Isolation of an organic solvent-tolerant bacterium Bacillus licheniformis PAL05 that is able to secrete solvent-stable lipase.

In this study, seven lipase-producing bacterial strains were isolated from salt-enriched and cattle farm soil samples after incubation in toluene- and benzene-enriched media. One strain (PAL05) showed significantly greater lipase activity on spirit blue agar medium and stability in organic solvents. The positive strain (PAL05) was identified as Bacillus licheniformis by 16S rRNA gene sequencing. Lipase production was optimized in a medium containing glycerol as the carbon source and Tween 80 as an inducer (0.5% glycerol+0.5% Tween 80) at pH 8.0 and a temperature of 30 °C. In addition, the enzyme was moderately halotolerant as it exhibited increased activity in the presence of 2.5% NaCl. Optimized conditions increased the lipase production threefold. Crude lipase retained its activity for 14 days of incubation in the presence of various organic solvents at a level of 25% and 50%. The enzyme was stable at 25% in most solvents; some of the solvents such as hexane, benzene, and ethanol actually stimulated enzyme activity. The organic solvent stability of the lipase produced by the strain PAL05 enables the enzyme to be used as a potential biocatalyst for ester synthesis and other applications in nonaqueous conditions.

Isolation and characterization of a novel oxidant- and surfactant-stable extracellular alkaline protease from Exiguobacterium profundum BK-P23.

The novel protease from Exiguobacterium profundum BK-P23 was partially purified by ammonium sulfate precipitation and further purified Mono Q 5/50 and Superdex 200 10/300 column chromatography. The enzyme was purified 10.23-fold with a yield of 14%. The molecular weight was estimated to be 52 kDa by SDS-PAGE. The enzyme was most active at a pH of 8.0 and temperature of 40°C and the enzyme was stable between a pH of 7 and 10 and up to a temperature of 50°C. The enzyme activity was enhanced by CaCl2 but was slightly inhibited by CoCl2 , MgSO4 , and AgNO3 . In addition, this enzyme was completely inhibited by phenylmethylsulfonyl fluoride, indicating that this enzyme was a serine protease. Furthermore, the alkaline protease was more stable in the presence of surfactants such as Triton X-100, which was followed by Tween 80 and SDS. Moreover, the enzyme was highly stable in the presence of 1% oxidizing agent (H2 O2 ). The enzyme also has significant stability (70%-80%) in a few organic solvents. Thus, the increased stability of the enzyme in the presence of oxidizing agent, surfactants, and organic solvents may find potential applications in the detergent industry and peptide synthesis in nonaqueous media.

Growth of the oleaginous microalga Aurantiochytrium sp. KRS101 on cellulosic biomass and the production of lipids containing high levels of docosahexaenoic acid.

We examined the growth of a novel oleaginous microalga, Aurantiochytrium sp. KRS101, using cellulosic materials as nutrients, and the resultant production of lipids containing high levels of docosahexaenoic acid (DHA). The microalgal strain could grow using either carboxymethylcellulose or cellobiose as a carbon source, and produced lipids containing high levels of DHA (49-58% of total fatty acids). In line with this growth behavior, carboxymethylcellulase and cellobiohydrolase activities were evident in both cell-free lysates and culture broths. Additionally, an industrial cellulosic biomass, palm oil empty fruit bunches (POEFB), a by-product of the palm oil industry, were utilized by the microalgal strain for cell growth and lipid production.

Production of lipids containing high levels of docosahexaenoic acid by a newly isolated microalga, Aurantiochytrium sp. KRS101.

In the present study, a novel oleaginous Thraustochytrid containing a high content of docosahexaenoic acid (DHA) was isolated from a mangrove ecosystem in Malaysia. The strain identified as an Aurantiochytrium sp. by 18S rRNA sequencing and named KRS101 used various carbon and nitrogen sources, indicating metabolic versatility. Optimal culture conditions, thus maximizing cell growth, and high levels of lipid and DHA production, were attained using glucose (60 g l⁻¹) as carbon source, corn steep solid (10 g l⁻¹) as nitrogen source, and sea salt (15 g l⁻¹). The highest biomass, lipid, and DHA production of KRS101 upon fed-batch fermentation were 50.2 g l⁻¹ (16.7 g l⁻¹ day⁻¹), 21.8 g l⁻¹ (44% DCW), and 8.8 g l⁻¹ (40% TFA), respectively. Similar values were obtained when a cheap substrate like molasses, rather than glucose, was used as the carbon source (DCW of 52.44 g l⁻¹, lipid and DHA levels of 20.2 and 8.83 g l⁻¹, respectively), indicating that production of microbial oils containing high levels of DHA can be produced economically when the novel strain is used.

Taxol-producing [corrected] fungal endophyte, Pestalotiopsis species isolated from Taxus cuspidata.

The endophytic fungi, Pestalotiopsis versicolor and Pestalotiopsis neglecta, were isolated from the healthy leaves and bark of the Japanese Yew tree, Taxus cuspidata. The fungal species were identified by their characteristic culture morphology and molecular analysis. For the first time, the test fungi were screened for the production of taxol in modified liquid medium. The presence of taxol was confirmed by HPLC, (1)H NMR, and LC-MS methods of analysis. The maximum amount of taxol production in P. versicolor was recorded as 478 µg/l. The production rate was increased to 9560-fold than that found in the culture broth of earlier reported fungus, Taxomyces andreanae. The extracted fungal taxol showed a strong cytotoxic activity in the in vitro culture of tested human cancer cells by apoptotic assay indicating that the increase in taxol concentration induces increased cell death. A PCR-based screening for ts, a unique gene in the formation of the taxane skeleton, confirmed the molecular blueprint for taxol biosynthesis. The results designate that the fungal endophyte, P. versicolor, is an excellent candidate for an alternate source of taxol supply and can also serve as a potential species for genetic engineering to enhance the production of taxol to a higher level.

Isolation and characterization of the eicosapentaenoic acid biosynthesis gene cluster from Shewanella sp. BR-2.

Forty-four eicosapentaenoic acid (EPA)-producing microbial strains were isolated from the intestines of marine fishes. Among them, one strain showing a maximum level of EPA (4.78%of total fatty acids) was identified as Shewanella sp. BR-2 on the basis of its 16S rRNA sequence. The EPA content reached a maximum level during the mid-exponential phase of cell growth, and gradually decreased with further growth of the cells. A cosmid DNA including the EPA biosynthesis gene cluster consisting of pfaA-E was isolated from a cosmid library of genomic DNA of Shewanella sp. BR-2, named pCosEPA-BR2. An E. coli clone harboring pCosEPA-BR2 produced EPA at a maximum level of 7.5%of total fatty acids, confirming the EPA biosynthesis activity of the cloned gene cluster.

Isolation and identification of an anticancer drug, taxol from Phyllosticta tabernaemontanae, a leaf spot fungus of an angiosperm, Wrightia tinctoria.

Phyllosticta tabernaemontanae, a leaf spot fungus isolated from the diseased leaves of Wrightia tinctoria, showed the production of taxol, an anticancer drug, on modified liquid medium (M1D) and potato dextrose broth (PDB) medium in culture for the first time. The presence of taxol was confirmed by spectroscopic and chromatographic methods of analysis. The amount of taxol produced by this fungus was quantified using high performance liquid chromatography (HPLC). The maximum amount of taxol production was recorded in the fungus grown on M1D medium (461 microg/L) followed by PDB medium (150 microg/L). The production rate was increased to 9.2 x 10(3) fold than that found in the culture broth of earlier reported fungus, Taxomyces andreanae. The results designate that P. tabernaemontanae is an excellent candidate for taxol production. The fungal taxol extracted also showed a strong cytotoxic activity in the in vitro culture of tested human cancer cells by apoptotic assay.

Screening of species of the endophytic fungus Phomopsis for the production of the anticancer drug taxol.

Three different strains of the endophytic fungus Phomopsis were isolated from the healthy leaves of Taxus cuspidata (Japanese yew), Ginkgo biloba (ginkgo or maidenhair tree) and Larix leptolepis (Japanese larch) and screened for the production of taxol on a modified liquid medium for the first time. The presence of taxol was confirmed by spectroscopic and chromatographic analyses. The amount of taxol produced by this fungus was quantified by HPLC. The maximum amount of fungal taxol production was recorded as 418 microg/litre in the strain BKH 27. The yield was increased to 8360-fold that found for the fungus Taxomyces andreanae reported previously [Stierle, Strobel and Stierle (1993) Science 260, 214-216]. The fungal taxol extracted also showed a strong cytotoxicity towards the human cancer cells in an apoptosis assay. All the three isolates showed positive sign towards PCR for the conserved sequence of the taxadiene synthase gene. The results suggest that Phomopsis could be an excellent alternative source for taxol and may serve as a potential genetic-engineered species for the enhanced production of taxol.

Purification, characterization and crystallization of an extracellular alkaline protease from Aspergillus nidulans HA-10.

Aspergillus nidulans is a highly potent fungus used in the production of alkaline protease. Extracellular alkaline protease was purified from A. nidulans in a two-step procedure involving ammonium sulphate precipitation and Sephadex G-100 column chromatography. The molecular mass of the enzyme was determined to be 42 kDa by SDS-PAGE. The enzyme activity was also analyzed by zymogram with gelatin. The enzyme was more stable over a wide range of pH (6-10) and the temperatures up to 50 degrees C. It showed optimum enzyme activity at pH 8.0 and a temperature of 35 degrees C. The protease enzyme was completely inhibited by the serine protease inhibitor of phenylmethylsulfonyl fluoride (PMSF). The crystallization of the purified enzyme was performed by hanging drop vapour diffusion method using PEG 6000 as the precipitant. The micro crystals occurred in 40% of PEG 6,000.

Taxol from Phyllosticta citricarpa, a leaf spot fungus of the angiosperm Citrus medica.

Phyllosticta citricarpa, a leaf spot fungus isolated from the diseased leaves of Citrus medica, displayed the production of taxol, an anticancer drug on M1D and potato dextrose broth medium in culture for the first time. The presence of taxol was confirmed by spectroscopic and chromatographic methods of analysis. The maximum amount of taxol production was recorded in the fungus grown on M1D medium (265 microg/l) followed by PDB medium (137 microg/l). The production rate was increased to 5.3 x 10(3) fold than that observed in the culture broth of an earlier reported fungus, Taxomyces andreanae.

Enhancement of heterologous production of eicosapentaenoic acid in Escherichia coli by substitution of promoter sequences within the biosynthesis gene cluster.

To enhance the heterologous production of eicosapentaenoic acid (EPA) in Escherichia coli, the EPA biosynthesis gene cluster from Shewanella oneidensis MR-1 was cloned under the lacZ promoter on a high-copy number plasmid, pBluescript SK+. The production of EPA was remarkably enhanced yielding levels of up to 7.5% of the total fatty acid content in the recombinant E. coli strain by induction with IPTG, whereas the stimulation of EPA production was abolished by adding glucose into the culture medium, probably due to glucose repression acting on the promoter activity.

Study on persistent infection of Japanese encephalitis virus Beijing-1 strain in serum-free Sf9 cell cultures.

Sf9 cells have obvious advantages for the conventional production technology of vaccine. They are useful tools for high concentration and large-scale cultures. Sf9 cells were grown to maximal concentration, 8 x 10(6) cells/ml in a 500ml spinner flask, with a doubling time at the exponentially growing phase of 24.5 hours, using serum-free media. To explore the ability of Sf9 cells to be infected by the Japanese encephalitis (JE) virus Beijing-1 strain, Sf9 cells were infected with the virus. By 4-5 days post-infection, 10-15% of the Sf9 cells showed cytopathic effect (CPE), from granularity to the formation of syncytia and multinucleated giant cells continuously observed over a period of 35 days. Positive fluorescent reactions were detected in 30-40% of cells infected with the JE virus Beijing-1 strain, and the uninfected Sf9 cells were completely negative. Virus particles, propagated in Sf9 and Vero cells, were concentrated by sedimentation on 40% trehalose cushions by ultracentrifugation, and showed identical patterns of viral morphogenesis. Complete virus particles, 40 to 50 nm in diameter, were observed, and JE virus envelope (E) proteins, at 53 kDa, were found in the western blot analysis to the anti-JE virus E protein monoclonal antibody and reacted as a magenta band in the same position to the glycoprotein staining. To evaluate whether the infectious virus was produced in Sf9 cells inoculated with the JE virus Beijing-1 stain, Sf9 cells were inoculated with the virus, and sample harvested every 5 days. The titers of the JE virus Beijing-1 strain rose from 1.0 x 10(5) to 1.5 x 10(6) pfu/ml. The infected Sf9 cells could be sub-cultured in serum-free medium, with no change in the plaque sizes formed by the JE virus Beijing-1 strain in the plaque assay. It is suggested that the ability of the JE virus Beijing-1 strain to infect Sf9 cells in serum-free media will provide a useful insect cell system, where the JE virus replication, cytopathogenicity and vaccine immunogen can be studied.