scholarly journals In vitro and In Silico Approach For Characterization of Antimicrobial Peptide From Probiotics Against Staphylococcus Aureus and Escherichia Coli

2021 ◽  
Author(s):  
Amrutha Bindu ◽  
Lakshmi Devi
Keyword(s):  
Amino Acid ◽  
Antimicrobial Peptide ◽  
Lactobacillus Plantarum ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Proteinase K ◽  
Kinetic Assay ◽  
Wide Range

Abstract The focus of present study was to characterize antimicrobial peptide produced by probiotic cultures, Enterococcus durans DB-1aa (MCC4243), Lactobacillus plantarum Cu2-PM7 (MCC4246) and Lactobacillus fermentum Cu3-PM8 (MCC4233) against Staphylococus aureus and E. coli. The growth kinetic assay revealed 24 h of incubation to be optimum for bacteriocin production. The partially purified compound after ion-exchange chromatography was found to be thermoresistant and stable under wide range of pH. The compound was sensitive to proteinase-K, but resistant to trypsin, a-amylase and lipase. The apparent molecular weight of bacteriocin from MCC4243 and MCC4246 was found to be 3.5 KDa. Translated partial amino acid sequence of plnA gene in MCC4246 displayed 48 amino acid sequences showing 100% similarity with plantaricin A of Lactobacillus plantarum (WP_0036419). The sequence revealed 7 β sheets, 6 α sheets, 6 predicted coils and 9 predicted turns. The functions on cytoplasm show 10.82 isoelectric point and 48.6% hydrophobicity. The molecular approach of using Geneious Prime software and protein prediction data base for characterization of bacteriocin is novel and predicts “KSSAYSLQMGATAIKQVKKLFKKWGW” as peptide responsible for antimicrobial activity. The study provides information about broad spectrum bacteriocin in native probiotic culture and paves a way towards its application in functional foods as biopreservative agents.

scholarly journals In-vitro and In-silico approach for characterization of antimicrobial-peptide from probiotics against Staphylococcus aureus and Escherichia coli

2021 ◽  
Author(s):  
Amrutha Bindu ◽  
Lakshmidevi N
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Lactobacillus Plantarum ◽  
Exchange Chromatography ◽  
Proteinase K ◽  
Kinetic Assay ◽  
Wide Range

Abstract The present aim was to determine the characteristic feature and stability of antimicrobial compound (AMC) produced by probiotic strains of Enterococcus durans MCC4243, Lactiplantibacillus plantarum (Basanym: Lactobacillus plantarum MCC4246) and Limosilactobacillus fermentum (Basonym: Lactobacillus fermentum MCC4233) against Staphylococcus aureus MTCC96 and Escherichia coli MTCC118. Growth kinetic assay revealed 24h of incubation to be optimum for bacteriocin production. Ammonium sulphate precipitation-dialysis was found to be favorable method for extraction of AMC compared to other methods employed. The partially purified compound after ion-exchange chromatography was found to be thermo-resistant upto 90°C and stable under wide range of pH. The compound was sensitive to proteinase-K, but resistant to trypsin, α-amylase and lipase. The apparent molecular weight of AMC from MCC4243 and MCC4246 was found to be 3.5KDa. PCR confirmed the presence of plantaricinA gene in MCC4246. Translated partial amino acid sequence of plnA gene of MCC4246 displayed 48 amino acid sequence which had 100% similarity with plantaricinA of Lactobacillus plantarum (WP 0036419). The sequence revealed 7 β sheets, 6 α sheets, 6 predicted coils and 9 predicted turns and functions on cytoplasm with 10.82 isoelectric point and 48.6% hydrophobicity. From the study, the amino acid sequence “KSSAYSLQMGATAIKQVKKLFKKWGW” of peptide was predicted to be responsible for antimicrobial activity.

Differentiating closely affiliated Dehalococcoides lineages by a novel genetic marker identified via computational pangenome analysis

2021 ◽  
Author(s):  
Siyan Zhao ◽  
Chen Zhang ◽  
Matthew J. Rogers ◽  
Xuejie Zhao ◽  
Jianzhong He
Keyword(s):  
Amino Acid ◽  
Microbial Communities ◽  
Genetic Marker ◽  
Genetic Markers ◽  
Unknown Function ◽  
Computational Approach ◽  
Amino Acid Sequences ◽  
Reductive Dehalogenation ◽  
Wide Range

As a group, Dehalococcoides dehalogenate a wide range of organohalide pollutants but the range of organohalide compounds that can be utilized for reductive dehalogenation differs among the Dehalococcoides strains. Dehalococcoides lineages cannot be reliably disambiguated in mixed communities using typical phylogenetic markers, which often confounds bioremediation efforts. Here, we describe a computational approach to identify Dehalococcoides genetic markers with improved discriminatory resolution. Screening core genes from the Dehalococcoides pangenome for degree of similarity and frequency of 100% identity found a candidate genetic marker encoding a bacterial neuraminidase repeat (BNR)-containing protein of unknown function. This gene exhibits the fewest completely identical amino acid sequences and among the lowest average amino acid sequence identity in the core pangenome. Primers targeting BNR could effectively discriminate between 40 available BNR sequences ( in silico ) and 10 different Dehalococcoides isolates ( in vitro ). Amplicon sequencing of BNR fragments generated from 22 subsurface soil samples revealed a total of 109 amplicon sequence variants, suggesting a high diversity of Dehalococcoides distributed in environment. Therefore, the BNR gene can serve as an alternative genetic marker to differentiate strains of Dehalococcoides in complicated microbial communities. Importance The challenge of discriminating between phylogenetically similar but functionally distinct bacterial lineages is particularly relevant to the development of technologies seeking to exploit the metabolic or physiological characteristics of specific members of bacterial genera. A computational approach was developed to expedite screening of potential genetic markers among phylogenetically affiliated bacteria. Using this approach, a gene encoding a bacterial neuraminidase repeat (BNR)-containing protein of unknown function was selected and evaluated as a genetic marker to differentiate strains of Dehalococcoides , an environmentally relevant genus of bacteria whose members can transform and detoxify a range of halogenated organic solvents and persistent organic pollutants, in complex microbial communities to demonstrate the validity of the approach. Moreover, many apparently phylogenetically distinct, currently uncharacterized Dehalococcoides were detected in environmental samples derived from contaminated sites.

scholarly journals Isolation and characterization of pig enamelins

1987 ◽  
Vol 243 (2) ◽  
pp. 385-390 ◽  
Author(s):  
H Limeback
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Binding Studies ◽  
Isolation And Characterization ◽  
Sequential Extraction Procedures ◽  
Enamel Proteins

Enamel proteins were extracted from pig developing enamel by sequential extraction procedures. Two proteins identified as enamelins by slab-gel electrophoresis (Mr 67,000 and 63,000) were separated from amelogenins by gel sieving and ion-exchange chromatography. Their enamelin characteristic was confirmed by hydroxyapatite-binding studies and amino acid analysis. Degradation of extracted enamel proteins was also studied in vitro. The larger of the two enamelins appeared to be resistant to degradation by endogenous enamel proteinases. Hydroxyapatite showed strong binding with the enamelins, but did not prevent the degradation of the Mr-63,000 enamelin. These results indicate that at least one high-Mr enamelin in pig developing enamel is a source of enamelin breakdown products.

scholarly journals Purification and Characterization of AsES Protein

2013 ◽  
Vol 288 (20) ◽  
pp. 14098-14113 ◽  
Author(s):  
Nadia R. Chalfoun ◽  
Carlos F. Grellet-Bournonville ◽  
Martín G. Martínez-Zamora ◽  
Araceli Díaz-Perales ◽  
Atilio P. Castagnaro ◽  
...  
Keyword(s):  
Amino Acid ◽  
Proteolytic Activity ◽  
Foliar Spray ◽  
Amino Acid Sequences ◽  
Purification And Characterization ◽  
Degenerate Primers ◽  
Acremonium Strictum ◽  
Rapid Amplification

In this work, the purification and characterization of an extracellular elicitor protein, designated AsES, produced by an avirulent isolate of the strawberry pathogen Acremonium strictum, are reported. The defense eliciting activity present in culture filtrates was recovered and purified by ultrafiltration (cutoff, 30 kDa), anionic exchange (Q-Sepharose, pH 7.5), and hydrophobic interaction (phenyl-Sepharose) chromatographies. Two-dimensional SDS-PAGE of the purified active fraction revealed a single spot of 34 kDa and pI 8.8. HPLC (C2/C18) and MS/MS analysis confirmed purification to homogeneity. Foliar spray with AsES provided a total systemic protection against anthracnose disease in strawberry, accompanied by the expression of defense-related genes (i.e. PR1 and Chi2-1). Accumulation of reactive oxygen species (e.g. H2O2 and O2̇̄) and callose was also observed in Arabidopsis. By using degenerate primers designed from the partial amino acid sequences and rapid amplification reactions of cDNA ends, the complete AsES-coding cDNA of 1167 nucleotides was obtained. The deduced amino acid sequence showed significant identity with fungal serine proteinases of the subtilisin family, indicating that AsES is synthesized as a larger precursor containing a 15-residue secretory signal peptide and a 90-residue peptidase inhibitor I9 domain in addition to the 283-residue mature protein. AsES exhibited proteolytic activity in vitro, and its resistance eliciting activity was eliminated when inhibited with PMSF, suggesting that its proteolytic activity is required to induce the defense response. This is, to our knowledge, the first report of a fungal subtilisin that shows eliciting activity in plants. This finding could contribute to develop disease biocontrol strategies in plants by activating its innate immunity.

scholarly journals Enzymic and structural characterization of nepenthesin, a unique member of a novel subfamily of aspartic proteinases

2004 ◽  
Vol 381 (1) ◽  
pp. 295-306 ◽  
Author(s):  
Senarath B. P. ATHAUDA ◽  
Koji MATSUMOTO ◽  
Sanath RAJAPAKSHE ◽  
Masayuki KURIBAYASHI ◽  
Masaki KOJIMA ◽  
...  
Keyword(s):  
Amino Acid ◽  
Computer Modelling ◽  
Structural Characteristics ◽  
Amino Acid Sequences ◽  
Cysteine Residues ◽  
Aspartic Proteinases ◽  
Protein Databases ◽  
Wide Range ◽  
First Time

Carnivorous plants are known to secrete acid proteinases to digest prey, mainly insects, for nitrogen uptake. In the present study, we have purified, for the first time, to homogeneity two acid proteinases (nepenthesins I and II) from the pitcher fluid of Nepenthes distillatoria (a pitcher-plant known locally as badura) and investigated their enzymic and structural characteristics. Both enzymes were optimally active at pH approx. 2.6 towards acid-denatured haemoglobin; the specificity of nepenthesin I towards oxidized insulin B chain appears to be similar, but slightly wider than those of other APs (aspartic proteinases). Among the enzymic properties, however, the most notable is their unusual stability: both enzymes were remarkably stable at or below 50 °C, especially nepenthesin I was extremely stable over a wide range of pH from 3 to 10 for over 30 days. This suggests an evolutionary adaptation of the enzymes to their specific habitat. We have also cloned the cDNAs and deduced the complete amino acid sequences of the precursors of nepenthesins I and II (437 and 438 residues respectively) from the pitcher tissue of N. gracilis. Although the corresponding mature enzymes (each 359 residues) are homologous with ordinary pepsin-type APs, both enzymes had a high content of cysteine residues (12 residues/molecule), which are assumed to form six unique disulphide bonds as suggested by computer modelling and are supposed to contribute towards the remarkable stability of nepenthesins. Moreover, the amino acid sequence identity of nepenthesins with ordinary APs, including plant vacuolar APs, is remarkably low (approx. 20%), and phylogenetic comparison shows that nepenthesins are distantly related to them to form a novel subfamily of APs with a high content of cysteine residues and a characteristic insertion, named ‘the nepenthesin-type AP-specific insertion’, that includes a large number of novel, orthologous plant APs emerging in the gene/protein databases.

scholarly journals Molecular characterization of three newly recognized rat parvoviruses

2002 ◽  
Vol 83 (8) ◽  
pp. 2075-2083 ◽  
Author(s):  
Cho-Hua Wan ◽  
Maria Söderlund-Venermo ◽  
David J. Pintel ◽  
Lela K. Riley
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Molecular Characterization ◽  
Amino Acid Sequences ◽  
Minute Virus ◽  
Genomic Nucleotide Sequence ◽  
Kilham Rat Virus

Rodent parvoviruses have been documented to interfere with both in vivo and in vitro research. In this study, three rat parvoviruses distinct from previously characterized rodent parvoviruses were identified from naturally infected rats obtained from four discrete sources. These three newly recognized parvoviruses were designated rat minute virus (RMV)-1a, -1b and -1c. In this study, the genomic nucleotide sequence and the predicted amino acid sequences of proteins for each of the three RMV-1 variants and Kilham rat virus (KRV) were determined and compared with previously characterized rodent parvoviruses. The three RMV-1 variants were shown to be closely related to each other, to be distinct from but closely related to KRV and H-1 virus, and to be significantly different from the previously identified rat parvovirus isolate, RPV-1a.

scholarly journals Biochemical and Molecular Characterization of Three New Variants of AmpC β-Lactamases from Morganella morganii

2006 ◽  
Vol 50 (3) ◽  
pp. 962-967 ◽  
Author(s):  
Pablo Power ◽  
Moreno Galleni ◽  
Juan A. Ayala ◽  
Gabriel Gutkind
Keyword(s):  
Amino Acid ◽  
Catalytic Properties ◽  
Amino Acid Sequences ◽  
Major Influence ◽  
Morganella Morganii ◽  
Encoding Gene ◽  
Hydrolysis Of ◽  
Similar Kinetic

ABSTRACT Morganella morganii produces an inducible, chromosomally encoded AmpC β-lactamase. We describe in this study three new variants of AmpC within this species with apparent pIs of 6.6 (M19 from M. morganii strain PP19), 7.4 (M29 from M. morganii strain PP29), and 7.8 (M37 from M. morganii strain PP37). After gene sequencing, deduced amino acid sequences displayed one to six substitutions when compared to the available Morganella AmpC sequences. An AmpR-encoding gene was also found upstream of ampC, including the LysR regulators' helix-turn-helix DNA-binding domain and the putative T-N11-A-protected region in the ampR-ampC intercistronic sequence. All three AmpC variants were purified from in vitro-generated derepressed mutants and showed overall similar kinetic parameters. None of the observed amino acid changes, occurring at the surface of the protein, appear to have a major influence in their catalytic properties. Morganella AmpCs exhibit the highest catalytic efficiencies (kcat /Km ) on classical penicillins, cefoxitin, narrow-spectrum cephalosporins, and cefotaxime. Cefotaxime was more effectively hydrolyzed than other oxyimino-cephalosporins, whereas cefepime was 3 log-fold less efficiently hydrolyzed than other cephalosporins such as cephalothin. Several differences with other AmpC β-lactamases were found. Ampicillin was more efficiently hydrolyzed than benzylpenicillin. High kcat /Km values were observed for oxacillin and piperacillin, which are usually poor substrates for AmpC. A fairly efficient hydrolysis of imipenem was detected as well. Aztreonam, carbenicillin, and tazobactam were effective transient inactivators of these variants.

scholarly journals Discovery and characterisation of novel circular bacteriocin plantacyclin B21AG from Lactobacillus plantarum B21

2020 ◽  
Author(s):  
Aida Golneshin ◽  
Mian Chee Gor ◽  
Ben Vezina ◽  
Nicholas Williamson ◽  
Thi Thu Hao Van ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Amino Acid ◽  
Lactobacillus Plantarum ◽  
De Novo ◽  
Peptide Sequencing ◽  
Exchange Chromatography ◽  
Leader Peptide ◽  
Proteolytic Resistance ◽  
Wide Range ◽  
Circular Bacteriocin

AbstractLactobacillus plantarum B21 isolated from Vietnamese sausage (nem chua) has previously displayed broad antimicrobial activity against gram positive bacteria including foodborne pathogens Listeria monocytogenes and Clostridium perfringens. This study successfully identified the antimicrobial agent as plantacyclin B21AG, a 5668 Da circular bacteriocin demonstrating high thermostability, resistance to a wide range of pH, proteolytic resistance and temporal stability. We report a reverse genetics approach used to identify and characterise plantacyclin B21AG. The bacteriocin was purified from culture supernatant by a short process consisting of concentration, n-butanol extraction and cation exchange chromatography. A de novo peptide sequencing using LC-MS/MS techniques identified two putative peptide fragments which were mapped to the genome of Lactobacillus plantarum B21. This revealed an ORF corresponding to a putative circular bacteriocin with a 33-amino acid leader peptide and 58-amino acid mature peptide found on native plasmid pB21AG01. The corresponding gene cluster, consisted of seven genes associated with post-translational circularisation, immunity and secretion. The robust nature of plantacyclin B21AG, its antimicrobial activity and associated machinery for cyclisation make it an interesting biotechnological target for further development, and application as a food-safe antimicrobial.

scholarly journals Functional Characterization of BrF3'H, Which Determines the Typical Flavonoid Profile of Purple Chinese Cabbage

2021 ◽  
Vol 12 ◽  
Author(s):  
Sangkyu Park ◽  
Hyo Lee ◽  
Myung Ki Min ◽  
Jihee Ha ◽  
Jaeeun Song ◽  
...  
Keyword(s):  
Chinese Cabbage ◽  
Expression Patterns ◽  
Functional Characterization ◽  
Phenylpropanoid Pathway ◽  
Amino Acid Sequences ◽  
In Planta ◽  
Normal Green ◽  
Wide Range

Flavonols and anthocyanins are the two major classes of flavonoids in Brassica rapa. To elucidate the flavonoid biosynthetic pathway in Chinese cabbage (B. rapa L. subsp. pekinensis), we analyzed flavonoid contents in two varieties of Chinese cabbage with normal green (5546) and purple (8267) leaves. The 8267 variety accumulates significantly higher levels of quercetin, isorhamnetin, and cyanidin than the 5546 variety, indicating that 3′-dihydroxylated flavonoids are more prevalent in the purple than in the green variety. Gene expression analysis showed that the expression patterns of most phenylpropanoid pathway genes did not correspond to the flavonoid accumulation patterns in 5546 and 8267 varieties, except for BrPAL1.2 while most early and late flavonoid biosynthetic genes are highly expressed in 8267 variety. In particular, the flavanone 3′-hydroxylase BrF3′H (Bra009312) is expressed almost exclusively in 8267. We isolated the coding sequences of BrF3′H from the two varieties and found that both sequences encode identical amino acid sequences and are highly conserved with F3'H genes from other species. An in vitro enzymatic assay demonstrated that the recombinant BrF3′H protein catalyzes the 3′-hydroxylation of a wide range of 4′-hydroxylated flavonoid substrates. Kinetic analysis showed that kaempferol is the most preferred substrate and dihydrokaempferol (DHK) is the poorest substrate for recombinant BrF3′H among those tested. Transient expression of BrF3′H in Nicotiana benthamiana followed by infiltration of naringenin and DHK as substrates resulted in eriodictyol and quercetin production in the infiltrated leaves, demonstrating the functionality of BrF3′H in planta. As the first functional characterization of BrF3′H, our study provides insight into the molecular mechanism underlying purple coloration in Chinese cabbage.

An Antimicrobial Peptide from the Australian Native Hardenbergia violacea Provides the First Functionally Characterised Member of a Subfamily of Plant Defensins

1997 ◽  
Vol 24 (5) ◽  
pp. 571 ◽  
Author(s):  
Stuart J. Harrison ◽  
John P. Marcus ◽  
Kenneth C. Goulter ◽  
Jodie L. Green ◽  
Donald J. Maclean ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Antimicrobial Peptide ◽  
Mammalian Cells ◽  
Amino Acid Sequences ◽  
Plant Defensin ◽  
Defensive Role ◽  
Plant Defensins

An antimicrobial peptide (HvAMP1) was isolated from seeds of the Australian native legume Hardenbergia violacea (Schneev.) Stearn. The peptide is 47 amino acid residues in length, contains 8 cysteines, and has a molecular weight of 5392 and a predicted pI of 10.41. HvAMP1 inhibited the growth of several plant pathogenic fungi at concentrations as low as 1 µM in vitro and produced distinct hyphal distortion and increased branching. This antimicrobial activity was greatly diminished in the presence of 1 mM CaCl2 and 50 mM KCl. The purified peptide at 40 µM did not inhibit three different a-amylase enzymes. Aeukaryotic cell-free translation system showed inhibition approaching 50% in the presence of ~100 µM of HvAMP1. The viability of plant and mammalian cells cultured in vitro was not adversely affected by concentrations of HvAMP1 as high as 40 mM. The amino acid sequence of HvAMP1 contained the consensus amino acids that define the plant defensin family of peptides. The HvAMP1 amino acid sequence showed 87% and 57% identity with the amino acid sequences deduced from cDNA sequences from defensins of Vigna unguiculata and Pisum sativum, respectively. Other plant defensin sequences showed less than 33% amino acid identity to the peptide. Therefore, HvAMP1 and the putative plant defensins of cowpea and pea define a distinct sequence subfamily of plant defensins which is at present limited to members of the Fabaceae. HvAMP1 is the first member of this subfamily to be purified and functionally characterised. The antimicrobial activity of HvAMP1 suggests a defensive role for this subfamily of peptides.

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