Microbiology and Immunology Department, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt.
Pharmaceutics and Pharmaceutical Technology Department, College of Pharmacy, PO Box 30040, Taibah University, Al-Madinah Al-Munawarah, Saudi Arabia.
Curr Pharm Biotechnol. 2019;20(6):497-505. doi: 10.2174/1389201020666190416144650.
Bacterial lipases especially Pseudomonas lipases are extensively used for different biotechnological applications.
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.
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.
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.
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.
细菌脂肪酶，特别是假单胞菌脂肪酶，被广泛应用于不同的生物技术领域。
随着人们对其活性的理解不断深入，并且为了满足不断增长的市场需求，我们旨在通过将其固定在金纳米粒子表面来提高脂肪酶的重组生产和生物催化活性。
从铜绿假单胞菌 PA14 的基因组 DNA 中扩增全长编码序列（lipA）、脂肪酶特异性折叠酶基因（lipf）和双盒式基因（lipAf），并将其克隆到细菌表达载体 pRSET-B 中。重组脂肪酶在大肠杆菌 BL-21（DE3）pLysS 中表达，然后使用镍亲和层析进行纯化，并通过 SDS-PAGE 和 Western blot 分析确认蛋白质的身份。纯化的重组脂肪酶通过表面共轭固定在金纳米粒子上，并通过比色法定量测定酶活性。
成功构建了两个单表达质粒系统 pRSET-B-lipA 和 pRSET-B-lipf 以及一个双盒式表达质粒系统 pRSET-B-lipAf。重组脂肪酶 LipA、Lipf 和 LipAf 的脂肪酶活性分别为 4870、426 和 6740 IUmg-1。然而，当这些重组脂肪酶固定在制备好的金纳米粒子（GNPs）上时，LipA-GNPs、Lipf-GNPs 和 LipAf-GNPs 的活性分别为 7417、822 和 13035 IUmg-1。固定化后，LipA 和 LipAf 的活性分别提高了 1.52 和 1.93 倍。
与游离重组酶相比，生物缀合物中重组脂肪酶的脂肪酶活性显著提高，固定化使酶达到了最佳性能。
