scholarly journals Purification and characterization of the class-II D-fructose 1,6-bisphosphate aldolase from Escherichia coli (Crookes' strain)

1978 ◽  
Vol 169 (3) ◽  
pp. 633-641 ◽  
Author(s):  
S A Baldwin ◽  
R N Perham ◽  
D Stribling
Keyword(s):  
Escherichia Coli ◽  
Common Ancestor ◽  
Class Ii ◽  
Purification And Characterization ◽  
Thiol Compounds ◽  
E Coli ◽  
Optimum Ph ◽  
Class Ii Aldolase ◽  
Fructose 1,6 Bisphosphate

A new form of the class-II D-fructose 1,6-bisphosphate aldolase (EC 4.1.2.13) of Escherichia coli (Crookes' strain) was isolated from an extract of glycerol-grown bacteria. It has a higher molecular weight (approx. 80000)than previous preparations of the enzyme and closely resembles the typical class-II aldolase from yeast in size and amino acid composition. On the other hand, its kinetic behaviour is not typical of a class-II aldolase. The enzyme has no requirement for thiol compounds either for stability or activity, added K+ ions have no effect, and the optimum pH for the cleavage activity is unusually high. The class-II enzymes from the prokaryote E. coli and the eukaryote yeast show no immunological identity. However, the similarity of their structures suggests that they have evolved from a common ancestor.

scholarly journals Cloning, sequence analysis and over-expression of the gene for the class II fructose 1,6-bisphosphate aldolase of Escherichia coli

1989 ◽  
Vol 257 (2) ◽  
pp. 529-534 ◽  
Author(s):  
P R Alefounder ◽  
S A Baldwin ◽  
R N Perham ◽  
N J Short
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Recombinant Expression ◽  
Class Ii ◽  
Nucleotide Sequence Analysis ◽  
Chromosomal Dna ◽  
Expression Plasmid ◽  
E Coli ◽  
Fructose 1,6 Bisphosphate

Nucleotide sequence analysis of the Escherichia coli chromosomal DNA inserted in the plasmid pLC33-5 of the Clarke and Carbon library [Clarke & Carbon (1976) Cell 9, 91-99] revealed the existence of the gene, fda, encoding the Class II (metal-dependent) fructose 1,6-bisphosphate aldolase of E. coli. The primary structure of the polypeptide chain inferred from the DNA sequence of the fda gene comprises 359 amino acids, including the initiating methionine residue, from which an Mr of 39,146 could be calculated. This value is in good agreement with that of 40,000 estimated from sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified dimeric enzyme. The amino acid sequence of the Class II aldolase from E. coli showed no homology with the known amino acid sequences of Class I (imine-forming) fructose 1,6-bisphosphate aldolases from a wide variety of sources. On the other hand, there was obvious homology with the N-terminal sequence of 40 residues already established for the Class II fructose 1,6-bisphosphate aldolase of Saccharomyces cerevisiae. These Class II aldolases, one from a prokaryote and one from a eukaryote, evidently are structurally and evolutionarily related. A 1029 bp-fragment of DNA incorporating the fda gene was excised from plasmid pLC33-5 by digestion with restriction endonuclease HaeIII and subcloned into the expression plasmid pKK223-3, where the gene came under the control of the tac promoter. When grown in the presence of the inducer isopropyl-beta-D-thiogalactopyranoside, E. coli JM101 cells transformed with this recombinant expression plasmid generated the Class II fructose 1,6-bisphosphate aldolase as approx. 70% of their soluble protein. This unusually high expression of an E. coli gene should greatly facilitate purification of the enzyme for any future structural or mechanistic studies.

scholarly journals Epidemiological Study of pap Genes among Diarrheagenic or Septicemic Escherichia coli Strains Producing CS31A and F17 Adhesins and Characterization of Pap31AFimbriae

2000 ◽  
Vol 38 (4) ◽  
pp. 1502-1509 ◽  
Author(s):  
Yolande Bertin ◽  
Jean-Pierre Girardeau ◽  
Arlette Darfeuille-Michaud ◽  
Christine Martin
Keyword(s):  
Escherichia Coli ◽  
Reference Strain ◽  
Class Ii ◽  
Intestinal Cells ◽  
Growth Media ◽  
Minimal Growth ◽  
E Coli ◽  
Structural Subunit ◽  
Tract Infections

The association of the pap operon with the CS31A and F17 adhesins was studied with 255 Escherichia coli strains isolated from calves, lambs, or humans with diarrhea. The three classes of PapG adhesin with different receptor binding preferences were also screened. The pap operon was associated with 50 and 36% of human strains that produced CS31A and ovine strains that produced F17, respectively. Among the bovine isolates, the pap operon was detected in 61% of the CS31A-positive isolates and 72% of the strains that produce both CS31A and F17. The class II adhesin gene was present in bovine (20%) and ovine (71%) isolates. Both class II and III adhesins were genetically associated with 36% of the human strains. The highest prevalence of the pap operon was observed amongE. coli strains that produce additional adhesins involved in the binding of bacteria to intestinal cells. Among the bovine isolates, the reference strain for CS31A and F17c was found to be positive for the pap operon. Phenotypic and genotypic characterizations were undertaken. Pap31A appeared as fine and flexible fimbriae surrounding the bacteria but did not mediate adhesion to calf intestinal villi. Pap31A production was optimal with bacteria cultured on minimal growth media and repressed by addition of exogenous leucine. The deduced amino acid sequence of the PapA31A structural subunit showed 57 to 97% identity with the different P-related structural subunits produced by E. coli strains isolated from pigs with septicemia or humans with urinary tract infections. None of the three papG allelic variants was detected, but a homologous papG gene was present in the chromosome of strain 31A.

scholarly journals Characterization of Methylglyoxal Synthase fromClostridium acetobutylicum ATCC 824 and Its Use in the Formation of 1,2-Propanediol

1999 ◽  
Vol 65 (7) ◽  
pp. 3244-3247 ◽  
Author(s):  
Ke-xue Huang ◽  
Frederick B. Rudolph ◽  
George N. Bennett
Keyword(s):  
Escherichia Coli ◽  
Clostridium Acetobutylicum ◽  
Glycerol Dehydrogenase ◽  
Dihydroxyacetone Phosphate ◽  
Gene Encoding ◽  
E Coli ◽  
Native Molecular Mass ◽  
Optimum Ph ◽  
Methylglyoxal Synthase

ABSTRACT A gene encoding a putative 150-amino-acid methylglyoxal synthase was identified in Clostridium acetobutylicum ATCC 824. The enzyme was overexpressed in Escherichia coli and purified. Methylglyoxal synthase has a native molecular mass of 60 kDa and an optimum pH of 7.5. The Km andV max values for the substrate dihydroxyacetone phosphate were 0.53 mM and 1.56 mmol min−1μg−1, respectively. When E. coli glycerol dehydrogenase was coexpressed with methylglyoxal synthase in E. coli BL21(DE3), 3.9 mM 1,2-propanediol was produced.

scholarly journals Purification and characterization of a diptericin homologue from Sarcophaga peregrina (flesh fly)

1992 ◽  
Vol 287 (2) ◽  
pp. 573-578 ◽  
Author(s):  
M Ishikawa ◽  
T Kubo ◽  
S Natori
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Bactericidal Activity ◽  
Fat Body ◽  
Amino Acid Sequencing ◽  
Purification And Characterization ◽  
E Coli ◽  
Escherichia Coli Cells

A protein with a molecular mass of 8 kDa was found to be synthesized specifically when the fat-body from injured Sarcophaga peregrina larvae was cultured in vitro. This protein was purified from the haemolymph of the injured larvae to near-homogeneity. Partial amino acid sequencing revealed that this protein is a diptericin homologue. It showed bactericidal activity on growing, but not resting Escherichia coli cells. E. coli cells become elongated on treatment with this protein.

Purification and characterization of D-β-hydroxybutyrate dehydrogenase expressed in Escherichia coli

1993 ◽  
Vol 71 (7-8) ◽  
pp. 406-410
Author(s):  
Les Jones ◽  
Sharon Churchill ◽  
Perry Churchill
Keyword(s):  
Escherichia Coli ◽  
Kinetic Parameters ◽  
Functional Properties ◽  
Rat Liver ◽  
Specific Activity ◽  
Purification And Characterization ◽  
E Coli ◽  
Specific Activities ◽  
Viable Method

D-β-Hydroxybutyrate dehydrogenase (BDH), a lipid-requiring enzyme, has been cloned into pUC18, expressed in Escherichia coli, and purified to homogeneity. The apoenzyme, i.e., the enzyme devoid of phospholipid, has no activity, but can be activated by phospholipid to a specific activity of 129 μmol/(min∙mg). The functional properties of the enzyme expressed in E. coli were compared with the enzyme purified from rat liver. The specific activities, kinetic parameters, and phospholipid activation profiles were virtually identical. These results indicate that the expression of the enzyme in E. coli is a viable method for producing active functional BDH and should allow for the production of specifically altered BDH molecules.Key words: D-β-hydroxybutyrate dehydrogenase, cloning, expression, lipid requiring.

scholarly journals Purification, crystallization and characterization of N-acetylneuraminate lyase from Escherichia coli

1991 ◽  
Vol 276 (2) ◽  
pp. 541-546 ◽  
Author(s):  
K Aisaka ◽  
A Igarashi ◽  
K Yamaguchi ◽  
T Uwajima
Keyword(s):  
Escherichia Coli ◽  
Gel Filtration ◽  
Genetically Engineered ◽  
E Coli ◽  
Sds Page ◽  
Optimum Ph ◽  
Molecular Masses ◽  
Related Compounds ◽  
Optimum Ph And Temperature

N-Acetylneuraminate lyase produced by Escherichia coli was purified and crystallized from a genetically engineered strain (E. coli SF8/pNAL1). The enzyme showed apparent molecular masses of 105,000 Da on gel filtration and 35,000 Da on SDS/PAGE, suggesting that the enzyme is a trimer. The apparent optimum pH and temperature were found to be 6.5-7.0 and 80 degrees C respectively. The Km values for N-acetylneuraminate and N-glycollylneuraminate were 3.3 and 3.3 mM respectively. The enzyme was inhibited by reduction with NaBH4 in the presence of the substrate, indicating that the enzyme belongs to the Schiff-base-forming Class I aldolases. The enzyme was strongly inhibited by Cu2+ ions, p-chloromercuribenzoate and N-bromosuccinimide, and also inhibited competitively by the reaction product, pyruvate, and its structurally related compounds, dihydroxyacetone and DL-glyceraldehyde.

Sign in / Sign up

Export Citation Format

Share Document