Abeer M. Abd El-Aziz, Mohamed A. Shaker* and Mona I. Shaaban Pages 497 - 505 ( 9 )
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.
Recombinant lipase, Pseudomonas aeruginosa, chaperone immobilization, gold nanoparticles, enhanced biocatalytic activity.
Microbiology and Immunology Department, Faculty of Pharmacy, Mansoura University, Mansoura, Pharmaceutics and Pharmaceutical Technology Department, College of Pharmacy, PO Box 30040, Taibah University, Al-Madinah Al-Munawarah, Microbiology and Immunology Department, Faculty of Pharmacy, Mansoura University, Mansoura