ABSTRACT:The current study was carried out to evaluate the effects of gamma irradiation on the epiphytic microflora and ripening process of the green Dwarf Cavendish bananas harvested at the three-quarter stage of the maturity. The mature green bananas were irradiated using Cobalt-60 as the source of irradiation at different dosages of 0.5, 0.75 and 1.0 kGy. The mean life of both the experimental and control group of fruits was analyzed under ambient conditions. For all the treatments the microbial potential, the decay percent and the ripening behavior of the fruits were recorded. Results revealed that the applied radiation doses reduced the decay incidence, delayed ripening process and greatly inhibit the microbial growth (total bacterial and fungal count) thereby enhancing the shelf life of bananas. Irradiation dose of 1.0 kGy was found to be the most effective dose to positively maintain the stored bananas under ambient conditions. The mean life of bananas was extended by 14 days. The identification of the enteric bacteriaeaceae through API 20 E strips revealed the presence of Shigella sonnie on the fruit surface along with Escherichia coli and a nonfermentor spp. The dominant spoilage causing fungi identified were Aspergillus niger, Aspergillus flavus, Collotrichum musae, Fusarium oxysporum,Mucor spp, Lasiodiplodia theobromea and Rhizopus stolonifer.
Pseudomonas aeruginosa exotoxin A (PE) is a bacterial toxin composed of three domains namely: cell binding, translocation and enzymatic domain. The cytotoxic activity of PE is attributed to the enzymatic domain, which inhibits protein synthesis through ADP-ribosylation of EF-2. PE can be genetically modified to fight cancer. In this regard, a truncated and modified form of PE was produced that could be used for more potent immunotoxins. This modified form termed PE38KDEL was completely devoid of cell binding domain and parts of translocation domain II and Ib which are reported to be inessential for cytotoxicity of the toxin. The resultant expressed protein consisted of the essential translocation domain II and catalytic subunit (domain Ib, III). The deletions in the exotoxin A gene for truncated protein production were made via overlapping PCR extension. The amplicon was cloned in pTZ57r-T vector for DNA works and sub cloned in pET22b expression vector. It is demonstrated here that PE38KDEL can be expressed in huge quantities in Escherichia coli by using the recombinant vector PE38KDEL/pET under control of T7 promoter and E. coli host strain BL21 (DE3) CodonPlus. The protein expression was optimized at 0.5 mM IPTG concentration for induction as soon as the OD600 nm reached 0.6 with 6 hours of post induction culturing at 37°C. The recombinant protein was expressed both as soluble and inclusion body forms however the expression of the soluble form was more pronounced.
The present study focused on the optimized expression of Pseudomonas aeruginosa truncated Exotoxin A (PE38KDEL) gene. This protein is considered as apotent toxin and is largely being used in the construction of immunotoxins for targeted cancer therapy. Escherichia coli BL21 DE3 Codon Plus was used as the expression host and the protein was expressed under the control of T7 promoter system. The effects of inducer type (IPTG and lactose), inducer concentration (IPTG: 0.2, 0.5 and 1 mM/lactose: 2.0, 5.0, 10, 15 and 20 mM) and incubation period (2, 4, 6, 8 and 10 hrs) were evaluated for the expressed protein. The maximum protein production was observed when IPTG at a concentration of 0.5 mM was used as an inducer however, lactose also showed substantial protein expression at 20 mM concentration. The most suitable incubation period for optimum protein expression was 6 hrs post induction.
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