Molecular and biochemical characterization of Iranian surfactin-producingBacillus subtilisisolates and evaluation of their biocontrol potential againstAspergillus flavusandColletotrichum gloeosporioides

2009 ◽  
Vol 55 (4) ◽  
pp. 395-404 ◽  
Author(s):  
Matin Mohammadipour ◽  
Maryam Mousivand ◽  
Gholamreza Salehi Jouzani ◽  
Saeed Abbasalizadeh
Keyword(s):  
High Performance ◽  
Biochemical Characterization ◽  
Blood Agar ◽  
Dual Culture ◽  
Ecological Zones ◽  
Gene Coding ◽  
Mycelia Growth ◽  
Biocontrol Potential ◽  
Surfactin Production

The characterization of surfactin-producing Bacillus subtilis isolates collected from different ecological zones of Iran is presented. Characterization was performed using blood agar, PCR, drop-collapse, and reverse-phase high-performance liquid chromatography (HPLC) analyses, and the isolates’ biocontrol effects against the aflatoxin-producing agent Aspergillus flavus and the citrus antracnosis agent Colletotrichum gloeosporioides were studied. In total, 290 B. subtilis isolates were isolated from phylosphere and rhizosphere samples collected from fields and gardens of 5 provinces of Iran. Blood agar assays showed that 185 isolates produced different biosurfactants. Isolates containing the sfp gene, coding for surfactin, were detected using the PCR method. It was found that 14 different isolates contained the sfp gene. Drop-collapse assays, which detect isolates with high production of surfactin, showed that 7 isolates produced high levels of surfactin. It was found from HPLC analysis that the isolates containin the sfp gene produced between 55 and 1610 mg of surfactin per litre of broth medium. Four isolates, named BS119m, BS116l, N3dn, and BS113c, produced more than 1000 mg of surfactin per litre of broth. The highest surfactin production level was observed for isolate BS119m (1610 mg/L). The antagonistic potential of the sfp gene-containing isolates was determined using dual culture and chloroform vapour methods. Our bioassay results indicated that isolate BS119m showed high inhibitory effects against A. flavus (100%) and C. gloeosporioides (88%). Furthermore, the effect of purified surfactin on the growth of A. flavus was evaluated. Mycelia growth was considerably reduced with increasing concentration of surfactin, and 36%, 54%, 84%, and 100% inhibitions of mycelia growth were, respectively, observed at 20, 40, 80, and 160 mg/L after 7 days of incubation.

scholarly journals Purification and partial biochemical characterization of normal human interleukin 1.

1984 ◽  
Vol 160 (3) ◽  
pp. 772-787 ◽  
Author(s):  
J A Schmidt
Keyword(s):  
High Performance ◽  
Biochemical Characterization ◽  
Interleukin 1 ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Thymocyte Proliferation ◽  
Exclusion Chromatography ◽  
Normal Human

A protocol for the rapid, efficient purification of the major charged species of human interleukin 1 (IL-1) has been developed using high performance anion exchange and size exclusion chromatography. The isolated material is pure as determined by sodium dodecyl sulfate (SDS) gradient polyacrylamide gel electrophoresis (PAGE) and analytical isoelectric focusing (IEF). The molecular weight of the purified material is 15,000 and the isoelectric point (pI) is 6.8, values that are in good agreement with those previously reported for human IL-1. 10(-10) M concentrations of the purified material give half-maximal stimulation in the thymocyte proliferation assay. Amounts of IL-1 sufficient for receptor studies and detailed biochemical analysis can now be produced on a regular basis.

Identification of ß-carotene producing bacteria from terrestrial habitats of Jaipur, Rajasthan

2019 ◽  
Vol 48 (4) ◽  
pp. 329-336
Author(s):  
Ankit Chokriwal ◽  
Bjorn John Stephen ◽  
Abhijeet Singh
Keyword(s):  
High Performance ◽  
Design Methodology ◽  
Biochemical Characterization ◽  
Dpph Assay ◽  
Content Type ◽  
Isolation And Purification ◽  
Ic50 Value ◽  
Terrestrial Habitats ◽  
Layer Chromatography

Purpose Carotenoids are pigments that have significant value as colorants and antioxidants in pharmaceutical, food, textile and cosmetic industries. Owing to its high demand, this study aims to identify ß-carotene producing bacteria from different terrestrial habitats of Jaipur region. Design/methodology/approach In this paper, standard isolation and purification process was used, followed by colony morphology and biochemical characterization of ß-carotene producing bacteria. ß-carotene concentration was determined quantitatively using spectrophotometric method. Findings Out of 43 isolates, 21 isolates showed peak range between 400 and 500 nm confirming the presence of carotenoids. Only one bacteria SAN-A has capacity to produce ß-carotene confirmed by the thin layer chromatography and high performance liquid chromatography (HPLC) with a yield of 1.68 mg/l. The 2, 2-Diphenyl-2-picrylhydrazil (DPPH) assay showed an IC50 value of 4.0 mg/ml. Originality/value The present study revealed the presence of ß-carotene producing bacteria in the soil of different terrestrial habitat of Jaipur region which can be exploited as an economical source for ß-carotene production.

scholarly journals Characterization of an Arginine:Pyruvate Transaminase in Arginine Catabolism of Pseudomonas aeruginosa PAO1

2007 ◽  
Vol 189 (11) ◽  
pp. 3954-3959 ◽  
Author(s):  
Zhe Yang ◽  
Chung-Dar Lu
Keyword(s):  
Pseudomonas Aeruginosa ◽  
High Performance ◽  
Biochemical Characterization ◽  
Physiological Function ◽  
Catalytic Efficiency ◽  
Kinetic Mechanism ◽  
Arginine Catabolism ◽  
Ping Pong ◽  
Coupled Reaction

ABSTRACT The arginine transaminase (ATA) pathway represents one of the multiple pathways for l-arginine catabolism in Pseudomonas aeruginosa. The AruH protein was proposed to catalyze the first step in the ATA pathway, converting the substrates l-arginine and pyruvate into 2-ketoarginine and l-alanine. Here we report the initial biochemical characterization of this enzyme. The aruH gene was overexpressed in Escherichia coli, and its product was purified to homogeneity. High-performance liquid chromatography and mass spectrometry (MS) analyses were employed to detect the presence of the transamination products 2-ketoarginine and l-alanine, thus demonstrating the proposed biochemical reaction catalyzed by AruH. The enzymatic properties and kinetic parameters of dimeric recombinant AruH were determined by a coupled reaction with NAD+ and l-alanine dehydrogenase. The optimal activity of AruH was found at pH 9.0, and it has a novel substrate specificity with an order of preference of Arg > Lys > Met > Leu > Orn > Gln. With l-arginine and pyruvate as the substrates, Lineweaver-Burk plots of the data revealed a series of parallel lines characteristic of a ping-pong kinetic mechanism with calculated V max and k cat values of 54.6 ± 2.5 μmol/min/mg and 38.6 ± 1.8 s−1. The apparent Km and catalytic efficiency (k cat/Km ) were 1.6 ± 0.1 mM and 24.1 mM−1 s−1 for pyruvate and 13.9 ± 0.8 mM and 2.8 mM−1 s−1 for l-arginine. When l-lysine was used as the substrate, MS analysis suggested Δ1-piperideine-2-carboxylate as its transamination product. These results implied that AruH may have a broader physiological function in amino acid catabolism.

scholarly journals Cloning and expression in Bacillus subtilis of the npr gene from Bacillus thermoproteolyticus Rokko coding for the thermostable metalloprotease thermolysin

1994 ◽  
Vol 300 (2) ◽  
pp. 599-603 ◽  
Author(s):  
M J O'Donohue ◽  
B P Roques ◽  
A Beaumont
Keyword(s):  
Bacillus Subtilis ◽  
Bacillus Stearothermophilus ◽  
Biochemical Characterization ◽  
Signal Sequence ◽  
Cloning And Expression ◽  
Bacillus Species ◽  
Gene Coding ◽  
Strong Homology ◽  
Different Strains

We report the isolation, cloning and expression, in Bacillus subtilis, of the gene coding for thermolysin, a thermostable metalloprotease which is produced by Bacillus thermoproteolyticus Rokko. The nucleotide sequence has revealed that, like neutral proteases produced by other members of the Bacillus species, thermolysin is probably produced as a preproenzyme carrying a typical N-terminal membrane signal sequence. Further, the thermolysin gene shares a strong homology with two other previously cloned genes from two different strains of Bacillus stearothermophilus. The sequence of the mature secreted protease, inferred from the DNA sequence, is, with two exceptions, identical with the previously published protein sequence of thermolysin [Titani, Hermodson, Ericsson, Walsh and Neurath (1972) Nature (London) 238, 35-37]. The exceptions are Asn37 and Gln119, originally reported to be Asp and Glu respectively. The biochemical characterization of the secreted recombinant protein shows that it is indistinguishable from the wild-type thermolysin.

scholarly journals Biochemical Characterization of Bovine Brain Myristoyl-CoA:Protein N-Myristoyltransferase Type 2

2009 ◽  
Vol 2009 ◽  
pp. 1-9 ◽  
Author(s):  
Ponniah Selvakumar ◽  
Ashakumary Lakshmikuttyamma ◽  
Rajendra K. Sharma
Keyword(s):  
Metal Ion ◽  
Biochemical Characterization ◽  
Kinetic Studies ◽  
Bovine Brain ◽  
Covalent Attachment ◽  
Reading Frame ◽  
Gene Coding ◽  
Acid Protein

Protein N-myristoylation is a lipidic modification which refers to the covalent attachment of myristate, a 14-carbon saturated fatty acid, to the N-terminal glycine residue of a number of mammalian, viral, and fungal proteins. In this paper, we have cloned the gene coding for myristoyl-CoA:protein N-myristoyltransferase (NMT) fromBos tarusbrain. The open reading frame codes for a 410-amino-acid protein and overexpressed inEscherichia coli. Kinetic studies suggested that bovine brain NMT2 and human NMT1 show significant differences in their peptide substrate specificities. The metal ionCa2+had stimulatory effects on NMT2 activity whileMn2+andZn2+inhibited the enzyme activity. In addition, NMT2 activity was inhibited by various organic solvents and other detergents while NMT1 had a stimulatory effect. Biochemical characterization suggested that both forms of NMT have unique characteristics. Further analysis towards functional role NMT2 will lead the development of therapeutic target for the progression of various diseases such as cancer, cardiovascular diseases, and neurodegenerative diseases.

Isolation, Purification and Biochemical Characterization of Human Placental Interferons by Tandem High-Performance Affinity Chromatography

1992 ◽  
Vol 22 (2) ◽  
pp. 105-121 ◽  
Author(s):  
George Aboagye-Mathiesen ◽  
Ferenc D. Tóth ◽  
Anne Mette Dalsgaard ◽  
Peter M. Petersen ◽  
Vladimir Zachar ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
High Performance ◽  
Biochemical Characterization ◽  
High Performance Affinity Chromatography

scholarly journals The cbiS Gene of the Archaeon Methanopyrus kandleri AV19 Encodes a Bifunctional Enzyme with Adenosylcobinamide Amidohydrolase and α-Ribazole-Phosphate Phosphatase Activities

2006 ◽  
Vol 188 (12) ◽  
pp. 4227-4235 ◽  
Author(s):  
Jesse D. Woodson ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
High Performance ◽  
Biochemical Characterization ◽  
The Other ◽  
Bifunctional Enzyme ◽  
Visible Spectroscopy ◽  
Uv Visible ◽  
Cobyric Acid ◽  
Methanopyrus Kandleri

ABSTRACT Here we report the initial biochemical characterization of the bifunctional α-ribazole-P (α-RP) phosphatase, adenosylcobinamide (AdoCbi) amidohydrolase CbiS enzyme from the hyperthermophilic methanogenic archaeon Methanopyrus kandleri AV19. The cbiS gene encodes a 39-kDa protein with two distinct segments, one of which is homologous to the AdoCbi amidohydrolase (CbiZ, EC 3.5.1.90) enzyme and the other of which is homologous to the recently discovered archaeal α-RP phosphatase (CobZ, EC 3.1.3.73) enzyme. CbiS function restored AdoCbi salvaging and α-RP phosphatase activity in strains of the bacterium Salmonella enterica where either step was blocked. The two halves of the cbiS genes retained their function in vivo when they were cloned separately. The CbiS enzyme was overproduced in Escherichia coli and was isolated to >95% homogeneity. High-performance liquid chromatography, UV-visible spectroscopy, and mass spectroscopy established α-ribazole and cobyric acid as the products of the phosphatase and amidohydrolase reactions, respectively. Reasons why the CbiZ and CobZ enzymes are fused in some archaea are discussed.

Purification and Biochemical Characterization of Trypsin Inhibitor from Oyster

2012 ◽  
Vol 577 ◽  
pp. 119-124
Author(s):  
Yuan Hui Zhao ◽  
Ming Yong Zeng ◽  
Xia Li
Keyword(s):  
Liquid Chromatography ◽  
Trypsin Inhibitor ◽  
High Performance ◽  
Biochemical Characterization ◽  
Gel Filtration ◽  
Reversed Phase ◽  
Phase Liquid ◽  
Lung Adenocarcinoma A549 ◽  
Head Inhibitor

In this paper, the purification and biochemical characterization of the endogenous oyster (Crassostrea gigas) trypsin inhibitor were researched. A oyster trypsin inhibitor（OTI）has been purified by successive ammonium sulfate precipitation, gel filtration, affinity chromatography and high performance reversed-phase liquid chromatography. OTI has a molecular weight of approximately 5036 Da estimated by high performance size exclusive liquid chromatography. OTI was heat-, acid- and basic-stable competitive trypsin inhibitor. And OTI was double-head inhibitor with the inhibition constant (Ki) value of 1.644×10-2 mmol L-1. OTI was composed of nine kinds of amino acid, and rich in cysteine, alanine and glutamic acid. Furthermore, OTI can inhibit the proliferations of human lung adenocarcinoma A549 cell and human cervical cancer Hela cell

scholarly journals Discovery and Biochemical Characterization of a Novel Polyesterase for the Degradation of Synthetic Plastics

2020 ◽  
Vol 2 (1) ◽  
pp. 33
Author(s):  
Efstratios Nikolaivits ◽  
Phaedra Dimopoulou ◽  
Veselin Maslak ◽  
Jasmina Nikodinovic-Runic ◽  
Evangelos Topakas
Keyword(s):  
Food Web ◽  
Polyethylene Terephthalate ◽  
Enzymatic Degradation ◽  
Environmental Problem ◽  
Biochemical Characterization ◽  
Plastic Waste ◽  
Degrading Enzyme ◽  
Gene Coding

Plastic waste poses an enormous environmental problem as a result of soil and ocean contamination, causing the release of microplastics that end up in humans through the food web. Enzymatic degradation of plastics has emerged as an alternative to traditional recycling processes. In the present work, we used bioinfomatics tools to discover a gene coding for a putative polyester degrading enzyme (polyesterase). The gene was heterologously expressed, purified and biochemically characterized. Furthermore, its ability to degrade polyethylene terephthalate (PET) model substrates and synthetic plastics was assessed.

scholarly journals Screening of Fungal Endophytes for their Biocontrol Potential against Rhizopus sp. Isolated from Diseased Cassava (Manihot esculenta Crantz)

2021 ◽  
Vol 4 (2) ◽  
pp. 25-37
Author(s):  
Onyemaechi H.O. ◽  
Obehi V.O. ◽  
Felix O.
Keyword(s):  
Culture Filtrate ◽  
Fungal Endophytes ◽  
Dual Culture ◽  
Yeast Isolate ◽  
Manihot Esculenta Crantz ◽  
Mycelia Growth ◽  
Dual Culture Assay ◽  
Fusarium Sp ◽  
Biocontrol Potential ◽  
Rhizopus Sp

The aim of this study was to screen for the bio-control potential of fungal endophytes isolated from cassava against a test pathogen of cassava. Fungal endophytes and pathogen were isolated and identified from healthy and diseased cassava respectively. The isolated fungal endophytes were screened for their biocontrol potential against a test pathogen using the dual culture and culture filtrate assay. Fusarium sp., Botryosphaeria sp., Colletotrichum sp., yeast isolate 1 and 2 were the isolated fungal endophytes while the pathogen was Rhizopus sp. The effect of endophytes on the mycelia growth of Rhizopus sp. using the dual culture assay indicated that yeast isolate 1 & 2 and Colletotrichum sp. were effective in inhibiting the mycelia growth of the test pathogen while Fusarium sp. and Botryosphaeria sp. were not effective. The co-culture of yeast isolate 2 with the test pathogen gave the lowest mycelia growth (1.66a±0.09) at day 2. The effect of endophytic culture filtrate on the mycelia growth of Rhizopus sp. showed that Fusarium sp. gave the lowest mycelia growth in the three days observed. The findings from this study suggested that the test endophytes have biocontrol potential against Rhizopus sp. The biocontrol abilities of the test endophytes vary using the dual culture and culture filtrate assay.

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