BackgroundDue to broad range of clinical and industrial applications of cholesterol oxidase, isolation and screening of bacterial strains producing extracellular form of cholesterol oxidase is of great importance.ResultsOne hundred and thirty actinomycete isolates were screened for their cholesterol oxidase activity. Among them, a potential culture, strain NEAE-42 is displayed the highest extracellular cholesterol oxidase activity. It was selected and identified as Streptomyces cavourensis strain NEAE-42. The optimization of different process parameters for cholesterol oxidase production by Streptomyces cavourensis strain NEAE-42 using Plackett–Burman experimental design and response surface methodology was carried out. Fifteen variables were screened using Plackett–Burman experimental design. Cholesterol, initial pH and (NH4)2SO4 were the most significant positive independent variables affecting cholesterol oxidase production. Central composite design was chosen to elucidate the optimal concentrations of the selected process variables on cholesterol oxidase production. It was found that, cholesterol oxidase production by Streptomyces cavourensis strain NEAE-42 after optimization process was 20.521U/mL which is higher than result obtained from the basal medium before screening process using Plackett-Burman (3.31 U/mL) with a fold of increase 6.19.ConclusionsThe cholesterol oxidase level production obtained in this study (20.521U/mL) by the statistical method is higher than many of the reported values.

Among the antitumor drugs, bacterial enzyme L-asparaginase has been employed as the most effective chemotherapeutic agent in pediatric oncotherapy especially for acute lymphoblastic leukemia. Glutaminase free L-asparaginase producing actinomycetes were isolated from soil samples collected from Egypt. Among them, a potential culture, strain NEAE-119, was selected and identified on the basis of morphological, cultural, physiological, and biochemical properties together with 16S rRNA sequence as Streptomyces olivaceus NEAE-119 and sequencing product (1509 bp) was deposited in the GenBank database under accession number KJ200342. The optimization of different process parameters for L-asparaginase production by Streptomyces olivaceus NEAE-119 using Plackett-Burman experimental design and response surface methodology was carried out. Fifteen variables (temperature, pH, incubation time, inoculum size, inoculum age, agitation speed, dextrose, starch, L-asparagine, KNO3, yeast extract, K2HPO4, MgSO4·7H2O, NaCl, and FeSO4·7H2O) were screened using Plackett-Burman experimental design. The most positive significant independent variables affecting enzyme production (temperature, inoculum age, and agitation speed) were further optimized by the face-centered central composite design-response surface methodology.

In recent years, microbial cholesterol oxidases have gained great attention due to its widespread use in medical applications for serum cholesterol determination. Streptomyces aegyptia strain NEAE-102 exhibited high level of extracellular cholesterol oxidase production using a minimum medium containing cholesterol as the sole source of carbon. Fifteen variables were screened using Plackett–Burman design for the enhanced cholesterol oxidase production. The most significant variables affecting enzyme production were further optimized by using the face-centered central composite design. The statistical optimization resulted in an overall 4.97-fold increase (15.631 UmL−1) in cholesterol oxidase production in the optimized medium as compared with the unoptimized medium before applying Plackett Burman design (3.1 UmL−1). The purified cholesterol oxidase was evaluated for its in vitro anticancer activities against five human cancer cell lines. The selectivity index values on rhabdomyosarcoma and breast cancer cell lines were 3.26 and 2.56; respectively. The in vivo anticancer activity of cholesterol oxidase was evaluated against Ehrlich solid tumor model. Compared with control mice, tumors growth was significantly inhibited in the mice injected with cholesterol oxidase alone, doxorubicin alone and cholesterol oxidase/doxorubicin combination by 60.97%, 72.99% and 97.04%; respectively. These results demonstrated that cholesterol oxidase can be used as a promising natural anticancer drug.

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Bioprocess development for L-asparaginase production by Streptomyces rochei, purification and in-vitro efficacy against various human carcinoma cell lines noura el-Ahmady el-naggar ✉ & nancy M. el-Shweihy in the near future, the demand for L-asparaginase is expected to rise several times due to an increase in its clinical and industrial applications in various industrial sectors, such as food processing. Streptomyces sp. strain NEAE-K is potent L-asparaginase producer, isolated and identified as new subsp. Streptomyces rochei subsp. chromatogenes neAe-K and the sequence data has been deposited under accession number KJ200343 at the GenBank database. Sixteen different independent factors were examined for their effects on L-asparaginase production by Streptomyces rochei subsp. chromatogenes NEAE-K under solid state fermentation conditions using Plackett-Burman design. pH, dextrose and yeast extract were the most significant factors affecting L-asparaginase production. Thus, using central composite design, the optimum levels of these variables were determined. L-asparaginase purification was carried out by ammonium sulfate followed by DEAE-Sepharose CL-6B ion exchange column with a final purification fold of 16.18. The monomeric molecular weight of the purified L-asparaginase was 64 kD as determined by SDS-PAGE method. The in vitro effects of L-asparaginase were evaluated on five human tumor cell lines and found to have a strong anti-proliferative effects. The results showed that the strongest cytotoxic effect of L-asparaginase was exerted on the HeLa and HepG-2 cell lines (ic 50 = 2.16 ± 0.2 and 2.54 ± 0.3 U/mL; respectively). In addition, the selectivity index of L-asparaginase against HeLa and HepG-2 cell lines was 3.94 and 3.35; respectively. L-asparaginase is one of the amidase group, that catalyzes the hydrolysis of L-asparagine and releases L-aspartic acid and ammonia 1. L-asparaginase is an effective therapeutic enzyme used in combination with other drugs for treating melanosarcoma, reticulosarbom, lymphocytic leukemia, Hodgkin disease, chronic lymphosarcoma, acute myelomonocytic leukemia, acute myelocytic leukemia and acute lymphoblastic leukemia in children and adults 2,3. In pediatric oncology, L-asparaginase is the recommended drug used for acute lymphoblastic leukemia therapy, resulting in a total recovery of more than 90 percent of kids within a period of four weeks 3. The anti-leukemic impact of L-asparaginase based on the fact that the tumor cells need a huge quantity of L-asparagine to survive. The tumor cells cannot synthesize L-asparagine for their needs because of the lack of L-asparagine synthetase and thus cannot synthesize the necessary proteins that rely on L-asparagine 4. The survival and growth of tumor cells rely on the exogenous source of L-asparagine (consumed in a diet, absorbed and accessible in the circulating pool) 5. Thus, injection of the enzyme into cancer patients intravenously decreases L-asparagine blood levels and selectively damages tumor cells 6. Conversely, the normal cell...

BackgroundThere is an increasing demand on cholesterol oxidase for its various industrial and clinical applications. The current research was focused on extracellular cholesterol oxidase production under submerged fermentation by a local isolate previously identified as Streptomyces aegyptia NEAE 102. The crude enzyme extract was purified by two purification steps, protein precipitation using ammonium sulfate followed by ion exchange chromatography using DEAE Sepharose CL-6B. The kinetic parameters of purified cholesterol oxidase from Streptomyces aegyptia NEAE 102 were studied.ResultsThe best conditions for maximum cholesterol oxidase activity were found to be 105 min of incubation time, an initial pH of 7 and temperature of 37 °C. The optimum substrate concentration was found to be 0.4 mM. The higher thermal stability behavior of cholesterol oxidase was at 50 °C. Around 63.86% of the initial activity was retained by the enzyme after 20 min of incubation at 50 °C. The apparent molecular weight of the purified enzyme as sized by sodium dodecyl sulphate-polyacryalamide gel electrophoresis was approximately 46 KDa. On DEAE Sepharose CL-6B column cholesterol oxidase was purified to homogeneity with final specific activity of 16.08 U/mg protein and 3.14-fold enhancement. The amino acid analysis of the purified enzyme produced by Streptomyces aegyptia NEAE 102 illustrated that, cholesterol oxidase is composed of 361 residues with glutamic acid as the most represented amino acid with concentration of 11.49 μg/mL.ConclusionsTaking into account the extracellular production, wide pH tolerance, thermal stability and shelf life, cholesterol oxidase produced by Streptomyces aegyptia NEAE 102 suggested that the enzyme could be industrially useful.

L-asparaginase is a promising enzyme that has a wide range of significant applications including cancer therapy and starchy food industries. The statistical design of Plackett–Burman and face centered central composite design were employed to optimize L-asparaginase production by Streptomyces brollosae NEAE-115. As a result, a medium of the following formula is the optimum for producing L-asparaginase in the culture filtrate of Streptomyces brollosae NEAE-115: Dextrose 2 g, starch 20 g, L-asparagine 10 g, KNO3 1 g, K2HPO4 1 g, MgSO4.7H2O 0.5 g, NaCl 0.1 g, pH 7, fermentation period 7 days, temperature 30 °C, inoculum size 4%, v/v, agitation speed 150 rpm and inoculum age 48 h. The kinetics of cell growth, carbohydrates consumption and L- asparaginase production were studied in 7-L stirred tank bioreactor under different cultivation conditions. A significant increase in both cell growth and carbohydrate consumption was observed as the stirring speed increased from 200 to 600 rpm under uncontrolled pH. The highest L- asparaginase activity of 108.46 U/mL was obtained after 96 h at 400 rpm. On the other hand, the specific enzyme production (Yp/x) under uncontrolled pH reached its maximal value of about 20.3 U/mg cells. Further improvement of enzyme production was attained by controlling pH at 7 using the selected stirring speed of 400 rpm. Enzyme production of 162.11 U/mL obtained from the controlled pH cultures exceeded this value gained from uncontrolled pH (108.46 U/mL) by about 50%.

Background: Cholesterol oxidase biosensors have been used to determine the level of cholesterol in different serum and food samples. Due to a wide range of industrial and clinical applications of microbial cholesterol oxidase, isolation and identification of a new microbial source (s) of cholesterol oxidase are very important. Results: The local isolate Streptomyces sp. strain NEAE-94 is a promising source of cholesterol oxidase. It was identified based on cultural, morphological and physiological characteristics; in addition to the 16S rRNA sequence. The sequencing product had been deposited in the GenBank database under the accession number KC354803. Cholesterol oxidase production by Streptomyces anulatus strain NEAE-94 in shake flasks was optimized using surface response methodology. The different process parameters were first screened using a Plackett-Burman design and the parameters with significant effects on the production of cholesterol oxidase were identified. Out of the 15 factors screened, agitation speed, cholesterol and yeast extract concentrations had the most significant positive effects on the production of cholesterol oxidase. The optimal levels of these variables and the effects of their mutual interactions on cholesterol oxidase production were determined using Box-Behnken design. Cholesterol oxidase production by Streptomyces anulatus strain NEAE-94 was 11.03, 27.31 U/mL after Plackett-Burman Design and Box-Behnken design; respectively, with a fold of increase of 6.06 times compared to the production before applying the Plackett-Burman design (4.51 U/mL). Conclusions: Maximum cholesterol oxidase activity was obtained at the following fermentation conditions: g/L (cholesterol 4, yeast extract 5, NaCl 0.5, K 2 HPO 4 1, FeSO 4 .7H 2 O 0.01, MgSO 4 .7H 2 O 0.5), pH 7, inoculum size 4% (v/v), temperature 37°C, agitation speed of 150 rpm, medium volume 50 mL and incubation time 5 days.