scholarly journals Isolation and Characterization of a Broad Spectrum Bacteriocin fromBacillus amyloliquefaciensRX7

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Kong Boon Lim ◽  
Marilen P. Balolong ◽  
Sang Hoon Kim ◽  
Ju Kyoung Oh ◽  
Ji Yoon Lee ◽  
...  
Keyword(s):  
Amino Acids ◽  
Broad Spectrum ◽  
Inhibitory Activity ◽  
Direct Detection ◽  
Sodium Dodecyl ◽  
Proteinase K ◽  
Bacillus Halodurans ◽  
Rrna Gene ◽  
Isolation And Characterization ◽  
Wide Range

We isolated aBacillusstrain, RX7, with inhibitory activity againstListeria monocytogenesfrom soil and identified it asBacillus amyloliquefaciensbased on 16S rRNA gene sequencing. The inhibitory activity was stable over a wide range of pH and was fully retained after 30 min at 80°C, after which it decreased gradually at higher temperatures. The activity was sensitive to the proteolytic action ofα-chymotrypsin, proteinase-K, and trypsin, indicating its proteinaceous nature. This bacteriocin was active against a broad spectrum of bacteria and the fungusCandida albicans. Direct detection of antimicrobial activity on a sodium dodecyl sulfate-polyacrylamide gel suggested an apparent molecular mass of approximately 5 kDa. Ammonium sulfate precipitation and anion-exchange and gel permeation chromatography integrated with reverse phase-high-performance liquid chromatography were used for bacteriocin purification. Automated N-terminal Edman degradation of the purified RX7 bacteriocin recognized the first 15 amino acids as NH2-X-Ala-Trp-Tyr-Asp-Ile-Arg-Lys-Leu-Gly-Asn-Lys-Gly-Ala, where the letter X in the sequence indicates an unknown or nonstandard amino acid. Based on BLAST similarity search and multiple alignment analysis, the obtained partial sequence showed high homology with the two-peptide lantibiotic haloduracin (HalA1) fromBacillus halodurans, although at least two amino acids differed between the sequences. A time-kill study demonstrated a bactericidal mode of action of RX7 bacteriocin.

scholarly journals Isolation and characterization of partially furified bacteriocin of Bacillus cereus (CF1) soil isolate

2020 ◽  
Vol 12 (1) ◽  
pp. 372-379
Author(s):  
I. Bala ◽  
A.H. Arzai ◽  
M.D. Mukhtar ◽  
S.B. Manga
Keyword(s):  
Bacillus Cereus ◽  
Broad Spectrum ◽  
Inhibitory Activity ◽  
Molecular Conformation ◽  
Ph Value ◽  
Bacterial Species ◽  
Sodium Dodecyl ◽  
Tween 80 ◽  
Antimicrobial Substances ◽  
Isolation And Characterization

Bacteriocins are proteinaceous antimicrobial substances produce by bacteria against closely related bacterial species. However their broad spectrum inhibitory activity has been observed against other microorganisms such as fungi. The aim of this study was to isolate and determine physical, chemical and biological characteristics of bacteriocin from B. cereus of soil origin. A Gram-positive spore forming bacilli coded as CF1 was isolated by pour plating technique from the soil sample of the cereal farm land of Kura local government area, Kano State Nigeria and identified as Bacillus cereus based on cultural, microscopic and biochemical characteristics. Bacteriocin was isolated from this B. cereus and partially purified by solvent method (cold acetone) and had broad spectrum inhibitory activity with MIC of 64 AU/ ml. This bacteriocin was thermo stable up to 121°C for 15 minutes and its activity was maintained at wider pH value of 2-12. Although, the bacteriocin isolated by this B. cereus was not affected by some chemical such as Urea, EDTA and Tween 80, other chemical such as SDS and trypsin enzyme deactivated its activity. It is therefore recommended that further studies be carryout on this bacteriocin to purify the bacteriocin using Poly Acryl Amide Gel Electrophoresis PAGE and to elucidate its sequence, to determine its molecular conformation and antibacterial activity on multi drug resistant pathogens. Key words: Bacteriocin, Bacillus cereus, Broad spectrum, Tween 80 and Sodium Dodecyl Sulphate.

scholarly journals Isolation and characterization of a specific enterokinase inhibitor from kidney bean (Phaseolus vulgaris)

1983 ◽  
Vol 209 (1) ◽  
pp. 91-97 ◽  
Author(s):  
R T Jacob ◽  
P G Bhat ◽  
T N Pattabiraman
Keyword(s):  
Phaseolus Vulgaris ◽  
Inhibitory Activity ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Kidney Bean ◽  
Gel Chromatography ◽  
Isolation And Characterization ◽  
Wide Range ◽  
Arginine Residues

A specific enterokinase inhibitor from kidney bean (Phaseolus vulgaris) was purified to homogeneity. It showed a single protein band on sodium dodecyl sulphate/polyacryl-amide-gel electrophoresis in the presence of mercaptoethanol, and the Mr was 31000. Aspartic acid was identified as the N-terminus of the inhibitor. The Mr by gel chromatography on Sephadex G-200 was found to be 60000, indicating the dimeric nature of the inhibitor. The inhibitor was found to be a glycoprotein. The monosaccharide moieties were glucose, mannose, glucuronic acid and glucosamine in the proportions 3.15%, 5.0%, 0.85% and 1.3% respectively. The inhibitor was most active on pig enterokinase, followed by bovine and human enterokinases. Maximal inhibitory activity was elicited by preincubation of the inhibitor with the enzyme for 15 min. Digestion with pepsin resulted in loss of inhibitory activity. The inhibitor was stable to exposure to a wide range of pH values (2-10), and exposure to pH above 10 resulted in loss of inhibitory activity. Modification of arginine residues by cyclohexane 1,2-dione and ninhydrin led to complete loss of enterokinase-inhibitory activity.

scholarly journals Isolation and Characterization of Streptomycetes with Plant Growth Promoting Potential from Mangrove Ecosystem

2015 ◽  
Vol 64 (4) ◽  
pp. 339-349 ◽  
Author(s):  
Pooja Shrivastava ◽  
Rajesh Kumar ◽  
Mahesh Yandigeri ◽  
Nityanand Malviya ◽  
Dilip Arora
Keyword(s):  
Plant Growth ◽  
Sodium Dodecyl ◽  
16S Rrna Gene Sequencing ◽  
Mangrove Ecosystem ◽  
Rrna Gene ◽  
Population Count ◽  
Isolation And Characterization ◽  
Plant Growth Promoting ◽  
Growth Promoting ◽  
Enrichment Techniques

A total of 66 actinomycetes isolates were isolated from mangroves of Andhra Pradesh, India, using various enrichment techniques and pre-treatments. The samples were collected from Coringa mangrove ecosystem and pre-treated by enrichment with CaCO3, sodium dodecyl sulphate and phenol, plated on the media supplemented with cycloheximide (50 mg/ml), nystatin (25 mg/ml) and nalidixic acid (50 mg/ml). The population count of actinomycetes fluctuated from 1.9×105 to 8.0×105/g soil. Out of the isolated 66 actinomycetes, 8 isolates possessing plant growth promoting potential were further studied and characterized by physiological and biochemical traits and identified by 16S rRNA gene sequencing as different species of Streptomycetes genera.

Isolation and characterization of calcineurin from bovine brain

1985 ◽  
Vol 63 (8) ◽  
pp. 1000-1006 ◽  
Author(s):  
Ramesh C. Gupta ◽  
Ramji L. Khandelwal ◽  
Prakash V. Sulakhe
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Myelin Basic Protein ◽  
Phosphatase Activity ◽  
Inhibitory Activity ◽  
Gel Electrophoresis ◽  
Basic Protein ◽  
Sodium Dodecyl ◽  
Subunit A ◽  
Isolation And Characterization ◽  
Dodecyl Sulfate

Calcineurin was isolated from bovine cerebrum extracts by sequential chromatography on Affi-Gel blue and calmodulin affinity columns. Calcineurin so isolated was ~90% pure and was composed of equimolar amounts of subunit A (Mr = 61 000 – 63 000) and subunit B (Mr = 15 000 – 17 000) when examined by sodium dodecyl sulfate gel electrophoresis. A polypeptide (<10%) with Mr = 71 000 whose function and role remains to be investigated, was routinely detected in the calcineurin preparation. Both inhibitory activity (towards calmodulin-dependent cAMP phosphodiesterase) and phosphatase activity (with 32P-labelled myelin basic protein as substrate) were associated with calcineurin as evidenced by (i) coelution from Affi-Gel blue, Affi-Gel calmodulin, diethythaminoethyl-Sepharose, and Sephacryl S-200 chromatography columns; (ii) association with the same protein band on nondenaturing gels; (iii) similar stability upon storage at 4 °C and with repeated freezing and thawing; and (iv) parallel heat inactivation. Phosphatase activity of calcineurin was maximal with 32P-labelled myelin basic protein as the substrate. Using this substrate, enzyme activity was generally stimulated 5- to 10-fold in the presence of Ca2+ and calmodulin; half-maximal activation (A0.5) was observed with 25 nM calmodulin. Calmodulin increased the Vmax of the reaction without affecting the Km for the substrate. Optimum temperature and pH for the reaction were 45 °C and 7, respectively, in both the absence and presence of Ca2+ and calmodulin. The presence of sodium dodecyl sulfate in the assay mixtures caused a decrease in Ca2+ –calmodulin dependent phosphatase but an increase in Mn2+ –calmodulin dependent phosphatase; inhibitory activity was decreased, although to a lesser extent. When A and B subunits of calcineurin were resolved by polyacrylamide gel electrophoresis in the presence of MnCl2 and a low concentration of sodium dodecyl sulfate, the phosphatase activity was found to be associated with subunit A and was stimulated by MnCl2 or MnCl2 + calmodulin.

scholarly journals Heterogeneity of rabbit muscle creatine kinase and limited proteolysis by proteinase K

1977 ◽  
Vol 167 (3) ◽  
pp. 731-737 ◽  
Author(s):  
J Williamson ◽  
J Greene ◽  
S Chérif ◽  
E J Milner-White
Keyword(s):  
Amino Acids ◽  
Creatine Kinase ◽  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Proteinase K ◽  
Rabbit Muscle ◽  
Muscle Creatine Kinase ◽  
Muscle Creatine

By using sodium dodecyl sulphage/polyacrylamide-gel electrophoresis it was shown that rabbit muscle creatine kinase, both in a homogenate and purified, appears to be composed of a mixture of two peptides (mol.wts. 42100 and 40300) differing in length by about 15 amino acids. It is found that low concentrations of proteinase K from the fungus Tritirachium album can cleave about 38 amino acids from each chain of creatine kinase, leaving two large fragments (mol.wts 37700 and 35500). Scission of the whole enzyme was found to be concomitant with complete loss of enzyme activity. MgADP in the presence of absence of creatine slowed the rate of proteolysis by about 50%, but the transition-state analogue complex creatine-NO3—MgADP appeared to protect completely. The time course for the proteolytic inactivation in the presence of this complex, but not in its absence, was biphasic.

Characterization of a human–pathogenicAcanthamoeba griffiniisolated from a contact lens-wearing keratitis patient in Spain

Parasitology ◽  
2014 ◽  
Vol 142 (2) ◽  
pp. 363-373 ◽  
Author(s):  
I. HEREDERO-BERMEJO ◽  
A. CRIADO-FORNELIO ◽  
I. DE FUENTES ◽  
J. SOLIVERI ◽  
J. L. COPA-PATIÑO ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Protease Activity ◽  
Mammalian Cells ◽  
Proteinase Inhibitors ◽  
Proteolytic Enzymes ◽  
Sodium Dodecyl ◽  
Cysteine Proteases ◽  
Rrna Gene ◽  
Significant Activity ◽  
Wide Range

SUMMARYAmoebae were isolated from contact lenses of a symptomatic lens wearer in Spain. Protozoa were characterized by studying their morphology, biology, protease activity and the 18S rRNA gene sequence. Morphology of the organism was observed by light microscopy, scanning electron microscopy and transmission electron microscopy. Its structure corresponded to an amphizoic amoeba. The protozoa grew well at 37 °C and poorly at lower temperatures. In addition, it was capable of lysing mammalian cellsin vitro. A major 56 kDa proteolytic enzyme was observed in amoeba crude extracts by gelatin–sodium dodecyl sulphate–polyacrylamide gel electrophoresis. Most proteolytic enzymes in protozoa extracts showed significant activity over a wide range of pH (3–9) and temperature (8–45 °C) values. The assays on inhibition of protease activity indicated strongly that enzymes detected in amoeba extracts corresponded to serine proteases and, to a lesser extent, cysteine proteases. The use of proteinase inhibitors on a tissue culture model proved that the proteinase activity is critical for developing focal lesions in HeLa cell monolayers. Finally, partial sequencing of the 18S ribosomal RNA gene and phylogenetic analyses indicated that the isolate is closely related toAcanthamoeba griffiniH37 from the UK (T3 genotype).

scholarly journals Improved Method for Purification of Bacterial DNA from Bovine Milk for Detection of Brucella spp. by PCR

1999 ◽  
Vol 65 (8) ◽  
pp. 3735-3737 ◽  
Author(s):  
C. Romero ◽  
I. Lopez-Goñi
Keyword(s):  
Limit Of Detection ◽  
Direct Detection ◽  
Sodium Dodecyl ◽  
Bovine Milk ◽  
Proteinase K ◽  
Improved Method ◽  
Bacterial Dna ◽  
Efficient Extraction ◽  
Lysis Buffer ◽  
High Concentrations

ABSTRACT Different methods of extraction of bacterial DNA from bovine milk to improve the direct detection of Brucella by PCR were evaluated. We found that the use of a lysis buffer with high concentrations of Tris, EDTA, and NaCl, high concentrations of sodium dodecyl sulfate and proteinase K, and high temperatures of incubation was necessary for the efficient extraction of Brucella DNA. The limit of detection by PCR was 5 to 50 Brucella CFU/ml of milk.

Identification and Partial Characterization of Lacticin BH5, a Bacteriocin Produced by Lactococcus lactis BH5 Isolated from Kimchi

2000 ◽  
Vol 63 (12) ◽  
pp. 1707-1712 ◽  
Author(s):  
JI-WOON HUR ◽  
HYUNG-HWAN HYUN ◽  
YU-RYANG PYUN ◽  
TAE-SEOK KIM ◽  
ICK-HYUN YEO ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Lactococcus Lactis ◽  
Bactericidal Activity ◽  
Broad Spectrum ◽  
Direct Detection ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Pathogenic Microorganisms ◽  
Bacteriocin Producer ◽  
Ph Range

Strain BH5 was isolated from naturally fermented Kimchi and identified as a bacteriocin producer that has bactericidal activity against Micrococcus flavus ATCC 10240. Strain BH5 was identified tentatively as Lactococcus lactis by API test. Lactococcus lactis BH5 showed a broad spectrum of activity against most of the nonpathogenic and pathogenic microorganisms tested by the modified deferred method. The activity of lacticin BH5, named tentatively as the bacteriocin produced by L. lactis BH5, was detected at the mid-log growth phase, reached its maximum during the early stationary phase, and decreased after the late stationary phase. Lacticin BH5 also showed a relatively broad spectrum of activity against nonpathogenic and pathogenic microorganisms as tested by the spot-on-lawn method. Its antimicrobial activity on sensitive indicator cells was completely destroyed by protease XIV. The inhibitory activities of lacticin BH5 were detected during treatments up to 100°C for 30 min. Lacticin BH5 was very stable over a pH range of 2.0 to 9.0 and was stable with all the organic solvents examined. It demonstrated a typical bactericidal mode of inhibition against M. flavus ATCC 10240. The apparent molecular mass of lacticin BH5 was estimated to be in the region of 3 to 3.5 kDa, by the direct detection of bactericidal activity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

scholarly journals Isolation and characterization of novel phage (Podoviridae ɸParuNE1) and its efficacy against multi-drug-resistant Pseudomonas aeruginosa planktonic cells and biofilm

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Nkechi V. Enwuru ◽  
Jason J. Gill ◽  
Katri P. Anttonen ◽  
Christian A. Enwuru ◽  
Ry. Young ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Host Range ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Multi Drug Resistance ◽  
Broad Host Range ◽  
Drug Resistant ◽  
Isolation And Characterization ◽  
Biofilm Removal ◽  
Wide Range

Abstract Background Bacterial pathogen (Pseudomonas aeruginosa) could form biofilm that conveys multi-drug resistance. Bacteriophage as an alternative to antibacterial resistance is useful against biofilm complications. This study evaluated antibacterial and biofilm removal activities of lytic phage, specific against multi-drug-resistant clinical P. aeruginosa. Results The phage showed a wide range of pH (5–10) and heat (7–44 °C) stability. Electron microscopy showed ɸPauNE1 phage head (60 nm in diameter) and non-contractile tail (12 nm in length by 8 nm in width); hence, the family Podoviridae and the order Caudovirales. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed structured protein of 55 kDa and double-stranded DNA of 45 kb. The phage was species specific and had broad host range activity. It inhibited bacterial growth at multiplicity of infection (MOI) 1–0.000001 pfu/ml. Inhibition was maximal at both low (1 × 105) and high (1 × 109) bacterial CFU/ml. Biofilm removal test showed that the phage removed more than 60% cell biomass within CFU/ml of 1.5 × 108, 6.0 × 108 and l.0 × 109. Conclusion Phage (ɸPauNE1) was unique and had broad host range activity. The phage exhibited strong bacteriolytic activity against biofilm forming multi-drug-resistant strains. It had no lytic effect on the heterogeneous strains and so a promising bioagent.

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