Background:
Bacterial lipases especially Pseudomonas lipases are extensively used for different
biotechnological applications.
Objectives:
With the better understanding and progressive needs for improving its activity in accordance
with the growing market demand, we aimed in this study to improve the recombinant production
and biocatalytic activity of lipases via surface conjugation on gold nanoparticles.
Methods:
The full length coding sequences of lipase gene (lipA), lipase specific foldase gene (lipf) and
dual cassette (lipAf) gene were amplified from the genomic DNA of Pseudomonas aeruginosa PA14
and cloned into the bacterial expression vector pRSET-B. Recombinant lipases were expressed in
E. coli BL-21 (DE3) pLysS then purified using nickel affinity chromatography and the protein identity
was confirmed using SDS-PAGE and Western blot analysis. The purified recombinant lipases were
immobilized through surface conjugation with gold nanoparticles and enzymatic activity was
colorimetrically quantified.
Results:
Here, two single expression plasmid systems pRSET-B-lipA and pRSET-B-lipf and one dual
cassette expression plasmid system pRSET-B-lipAf were successfully constructed. The lipolytic activities
of recombinant lipases LipA, Lipf and LipAf were 4870, 426 and 6740 IUmg-1, respectively.
However, upon immobilization of these recombinant lipases on prepared gold nanoparticles (GNPs),
the activities were 7417, 822 and 13035 IUmg-1, for LipA-GNPs, Lipf-GNPs and LipAf-GNPs, respectively.
The activities after immobilization have been increased 1.52 and 1.93 -fold for LipA and LipAf,
respectively.
Conclusion:
The lipolytic activity of recombinant lipases in the bioconjugate was significantly increased
relative to the free recombinant enzyme where immobilization had made the enzyme attain its
optimum performance.
Plasma decay of chylomicron remnants is not affected by heparin-stimulated plasma lipolytic activity in normal fasting man.
