scholarly journals Structural Modelling and in-silico characterization of a Novel Thermophilic β-amylase from Clostridium thermosulfuregenes

2021 ◽  
pp. 1-22
Author(s):  
Shafiqa Nayel ◽  
Mohd Shahir Shamsir ◽  
Farid Ahmad Danishfar
Keyword(s):  
Amino Acid ◽  
Binding Sites ◽  
Hydrolytic Enzyme ◽  
Beta Sheets ◽  
Amino Acid Residues ◽  
Structural Domains ◽  
Structural Conformation ◽  
In Silico Characterization

β-amylase is a hydrolytic enzyme that is involved in breaking down starch and producing energy. Since the discovery of β-amylase, it has been applied in various applications especially in the food industry. In this study, a novel β-amylase from Clostridium thermosuluregen, a thermophilic anaerobic bacterium that ferments its extracellular emulsion to ethanol at 62 ℃ was modelled and studied using bioinformatics tools and compared with B. cereus β-amylases that functions at mesophilic conditions. The results showed that the overall structural conformations, secondary structures, and important residues involved in active and binding sites were identified in both proteins. The results revealed that the modelled β-amylase of C. thermosulfuregen is very similar with respect to the global conformation, location of active and binding sites. Both proteins showed identical structural domains with the thermophilic variant possessing a high percentage of hydrophobic amino acid residues, polar amino acid residues, and differences in secondary composition such as loops and beta sheets as the potential evolutionary thermal adaptations that make it stable enzyme that functions up to 70 ℃. The results suggest that the thermal stability are not dependent on one single unique mechanism and may use one or a combination of the mechanisms to sustain its structural conformation at a higher operating temperature. Overall, considering the common properties of this modelled protein with the β-amylase of B. cereus, it can be assumed that if the β-amylase of C. thermosulfuregen were expressed in-vitro, it would produce a stable protein that possesses the hydrolysis function for C. thermosulfuregen to break down the starch and sugar formation.

Molecular cloning and in silico characterization of two alpha-like neurotoxins and one metalloproteinase from the maxilllipeds of the centipede Scolopendra subspinipes mutilans

2018 ◽  
Vol 43 (6) ◽  
pp. 651-661
Author(s):  
Xichao Xia ◽  
Yang Liu ◽  
Jianxin Huang ◽  
Xiaozhu Yu ◽  
Zhiguo Chen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Beta Sheets ◽  
Binding Motif ◽  
Amino Acid Residues ◽  
Primary Sequence Analysis ◽  
Rapid Amplification ◽  
Scolopendra Subspinipes Mutilans ◽  
In Silico Characterization ◽  
Zinc Binding Motif

Abstract Aims In order to shed light of characterizations of centipede Scolopendra subspinipes mutilans venom, a two novel full-lengths of alpha-like-neurotoxin and one metalloproteinase cDNAs derived from the maxilllipeds RNA of centipede S. subspinipes mutilans were isolated, and, respectively, named as SsuTA1, SsuTA2 and SsuMPs. Materials and methods The SsuTA1, SsuTA2 and SsuMPs were cloned from the S. subspinipes mutilans using the rapid amplification of cDNA ends methods. Results In the current study, SsuTA1 and SsuTA2 were, respectively, composed of 82 amino acid residues and 106 amino acid residues. Deduced protein sequence of SsuTA1 shared high homology with that of SsuTA2, one major difference was the C-terminal 24-residue extension in SsuTA2. An abundance of cysteine residues and several adjacent beta-sheets were observed in SsuTA1 and SsuTA2. SsuMPs had 594 amino acid residues containing with a molecular mass of 68.29 kDa. The primary sequence analysis indicated that the SsuMPs contains a zinc-binding motif (HEIGHSLGLAHS) and methionine-turn motif (YIM). Phylogenetic analysis revealed early divergence and independent evolution of SsuTA1 and SsuTA2 from other α-neurotoxins. Conclusion The results suggested that centipede S. subspinipes mutilans is an ancient member of venomous arthropods, but its venom exhibits novel scenario.

scholarly journals In vitro Characterization of Fitness and Convalescent Antibody Neutralization of SARS-CoV-2 Cluster 5 Variant Emerging in Mink at Danish Farms

2021 ◽  
Vol 12 ◽  
Author(s):  
Ria Lassaunière ◽  
Jannik Fonager ◽  
Morten Rasmussen ◽  
Anders Frische ◽  
Charlotta Polacek ◽  
...  
Keyword(s):  
Amino Acid ◽  
Reference Strain ◽  
Rhesus Macaques ◽  
Amino Acid Residues ◽  
Susceptible Animal ◽  
In Vitro Characterization ◽  
Cynomolgus Macaques ◽  
Animal Hosts

In addition to humans, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit to animals that include hamsters, cats, dogs, mink, ferrets, tigers, lions, cynomolgus macaques, rhesus macaques, and treeshrew. Among these, mink are particularly susceptible. Indeed, 10 countries in Europe and North America reported SARS-CoV-2 infection among mink on fur farms. In Denmark, SARS-CoV-2 spread rapidly among mink farms and spilled-over back into humans, acquiring mutations/deletions with unknown consequences for virulence and antigenicity. Here we describe a mink-associated SARS-CoV-2 variant (Cluster 5) characterized by 11 amino acid substitutions and four amino acid deletions relative to Wuhan-Hu-1. Temporal virus titration, together with genomic and subgenomic viral RNA quantitation, demonstrated a modest in vitro fitness attenuation of the Cluster 5 virus in the Vero-E6 cell line. Potential alterations in antigenicity conferred by amino acid changes in the spike protein that include three substitutions (Y453F, I692V, and M1229I) and a loss of two amino acid residues 69 and 70 (ΔH69/V70), were evaluated in a virus microneutralization assay. Compared to a reference strain, the Cluster 5 variant showed reduced neutralization in a proportion of convalescent human COVID-19 samples. The findings underscore the need for active surveillance SARS-CoV-2 infection and virus evolution in susceptible animal hosts.

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 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.

scholarly journals Characterization of a minus end-directed kinesin-like motor protein from cultured mammalian cells.

1995 ◽  
Vol 129 (4) ◽  
pp. 1049-1059 ◽  
Author(s):  
R Kuriyama ◽  
M Kofron ◽  
R Essner ◽  
T Kato ◽  
S Dragas-Granoic ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Mammalian Cells ◽  
Amino Acid Residues ◽  
Central Stalk ◽  
Vitro Analysis ◽  
Kinesin Superfamily ◽  
In Vitro Analysis

Using the CHO2 monoclonal antibody raised against CHO spindles (Sellitto, C., M. Kimble, and R. Kuriyama. 1992. Cell Motil. Cytoskeleton. 22:7-24) we identified a 66-kD protein located at the interphase centrosome and mitotic spindle. Isolated cDNAs for the antigen encode a 622-amino acid polypeptide. Sequence analysis revealed the presence of 340-amino acid residues in the COOH terminus, which is homologous to the motor domain conserved among other members of the kinesin superfamily. The protein is composed of a central alpha-helical portion with globular domains at both NH2 and COOH termini, and the epitope to the monoclonal antibody resides in the central alpha-helical stalk. A series of deletion constructs were created for in vitro analysis of microtubule interactions. While the microtubule binding and bundling activities require both the presence of the COOH terminus and the alpha-helical domain, the NH2-terminal half of the antigen lacked the ability to interact with microtubules. The full-length as well as deleted proteins consisting of the COOH-terminal motor and the central alpha-helical stalk supported microtubule gliding, with velocity ranging from 1.0 to 8.4 microns/minute. The speed of microtubule movement decreased with decreasing lengths of the central stalk attached to the COOH-terminal motor. The microtubules moved with their plus end leading, indicating that the antigen is a minus end-directed motor. The CHO2 sequence shows 86% identify to HSET, a gene located at the centromeric end of the human MHC region in chromosome 6 (Ando, A., Y. Y. Kikuti, H. Kawata, N. Okamoto, T. Imai, T. Eki, K. Yokoyama, E. Soeda, T. Ikemura, K. Abe, and H. Inoko. 1994. Immunogenetics. 39:194-200), indicating that HSET might represent a human homologue of the CHO2 antigen.

scholarly journals Purification of a high-molecular-weight somatoliberin (growth-hormone-releasing factor) from pig hypothalami

1983 ◽  
Vol 209 (3) ◽  
pp. 643-651 ◽  
Author(s):  
J E C Sykes ◽  
P J Lowry
Keyword(s):  
Molecular Weight ◽  
Growth Hormone ◽  
Amino Acid ◽  
Response Curve ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Amino Acid Residues ◽  
Growth Hormone Releasing Factor

Preliminary observations [Sykes & Lowry (1980) J. Endocrinol. 85, 42P-43P] had suggested that the major hypothalamic somatoliberin (growth-hormone-releasing factor) was a larger peptide than the other characterized hypothalamic factors, with an elution position on Sephadex G-50 between those of neurophysin and corticotropin. The present paper reports the isolation and preliminary characterization of pig hypothalamic somatoliberin. Acid extracts of pig stalk median eminence were purified by gel filtration and preparative and analytical high-pressure liquid chromatography to yield a preparation that was specific in the release of somatotropin (growth hormone) in vitro, giving a steep dose-response curve at doses in the range 0.20-3.0 ng. Amino acid analysis revealed a non-cysteine-containing peptide with a high number of glutamate (or glutamine) and aspartate (or asparagine) residues. The peptide had about 56-57 amino acid residues and an apparent molecular weight of 6400, in keeping with its elution position on a column of Sephadex G-50.

IN VITRO/IN SILICO CHARACTERIZATION OF ACTIVE AND PASSIVE STRESSES IN CARDIAC MUSCLE

2012 ◽  
Vol 10 (2) ◽  
pp. 171-188 ◽  
Author(s):  
Markus Boel ◽  
Oscar J. Abilez ◽  
Ahmed N Assar ◽  
Christopher K. Zarins ◽  
Ellen Kuhl
Keyword(s):  
Cardiac Muscle ◽  
In Silico ◽  
In Silico Characterization

In-Vitro and In-Silico Characterization of Xylose Reductase from Emericella nidulans

2019 ◽  
Vol 13 (2) ◽  
pp. 159-170 ◽  
Author(s):  
Vishal Ahuja ◽  
Aashima Sharma ◽  
Ranju Kumari Rathour ◽  
Vaishali Sharma ◽  
Nidhi Rana ◽  
...  
Keyword(s):  
Production Process ◽  
In Silico ◽  
Xylose Reductase ◽  
In Silico Analysis ◽  
Emericella Nidulans ◽  
Higher Temperature ◽  
In Silico Characterization ◽  
Silico Analysis

Background: Lignocellulosic residues generated by various anthropogenic activities can be a potential raw material for many commercial products such as biofuels, organic acids and nutraceuticals including xylitol. Xylitol is a low-calorie nutritive sweetener for diabetic patients. Microbial production of xylitol can be helpful in overcoming the drawbacks of traditional chemical production process and lowring cost of production. Objective: Designing efficient production process needs the characterization of required enzyme/s. Hence current work was focused on in-vitro and in-silico characterization of xylose reductase from Emericella nidulans. Methods: Xylose reductase from one of the hyper-producer isolates, Emericella nidulans Xlt-11 was used for in-vitro characterization. For in-silico characterization, XR sequence (Accession No: Q5BGA7) was used. Results: Xylose reductase from various microorganisms has been studied but the quest for better enzymes, their stability at higher temperature and pH still continues. Xylose reductase from Emericella nidulans Xlt-11 was found NADH dependent and utilizes xylose as its sole substrate for xylitol production. In comparison to whole cells, enzyme exhibited higher enzyme activity at lower cofactor concentration and could tolerate higher substrate concentration. Thermal deactivation profile showed that whole cell catalysts were more stable than enzyme at higher temperature. In-silico analysis of XR sequence from Emericella nidulans (Accession No: Q5BGA7) suggested that the structure was dominated by random coiling. Enzyme sequences have conserved active site with net negative charge and PI value in acidic pH range. Conclusion: Current investigation supported the enzyme’s specific application i.e. bioconversion of xylose to xylitol due to its higher selectivity. In-silico analysis may provide significant structural and physiological information for modifications and improved stability.

Oxytocin analogues with non-coded amino acid residues in position 8: [8-Neopentylglycine]oxytocin and [8-cycloleucine]oxytocin

1987 ◽  
Vol 52 (9) ◽  
pp. 2317-2325 ◽  
Author(s):  
Jan Hlaváček ◽  
Jan Pospíšek ◽  
Jiřina Slaninová ◽  
Walter Y. Chan ◽  
Victor J. Hruby
Keyword(s):  
Amino Acid ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
The Other ◽  
Amino Acid Residues ◽  
Phase Synthesis ◽  
Other Hand ◽  
Fragment Condensation

[8-Neopentylglycine]oxytocin (II) and [8-cycloleucine]oxytocin (III) were prepared by a combination of solid-phase synthesis and fragment condensation. Both analogues exhibited decreased uterotonic potency in vitro, each being about 15-30% that of oxytocin. Analogue II also displayed similarly decreased uterotonic potency in vivo and galactogogic potency. On the other hand, analogue III exhibited almost the same potency as oxytocin in the uterotonic assay in vivo and in the galactogogic assay.

Synthesis of Fragments of the Vasoactive Intestinal Peptide (VIP) and Analysis of Their Residual Biological Activities

1995 ◽  
Vol 60 (7) ◽  
pp. 1229-1235 ◽  
Author(s):  
Ivana Zoulíková ◽  
Ivan Svoboda ◽  
Jiří Velek ◽  
Václav Kašička ◽  
Jiřina Slaninová ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biological Activities ◽  
Residual Activity ◽  
Detailed Examination ◽  
Amino Acid Residues ◽  
Linear Peptide ◽  
Zone Electrophoresis ◽  
Capillary Zone

The vasoactive intestinal (poly)peptide (VIP) is a linear peptide containing 28 amino acid residues, whose primary structure indicates a low metabolic stability. The following VIP fragments, as potential metabolites, and their analogues were prepared by synthesis on a solid: [His(Dnp)1]VIP(1-10), VIP(11-14), [D-Arg12]VIP(11-14), [Lys(Pac)15,21,Arg20]VIP(15-22), and VIP(23-28). After purification, the peptides were characterized by amino acid analysis, mass spectrometry, RP HPLC, and capillary zone electrophoresis. In some tests, detailed examination of the biological activity of the substances in vivo and in vitro gave evidence of a low, residual activity of some fragments, viz. a depressoric activity in vivo for [His(Dnp)1]VIP(1-10) and a stimulating activity for the release of α-amylase in vitro and in vivo for [Lys(Pac)15,21,Arg20]VIP(15-22) and VIP(23-28).

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