Biocatalytic lipoprotein bioamphiphile induced treatment of recalcitrant hydrocarbons in petroleum refinery oil sludge through transposon technology.

The present investigation employed transposon technology to enhance the degradation of recalcitrant petroleum hydrocarbons present in petroleum oil sludge by using biosurfactant hyper-producing strain Enterobacter xiangfangensis STP-3. Out of 2500 transposon induced mutants, mutants M257E.xiangfangensis and M916E.xiangfangensis hyper-produce biocatalytic lipoprotein biosurfactant by1.98 and 2.34 fold higher than wild-type strain. Transposon induced mutation also modified the amino acid composition which improved the hydrophobicity and thermal stability of the biosurfactants produced by mutants, compared to the wild-type biosurfactant. GC-MS and LC-MS-MS revealed that biosurfactants have pentameric lipid moiety and esterase as protein moiety. Increased biosurfactant hydrophobicity and yield by the mutants resulted in the enhanced bioavailability of petroleum hydrocarbons, thereby mutants M257E.xiangfangensis and M916E.xiangfangensis demonstrated better petroleum oil sludge degradation by 82% and 88% respectively, than wild-type (72%). Disrupted genes vgr G and pgm M in M257E.xiangfangensis and M916E.xiangfangensis respectively hyper-produce biosurfactant by competitive pathway inhibition and increased precursor availability mechanism. Hyper-production of biosurfactant was also validated by comparing the expression of biosynthetic genes ent E, ent F and est using qPCR. This is the first report on the application of transposon technology to hyper-produce biosurfactant for the effective bioremediation of hydrocarbon contaminated environments.

Recombinant l-Asparaginase II from Lactobacillus casei subsp. casei ATCC 393 and Its Anticancer Activity.

l-asparaginases from bacterial origin are employed extensively in leukemic treatment and food industry. The present study focuses on the characterization of the recombinant l-asparaginase II from Lactobacillus casei subsp. casei ATCC 393 cloned into Escherichia coli expression system and purified using Ni-NTA chromatography. The recombinant l-asparaginase as a monomer had a molecular weight of 35 kDa. The enzyme was active from 10 to 80 °C with the optimum at 40 °C. The enzyme retained its activity at 28 °C and 37 °C up to 24 h. The enzyme had optimum pH of 6 and retained 50% activity till 18 h. The Km of the recombinant enzyme was 0.01235 mM and Vmax 1.576 mM/min. The half life of recombinant l-asparaginase II in human serum was 44 h and trypsin was for 15 min. The LC-MS/MS analysis revealed the molecular weight of 35,050 and pI of 5.64. The secondary structure prediction using CD spectroscopy for the recombinant enzyme showed 33.5% α-helix, 66.5% turn and 0% ß sheets. The cytotoxicity of the recombinant enzyme was analysed against MOLT 3, Jurkat E6.1 and K-562 with the IC 50 value of 30, 62.5 and 50 µg/ml.