School of Pharmacy and Life Sciences, Jiujiang University, Jiujiang, China.
School of Nursing, Jiujiang University, Jiujiang, China.
Lett Appl Microbiol. 2021 Nov;73(5):652-657. doi: 10.1111/lam.13548. Epub 2021 Aug 30.
Two polyurethanases PueA and PueB from Pseudomonas protegens Pf-5 have been reported to have hydrolytic activity against synthetic p-nitrophenyl palmitate of lipase substrate, and PueA may play a more effective role in this activity. However, it is still unknown whether PueA and PueB play similar parts in the lipase activity against natural acylglycerols and achieve the extracellular secretion via their cognate ABC exporter AprDEF. In this study, we investigated these questions through the construction of four markerless deletion mutants in Pf5139 (Δupp derivative of Pf-5), two heterologous co-expression strains and their three control strains in lipase-free Escherichia coli BL21(DE3), and detected their lipase activities by the tributyrin plate assay and the liquid culture assay. The results showed that PueA and PueB, classified as subfamily I.3 lipases, are major extracellular lipases involved in the uptake of oil in Pf-5, and PueA plays a leading role in extracellular lipase activity. In addition, the extracellular secretion of PueA and PueB can be partly mediated via AprDEF in Pf-5 and BL21(DE3). Finally, PueA and PueB are also able to achieve the extracellular secretion without the assistance of AprDEF in Pf-5 and BL21(DE3).
已报道来自假单胞菌 Pf-5 的两种尿烷酶 PueA 和 PueB 具有水解合成的脂肪酶底物对硝基苯棕榈酸酯的活性，并且 PueA 可能在这种活性中发挥更有效的作用。然而，目前尚不清楚 PueA 和 PueB 在针对天然酰基甘油的脂肪酶活性中是否发挥类似的作用，并通过其同源 ABC 外排泵 AprDEF 实现细胞外分泌。在这项研究中，我们通过在 Pf5139（Pf-5 的Δupp 衍生物）中构建四个无标记缺失突变体、两种异源共表达菌株及其三个对照菌株在脂肪酶缺乏的大肠杆菌 BL21(DE3)中进行了研究，并通过三丁酸甘油酯平板测定法和液体培养测定法检测了它们的脂肪酶活性。结果表明，属于亚家族 I.3 脂肪酶的 PueA 和 PueB 是 Pf-5 中参与油脂摄取的主要细胞外脂肪酶，并且 PueA 在细胞外脂肪酶活性中起主导作用。此外，Pf-5 和 BL21(DE3)中的 AprDEF 可部分介导 PueA 和 PueB 的细胞外分泌。最后，PueA 和 PueB 也能够在 Pf-5 和 BL21(DE3)中无需 AprDEF 的协助即可实现细胞外分泌。
