Efficient media for high lipase production: one variable at a time approach

Background: lipase enzymes have applications in a wide range of industries. A crucial determining factor of industrial prices of these enzymes is the culture media composition that is constantly under review by researchers. In this work, for maximum lipase production by Bacillus sp. ZR-5, culture media compositions were optimized using "one variable at a time" strategy

Methods: for this purpose, the culture medium parameters such as low and high cost carbon and nitrogen sources, substrates and incubation times were evaluated

Results: maximum lipase activity was achieved after 24 hr of incubation with 1.5% of glucose syrup [1600+/-69.1u/mg], 1% of fish powder [1238+/-36.7 u/mg] and olive oil [1407+/-2.1 u/mg] as low cost carbon and nitrogen sources and substrate, respectively

Conclusion: our results show a significant increase in lipase activity with usage of low cost sources; this could help in reducing the media prices for industrial application of lipase enzyme

Inactivation of aprE gene in bacillus subtilis 168 by homologus recombination

One of the most important producers of high quality industrial enzymes is the Gram-positive bacterium, Bacillus subtilis [B. Subtilis]. One major limitation that hinders the wide application of B. subtilis is the secretion of high levels of extracellular proteases which degrade the secreted foreign proteins. In this study, homologus recombination technique was used to knock out its protease gene, aprE. The internal segment of the pro-sequence of aprE gene of B. subtilis 168 with a length of 80 bps and its complementary sequence were synthesized and ligated into pUB110 at EcoR1 and XbaI restriction sites. Competent cells of B. subtilis 168 were prepared and transformed by electroporation using Bio Rad gene pulser as explained in the methods section. Transformants carrying the recombinant plasmid were selected for resistance to neomycin. The success of homologous recombination was checked by PCR amplification of the neomycin gene which was part of the vector and did not exist in the genome of B. subtilis 168. The protease activity was measured using the Protease Fluorescent Detection Kit based on the proteolytic hydrolysis of fluorescein isothiocyanate [FITC]-labeled casein-substrate. The results demonstrated that aprE gene would not be able to produce further active subtilisin E. The reduction of protease activity also confirmed the efficacy of the induced mutation in this gene. It will therefore be a major challenge for future research to identify and modulate quality control systems of B. subtilis which limit the production of high quality protease- sensitive products such as lipase

Acute and chronic effects of nitrendipine on naloxone precipitated morphine withdrawal in mice

There is growing evidence indicating that neuronal calcium channels play an important role in the mechanism of morphine dependence. In this study, the effects of acute and chronic administration of nitrendipine on naloxone precipitated morphine withdrawal signs were investigated. Mice were rendered dependent to morphine by subcutaneous injection of morphine over a period of 5 days. In chronic studies, nitrendipine [25 and 50 mg/kg, i.p.], or vehicle injections were given once a day during the morphine treatment, and the last injection of nitrendipine was given 24 h before the morphine withdrawal. For acute studies, nitrendipine [25 and 50 mg/kg, i.p.] was given 1 h after the last dose of morphine [1 h before naloxone]. A single injection of nitrendipine at 25 mg/kg was ineffective in blocking most signs of morphine withdrawal, however, at 50 mg/kg nitrendipine blocked signs such as hair raising, sniffing, diarrhea and number of jumping. The concurrent injections of nitrendipine with morphine prevented most signs of morphine withdrawal. In agreement with previous findings, these results suggest that alterations in voltage-sensitive calcium channels play a role in the adaptations that occur on chronic treatment with morphine