Laccase- and peroxidase-free tyrosinase production by isolated microbial strain.

Laccase- and peroxidase-free tyrosinase has commercial importance in the production of L-3, 4-dihydroxyphenylalanine (L-DOPA), which is mainly used in the treatment of Parkinson's disease. In the present study, isolation of an actinomycetes microbial strain capable of producing only tyrosinase is reported. Among all soil isolates, three individual colonies revealed black color around the colony in the presence of tyrosine. Further screening for laccase and peroxidase activities using syringaldazine denoted that one of the isolates, designated as RSP-T1, is laccase and peroxidase negative and produces only tyrosinase. The microbe was authenticated as Streptomyces antibioticus based on 16S ribotyping. Effective growth of this isolate was noticed with the use of medium (pH 5.5) containing casein acid hydrolysate (10.0 g/l), K(2)HPO(4) (5.0 g/l), MgSO(4) (0.25 g/l), L-tyrosine (1.0 g/l), and agar (15 g/l). The scanning electron micrograph depicted that the microbe is highly branched and filamentous in nature. The enzyme production was positively regulated in the presence of copper sulfate. The impact of different fermentation parameters on tyrosinase production depicted that the maximized enzyme titer values were observed when this isolate was grown at 6.5 pH and at 30 degrees C temperature under agitated conditions (220 rpm). Among all the studied physiological parameters, agitation played a significant role on tyrosinase production. Upon optimization of the parameters, the yield of tyrosinase was improved more than 100% compared with the initial yield.

Cytological and biochemical evidence for an early cell dismantling event in surface cultures of Streptomyces antibioticus.

A process of programmed cell death taking place late in the aerial mycelium was previously reported in surface cultures of Streptomyces antibioticus ATCC11891. In this study, we present evidence for the occurrence of a similar process taking place early in the vegetative mycelium of surface cultures of the same strain. Several indicators, such as cell wall and membrane disruption, DNA degradation and release of the cytoplasmic content into the exocellular medium, support the existence of active, highly regulated cell suicide involving specific enzymes. Calcium-dependent proteolytic activation of a precursor of nucleases and the nucleolytic formation of a ladder of chromosomal bands are conspicuous events associated with the initiation of the death process.

Mycelium development in Streptomyces antibioticus ATCC11891 occurs in an orderly pattern which determines multiphase growth curves.

BACKGROUND: The current model for the developmental cycle of Streptomyces confluent cultures on agar surface is based on the assumption that the only differentiation takes place along the transverse axis (bottom-up): a vegetative (substrate) mycelium grows completely live and viable on the surface and inside the agar until it undergoes a death process and differentiates to a reproductive (aerial) mycelium which grows into the air. Hence, this vertical description assumes that the development in the pre-sporulating phases is more or less homogeneous in all zones of the plate surface. RESULTS: The work presents a detailed analysis of the differentiation cycle in Streptomyces antibioticus ATCC11891 considering a different spatial dimension: the longitudinal axes, represented by the plate surface. A previously unsuspected complexity during the substrate mycelial phase was detected. We have demonstrated that the young substrate hyphae suffer an early death round that has not been previously described. Subsequently, the remaining mycelium grows in successive waves which vary according to the density of the spore inoculum. In the presence of dense inocula (1.5 x 10(6) spores per plate), the hyphae develop in regular circles, approximately 0.5 cm in diameter. By contrast, with highly diluted inocula (6 x 10(3) spores per plate), aerial mycelium develops initially in the form of islands measuring 0.9 mm in diameter. Further mycelial development occurs between the circles or islands until the plate surface is totally covered. This pattern persists throughout the entire developmental cycle including the sporulation phases. CONCLUSION: An early death round during the substrate mycelial phase of Streptomyces antibioticus ATCC11891 takes place prior to successive growth periods in surface cultures. These developmental periods in turn, determine the shape of the complex multiphase growth curves observed. As shown here, these results also apply to other Streptomyces strains and species. Understanding these peculiarities of the Streptomyces developmental cycle is essential in order to properly interpret the morphological/biochemical data obtained from solid cultures and will expand the number of potential phenotypes subject to study.

Nuclease activities and cell death processes associated with the development of surface cultures of Streptomyces antibioticus ETH 7451.

The presence and significance of developmentally regulated nucleases in Streptomyces antibioticus ETH 7451 has been studied in relation to the lytic processes occurring during differentiation. The cell-death processes have been followed in surface cultures by a propidium iodide viability assay. This has allowed the visualization of dead (membrane-damaged, red fluorescent) and live (membrane-intact, green fluorescent) mycelium during development, and has facilitated the analysis of the role of nucleases in these processes. A parallel activity-gel analysis showed the appearance of 20-22 kDa, 34 kDa and 44 kDa nucleases, the latter appearing only when aerial mycelium is formed. The appearance of these nucleases shows a remarkable correlation with the death process of the mycelium during differentiation and with chromosomal DNA degradation. The 20-22 kDa enzymes are possibly related to the lytic phenomena taking place in the vegetative substrate mycelium before the emergence of the reproductive aerial mycelium, whereas the function of the 44 kDa nuclease seems to be related to the sporulation step. The 20-22 kDa nucleases require Ca2+ for activity and are inhibited by Zn2+. The nucleases are loosely bound to the cell wall from where they can be liberated by simple washing. Conceivably, these enzymes work together and co-ordinate to achieve an efficient hydrolysis of DNA from dying cells. The results show that the biochemical reactions related with the lytic DNA degradation during the programmed cell death are notably conserved in Streptomyces. Some of the features of the process and the biochemical characteristics of the enzymes involved are analogous to those taking place during the DNA fragmentation processes in eukaryotic apoptotic cells.

Actinomycin production persists in a strain of Streptomyces antibioticus lacking phenoxazinone synthase.

Truncated fragments of the phenoxazinone synthase gene, phsA, were prepared by the PCR. The resulting fragments were cloned into conjugative plasmid pKC1132 and transferred to Streptomyces antibioticus by conjugation from Escherichia coli. Two of the resulting constructs were integrated into the S. antibioticus chromosome by homologous recombination, and each of the resulting strains, designated 3720/pJSE173 and 3720/pJSE174, contained a disrupted phsA gene. Strain 3720/pJSE173 grew poorly, and Southern blotting suggested that genetic changes other than the disruption of the phsA gene might have occurred during the construction of that strain. Strain 3720/pJSE174 sporulated well and grew normally on the medium used to prepare inocula for antibiotic production. Strain 3720/pJSE174 also grew as well as the wild-type strain on antibiotic production medium containing either 1 or 5.7 mM phosphate. Strain 3720/pJSE174 was shown to be devoid of phenoxazinone synthase (PHS) activity, and PHS protein was undetectable in this strain by Western blotting. Despite the absence of detectable PHS activity, strain 3720/pJSE174 produced slightly more actinomycin than did the wild-type parent strain in medium containing 1 or 5.7 mM phosphate. The observation that strain 3720/pJSE174, lacking detectable PHS protein or enzyme activity, retained the ability to produce actinomycin supports the conclusion that PHS is not required for actinomycin biosynthesis in S. antibioticus.

Purification, characterization, and role of nucleases and serine proteases in Streptomyces differentiation. Analogies with the biochemical processes described in late steps of eukaryotic apoptosis.

Two exocellular nucleases with molecular masses of 18 and 34 kDa, which are nutritionally regulated and reach their maximum activity during aerial mycelium formation and sporulation, have been detected in Streptomyces antibioticus. Their function appears to be DNA degradation in the substrate mycelium, and in agreement with this proposed role the two nucleases cooperate efficiently with a periplasmic nuclease previously described in Streptomyces antibioticus to completely hydrolyze DNA. The nucleases cut DNA nonspecifically, leaving 5'-phosphate mononucleotides as the predominant products. Both proteins require Mg2+, and the additional presence of Ca2+ notably stimulates their activities. The two nucleases are inhibited by Zn2+ and aurin tricarboxylic acid. The 18-kDa nuclease from Streptomyces is reminiscent of NUC-18, a thymocyte nuclease proposed to have a key role in glucocorticoid-stimulated apoptosis. The 18-kDa nuclease was shown, by amino-terminal protein sequencing, to be a member of the cyclophilin family and also to possess peptidylprolyl cis-trans-isomerase activity. NUC-18 has also been shown to be a cyclophilin, and "native" cyclophilins are capable of DNA degradation. The S. antibioticus 18-kDa nuclease is produced by a proteolytic processing from a less active protein precursor. The protease responsible has been identified as a serine protease that is inhibited by Nalpha-p-tosyl-L-lysine chloromethyl ketone and leupeptin. Inhibition of both of the nucleases or the protease impairs aerial mycelium development in S. antibioticus. The biochemical features of cellular DNA degradation during Streptomyces development show significant analogies with the late steps of apoptosis of eukaryotic cells.

Hyphal death during colony development in Streptomyces antibioticus: morphological evidence for the existence of a process of cell deletion in a multicellular prokaryote.

During the life cycle of the streptomycetes, large numbers of hyphae die; the surviving ones undergo cellular differentiation and appear as chains of spores in the mature colony. Here we report that the hyphae of Streptomyces antibioticus die through an orderly process of internal cell dismantling that permits the doomed hyphae to be eliminated with minimum disruption of the colony architecture. Morphological and biochemical approaches revealed progressive disorganization of the nucleoid substructure, followed by degradation of DNA and cytoplasmic constituents with transient maintenance of plasma membrane integrity. Then the hyphae collapsed and appeared empty of cellular contents but retained an apparently intact cell wall. In addition, hyphal death occurred at specific regions and times during colony development. Analysis of DNA degradation carried out by gel electrophoresis and studies on the presence of dying hyphae within the mycelium carried out by electron microscopy revealed two rounds of hyphal death: in the substrate mycelium during emergence of the aerial hyphae, and in the aerial mycelium during formation of the spores. This suggests that hyphal death in S. antibioticus is somehow included in the developmental program of the organism.

A membrane-like structure envelopes substrate mycelium during colony development in Streptomyces.

Colonies of Streptomyces antibioticus were studied by transmission and scanning electron microscopy. The micrographs show that substrate mycelium growth takes place among an intercellular material and this mycelium is covered by a surface film. This structure could be a boundary between the aerial mycelium and the substrate mycelium.

Nutritional regulation of differentiation and synthesis of an exocytoplasmic deoxyriboendonuclease in Streptomyces antibioticus.

Streptomyces antibioticus produces a cell-wall-located deoxyriboendonuclease (DNAase) the synthesis of which in submerged and surface cultures is related to the growth rate. DNAase synthesis always preceded aerial mycelium formation in surface cultures. Production of aerial mycelium began at the end of exponential growth or in the early stationary phase; it was absent in cultures grown on nutrient agar/glucose or in media with a high concentration of casein hydrolysate. These nutritional conditions also impaired production of the DNAase. External DNA substrates were not degraded by mycelium producing the DNAase. These observations lead us to suggest a role for the enzyme in the developmental cycle of S. antibioticus.

Fusäo de protoplastos de Streptomyces capoamus visando a obtençäo de cepas produtoras de antibióticos "híbridos" / Protoplasts fusion of Streptomyces capoamus for obtention of strains productors of hybrid antibiotics

Pela fusäo protoplastos inativados por aquecimento a 60-C, de Streptomyces capoamus, cepa IA-M3122/182 (prototrófica, produtora de pigmento vermelho e do antibiótico antraciclínico ciclamicina) com protoplastos viáveis de Streptomyces melanochromogenes, cepa IA-M3183 (auxotrófica met- e produtora de actinomicina), foram obtidas cinco cepas com características sugestivas de recombinaçäo. Adicionalmente, foi observado que a simples produçäo e regeneraçäo de protoplastos do Streptomyces capoamus IA-M3122/182 levou à obtençäo de subculturas cuja produçäo de ciclamicina foi aumentada entre 3 e 7 vezes em relaçäo à cultura original

[Developmental cycle and the cytomorphological characteristics of the oleandomycin producer Streptomyces antibioticus]. / Tsikl razvitiia i tsitomorfologicheskie osobennosti produtsenta oleandomitsina Streptomyces antibioticus.

The developmental cycle and cytomorphological features of the industrial strain OL-1 and its variant 0968 of the oleandomycin-producing organism were studied. Variant 0968 was obtained as a result of exposure of the spores of strain OL-1 to UV light. When grown under submerged conditions in flasks with the rich medium, the strains were characterized by a complete developmental cycle consisting of three generations of the hyphae. Every generation had a tendency for formation of submerged spores. The UV-induced variant differed from the industrial strain by higher levels of the antibiotic accumulation which correlated with higher rates of the spore germination. The strains were characterized by polymorphism of the mycelium and formation of submerged spores during their cultivation which is likely to prolong the antibiotic synthesis from 120 to 216 hours from the inoculation moment. The long-term selection of the oleandomycin-producing organism on the rich medium markedly changed the culture genotype and resulted in significant changes in the developmental cycle under submerged cultivation conditions. The data may be used for the microscopic control of the process of oleandomycin production.