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영어

Enzymes can be tailored for optimal performance in industrial applications by directing their evolution in vitro. This approach is particularly attractive for engineering industrial enzymes. We have created an efficient para-nitrobenzyl esterase(PNBE) through immobilization. In this study, we cloned, over-expressed and purified the Bacillus subtilis pnbA gene product. The gene coding for pnbA was generated by amplifying the pnbA gene from B. subtilis by polymerase
chain reaction. it was inserted into the expression plasmid pET-21a. A BL21(DE3) E. coli strain transformed with the pnbA-expression vector pET -21a-pnbA accumulates large amounts of a soluble protein with a molecular mass of 54 kDa in SDS-PAGE. It was purified in NI-NTA resin columns.
Prepared PNBE was immobilized on the surface of electrospun polymer nanofibers by using cross-linking enzyme aggregation by glutaraldehyde. The activity of enzyme aggregates coated on PS(polystyrene)+PSMA(polystyreneco-maleic anhydride) nanofibers showed improved reusability and stability. When immobilized PNBE was stored at room temperature, the residual activity after 30 days was 60%. After 30th recycle of immobilized PNBE, the retained activity
was over 70%. also, according to comparison of thermal stability of both free and immobilized PNBE at 50 . Both preparations exhibited ℃ a similar trend, however, the immobilized PNBE was more stable than free one. The stability and reusability of the immobilized PNBE is very important for commercial application. Our experiments can be concluded that this immobilized PNBE provides an economical, efficient and selective system for enzyme immobilization.