scholarly journals Modification of the Sweetness and Stability of Sweet-Tasting Protein Monellin by Gene Mutation and Protein Engineering

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Qiulei Liu ◽  
Lei Li ◽  
Liu Yang ◽  
Tianming Liu ◽  
Chenggu Cai ◽  
...  
Keyword(s):  
Protein Modification ◽  
Bioinformatics Analysis ◽  
Acid Resistance ◽  
Site Directed Mutagenesis ◽  
Sequence Structure ◽  
Sweet Proteins ◽  
Sweet Protein ◽  
Food Applications ◽  
Ionizable Residues ◽  
Ionizable Residue

Natural sweet protein monellin has a high sweetness and low calorie, suggesting its potential in food applications. However, due to its low heat and acid resistance, the application of monellin is limited. In this study, we show that the thermostability of monellin can be improved with no sweetness decrease by means of sequence, structure analysis, and site-directed mutagenesis. We analyzed residues located in theα-helix as well as an ionizable residue C41. Of the mutants investigated, the effects of E23A and C41A mutants were most remarkable. The former displayed significantly improved thermal stability, while its sweetness was not changed. The mutated protein was stable after 30 min incubation at 85°C. The latter showed increased sweetness and slight improvement of thermostability. Furthermore, we found that most mutants enhancing the thermostability of the protein were distributed at the two ends ofα-helix. Molecular biophysics analysis revealed that the state of buried ionizable residues may account for the modulated properties of mutated proteins. Our results prove that the properties of sweet protein monellin can be modified by means of bioinformatics analysis, gene manipulation, and protein modification, highlighting the possibility of designing novel effective sweet proteins based on structure-function relationships.

scholarly journals Metabolic Effects of the Sweet Protein MNEI as a Sweetener in Drinking Water. A Pilot Study of a High Fat Dietary Regimen in a Rodent Model

Nutrients ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 2643
Author(s):  
Rosa Cancelliere ◽  
Serena Leone ◽  
Cristina Gatto ◽  
Arianna Mazzoli ◽  
Carmine Ercole ◽  
...  
Keyword(s):  
Metabolic Syndrome ◽  
Lipid Profile ◽  
Western Diet ◽  
Metabolic Effects ◽  
High Fat ◽  
Metabolic Derangement ◽  
Dietary Regimen ◽  
Sweet Proteins ◽  
Sweet Protein ◽  
Typical Western Diet

Sweeteners have become integrating components of the typical western diet, in response to the spreading of sugar-related pathologies (diabetes, obesity and metabolic syndrome) that have stemmed from the adoption of unbalanced dietary habits. Sweet proteins are a relatively unstudied class of sweet compounds that could serve as innovative sweeteners, but their introduction on the food market has been delayed by some factors, among which is the lack of thorough metabolic and toxicological studies. We have tried to shed light on the potential of a sweet protein, MNEI, as a fructose substitute in beverages in a typical western diet, by studying the metabolic consequences of its consumption on a Wistar rat model of high fat diet-induced obesity. In particular, we investigated the lipid profile, insulin sensitivity and other indicators of metabolic syndrome. We also evaluated systemic inflammation and potential colon damage. MNEI consumption rescued the metabolic derangement elicited by the intake of fructose, namely insulin resistance, altered plasma lipid profile, colon inflammation and translocation of lipopolysaccharides from the gut lumen into the circulatory system. We concluded that MNEI could represent a valid alternative to fructose, particularly when concomitant metabolic disorders such as diabetes and/or glucose intolerance are present.

scholarly journals Novel Lytic Enzyme of Prophage Origin from Clostridium botulinum E3 Strain Alaska E43 with Bactericidal Activity against Clostridial Cells

2021 ◽  
Vol 22 (17) ◽  
pp. 9536
Author(s):  
Agnieszka Morzywolek ◽  
Magdalena Plotka ◽  
Anna-Karina Kaczorowska ◽  
Monika Szadkowska ◽  
Lukasz P. Kozlowski ◽  
...  
Keyword(s):  
Clostridium Botulinum ◽  
Bioinformatics Analysis ◽  
Analytical Ultracentrifugation ◽  
Lipoteichoic Acid ◽  
Lytic Activity ◽  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Lytic Enzyme ◽  
Gram Positive ◽  
Genes Encoding

Clostridium botulinum is a Gram-positive, anaerobic, spore-forming bacterium capable of producing botulinum toxin and responsible for botulism of humans and animals. Phage-encoded enzymes called endolysins, which can lyse bacteria when exposed externally, have potential as agents to combat bacteria of the genus Clostridium. Bioinformatics analysis revealed in the genomes of several Clostridium species genes encoding putative N-acetylmuramoyl-l-alanine amidases with anti-clostridial potential. One such enzyme, designated as LysB (224-aa), from the prophage of C. botulinum E3 strain Alaska E43 was chosen for further analysis. The recombinant 27,726 Da protein was expressed and purified from E. coli Tuner(DE3) with a yield of 37.5 mg per 1 L of cell culture. Size-exclusion chromatography and analytical ultracentrifugation experiments showed that the protein is dimeric in solution. Bioinformatics analysis and results of site-directed mutagenesis studies imply that five residues, namely H25, Y54, H126, S132, and C134, form the catalytic center of the enzyme. Twelve other residues, namely M13, H43, N47, G48, W49, A50, L73, A75, H76, Q78, N81, and Y182, were predicted to be involved in anchoring the protein to the lipoteichoic acid, a significant component of the Gram-positive bacterial cell wall. The LysB enzyme demonstrated lytic activity against bacteria belonging to the genera Clostridium, Bacillus, Staphylococcus, and Deinococcus, but did not lyse Gram-negative bacteria. Optimal lytic activity of LysB occurred between pH 4.0 and 7.5 in the absence of NaCl. This work presents the first characterization of an endolysin derived from a C. botulinum Group II prophage, which can potentially be used to control this important pathogen.

scholarly journals SARS-CoV-2 Spike Protein Evolution may Cause Difficulties for Vaccine

2020 ◽  
Author(s):  
Jian Zhou ◽  
Sun Jingjing ◽  
Gang Lu ◽  
Wanchun Wang ◽  
Lin Wang
Keyword(s):  
T Cell ◽  
B Cell ◽  
3D Model ◽  
Bioinformatics Analysis ◽  
Cell Epitope ◽  
Amino Acid Sequences ◽  
T Cell Epitopes ◽  
Sequence Structure ◽  
B Cell Epitopes ◽  
S Protein

Abstract Background: Coronavirus disease 2019 (COVID-19) poses a great threat to human health and life. We performed a bioinformatics analysis to compare the sequence, structure, and epitopes of SARS-CoV-2 spike (S) protein in 10 different countries. Methods: The amino acid sequences of SARS-CoV-2 S protein were obtained from the NCBI database. We used DNASTAR Lasergene software to analyze the protein’s secondary structures. SWISS-MODEL combined with VMD software was used to construct a 3D model of SARS-CoV-2 S protein. DNASTAR Protean and the IEDB database were used to analyze the B cell epitopes and T cell epitopes, respectively. Results: The results of B cell epitopes analysis indicated that the epitopes of SARS-CoV-2 S protein in Korea and American increased, which suggested that the antigenicity of SARS-CoV-2 in Czech, Korea and American might be enhanced. A small number of B cell epitopes disappeared in the SARS-CoV-2 S protein sequence from Greece, Australia, Sweden and India, which suggested that the antigenicity of SARS-CoV-2 in Greece, Australia, Sweden and India may be weakened. T cell epitope analysis indicated that the antigenicity of SARS-CoV-2 in Czech, Korea and American was enhanced, while antigenicity of SARS-CoV-2 in Greece, Australia, Inida, Sweden and Thailand may be weakened. The sequence of SARS-CoV-2 S protein has changed as the virus has spread, and the structures and epitopes have changed accordingly. Conclusion: The mutation leads to a decrease in the antigenicity of SARS-CoV-2, which may be a mechanism for the virus to evade surveillance by the immune system.

scholarly journals ChiIV3 Acts as a Novel Target of WRKY40 to Mediate Pepper Immunity Against Ralstonia solanacearum Infection

2019 ◽  
Vol 32 (9) ◽  
pp. 1121-1133 ◽  
Author(s):  
Zhiqin Liu ◽  
Lanping Shi ◽  
Yahong Weng ◽  
Huasong Zou ◽  
Xia Li ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Ralstonia Solanacearum ◽  
Bioinformatics Analysis ◽  
Wilt Disease ◽  
Site Directed Mutagenesis ◽  
Defense Gene ◽  
Bacterial Wilt Disease ◽  
Cis Element ◽  
Novel Target ◽  
Pepper Plants

ChiIV3, a chitinase of pepper (Capsicum annuum), stimulates cell death in pepper plants. However, there are only scarce reports on its role in resistance against bacterial wilt disease such as that caused by Ralstonia solanacearum and their transcriptional regulation. In this study, the silencing of ChiIV3 in pepper plants significantly reduced the resistance to R. solanacearum. The transcript of ChiIV3 was induced by R. solanacearum inoculation (RSI) as well as exogenous application of methyl jasmonate and abscisic acid. The bioinformatics analysis revealed that the ChiIV3 promoter consists of multiple stress-related cis elements, including six W-boxes and one MYB1AT. With the 5′ deletion assay in the ChiIV3 promoter, the W4-box located from −640 to −635 bp was identified as the cis element that is required for the response to RSI. In addition, the W4-box element was shown to be essential for the binding of the ChiIV3 promoter by the WRKY40 transcription factor, which is known to positively regulate the defense response to R. solanacearum. Site-directed mutagenesis in the W4-box sequence impaired the binding of WRKY40 to the ChiIV3 promoter. Subsequently, the transcription of ChiIV3 decreased in WRKY40-silenced pepper plants. These results demonstrated that the expression of the defense gene ChiIV3 is controlled through multiple modes of regulation, and WRKY40 directly binds to the W4-box element of the ChiIV3 promoter region for its transcriptional regulation.

Sequence-structure-function relationships of a tRNA (m7G46) methyltransferase studied by homology modeling and site-directed mutagenesis

2005 ◽  
Vol 59 (3) ◽  
pp. 482-488 ◽  
Author(s):  
Elżbieta Purta ◽  
Françoise van Vliet ◽  
Catherine Tricot ◽  
Lara G. De Bie ◽  
Marcin Feder ◽  
...  
Keyword(s):  
Homology Modeling ◽  
Structure Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Sequence Structure

scholarly journals A novel amidase signature family amidase from the marine actinomyceteSalinispora arenicolaCNS-205

2018 ◽  
Author(s):  
Ma Yanling ◽  
Zhang Xinfeng ◽  
Zeng Rong
Keyword(s):  
Bioinformatics Analysis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Acyl Transferase ◽  
Optimal Temperature ◽  
Aryl Acylamidase ◽  
Marine Actinomycete ◽  
Potential Applications ◽  
New Gene ◽  
Substrate Spectrum

AbstractWe cloned a new gene from the amidase signature (AS) family, designatedam, from the marine actinomyceteSalinispora arenicolaCNS-205. As indicated by bioinformatics analysis and site-directed mutagenesis, the AM protein belonged to the AS family. AM was expressed, purified, and characterised inEscherichia coliBL21 (DE3), and the AM molecular mass was determined to be 51 kDa. The optimal temperature and pH were 40 °C and pH 8.0, respectively. AM exhibited a wide substrate spectrum and showed amidase, aryl acylamidase, and acyl transferase activities. AM had high activity towards aromatic and aliphatic amides. The AM substrate specificity for anilides was very narrow; only propanil could be used as an effective substrate. The extensive substrate range of AM indicates it may have broad potential applications in biosynthetic processes and biodegradation.

scholarly journals Evolutionary relationships of microbial transglutaminases

2017 ◽  
Author(s):  
Deborah Giordano ◽  
Angelo Facchiano
Keyword(s):  
Food Processing ◽  
Protein Modification ◽  
Bioinformatics Analysis ◽  
Acyl Transfer ◽  
Transfer Reactions ◽  
Protein Modelling ◽  
Sequence Comparisons ◽  
Functional Features ◽  
Streptomyces Mobaraensis ◽  
Acyl Transfer Reactions

Transglutaminases (TGases) are a class of enzyme widely spread in nature, and observed in plants, microorganisms, vertebrates and invertebrates. This enzyme catalyzes post-translational protein modification, by acyl transfer reactions, deamidation and crosslinking (polymerisation). There is a large interest for TGases functions, in particular for human TGases and their involvement in physiopathological processes. In bacteria, TGases appear largely present, although the function of this enzyme is still unknown. Microbial TGases (MTG, or MTGase) are of extreme interest, in particular MTGase from Streptomyces mobaraensis, used in biopolymers industry, in cosmetics production, in wool textiles, and in the food processing. We present the results of bioinformatics analysis on MTGases sequences, based on database searching, sequence comparisons and alignments, phylogenetic tree constructions, with the aim of improving the knowledge of MTGases, in the perspective of investigating by protein modelling and simulations techniques the functional features.

scholarly journals Insights into the molecular basis for substrate binding and specificity of the wild-type L-arginine/agmatine antiporter AdiC

2016 ◽  
Vol 113 (37) ◽  
pp. 10358-10363 ◽  
Author(s):  
Hüseyin Ilgü ◽  
Jean-Marc Jeckelmann ◽  
Vytautas Gapsys ◽  
Zöhre Ucurum ◽  
Bert L. de Groot ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Radioligand Binding ◽  
Substrate Binding ◽  
Acid Resistance ◽  
Enteric Bacteria ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Functional Roles ◽  
Dynamics Simulations ◽  
Substrate Binding Sites

Pathogenic enterobacteria need to survive the extreme acidity of the stomach to successfully colonize the human gut. Enteric bacteria circumvent the gastric acid barrier by activating extreme acid-resistance responses, such as the arginine-dependent acid resistance system. In this response, l-arginine is decarboxylated to agmatine, thereby consuming one proton from the cytoplasm. In Escherichia coli, the l-arginine/agmatine antiporter AdiC facilitates the export of agmatine in exchange of l-arginine, thus providing substrates for further removal of protons from the cytoplasm and balancing the intracellular pH. We have solved the crystal structures of wild-type AdiC in the presence and absence of the substrate agmatine at 2.6-Å and 2.2-Å resolution, respectively. The high-resolution structures made possible the identification of crucial water molecules in the substrate-binding sites, unveiling their functional roles for agmatine release and structure stabilization, which was further corroborated by molecular dynamics simulations. Structural analysis combined with site-directed mutagenesis and the scintillation proximity radioligand binding assay improved our understanding of substrate binding and specificity of the wild-type l-arginine/agmatine antiporter AdiC. Finally, we present a potential mechanism for conformational changes of the AdiC transport cycle involved in the release of agmatine into the periplasmic space of E. coli.

scholarly journals Biochemical insight into redox regulation of plastidial 3-phosphoglycerate dehydrogenase from Arabidopsis thaliana

2020 ◽  
Vol 295 (44) ◽  
pp. 14906-14915
Author(s):  
Keisuke Yoshida ◽  
Kinuka Ohtaka ◽  
Masami Yokota Hirai ◽  
Toru Hisabori
Keyword(s):  
Arabidopsis Thaliana ◽  
Enzyme Activity ◽  
Disulfide Bond ◽  
Protein Modification ◽  
Redox Regulation ◽  
Site Directed Mutagenesis ◽  
Reductive Activation ◽  
Wide Range ◽  
Phosphoglycerate Dehydrogenase

Thiol-based redox regulation is a post-translational protein modification for controlling enzyme activity by switching oxidation/reduction states of Cys residues. In plant cells, numerous proteins involved in a wide range of biological systems have been suggested as the target of redox regulation; however, our knowledge on this issue is still incomplete. Here we report that 3-phosphoglycerate dehydrogenase (PGDH) is a novel redox-regulated protein. PGDH catalyzes the first committed step of Ser biosynthetic pathway in plastids. Using an affinity chromatography-based method, we found that PGDH physically interacts with thioredoxin (Trx), a key factor of redox regulation. The in vitro studies using recombinant proteins from Arabidopsis thaliana showed that a specific PGDH isoform, PGDH1, forms the intramolecular disulfide bond under nonreducing conditions, which lowers PGDH enzyme activity. MS and site-directed mutagenesis analyses allowed us to identify the redox-active Cys pair that is mainly involved in disulfide bond formation in PGDH1; this Cys pair is uniquely found in land plant PGDH. Furthermore, we revealed that some plastidial Trx subtypes support the reductive activation of PGDH1. The present data show previously uncharacterized regulatory mechanisms of PGDH and expand our understanding of the Trx-mediated redox-regulatory network in plants.

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