scholarly journals Expression of cmg1, an Exo-β-1,3-Glucanase Gene from Coniothyrium minitans, Increases during Sclerotial Parasitism

2001 ◽  
Vol 67 (2) ◽  
pp. 865-871 ◽  
Author(s):  
Gábor Giczey ◽  
Zoltán Kerényi ◽  
László Fülöp ◽  
László Hornok
Keyword(s):  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Single Copy ◽  
Amino Acid Sequences ◽  
Signal Peptidase ◽  
Coniothyrium Minitans ◽  
Calculated Molecular Mass ◽  
Secretion Signal

ABSTRACT During sclerotial infection of Sclerotinia sclerotiorumthe mycoparasite Coniothyrium minitans penetrates through the host cell wall, which contains β-1,3-glucan as its major component. A PCR-based strategy was used to clone a β-1,3-glucanase-encoding gene, designated cmg1, from a cDNA library of the fungus. The nucleotide and deduced amino acid sequences of this gene showed high levels of similarity to the sequences of other fungal exo-β-1,3-glucanase genes. The calculated molecular mass of the deduced protein (without the predicted 24-amino-acid N-terminal secretion signal peptide) was 83,346 Da, and the estimated pI was 4.73. Saccharomyces cerevisiaeINVSc1 expressing the cmg1 gene secreted a ∼100-kDa β-1,3-glucanase enzyme (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) into the culture medium. N-terminal sequence analysis of the purified recombinant enzyme revealed that the secreted enzyme starts at Ala-32, seven amino acids downstream from the predicted signal peptidase cleavage site. The purified recombinant glucanase inhibited in vitro mycelial growth ofS. sclerotiorum by 35 and 85% at concentrations of 300 and 600 μg ml−1, respectively. A single copy of thecmg1 gene is present in the genome of C. minitans. Northern analyses indicated increases in the transcript levels of cmg1 due to both carbon starvation and the presence of ground sclerotia of S. sclerotiorum; only slight repression was observed in the presence of 2% glucose. Expression of cmg1 increased during parasitic interaction with S. sclerotiorum.

scholarly journals Cloning and Disruption of pgx4 Encoding an In Planta Expressed Exopolygalacturonase from Fusarium oxysporum

2000 ◽  
Vol 13 (4) ◽  
pp. 359-365 ◽  
Author(s):  
F. I. García-Maceira ◽  
Antonio Di Pietro ◽  
M. Isabel G. Roncero
Keyword(s):  
Amino Acid ◽  
Fusarium Oxysporum ◽  
Vascular Tissue ◽  
Single Copy ◽  
Gene Replacement ◽  
Amino Acid Sequences ◽  
In Planta ◽  
Calculated Molecular Mass ◽  
Tomato Plants

Fusarium oxysporum f. sp. lycopersici, the causal agent of tomato vascular wilt, produces an array of pectinolytic enzymes, including at least two exo-α1,4-polygalac-turonases (exoPGs). A gene encoding an exoPG, pgx4, was isolated with degenerate polymerase chain reaction primers derived from amino acid sequences conserved in two fungal exoPGs. pgx4 encodes a 454 amino acid polypeptide with nine potential N-glycosylation sites and a putative 21 amino acid N-terminal signal peptide. The deduced mature protein has a calculated molecular mass of 47.9 kDa, a pI of 8.0, and 51 and 49% identity with the exoPGs of Cochliobolus carbonum and Aspergillus tubingensis, respectively. The gene is present in a single copy in different formae speciales of F. oxysporum. Expression of pgx4 was detected during in vitro growth on pectin, polygalacturonic acid, and tomato vascular tissue and in roots and stems of tomato plants infected by F. oxysporum f. sp. lycopersici. Two mutants of F. oxy-sporum f. sp. lycopersici with a copy of pgx4 inactivated by gene replacement were as virulent on tomato plants as the wild-type strain.

scholarly journals Molecular and Biochemical Analysis of Two β-Galactosidases from Bifidobacterium infantisHL96

2001 ◽  
Vol 67 (9) ◽  
pp. 4256-4263 ◽  
Author(s):  
Ming-Ni Hung ◽  
Zhicheng Xia ◽  
Nien-Tai Hu ◽  
Byong H. Lee
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Maximum Yield ◽  
Amino Acid Sequences ◽  
Nucleotide Sequence Analysis ◽  
Acid Base ◽  
Calculated Molecular Mass ◽  
Transcription Terminator

ABSTRACT Two genes encoding β-galactosidase isoenzymes,β-galI and β-galIII, fromBifidobacterium infantis HL96 were revealed on 3.6- and 2.4-kb DNA fragments, respectively, by nucleotide sequence analysis of the two fragments. β-galI (3,069 bp) encodes a 1,022-amino-acid (aa) polypeptide with a predicted molecular mass of 113 kDa. A putative ribosome binding site and a promoter sequence were recognized at the 5′ flanking region of β-galI. Further upstream a partial sequence of an open reading frame revealed a putative lactose permease gene transcribing divergently fromβ-galI. The β-galIII gene (2,076 bp) encodes a 691-aa polypeptide with a calculated molecular mass of 76 kDa. A rho-independent transcription terminator-like sequence was found 25 bp downstream of the termination codon. The amino acid sequences of β-GalI and β-GalIII are homologous to those found in the LacZ and the LacG families, respectively. The acid-base, nucleophilic, and substrate recognition sites conserved in the LacZ family were found in β-GalI, and a possible acid-base site proposed for the LacG family was located in β-GalIII, which featured a glutamate at residue 160. The coding regions of the β-galI andβ-galIII genes were each cloned downstream of a T7 promoter for overexpression in Escherichia coli. The molecular masses of the overexpressed proteins, as estimated by polyacrylamide gel electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, agree with their predicted molecular weights. β-GalI and β-GalIII were specific for β-d-anomer-linked galactoside substrates. Both are more active in response to ONPG (o-nitrophenyl-β-d-galactopyranoside) than in response to lactose, particularly β-GalIII. The galacto-oligosaccharide yield in the reaction catalyzed by β-GalI at 37°C in 20% (wt/vol) lactose solution was 130 mg/ml, which is more than six times higher than the maximum yield obtained with β-GalIII. The structure of the major trisaccharide produced by β-GalI catalysis was characterized asO-β-d-galactopyranosyl-(1-3)-O-β-d-galactopyranosyl-(1-4)-d-glucopyranose (3′-galactosyl-lactose).

scholarly journals Enzymatic Properties of a Novel Liquefying α-Amylase from an Alkaliphilic Bacillus Isolate and Entire Nucleotide and Amino Acid Sequences

1998 ◽  
Vol 64 (9) ◽  
pp. 3282-3289 ◽  
Author(s):  
Kazuaki Igarashi ◽  
Yuji Hatada ◽  
Hiroshi Hagihara ◽  
Katsuhisa Saeki ◽  
Mikio Takaiwa ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Molecular Mass ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Maximum Activity ◽  
Amino Acid Sequences ◽  
Ph Optimum ◽  
Calculated Molecular Mass

ABSTRACT A novel liquefying α-amylase (LAMY) was found in cultures of an alkaliphilic Bacillus isolate, KSM-1378. The specific activity of purified LAMY was approximately 5,000 U mg of protein−1, a value two- to fivefold greater between pH 5 and 10 than that of an industrial, thermostable Bacillus licheniformis enzyme. The enzyme had a pH optimum of 8.0 to 8.5 and displayed maximum activity at 55°C. The molecular mass deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 53 kDa, and the apparent isoelectric point was around pH 9. This enzyme efficiently hydrolyzed various carbohydrates to yield maltotriose, maltopentaose, maltohexaose, and maltose as major end products after completion of the reaction. Maltooligosaccharides in the maltose-to-maltopentaose range were unhydrolyzable by the enzyme. The structural gene for LAMY contained a single open reading frame 1,548 bp in length, corresponding to 516 amino acids that included a signal peptide of 31 amino acids. The calculated molecular mass of the extracellular mature enzyme was 55,391 Da. LAMY exhibited relatively low amino acid identity to other liquefying amylases, such as the enzymes from B. licheniformis (68.9%), Bacillus amyloliquefaciens (66.7%), and Bacillus stearothermophilus (68.6%). The four conserved regions, designated I, II, III, and IV, and the putative catalytic triad were found in the deduced amino acid sequence of LAMY. Essentially, the sequence of LAMY was consistent with the tertiary structures of reported amylolytic enzymes, which are composed of domains A, B, and C and which include the well-known (α/β)8 barrel motif in domain A.

scholarly journals Heterologous Expression and Characterization of A Novel Ochratoxin A Degrading Enzyme, N-acyl-L-amino Acid Amidohydrolase, from Alcaligenes faecalis

Toxins ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 518
Author(s):  
Honghai Zhang ◽  
Yunpeng Zhang ◽  
Tie Yin ◽  
Jing Wang ◽  
Xiaolin Zhang
Keyword(s):  
Amino Acid ◽  
Ochratoxin A ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Alcaligenes Faecalis ◽  
Degrading Enzyme ◽  
Sds Page ◽  
Ochratoxin Α ◽  
Acid Amidohydrolase

Ochratoxin A (OTA) is a well-known, natural contaminant in foods and feeds because of its toxic effects, such as nephrotoxicity in various animals. Recent studies have revealed that Alcaligenes faecalis could generate enzymes to efficiently degrade OTA to ochratoxin α (OTα) in vitro. In an effort to obtain the OTA degrading mechanism, we purified and identified a novel degrading enzyme, N-acyl-L-amino acid amidohydrolase (AfOTase), from A. faecalis DSM 16503 via mass spectrometry. The same gene of the enzyme was also encountered in other A. faecalis strains. AfOTase belongs to peptidase family M20 and contains metal ions at the active site. In this study, recombination AfOTase was expressed and characterized in Escherichia coli. The molecular mass of recombinant rAfOTase was approximately 47.0 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited a wide temperature range (30–70 °C) and pH adaptation (4.5–9.0) and the optimal temperature and pH were 50 °C and 6.5, respectively.

scholarly journals Cloning and Comparison of fliC Genes and Identification of Glycosylation in the Flagellin ofPseudomonas aeruginosa a-Type Strains

1998 ◽  
Vol 180 (12) ◽  
pp. 3209-3217 ◽  
Author(s):  
Cynthia D. Brimer ◽  
T. C. Montie
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Type Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Pcr Amplification ◽  
Amino Acid Sequences ◽  
Type Strains

ABSTRACT Pseudomonas aeruginosa a-type strains produce flagellin proteins which vary in molecular weight between strains. To compare the properties of a-type flagellins, the flagellin genes of severalPseudomonas aeruginosa a-type strains, as determined by interaction with specific anti-a monoclonal antibody, were cloned and sequenced. PCR amplification of the a-type flagellin gene fragments from five strains each yielded a 1.02-kb product, indicating that the gene size is not likely to be responsible for the observed molecular weight differences among the a-type strains. The flagellin amino acid sequences of several a-type strains (170018, 5933, 5939, and PAK) were compared, and that of 170018 was compared with that of PAO1, a b-type strain. The former comparisons revealed that a-type strains are similar in amino acid sequence, while the latter comparison revealed differences between 170018 and PAO1. Posttranslational modification was explored for its contribution to the observed differences in molecular weight among the a-type strains. A biotin-hydrazide glycosylation assay was performed on the flagellins of three a-type strains (170018, 5933, and 5939) and one b-type strain (M2), revealing a positive glycosylation reaction for strains 5933 and 5939 and a negative reaction for 170018 and M2. Deglycosylation of the flagellin proteins with trifluoromethanesulfonic acid (TFMS) confirmed the glycosylation results. A molecular weight shift was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis for the TFMS-treated flagellins of 5933 and 5939. These results indicate that the molecular weight discrepancies observed for the a-type flagellins can be attributed, at least in part, to glycosylation of the protein. Anti-a flagellin monoclonal antibody reacted with the TFMS-treated flagellins, suggesting that the glycosyl groups are not a necessary component of the epitope for the human anti-a monoclonal antibody. Comparisons between a-type sequences and a b-type sequence (PAO1) will aid in delineation of the epitope for this monoclonal antibody.

scholarly journals Conformational differences between two wheat (Triticum aestivum) ‘high-molecular-weight’ glutenin subunits are due to a short region containing six amino acid differences

1989 ◽  
Vol 263 (3) ◽  
pp. 837-842 ◽  
Author(s):  
A P Goldsbrough ◽  
N J Bulleid ◽  
R B Freedman ◽  
R B Flavell
Keyword(s):  
Molecular Weight ◽  
Triticum Aestivum ◽  
Amino Acid ◽  
High Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Amino Acid Sequences ◽  
Glutenin Subunits ◽  
Sds Page ◽  
Hmw Glutenin

‘High-molecular-weight’ (HMW, high-Mr) glutenin subunits are protein constituents of wheat (Triticum aestivum) seeds and are responsible in part for the viscoelasticity of the dough used to make bread. Two subunits, numbered 10 and 12, are the products of allelic genes. Their amino acid sequences have been derived from the nucleic acid sequences of the respective genes. Subunit 10 has fewer amino acids than subunit 12, but migrates more slowly on SDS/PAGE (polyacrylamide-gel electrophoresis). This anomaly is due to between one and six of the amino acid differences between the subunits, localized towards the C-terminal end of the proteins. This has been established by making chimaeric genes between the genes for subunits 10 and 12, transcribing and translating them in vitro and analysing the products by SDS/PAGE. The postulated conformational differences between subunits 10 and 12 are discussed in relation to current hypotheses for the structure of HMW glutenin subunits.

Isolation of Bacillus circulans and Paenibacillus polymyxa Strains Inhibitory to Campylobacter jejuni and Characterization of Associated Bacteriocins

2005 ◽  
Vol 68 (1) ◽  
pp. 11-17 ◽  
Author(s):  
EDWARD A. SVETOCH ◽  
NORMAN J. STERN ◽  
BORIS V. ERUSLANOV ◽  
YURI N. KOVALEV ◽  
LARISA I. VOLODINA ◽  
...  
Keyword(s):  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Paenibacillus Polymyxa ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Bacillus Circulans ◽  
Poultry Production ◽  
Molecular Masses ◽  
Campylobacter Infection

We evaluated anti-Campylobacter activity among 365 Bacillus and Paenibacillus isolates from poultry production environments. One novel antagonistic Bacillus circulans and three Paenibacillus polymyxa strains were identified and further studied. Cell-free ammonium sulfate precipitate (crude antimicrobial preparation) was obtained from each candidate culture. Zones of Campylobacter growth inhibition surrounding 10 μl of this crude antimicrobial preparation were quantified using a spot test. Campylobacter growth resumed when the preparation was preincubated with selected protease enzymes, demonstrating peptide characteristics consistent with a bacteriocin. These peptides were further purified using combinations of molecular mass resolution and ion exchange chromatography. Molecular masses of the peptides were estimated at approximately 3,500 Da by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Isoelectric focusing was used to determine the pI values of the peptides. Amino acid sequences of the bacteriocins and more precise molecular masses were obtained by matrix-assisted laser desorption and ionization–time of flight (MALDI-TOF) analysis. The bacteriocin from P. polymyxa NRRL B-30507 had a pI of 4.8, that from P. polymyxa NRRL B-30509 had a pI of 7.2, that from P. polymyxa NRRL B-30508 had a pI of 4.8, and that from B. circulans NRRL B-30644 had a pI of 7.8. The amino acid sequences were consistent with those of class IIa bacteriocins. These antagonists and the corresponding bacteriocins may be useful in the control of Campylobacter infection in poultry.

scholarly journals Isolation, by partial pepsin digestion, of the three collagen-like regions present in subcomponent Clq of the first component of human complement

1976 ◽  
Vol 155 (1) ◽  
pp. 5-17 ◽  
Author(s):  
K B M Reid
Keyword(s):  
Amino Acid ◽  
Sodium Dodecyl Sulphate ◽  
Sequence Data ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Sequences ◽  
Disulphide Bond ◽  
Small Peptides ◽  
Molecular Weights ◽  
A Chain

1. Digestion of human subcomponent C1q with pepsin at pH4.45 for 20h at 37 degrees C fragmented most of the non-collagen-like amino acid sequences in the molecule to small peptides, whereas the entire regions of collagen-like sequence that comprised 38% by weight of the subcomponent C1q were left intact. 2. The collagen-like fraction of the digest was eluted in the void volume of a Sephadex G-200 column, was was showm to be composed of two major fragments when examined by electrophoresis on polyacrylamide gels run in buffers containing sodium dodecyl sulphate. These fragments were separated on CM-cellulose at pH4.9 in buffers containing 7.5M-urea. 3. Human subcomponent C1q on reduction and alkylation yields equimolar amounnts of three chains, which have been designated A, B and C [Reid et al. (1972) Biochem. J. 130, 749-763]. One of the pepsin fragments was shown to be composed of the N-terminal 95 residues of the A chain linked, via residue A4, by a single disulphide bond to a residue in the sequence B2-B6 in the N-terminal 91 residues of the B chain. The second pepsin fragment was shown to be composed of a disulphide-linked dimer of the N-terminal 94 residues of the C chain, the only disulphide bond being located at residue C4.4. The mol. wts. of the unoxidized and oxidized pepsin fragments were estimated from their amino acid compositions to be 20 000 and 18 200 for the A-B and C-C dimers and 11 400, 8800 and 9600 for the collagen-like fragments of the A, B and C chains respectively. Estimation of the molecular weights of the peptic fragments by polyacrylamide-gel electrophoresis run in the presence of sodium dodecyl sulphate gave values that were approx. 50% higher than expected from the amino acid sequence data. This is probably due to the high collagen-like sequence content of these fragments.

scholarly journals Characterization of P40, a Cytadhesin of Mycoplasma agalactiae

2002 ◽  
Vol 70 (10) ◽  
pp. 5612-5621 ◽  
Author(s):  
Bénédicte Fleury ◽  
Dominique Bergonier ◽  
Xavier Berthelot ◽  
Ernst Peterhans ◽  
Joachim Frey ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Sodium Dodecyl ◽  
Mycoplasma Hominis ◽  
Single Copy ◽  
Signal Peptidase ◽  
Mycoplasma Species ◽  
Gene Encoding ◽  
Sequence Comparisons ◽  
Mycoplasma Agalactiae

ABSTRACT An immunodominant protein, P40, of Mycoplasma agalactiae was analyzed genetically and functionally. The gene encoding P40 was cloned from type strain PG2, sequenced, submitted to point mutagenesis in order to convert mycoplasma-specific TGATrp codon to the universal TGGTrp codon, and subsequently expressed in Escherichia coli. Nucleotide sequence-derived amino acid sequence comparisons revealed a similarity of P40 to the adhesin P50 of Mycoplasma hominis and to protein P89 of Spiroplasma citri, which is expected to be involved in adhesion. The amino acid sequence of P40 revealed a recognition site for a signal peptidase and strong antigenic and hydrophilic motifs in the C-terminal domain. Triton X-114 phase partitioning confirmed that P40 is a membrane protein. Fab fragments of antibodies directed against recombinant purified P40 significantly inhibited adherence of M. agalactiae strains PG2 to lamb joint synovial cells LSM 192. Sera taken sequentially from sheep infected with PG2 revealed that P40 induced a strong and persistent immune response that gave strong signals on immunoblots containing recombinant P40 even 3 months after infection. The gene encoding P40 was present in a single copy in all of the 26 field strains of M. agalactiae analyzed and was not detected in closely related mycoplasma species. P40 was expressed as a protein with an apparent molecular mass of 37 kDa on sodium dodecyl sulfate-acrylamide gels by all M. agalactiae strains except for serotype C strains, which showed nonsense mutations in their p40 genes.

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