) was purified from both fresh and aged pituitary glands of the ostrich Struthio camelus. 2. The isolation of corticotropin in pure form involved acid/acetone extraction, NaCl fractionation, CM-cellulose chromatography and Sephadex G-50 chromatography. 3. The hormone preparations from fresh and aged glands behaved as single substances on polyacrylamide-gel electrophoresis, and both preparations were found to consist of 39 amino acid residues, in identical molar proportions for the different amino acids. 4. The isoelectric points of the two hormone preparations were estimated to be in the range pH 8.3-8.7, indicating possible differences in amide content, and the N-terminal amino acid of both preparations appeared to be serine. 5. The hormone preparations from fresh and aged glands exhibited similar biological potencies (73 and 77 i.u./mg respectively), as measured by steroidogenesis in vitro. 6. Apart from possible differences in amide content, the corticotropin preparations obtained from fresh and aged glands appear to be indistinguishable.

scholarly journals Isolation and Characterization of a Novel Cold-Active, Halotolerant Endoxylanase from Echinicola rosea Sp. Nov. JL3085T

Marine Drugs ◽  
2020 ◽  
Vol 18 (5) ◽  
pp. 245
Author(s):  
Jianlong He ◽  
Le Liu ◽  
Xiaoyan Liu ◽  
Kai Tang
Keyword(s):  
Amino Acid ◽  
Marine Bacterium ◽  
Maximum Velocity ◽  
Maximum Activity ◽  
Glycoside Hydrolases ◽  
Amino Acid Residues ◽  
Xylanase Gene ◽  
Isolation And Characterization ◽  
Cold Active

We cloned a xylanase gene (xynT) from marine bacterium Echinicola rosea sp. nov. JL3085T and recombinantly expressed it in Escherichia coli BL21. This gene encoded a polypeptide with 379 amino acid residues and a molecular weight of ~43 kDa. Its amino acid sequence shared 45.3% similarity with an endoxylanase from Cellvibrio mixtus that belongs to glycoside hydrolases family 10 (GH10). The XynT showed maximum activity at 40 °C and pH 7.0, and a maximum velocity of 62 μmoL min−1 mg−1. The XynT retained its maximum activity by more than 69%, 51%, and 26% at 10 °C, 5 °C, and 0 °C, respectively. It also exhibited the highest activity of 135% in the presence of 4 M NaCl and retained 76% of its activity after 24 h incubation with 4 M NaCl. This novel xylanase, XynT, is a cold-active and halotolerant enzyme that may have promising applications in drug, food, feed, and bioremediation industries.

scholarly journals Acidianus Tailed Spindle Virus: a New Archaeal Large Tailed Spindle Virus Discovered by Culture-Independent Methods

2016 ◽  
Vol 90 (7) ◽  
pp. 3458-3468 ◽  
Author(s):  
Rebecca A. Hochstein ◽  
Maximiliano J. Amenabar ◽  
Jacob H. Munson-McGee ◽  
Eric S. Boyd ◽  
Mark J. Young
Keyword(s):  
Environmental Samples ◽  
Hot Spring ◽  
Metagenomic Data ◽  
Virus Discovery ◽  
Content Type ◽  
Isolation And Characterization ◽  
Virus Diversity ◽  
Culture Independent ◽  
Domains Of Life

ABSTRACTThe field of viral metagenomics has expanded our understanding of viral diversity from all three domains of life (Archaea,Bacteria, andEukarya). Traditionally, viral metagenomic studies provide information about viral gene content but rarely provide knowledge about virion morphology and/or cellular host identity. Here we describe a new virus,Acidianustailed spindle virus (ATSV), initially identified by bioinformatic analysis of viral metagenomic data sets from a high-temperature (80°C) acidic (pH 2) hot spring located in Yellowstone National Park, followed by more detailed characterization using only environmental samples without dependency on culturing. Characterization included the identification of the large tailed spindle virion morphology, determination of the complete 70.8-kb circular double-stranded DNA (dsDNA) viral genome content, and identification of its cellular host. Annotation of the ATSV genome revealed a potential three-domain gene product containing an N-terminal leucine-rich repeat domain, followed by a likely posttranslation regulatory region consisting of high serine and threonine content, and a C-terminal ESCRT-III domain, suggesting interplay with the host ESCRT system. The host of ATSV, which is most closely related toAcidianus hospitalis, was determined by a combination of analysis of cellular clustered regularly interspaced short palindromic repeat (CRISPR)/Cas loci and dual viral and cellular fluorescencein situhybridization (viral FISH) analysis of environmental samples and confirmed by culture-based infection studies. This work provides an expanded pathway for the discovery, isolation, and characterization of new viruses using culture-independent approaches and provides a platform for predicting and confirming virus hosts.IMPORTANCEVirus discovery and characterization have been traditionally accomplished by using culture-based methods. While a valuable approach, it is limited by the availability of culturable hosts. In this research, we report a virus-centered approach to virus discovery and characterization, linking viral metagenomic sequences to a virus particle, its sequenced genome, and its host directly in environmental samples, without using culture-dependent methods. This approach provides a pathway for the discovery, isolation, and characterization of new viruses. While this study used an acidic hot spring environment to characterize a new archaeal virus,Acidianustailed spindle virus (ATSV), the approach can be generally applied to any environment to expand knowledge of virus diversity in all three domains of life.

scholarly journals Isolation and characterization of phosphorylated bovine prolactin

1993 ◽  
Vol 296 (1) ◽  
pp. 41-47 ◽  
Author(s):  
B G Kim ◽  
C L Brooks
Keyword(s):  
Amino Acid ◽  
Phosphorylation Site ◽  
Growth Hormones ◽  
Amino Acid Residues ◽  
Placental Lactogens ◽  
Structural Conformation ◽  
Isolation And Characterization ◽  
Sequencing Studies ◽  
Phosphorylated Prolactin

Quantitative isolation of bovine prolactin was accomplished by immunoaffinity chromatography using clonal antibody as the stationary ligand. The phosphorylated and non-phosphorylated (native) prolactins contained in the immunopurified preparations were separated by chromatofocusing. Isolates from individual pituitaries revealed that phosphorylated prolactin represented between 20 and 80% of the total prolactin. The stoichiometry of phosphate in phosphorylated prolactin was 1.4:1 when determined by amino acid analysis after preparation of the S-ethylcysteine derivative. One major phosphorylation site, serine-90, and two minor sites, serine-26 and -34, were determined by mapping and sequencing studies. Serine-90 was conserved in prolactins, growth hormones and placental lactogens. Serine-26 and -34 were conserved in prolactins, but were not found in growth hormones or placental lactogens. Absorption spectroscopy of the aromatic amino acid residues indicated that phosphorylation of prolactin was associated with a unique structural conformation.

Sign in / Sign up

Export Citation Format

Share Document