Improvement of biodesulfurization activity of alginate immobilized cells in biphasic systems.

The immobilization of Pseudomonas delafieldii R-8 in calcium alginate beads has been studied in order to improve biodesulfurization activity in oil/water (O/W) biphasic systems. A gas jet extrusion technique was performed to produce immobilized beads. The specific desulfurization rate of 1.5 mm diameter beads was 1.4-fold higher than that of 4.0 mm. Some nonionic surfactants can significantly increase the activity of immobilized cells. The desulfurization rate with the addition of 0.5% Span 80 increased 1.8-fold compared with that of the untreated beads. The rate of biodesulfurization was markedly enhanced by decreasing the size of alginate beads and adding the surfactant Span 80, most likely resulting from the increasing mass transfer of substrate to gel matrix.

[Cloning and fusion expression of detoxifying gene in Escherichia coli].

In insects, esterases play an important role in the degradation of organophosphate (OP) and Carbamate (CB) insecticides. The ability of esterase B1 to degrading OP and CB insecticides opens broad prospect of using it in programs to the insecticide pollution. OPs can be detoxified by hydrolysis of their phosphoester bonds, pyrethroids and some OPs like malathion, by hydrolysis of their carboxylester bonds, and diverse carbamate insecticides, herbicides and fungicides by hydrolysis of amide or other similar bonds. Most of the candidate bioremediation enzymes identified to date have been hydrolases. In recent years, we have focused our work on the transfer of a detoxifying esterase B1 gene from mosquito into E. coli and explored the possibility of using the detoxified enzymes in environmental protection. 5' initial of esterase B1 cDNA was cloned by RT-PCR and sequenced consequently. After the combination of 5' initial and 3' terminal fragment of esterase B1 cDNA, the recombinant vector pET-ESTB1 was constructed and transformed into E. coli BL21. A 60 kD protein was induced by IPTG and its expression was temperature-dependent. After 12 h induction, the target protein occupied 27% of the total protein. A pure recombinant protein was obtained by purification, and was detected with 10% SDS-PAGE. The results showed that 22.1% of malathion was degraded by crude detoxification enzyme in 15 mins, demonstrated a high degradation property. This research provides a novel approach, which takes the advantages of eucaryotes for bioremediation of pesticide pollutions.

[Cloning and high level expression of mosquito detoxifying gene].

The full-length cDNA of mosquito esterase B1 had been isolated, and subcloned into pBV220. The recombinant vector pBV220B1 was constructed and transformed into E. coli DH5 alpha. A 60 kD protein was induced by 42 degrees C and its expression was temperature-dependent. After 6 h induction, the target protein occupied 50% of the total protein. The expressed product existed in both inclusion body and soluble proteins in the cells. The amount of the soluble detoxifying enzyme increased along with the induction time. The data of detoxifying experiments indicated that the detoxifying enzyme in expression strain of E. coli can detoxified toxicity of organophosphate insecticides, it showed a clear detoxifying affect on hens poisoned by organophosphate insecticides.