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.

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 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.

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.

Separation of bovine κ-casein glycomacropeptide from sweet whey protein products with undetectable level of phenylalanine by protein precipitation followed by anion exchange chromatography

2018 ◽  
Vol 85 (1) ◽  
pp. 110-113 ◽  
Author(s):  
Takuo Nakano ◽  
Lech Ozimek ◽  
Mirko Betti
Keyword(s):  
Heat Treatment ◽  
Amino Acid ◽  
Whey Protein ◽  
Anion Exchange ◽  
Large Scale ◽  
Dry Weight ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Shift ◽  
Sweet Whey

Bovine κ-casein glycomacropeptide (GMP) found in sweet whey is a 64 amino acid residue glycopeptide, which does not contain phenylalanine or other aromatic amino acids. There is, however, little information available concerning isolation of phenylalanine free GMP from sweet whey. In the study reported in this Research Communication, GMP was purified from three samples of sweet whey protein products (SWPP) by a procedure involving: (1) precipitation of protein by heat treatment; (2) precipitation of protein by pH shift to 4·6; and (3) diethylaminoethyl (DEAE)-Sephacel anion exchange chromatography of soluble portion of each sample obtained after removal of protein precipitates. The total protein precipitated with both heat treatment and pH shift accounted for average 61% of dry weight of SWPP. The GMP fraction obtained by DEAE-Sephacel chromatography accounted for average 7·5% of dry weight of SWPP. Amino acid analysis showed that there was no detectable level of phenylalanine in GMP fractions from all samples examined. The present method may help develop large scale methods of production of GMP.

Antithrombin Milano, Single Amino Acid Substitution at the Reactive Site, Arg393 to Cys

1988 ◽  
Vol 60 (03) ◽  
pp. 471-475 ◽  
Author(s):  
H Erdjument ◽  
D A Lane ◽  
H Ireland ◽  
V Di Marzo ◽  
M Panico ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Fast Atom Bombardment ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Reducing Conditions ◽  
Sds Page ◽  
Two Peaks

SummaryAntithrombin Milano is an unusual antithrombin variant, exhibiting an abnormal, fast moving component on crossed immunoelectrophoresis (in the absence of heparin). Antithrombin isolated from the propositus could be resolved into two peaks on anion-exchange chromatography; anti thrombin Milano peak 1 of Mr ∼60,000 which could inhibit thrombin, and antithrombin Milano peak 2 of Mr ∼120,000 which was inactive. The latter component also reacted with antisera to both antithrombin and albumin on immunoblotting. Under reducing conditions, the ∼120,000 Mr component migrated on SDS-PAGE as two distinct bands with Mr ∼60,000, one of which reacted with antiserum to antithrombin and the other (of slower mobility) of which reacted with antiserum to albumin only. These and other results established the ∼120,000 Mr component to be an inactive, disulphide-linked variant antithrombin and albumin complex. The variant antithrombin was isolated, following reduction and S-carboxy-methylation, by reverse-phase HPLC and then it was fragmented with CNBr. A major CNBr pool containing the sequence Gly339-Met423 was treated with trypsin, followed by V8 protease. The resulting peptides were analysed by fast atom bombardment mass spectrometry (Fab-MS) mapping. A peptide of molecular mass 1086, corresponding to the normal sequence Ala382-Arg393, was almost absent from the mass spectrum, but an additional peptide of mass number 1772 was present. These results are almost identical to those found in another variant antithrombin, North-wick Park (Erdjument et al., J Biol Chem, 262: 13381, 1987; Erdjument et al., J Biol Chem, 263: 5589-5593, 1988), indicating the same single amino acid substitution of Arg393 to Cys.

Molecular cloning of chicken calcyclin (S100A6) and identification of putative isoforms

1997 ◽  
Vol 75 (6) ◽  
pp. 733-738 ◽  
Author(s):  
Bruce G Allen ◽  
Jacquelyn E Andrea ◽  
Cindy Sutherland ◽  
Brett O Schönekess ◽  
Michael P Walsh
Keyword(s):  
Smooth Muscle ◽  
Amino Acid ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Chain Reaction ◽  
Proteolytic Fragment ◽  
Chicken Gizzard ◽  
Muscle Tissues ◽  
Bona Fide ◽  
Polymerase Chain

A full-length cDNA encoding smooth muscle calcyclin (S100A6) was cloned from chicken gizzard, using reverse transcription - polymerase chain reaction techniques. The deduced amino acid sequence contains 92 residues with 12 substitutions and a 2 amino acid C-terminal extension when compared with human calcyclin. Calcyclin was purified from chicken gizzard by Ca2+-dependent hydrophobic chromatography, heat treatment, and anion-exchange chromatography. N-terminal sequencing of two CNBr peptides confirmed its identity as calcyclin. Two isoforms of calcyclin (A and B), which differ with respect to the presence or absence of a C-terminal lysine, were identified and the native protein was shown to exist as noncovalently associated homodimers (AA and BB) and heterodimers (AB). Incubation of purified calcyclin AA with an extract of chicken gizzard did not result in degradation of calcyclin A or appearance of calcyclin B, suggesting that calcyclin B is a bona fide isoform rather than a proteolytic fragment generated during purification. Western blotting of chicken tissues with anti-(gizzard calcyclin) indicated abundant expression of calcyclin in smooth muscle tissues, including esophagus, large intestine, and trachea, with lower levels in lung, heart, kidney, and brain, and none detectable in liver or skeletal muscle.Key words: Ca2+-binding proteins, calcyclin, smooth muscle, cDNA cloning, isoforms.

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 Isolation of four forms of acetone-induced cytochrome P-450 in chicken liver by h.p.l.c. and their enzymic characterization

1990 ◽  
Vol 269 (1) ◽  
pp. 85-91 ◽  
Author(s):  
J F Sinclair ◽  
S Wood ◽  
L Lambrecht ◽  
N Gorman ◽  
L Mende-Mueller ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Chicken Liver ◽  
Terminal Amino Acid ◽  
Catalytic Activities ◽  
Terminal Amino ◽  
Cytochrome P 450 ◽  
Terminal Amino Acid Sequence

The purpose of this study was to purify and characterize the forms of cytochrome P-450 induced in chicken liver by acetone or ethanol. Using high performance liquid ion-exchange chromatography, we were able to isolate at least four different forms of cytochrome P-450 which were induced by acetone in chicken liver. All four forms of cytochrome P-450 proved to be distinct proteins, as indicated by their N-terminal amino acid sequences and their reconstituted catalytic activities. Two of these forms, also induced by glutethimide in chicken embryo liver, appeared to be cytochromes P450IIH1 and P450IIH2. Both of these cytochromes P-450 have identical catalytic activities towards benzphetamine demethylation. However, they differ in their abilities to hydroxylate p-nitrophenol and to convert acetaminophen into a metabolite that forms a covalent adduct with glutathione at the 3-position. Another form of cytochrome P-450 induced by acetone is highly active in the hydroxylation of p-nitrophenol and in the conversion of acetaminophen to a reactive metabolite, similar to reactions catalysed by mammalian cytochrome P450IIE. Yet the N-terminal amino acid sequence of this form has only 30-33% similarity with cytochrome P450IIE purified from rat, rabbit and human livers. A fourth form of cytochrome P-450 was identified whose N-terminal amino acid sequence and enzymic activities do not correspond to any mammalian cytochromes P-450 reported to be induced by acetone or ethanol.

Disulphide arrangement in bovine caseins: localization of intrachain disulphide bridges in monomers of κ- and αs2-casein from bovine milk

1994 ◽  
Vol 61 (4) ◽  
pp. 485-493 ◽  
Author(s):  
Lone K. Rasmussen ◽  
Peter Højrup ◽  
Torben E. Petersen
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Bovine Milk ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequence Analysis ◽  
Naturally Occurring ◽  
Localize Potential

SummaryNaturally occurring monomeric κ-casein and αs2-casein in bovine milk were purified by ion-exchange chromatography in order to localize potential intrachain disulphide bridges. Enzymic cleavage of the proteins followed by mass spectrometry and amino acid sequence analysis of cystine-containing peptides revealed the presence of an intrachain disulphide bond in both proteins.

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