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L-ASPARAGINASE is an anti-neoplasm agent. This agent is classified as an enzyme compound. L-asparaginase is used in medicine as well as in the food industry. Microbial strains that are producing L-Asparaginase can be found in many habitats such as soil. The aim of this study was to find isolates producing L-asparaginase enzyme with high ability to inhibit cancer cells. Bacteria of the Actinobacter family were isolated from the soil samples around the roots of the plants in the actinomycet isolation medium. Then, the bacteria were isolated and extracted by screening in M9 medium. Isolates were identified using16S PCR sequencing method. Three isolates of ASP and B and C showed the highest enzyme production, which showed a molecular weight of 43 kDa and in terms of enzymatic activity112. 572 IU/ml, 99.706 IU/ml, 138.572 IU/ml respectively. These three isolates with sequence analysis was similar to Streptomyces flavoviridis and Streptomyces griseus and Streptomyces hypolyticus respectively. Isolate C showed good effects in inhibiting cell MCF-7, HepG-2. There are various isolates enzyme production power in nature and Actinobacter family has shown more ability to produce various enzymes and metabolites and the isolates isolated in this study are used for industrial enzyme production.

Endophytic actinomycetes from medicinal plants were rarely reported as compared to those from soil and marine sources. The present results correlate the presence of endophytic actinomycetes in medicinal plants by isolating them from three medicinal plants i.e. Ocimum basillicum, Withania somnifera and Rauvolfia tetraphylla. In the present work surface sterilization method and media was standardized and 32 endophytic actinomycetes were isolated from three medicinal plants. We assessed the efficiency of four different surface sterilization methods and four medium for isolation of endophytic actinomycetes. The method with calcium hypochlorite, sodium hydrogen carbonate and sodium azide (New method) used was more effective in eliminating epiphytic microorganisms. Among four tested media starch casein agar (SCA) found to be best media for the isolation of endophytic actinomycetes. We succeeded in obtaining fast and luxurious growth of pure culture of endophytic actinomycetes on modified ISP-4 (M) (inorganic salt solution agar) in comparison with standard ISP-4(S). Preliminary antibacterial assay of all isolates were tested by confrontation test. The secondary screening of selected isolates were tested by disc diffusion test by using ethyl acetate extract which showed broad spectrum antibacterial activity against test human pathogens. Based on the morphological and phenotypic characters 12 isolates were identified as Streptomyces spp. Among 12 isolates A3 as a representative was characterized by SEM and identified by 16SrRNA analysis as Streptomyces flavoviridis A3WK, it showed significant antibacterial activity against test human pathogen. This is the first report of successful isolation of endophytic actinomycetes from the said medicinal plants, for using newly formulated surface sterilization method and new comparative study on ISP-4 medium.

The bleomycin (BLM) family of glycopeptide-derived antitumor antibiotics consists of BLMs, tallysomycins (TLMs), phleomycins (PLMs), and zorbamycin (ZBM). The self-resistant elements BlmB and TlmB, discovered from the BLM- and TLM-producing organisms Streptomyces verticillus ATCC15003 and Streptoalloteichus hindustanus E465-94 ATCC31158, respectively, are N-acetyltransferases that provide resistance to the producers by disrupting the metal-binding domain of the antibiotics required for activity. Although each member of the BLM family of antibiotics possesses a conserved metal-binding domain, the structural differences between each member, namely, the bithiazole moiety and C-terminal amine of BLMs, have been suggested to instill substrate specificity within BlmB. Here we report that BlmB and TlmB readily accept and acetylate BLMs, TLMs, PLMs, and ZBM in vitro but only in the metal-free forms. Kinetic analysis of BlmB and TlmB reveals there is no strong preference or rate enhancement for specific substrates, indicating that the structural differences between each member of the BLM family play a negligible role in substrate recognition, binding, or catalysis. Intriguingly, the zbm gene cluster from Streptomyces flavoviridis ATCC21892 does not contain an N-acetyltransferase, yet ZBM is readily acetylated by BlmB and TlmB. We subsequently established that S. flavoviridis lacks the homologue of BlmB and TlmB, and ZbmA, the ZBM-binding protein, alone is sufficient to provide ZBM resistance. We further confirmed that BlmB can indeed confer resistance to ZBM in vivo in S. flavoviridis, introduction of which into wild-type S. flavoviridis further increases the level of resistance.

To obtain basic information on how microbial cells absorb cadmium from aqueous solution, we examined cadmium absorption in various micro-organisms. Of 51 micro-organism strains tested, we found that some Gram-positive bacteria, such as, Arthrobacter nicotianae and Bacillus subtilis, and some actinomycetes, such as, Streptomyces flavoviridis and S. levoris were highly capable of absorbing cadmium from an aqueous solution. A. nicotianae absorbed the largest amount of cadmium, over 800 μmol cadmium per gram of dry wt. cells. However, cadmium absorption by A. nicotianae was affected by the solution pH, cadmium concentration, and cell density. The absorption of cadmium was very rapid. Some factors that affected cadmium absorption by A. nicotianae cells were also discussed.

The bleomycins (BLMs) are used clinically in combination with a number of other agents for the treatment of several types of tumors, and the BLM, etoposide, and cisplatin treatment regimen cures 90-95% of metastatic testicular cancer patients. BLM-induced pneumonitis is the most feared, dose-limiting side effect of BLM in chemotherapy, which can progress into lung fibrosis and affect up to 46% of the total patient population. There have been continued efforts to develop new BLM analogues in the search for anticancer drugs with better clinical efficacy and lower lung toxicity. We have previously cloned and characterized the biosynthetic gene clusters for BLMs from Streptomyces verticillus ATCC15003, tallysomycins from Streptoalloteichus hindustanus E465-94 ATCC31158, and zorbamycin (ZBM) from Streptomyces flavoviridis SB9001. Comparative analysis of the three biosynthetic machineries provided the molecular basis for the formulation of hypotheses to engineer novel analogues. We now report engineered production of three new analogues, 6'-hydroxy-ZBM, BLM Z, and 6'-deoxy-BLM Z and the evaluation of their DNA cleavage activities as a measurement for their potential anticancer activity. Our findings unveiled: (i) the disaccharide moiety plays an important role in the DNA cleavage activity of BLMs and ZBMs, (ii) the ZBM disaccharide significantly enhances the potency of BLM, and (iii) 6'-deoxy-BLM Z represents the most potent BLM analogue known to date. The fact that 6'-deoxy-BLM Z can be produced in reasonable quantities by microbial fermentation should greatly facilitate follow-up mechanistic and preclinical studies to potentially advance this analogue into a clinical drug.

The biosynthetic gene clusters for the glycopeptide antitumor antibiotics bleomycin (BLM), tallysomycin (TLM), and zorbamycin (ZBM) have been recently cloned and characterized from Streptomyces verticillus ATCC15003, Streptoalloteichus hindustanus E465-94 ATCC31158, and Streptomyces flavoviridis ATCC21892, respectively. The striking similarities and differences among the biosynthetic gene clusters for the three structurally related glycopeptide antitumor antibiotics prompted us to compare and contrast their respective biosynthetic pathways and to investigate various enzymatic elements. The presence of different numbers of isolated nonribosomal peptide synthetase (NRPS) domains in all three clusters does not result in major structural differences of the respective compounds. The seemingly identical domain organization of the NRPS modules responsible for heterocycle formation, on the other hand, is contrasted by the biosynthesis of two different structural entities, bithiazole and thiazolinyl-thiazole, for BLM/TLM and ZBM, respectively. Variations in sugar biosynthesis apparently dictate the glycosylation patterns distinct for each of the BLM, TLM, and ZBM glycopeptide scaffolds. These observations demonstrate nature’s ingenuity and flexibility in achieving structural differences and similarities via various mechanisms and will surely inspire combinatorial biosynthesis efforts to expand on natural product structural diversity.

The bleomycins (BLMs), tallysomycins (TLMs), phleomycin, and zorbamycin (ZBM) are members of the BLM family of glycopeptide-derived antitumor antibiotics. The BLM-producing Streptomyces verticillus ATCC15003 and the TLM-producing Streptoalloteichus hindustanus E465-94 ATCC31158 both possess at least two self-resistance elements, an N-acetyltransferase and a binding protein. The N-acetyltransferase provides resistance by disrupting the metal-binding domain of the antibiotic that is required for activity while the binding protein confers resistance by sequestering the metal-bound antibiotic and preventing drug activation via molecular oxygen. We recently established that the ZBM producer, Streptomyces flavoviridis ATCC21892, lacks the N-acetyltransferase resistance gene and that the ZBM-binding protein, ZbmA, is sufficient to confer resistance in the producing strain. To investigate the resistance mechanism attributed to ZbmA, we determined the crystal structures of apo and Cu(II)-ZBM-bound ZbmA at the high resolutions of 1.90 Å and 1.65 Å, respectively. Comparison and contrast with other structurally characterized members of the BLM-binding protein family revealed key differences in the protein-ligand binding environment that fine-tunes the ability of ZbmA to sequester metal-bound ZBM and supports drug sequestration as the primary resistance mechanism in the producing organisms of the BLM-family of antitumor antibiotics.

The biosynthetic gene cluster for the glycopeptide-derived antitumor antibiotic zorbamycin (ZBM) was cloned by screening a cosmid library of Streptomyces flavoviridis ATCC 21892. Sequence analysis revealed 40 ORFs belonging to the ZBM biosynthetic gene cluster. However, only 23 and 22 ORFs showed striking similarities to the biosynthetic gene clusters for the bleomycins (BLMs) and tallysomycins (TLMs), respectively; the remaining ORFs do not show significant homology to ORFs from the related BLM and TLM clusters. The ZBM gene cluster consists of 16 nonribosomal peptide synthetase (NRPS) genes encoding eight complete NRPS modules, three incomplete didomain NRPS modules, and eight freestanding single NRPS domains or associated enzymes, a polyketide synthase (PKS) gene encoding one PKS module, six sugar biosynthesis genes, as well as genes encoding other biosynthesis and resistance proteins. A genetic system using Escherichia coli-Streptomyces flavoviridis intergeneric conjugation was developed to enable ZBM gene cluster boundary determinations and biosynthetic pathway manipulations.