Plasmid pEA566 from Erwinia aroideae

A new plasmid designated pEA566 was isolated from Erwinia aroideae. The molecular weight of the plasmid, as determined by neutral and alkaline sucrose gradient centrifugation, electron microscopy, and agarose gel electrophoresis, was 6.6 × 10⁶. The plasmid replicated under relaxed control, had three cleavage sites for KpnI restriction endonuclease, and no sites for EcoRI, BamHI, SalI, PstI, and HindIII.     

L-Asparaginase Production by Erwinia aroideae

Maximum yields of 1,250 IU (international unit)/g (dry weight of cells) of L-asparaginase were obtained in 8 hr from Erwinia aroideae NRRL B-138. Partial purification and concentration of the extracted L-asparaginase yielded a preparation with an activity of 275 IU/ml. Only one L-asparaginase was present as determined by electrophoresis, and the enzyme exhibited a pH optimum of 7.5 and a K(m) of 3 x 10(-3) M.     

Exopectic Acid Transeliminase of an Erwinia

A species of Erwinia was found to produce no other pectolytic enzyme than the two transeliminases of exo-types, namely, an oligogalacturonide transeliminase and an exopectic acid transeliminase. Of the two enzymes, the exopectic acid transeliminase was isolated and its properties were investigated. The results are as follows: (1) Pectic acids having an unsaturated galacturonic acid residue at the non-reducing end of the molecule are susceptible but oxidized or reduced pectic acids resistant to the enzyme action. (2) The enzyme has no activity toward pectinic acid and polymethylpolygalacturonate methyl glycoside. The limit of the enzymatic degradation for citrus pectic acid is 43.8%. (3) The rate of the enzyme activity was maximal with tetragalacturonic acid and followed by acid-soluble pectic acid, acid-insoluble pectic acid, pectic acid and trigalacturonic acid. Unlike the oligogalacturonide transeliminases of Pseudomonas sp. (strain S2) and Erwinia aroideae, the present enzyme shows a considerably high activity toward pectic acids of high molecular weight. (4) The pH-activity curves vary with the buffer employed. (5) The enzyme is activated by Co²⁺ and Mn²⁺ but powerfully inhibited by Cu²⁺ and Hg²⁺. Ca²⁺ has no significant effect on the enzyme activity.     

L-Asparaginase Synthesis by Erwinia aroideae

Maximum L-asparaginase activity was obtained when 1.0% lactose and 1.5% yeast extract were supplied as carbon and nitrogen sources, respectively. Glucose inhibited the enzyme formation. The diauxie phenomenon was observed with Erwinia aroideae NRRL B-138 grown in a medium containing glucose and lactose.     

F'ColVColBtrpcys plasmid in Erwinia aroideae]

Erwinia aroideae carries a cryptic plasmid with 30 S sedimentation coefficient. Plasmid F'ColVColBtrpcys does not dissociate in E. aroideae and is replicated under stringent control since the number of plasmid copies per chromosome does not exceed one. The behaviour of F'ColVColBtrpcys plasmid in E. aroideae is characterized by (1) instability observed at both spontaneous and after EB treatment, (2) depression of plasmid genes that determine colicin synthesis.     

Factors Influencing L-Asparaginase Production by Erwinia aroideae

Increased yields of L-asparaginase from Erwinia aroideae NRRL B-138 were obtained by medium enrichment techniques coupled with aeration optimization.     

A Saccharifying Pectate trans-Eliminase of Erwinia aroideae

A saccharifying pectate trans-eliminase was found in the cells of Erwinia aroideae. This enzyme differs from known pectate trans-eliminase in the next two points. It degrades pectic acid liberating 4,5-unsaturated digalacturonic acid from the chain end of the molecule. It does not require calcium ion.

Some properties of 4,5-unsaturated digalacturonic acid, the main product of the saccharifying pectate trans-elimination, were also described in this paper.     

Purification and Properties of a Pectate Lyase in Erwinia aroideae

A strain of Erwinia aroideae produced an extracellular pectolytic enzyme under growth conditions with pectin or pectic acid as the inducer. This strain also produced a pectin lyase when nalidixic acid is added to a culture medium. The pectolytic enzyme produced under the growth conditions was purified approximately 40-fold from the culture fluid by carboxy- methyl cellulose and Sephadex G-75 gel column chromatographies. The purified enzyme was almost homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis, having a molecular weight of about 36,000 to 38,000. This enzyme, with optimal activity at pH 9.0 to 9.2, produced reaction products which had a strong absorption at 230 nm indicating a lyase type of the reaction. The enzyme activity was markedly stimulated by calcium ion and completely inhibited by cobalt and mercuric ions and by ethylenediaminetetraacetate. Pectic acid or pectin with lower methoxyl content was a good substrate for this enzyme, while no significant activity was observed when pectin with higher methoxyl content was used as a substrate. It was concluded that the enzyme produced under the normal growth conditions is an endo-pectate lyase and differs from the pectin lyase induced by nalidixic acid.     

Bacteriocins in Erwinia aroideae with Tail Like Structure of Bacteriophages

Diploid baker’s yeast capable of strongly activating a mouse macrophage was constructed based on haploid mutant AQ-37 obtained previously. The obtained strain BQ-55 activated also human immune cells. To clarify a factor for the activation, the cell wall structure, especially the β-glucan structure, was analyzed, suggesting that the length of branching, β-1,6-glucan, may be one of the factors.     

Oligogalacturonate hydrolase from carrot roots

The presence of multiple forms of enzyme with terminal action pattern on pectate was evaluated in the protein mixture obtained from carrot roots. The form with pH optimum 3.8 clearly preferred substrates with a lower degree of polymerization (oligogalacturonates). Its molecular mass, isoelectric point, glycosylation as well as cleavage of pectate from nonreducing end corresponded to an exopolygalacturonase [EC 3.2.2.67]. The affinity of this enzyme to the substrates increased with the increasing degree of polymerization, and the difference was observed only in the maximal ratio of catalysis of oligomeric and polymeric substrates. Sterical hindrance for substrates with more than six D-galactopyranuronic acid units is supposed and an oligogalacturonate hydrolase rather than exopolygalacturonase is considered.     

Effeet of Metal Ions on Activity of Exopectic Acid Transeliminase of Erwinia sp.

Contrary to the exopectic acid transeliminase of Clostridium multifermentans, that of Erwinia sp. was activated strongly by Na⁺ and to a much less extent by Ca²⁺. K⁺ had a small stimulating effect on the enzyme activity. Mn²⁺ and Co²⁺, like Ca²⁺, activated the enzyme weakly. Ba²⁺ and Mg²⁺ showed no and a slight inhibitory effect, respectively, on the activity.

An almost total loss of activity was caused by the addition of EDTA to the reaction mixture. In the presence of Na⁺ the enzyme activity was restored by addition of divalent cations. Individual monovalent cations or each of the divalent cations was ineffective in restoring the activity.     

A New Pectolytic Enzyme in Erwinia aroideae Formed in the Presence of Nalidixic Acid

Pectolytic enzyme formation by whole cells of Erwinia aroideae was markedly stimulated when nalidixic acid was added to a culture medium. The activity of pectolytic enzyme was markedly stimulated by nalidixic acid when the activity was measured by the decrease of viscosity of pectin, while activities of both polygalacturonic acid trans-eliminase and polygalacturonase which were measured respectively by the increase of optical density at 230 nm and the liberation of aldehyde groups, were not stimulated. The analysis of pectolytic enzyme by carboxymethyl cellulose column chromatography indicated that there was a significant difference in the elution profiles between the pectolytic enzyme induced by nalidixic acid and that synthesized under normal conditions. Therefore, we conclude that two enzymes are distinct protein species.     

Stimulation of Pectolytic Enzyme Formation of Erwinia aroideae by an Active Factor in Carrot Extracts

Formation of pectinase system in Erwinia aroideae was stimulated to a considerable extent when the cells were incubated in a pectin medium containing carrot extracts. The active factor in the extract was purified about 30 fold by ethanol precipitation, and further purification was achieved by ninhydrin treatment, charcoal adsorption, dialysis and gel filtration with Sephadex G-10. Although crude carrot extract preparation also stimulated protease formation in this organism, no stimulating activity for protease formation was found in the purified factor. Acetate and butyrate which had been shown to stimulate pectinase formation, were found to stimulated protease formation as well. Pectinase formation by this organism was also stimulated by polyamines and inorganic phosphate to a considerable extent.