scholarly journals Purification and Characterization of a Novel Antifungal Flagellin Protein from Endophyte Bacillus methylotrophicus NJ13 against Ilyonectria robusta

2019 ◽  
Vol 7 (12) ◽  
pp. 605 ◽  
Author(s):  
Yun Jiang ◽  
Chao Ran ◽  
Lin Chen ◽  
Wang Yin ◽  
Yang Liu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gene Sequence ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Inhibitory Effect ◽  
Exchange Chromatography ◽  
Antifungal Protein ◽  
Bacillus Methylotrophicus ◽  
Cloned Gene

Endophyte Bacillus methylotrophicus NJ13 was isolated from Panax ginseng. Its sterile fermentation liquid showed a significant inhibitory effect against Ilyonectria robusta, causing the rusty root rot of P. ginseng and P. quinquefolius. The antifungal protein was obtained after precipitation by 20% saturated ammonium sulfate, desalted by Sephadex G-25, weak anion exchange chromatography, and gel filtration chromatography. SDS-PAGE showed that the purified protein was approximately 29 KDa. The antifungal protein after desalting was not resistant to temperatures higher than 100 °C, resistant to acid conditions, and did not tolerate organic solvents and protease K. The amino acid sequence of purified antifungal protein had an identity of 76% to flagellin from Bacillus velezensis. The isoelectric point of the protein was 4.97 and its molecular mass was 27 KDa. Therefore, a specific primer G1 was designed based on the flagellin gene sequence, and a 770 bp gene sequence was cloned in NJ13 genomic DNA, which shared the same size of flagellin. There were ten base differences between the gene sequences of flagellin and the cloned gene, however, the amino acid sequence encoded by the cloned gene was identical to the flagellin. In conclusion, the antifungal protein produced by biocontrol agent NJ13 contained a flagellin protein.

Purification and Amino Acid Sequence of Fructose-1,6-bisphosphate Aldolase from the Electric Organ of Electrophorus electricus (L.)

2006 ◽  
Vol 61 (11-12) ◽  
pp. 884-888
Author(s):  
Salvatore G. De-Simone ◽  
Christiane M. Cardoso de Salles ◽  
Celia M. Batistae Silva ◽  
Aida Hassón-Voloch
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gel Filtration ◽  
Electric Organ ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
High Homology ◽  
Electrophorus Electricus ◽  
One Step ◽  
Fructose 1,6 Bisphosphate

Abstract A soluble fructose-1,6-bisphosphate aldolase enzyme has been purified 50.2-fold (2.36%) at the homogeneity from the electric organ of Electrophorus electricus by one step of DEAE- 52 anion exchange chromatography followed by Superose-12 gel filtration-FPLC. Like other aldolase enzymes the E. electricus protein is a dimer with two identical subunits of 45 kDa. The N-terminal (20 residues) revealed a high homology with S. aurata (75%, goldfish), R. ratus and M. musculus (mouse, 80%) enzymes.

scholarly journals Chemical identity of tryptensin with angiotensin

1980 ◽  
Vol 187 (3) ◽  
pp. 647-653 ◽  
Author(s):  
K Arakawa ◽  
M Yuki ◽  
M Ikeda
Keyword(s):  
Amino Acid ◽  
Thin Layer ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Human Plasma Protein ◽  
Plasma Protein Fraction ◽  
Layer Chromatography

Tryptensin, a vasopressor substance generated from human plasma protein fraction IV-4 by trypsin, has been isolated and the amino acid composition analysed. The procedures used for the isolation were: (a) adsorption of the formed tryptensin on Dowex 50W (X2; NH4+ form); (b) gel filtration through Sephadex G-25; (c) cation-exchange chromatography on CM-cellulose; (d) anion-exchange chromatography on DEAE-cellulose; (e) re-chromatography on CM-cellulose; (f) gel filtration on Bio-Gel P-2; (g) partition chromatography on high-pressure liquid chromatography. The homogeneity of the isolated tryptensin was confirmed by thin-layer chromatography and thin-layer electrophoresis. The amino acid analysis of the hydrolysate suggested the following proportional composition: Asp, 1; Val, 1; Ile, 1; Tyr, 1; Phe, 1; His, 1; Arg, 1; Pro, 1. This composition is identical with that of human angiotensin.

Physicochemical properties of a novel α-L-arabinofuranosidase fromRhizomucor pusillusHHT-1

2001 ◽  
Vol 47 (8) ◽  
pp. 767-772 ◽  
Author(s):  
A KM Shofiqur Rahman ◽  
Shinya Kawamura ◽  
Masahiro Hatsu ◽  
M M Hoq ◽  
Kazuhiro Takamizawa
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Rhizomucor Pusillus ◽  
Terminal Amino ◽  
Metal Cofactors

The zygomycete fungus Rhizomucor pusillus HHT-1, cultured on L(+)arabinose as a sole carbon source, produced extracellular α-L-arabinofuranosidase. The enzyme was purified by (NH4)2SO4fractionation, gel filtration, and ion exchange chromatography. The molecular mass of this monomeric enzyme was 88 kDa. The native enzyme had a pI of 4.2 and displayed a pH optimum and stability of 4.0 and 7.0–10.0, respectively. The temperature optimum was 65°C, and it was stable up to 70°C. The Kmand Vmaxfor p-nitrophenyl α-L-arabinofuranoside were 0.59 mM and 387 µmol·min–1·mg–1protein, respectively. Activity was not stimulated by metal cofactors. The N-terminal amino acid sequence did not show any similarity to other arabinofuranosidases. Higher hydrolytic activity was recorded with p-nitrophenyl α-L-arabinofuranoside, arabinotriose, and sugar beet arabinan; lower hydrolytic activity was recorded with oat–spelt xylan and arabinogalactan, indicating specificity for the low molecular mass L(+)-arabinose containing oligosaccharides with furanoside configuration.Key words: α-L-arabinofuranosidase, enzyme purification, amino acid sequence, Rhizomucor pusillus.

Serine protease isoforms of Deinagkistrodon acutus venom: cloning, sequencing and phylogenetic analysis

2001 ◽  
Vol 354 (1) ◽  
pp. 161-168 ◽  
Author(s):  
Ying-Ming WANG ◽  
Suei-Rong WANG ◽  
Inn-Ho TSAI
Keyword(s):  
Amino Acid ◽  
Serine Proteases ◽  
Parallel Evolution ◽  
Gel Filtration ◽  
Venom Gland ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Single Chain

The major coagulating fibrinogenase of Deinagkistrdon acutus venom, designated acutobin, was purified by anion-exchange chromatography, gel filtration and reverse-phase HPLC. Approximately 80% of its protein sequence was determined by sequencing the various fragments derived from CNBr cleavage and digestion with endoprotease. Extensive screening of the venom gland cDNA species after amplification by PCR resulted in the isolation of four distinct cDNA clones encoding acutobin and three other serine proteases, designated Dav-PA, Dav-KN and Dav-X. The complete amino acid sequences of these enzymes were deduced from the cDNA sequences. The amino-acid sequence of acutobin contains a single chain of 236 residues including four potential N-glycosylation sites. The purified acutobin (40kDa) contains approx. 30% carbohydrate by weight, which could be partly removed by N-glycanase. The phylogenetic tree of the complete amino acid sequences of 40 serine proteases from 18 species of Crotalinae shows functional clusters reflecting parallel evolution of the three major venom enzyme subtypes: coagulating enzymes, kininogenases and plasminogen activators. The possible structural elements responsible for the functional specificity of each subtype are discussed.

scholarly journals Studies on Marsupial Protein. II. Amino Acid Sequence of the -Chain of Haemoglobin from the Grey Kangaroo, Macropus Giganteus

1969 ◽  
Vol 22 (6) ◽  
pp. 1437 ◽  
Author(s):  
GM Air ◽  
EOP Thompson
Keyword(s):  
Amino Acid ◽  
Ion Exchange ◽  
Amino Acid Sequence ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Tryptic Digestion ◽  
Grey Kangaroo

The amino acid sequence of the jS-chain of haemoglobin from M. giganteus has been determined. The soluble peptides formed by tryptic digestion were isolated by gel filtration, ion-exchange chromatography, and paper ionophoresis, and amino acid sequences determined by the "dansyl"-Edman procedure. Special procedures were necessary for three peptides which were insoluble.

Purification, properties, and partial amino acid sequence of chitinase from a marine Alteromonas sp. strain O-7

1992 ◽  
Vol 38 (9) ◽  
pp. 891-897 ◽  
Author(s):  
Hiroshi Tsujibo ◽  
Yukio Yoshida ◽  
Katsushiro Miyamoto ◽  
Chiaki Imada ◽  
Yoshiro Okami ◽  
...  
Keyword(s):  
Amino Acid ◽  
Marine Bacterium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Residues ◽  
Amino Terminal

Chitinase (EC 3.2.1.14) was isolated from the culture supernatant of a marine bacterium, Alteromonas sp. strain O-7. The enzyme (Chi-A) was purified by anion-exchange chromatography (DEAE-Toyopearl 650 M) and gel filtration (Sephadex G-100). The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of Chi-A were 70 kDa and 3.9, respectively. The optimum pH and temperature of Chi-A were 8.0 and 50 °C, respectively. Chi-A was stable in the range of pH 5–10 up to 40 °C. Among the main cations, such as Na+, K+, Mg2+, and Ca2+, contained in seawater, Mg2+ stimulated Chi-A activity. N-Bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide inhibited Chi-A activity. The amino-terminal 27 amino acid residues of Chi-A were sequenced. This enzyme showed sequence homology with chitinases from terrestrial bacteria such as Serratia marcescens QMB1466 and Bacillus circulons WL-12. Key words: marine bacterium, Alteromonas sp., chitinase.

scholarly journals Myoglobins of Cartilaginous Fishes III. Amino Acid Sequence of Myoglobin of the Shark Galeorhinus Australis

1981 ◽  
Vol 34 (1) ◽  
pp. 5 ◽  
Author(s):  
WK Fisher ◽  
DD Koureas ◽  
EOP Thompson
Keyword(s):  
Amino Acid ◽  
Ion Exchange ◽  
Amino Acid Sequence ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Red Muscle ◽  
Cartilaginous Fishes ◽  
Port Jackson

Myoglobin isolated from the red muscle of the school shark Galeorhinus australis was purified by gel filtration and ion-exchange chromatography. The amino acid sequence was determined following digestion with trypsin and purification of the peptides by paper ionophoresis and chromatography. Sequences of purified peptides were determined by the dansyl-Edman procedure and the peptides aligned by homology with the sequence of the myoglobin of the gummy shark Mustelus antarcticus. The two myoglobin sequences showed a marked similarity (16 differences), but both sequences showed approximately the same number of differences (68) from myoglobin of the Port Jackson shark Heterodontus portusjacksoni. There are 19 residues unique to the three shark myoglobin sequences.

A novel bacteriocin-like substance (BLIS) from a pathogenic strain of Vibrio harveyi

Microbiology ◽  
2005 ◽  
Vol 151 (9) ◽  
pp. 3051-3058 ◽  
Author(s):  
Sathish Prasad ◽  
Peter C. Morris ◽  
Rasmus Hansen ◽  
Philip G. Meaden ◽  
Brian Austin
Keyword(s):  
Amino Acid ◽  
Vibrio Fischeri ◽  
Vibrio Harveyi ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Proteinase K ◽  
Broth Culture ◽  
Two Dimensional Gel Electrophoresis ◽  
Flight Mass Spectrometry

Inter-strain and inter-species inhibition mediated by a bacteriocin-like inhibitory substance (BLIS) from a pathogenic Vibrio harveyi strain VIB 571 was demonstrated against four isolates of the same species, and one culture each of a Vibrio sp., Vibrio fischeri, Vibrio gazogenes and Vibrio parahaemolyticus. The crude BLIS, which was obtained by ammonium-sulphate precipitation of the cell-free supernatant of a 72 h broth culture of strain VIB 571, was inactivated by lipase, proteinase K, pepsin, trypsin, pronase E, SDS and incubation at ≥60 °C for 10 min. The activity was stable between pH 2–11 for at least 5 h. Anion-exchange chromatography, gel filtration, SDS-PAGE and two-dimensional gel electrophoresis revealed the presence of a single major peak, comprising a protein with a pI of ∼5·4 and a molecular mass of ∼32 kDa. The N-terminal amino acid sequence of the protein comprised Asp-Glu-Tyr-Ile-Ser-X-Asn-Lys-X-Ser-Ser-Ala-Asp-Ile (with X representing cysteine or modified amino acid residues). A similarity search based on the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) generated peptide masses and the N-terminal sequence did not yield any significant matches.

scholarly journals Expression and Characterization of (R)-Specific Enoyl Coenzyme A Hydratase Involved in Polyhydroxyalkanoate Biosynthesis by Aeromonas caviae

1998 ◽  
Vol 180 (3) ◽  
pp. 667-673 ◽  
Author(s):  
Toshiaki Fukui ◽  
Naofumi Shiomi ◽  
Yoshiharu Doi
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Coenzyme A ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Pha Synthase ◽  
T7 Promoter ◽  
Aeromonas Caviae ◽  
One Step ◽  
Enoyl Coa Hydratase

ABSTRACT Complementation analysis of a polyhydroxyalkanoate (PHA)-negative mutant of Aeromonas caviae proved that ORF3 in thepha locus (a 402-bp gene located downstream of the PHA synthase gene) participates in PHA biosynthesis on alkanoic acids, and the ORF3 gene is here referred to as phaJAc. Escherichia coli BL21(DE3) carryingphaJAc under the control of the T7 promoter overexpressed enoyl coenzyme A (enoyl-CoA) hydratase, which was purified by one-step anion-exchange chromatography. The N-terminal amino acid sequence of the purified hydratase corresponded to the amino acid sequence deduced from the nucleotide sequence ofphaJAc except for the initial Met residue. The enoyl-CoA hydratase encoded by phaJAc exhibited (R)-specific hydration activity towardtrans-2-enoyl-CoA with four to six carbon atoms. These results have demonstrated that (R)-specific hydration of 2-enoyl-CoA catalyzed by the translated product ofphaJAc is a channeling pathway for supplying (R)-3-hydroxyacyl-CoA monomer units from fatty acid β-oxidation to poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis in A. caviae.

scholarly journals Cloning of a Beta-Hemolysin Gene ofBrachyspira (Serpulina) hyodysenteriaeand Its Expression in Escherichia coli

2001 ◽  
Vol 69 (2) ◽  
pp. 706-711 ◽  
Author(s):  
Tsungda Hsu ◽  
David L. Hutto ◽  
F. Chris Minion ◽  
Richard L. Zuerner ◽  
Michael J. Wannemuehler
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gene Sequence ◽  
Binding Motif ◽  
Terminal Amino Acid ◽  
Hemolytic Toxin ◽  
Terminal Amino ◽  
Cloned Gene ◽  
Terminal Amino Acid Sequence

ABSTRACT Brachyspira (Serpulina)hyodysenteriae induces a mucohemorrhagic diarrheal disease in pigs. The production of a beta-hemolysin has been considered a major virulence attribute of this organism. Previous reports have failed to correlate a specific cloned gene sequence with a purified beta-hemolytic protein sequence. Thus, questions still remain concerning the structural gene sequence of the hemolysin. To answer this question unequivocally, the beta-hemolytic toxin was purified from extracts of log-phase spirochetes, and the N-terminal amino acid sequence was determined (K-D-V-V-A-N-Q-L-N-I-S-D-K) and compared with the translated sequences of previously cloned genes, tlyAto tlyC. The lack of homology between tlyA totlyC translated sequences and the purified beta-hemolytic toxin sequence resulted in the study that is reported here. A degenerate probe was designed based on the N-terminal amino acid sequence of the purified beta-hemolysin and used to screen a B. hyodysenteriae genomic library. Three overlapping clones were identified, and one was sequenced to reveal an open reading frame coding for a putative 8.93-kDa polypeptide containing the N-terminal sequence of the purified beta-hemolysin. To distinguish this gene from the tlyA to tlyC genes, it has been designatedhlyA. A hemolysis-negative Escherichia colistrains containing hlyA was beta-hemolytic on blood agar media. Also, the hemolytic activity of the recombinant protein had identical protease and lipase sensitivities and electrophoretic mobility to those of native B. hyodysenteriaebeta-hemolysin. Based on sequence analysis, the translated protein had a pI of 4.3, an α-helical structure, and a phosphopantetheine binding motif. Hybridization analysis of genomic DNA indicated that thehlyA gene was present in B. hyodysenteriae andB. intermedia but was not detected in B. innocens, B. pilosicoli, or B. murdochiiunder high-stringency conditions. The location of hlyA on the chromosomal map was distinct from the locations oftlyA, tlyB, and tlyC.

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