The formation of cyclosporin A (Cy A) by immobilized spores and mycelia of Aspergillus terreus was investigated. Different carriers were tested as immobilizing carriers, whereby Ca-alginate was selected for further experimentation. The role of alginate concentration, biomass weight, pH value of the cultivation medium, repeated utilization of the immobilized fungus as well as the supplementation of different amino acid precursors were studied. Best Cy A outputs were attained with Ca-alginate 3% (w/v), mycelial weight 15% (w/v), pH 4.5 and four repeated cycles. Similarly, the Cy A productivity was markedly accelerated in the presence of L-valine and L-valine and L-leucine mixture.
This is the first report on the degradation of poly(3-hydroxybutyrate) (PHB), and its copolymers poly(3-hydroxyvalerate) P(3HB-co-10–20% HV) by Nocardiopsis aegyptia, a new species isolated from marine seashore sediments. The strain excreted an extracellular PHB depolymerase and grew efficiently on PHB or its copolymers as the sole carbon sources. The degradation activity was detectable by the formation of a transparent clearing zone around the colony on an agar Petri plate after 25 days, or a clearing depth under the colony in test tubes within 3 weeks. The previous techniques proved that the bacterium was able to assimilate the monomeric components of the shorter alkyl groups of the polymers. Nocardiopsis aegyptia hydrolyzed copolymers 10–20% PHBV more rapidly than the homopolymer PHB. The bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate), and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). The samples were degraded at the surface and proceeded to the inner part of the materials. Clear morphological alterations of the polymers were noticed, indicating the degradative capability of the bacterium. Plackett-Burman statistical experimental design has been employed to optimize culture conditions for maximal enzyme activity. The main factors that had significant positive effects on PHB depolymerase activity of Nocardiopsis aegyptia were sodium gluconate, volume of medium/flask and age of inoculum. On the other hand, MgSO4·7H2O, KH2PO4, K2HPO4 and NH4NO3 exhibited negative effects. Under optimized culture conditions, the highest activity (0.664 U/mg protein) was achieved in a medium predicted to be near optimum containing (in g/L): PHB, 0.5; C6H11O7Na, 7.5; MgSO4·7H2O, 0.35; K2HPO4, 0.35; NH4NO3, 0.5; KH2PO4, 0.35; malt extract, 0.5 and prepared with 50% seawater. The medium was inoculated with 1% (v/v) spore suspension of 7 days old culture. Complete clarity of the medium was achieved after 3 days at 30°C.
The bacteriophage Esc-A was isolated from sewage by using the intestinal pathogenic Escherichia coli 3-1 as the host. Toxicity in chickens showed its safety as a bio-product. Phage therapy against diarrhea in chickens indicated that Esc-A could decrease the death rate more efficiently compared with antibiotic treatments.
Growth of the heterotrophic marine bacterium, Alteromonas espejiana Bal-31 was inhibited in the presence of sucrose, maltose and even glucose, but not with starch. Extracellular α-amylase was induced with a lag phase of 2 h in the presence of starch. In contrast, cell growth of the S2a mutant was not affected by the addition of maltose, and starch was ineffective in the induction of extracellular α-amylase in this mutant. Activity of extracellular α-amylase was induced from the S2a mutant with a 4-h lag phase in the presence of maltose, and the high level of enzyme activity was maintained for at least 24 h. Activity of α-amylase induced by both wild type starch and S2a mutant maltose cultures were mainly observed in extracellular locations. This activity could be stopped by tetracycline treatment, indicating that enzyme induction was dependant on gene expression and not on enzyme protein secretory mechanisms. Our results showed that the mutation in S2a changed the growth and the modulation of the specific α-amylase in response to carbon nutrients.
Thermostable amylolytic enzymes are currently being investigated to improve industrial processes of starch degradation. A thermostable extracellular glucoamylase (exo-1, 4-α-D-glucanohydrolase, E.C.220.127.116.11) from the culture supernatant of a thermophilic fungus Chaetomium thermophilum was purified to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) homogeneity by using ammonium sulfate fraction, DEAE-Sepharose Fast Flow chromatography, and Phenyl-Sepharose Fast Flow chromatography. SDS-PAGE of the purified enzyme showed a single protein band of molecular weight 64 kDa. The glucoamylase exhibited optimum catalytic activity at pH 4.0 and 65°C. It was thermostable at 50°C and 60°C, and retained 50% activity after 60 min at 65°C. The half-life of the enzyme at 70°C was 20 min. N-terminal amino acid sequencing (15 residues) was AVDSYIERETPIAWN. Different metal ions showed different effects on the glucoamylase activity. Ca2+, Mg2+, Na+, and K+ enhanced the enzyme activity, whereas Fe2+, Ag+, and Hg2+ cause obvious inhibition. These properties make it applicable to other biotechnological purposes.
A novel thermophilic, aerobic, endospore-forming bacterium, designated strain PizzoT, was isolated from geothermal volcanic environment. Samples were collected from the Pizzo sopra la Fossa site at Stromboli Island (Eolian Islands, south of Italy) at the high altitude of 918 m. Cells of strain PizzoT were rod-shaped and stained Gram-positive. Growth was observed between 50 and 75°C (optimum 70°C) and at pH 5.0–8.0 (optimum pH 7.0). NaCl (0.4%, w/v) supported growth and among the hydrocarbons tested none induced growth. The G+C content of the DNA was 54.1 mol% and the sequence analysis of the 16S rRNA gene showed that the new isolate was phylogenetically closely related to the members of the Bacillus rRNA Group 5. DNA-DNA hybridization studies revealed a borderline similarity between the new isolate and Geobacillus thermoleovorans DSM 5366T (69.8%) and Geobacillus kaustophilus DSM 7263T (63.4%). On the basis of phylogenetic analysis and physiological traits of the isolate, it should be described as a new member of the Geobacillus thermoleovorans species and it is proposed that strain PizzoT can be classified as Geobacillus thermoleovorans subsp. stromboliensis, subsp. nov. (ATCC BAA-979T; DSM 15393T).