(Un)coupling the factors contributing to the interfacial activation of Streptomyces rimosus lipase: computational and spectrophotometric study

Abstract

Streptomyces rimosus lipases (SrLip) present an enzyme class that catalyzes the hydrolysis of triacylglycerols producing fatty acids and glycerols. The abrupt increase in catalytic activity of SrLip when found at water-oil interface is assigned as interfacial activation (IA). Since the latter is a complex function of interface charge, chemical composition, as well as substrate orientation and concentration, the role of individual factors and their combinations that trigger IA remains rather elusive. The aim of this work is to unravel the contribution of particular factor in IA by examining SrLip activity toward p-nitrophenylpalmitate (p-NPP) as a substrate incorporated in three chemically different self-assembled aggregates: gum Arabic, sodium deoxycholate and liposomes made of 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC) lipids, and at two different pH values (pH = 6.8 and pH = 10.0). Spectrophotometric (UV/Vis) study revealed that SrLip catalyzes p-NPP hydrolysis when the substrate is embedded in sodium deoxycholate at both pH values, suggesting that the coupling of favorable interface charge and substrate orientation drives the interfacial activation. Moreover, especially in alkaline conditions (pH = 10.0), hydrolysis of the ester bond of the substrate incorporated in DOPC bilayers occurs when appropriate orientation, concentration and arrangement are accomplished. All experiments were conducted in parallel on an inactive mutant SrLipS10/A as a reference. The molecular-level details of particular interface and accompanied substrates examined by all-atom molecular dynamics simulations revealed that the availability of p-NPP at these three interfaces decreases in the following order: sodium-deoxycholate > DOPC > model gum Arabic.

Graphical Abstract

Keywords:

• 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC) liposomes
• gum Arabic
• interfacial activation (IA)
• p-nitrophenylpalmitate (p-NPP)
• sodium deoxycholate
• streptomyces rimosus lipase (SrLip)

Acknowledgment

The authors gratefully acknowledge Dr. Darija Domazet Jurašin and Dr. Maja Dutour Sikirić (Laboratory for biocolloids and surface chemistry, Ruđer Bošković Institute) for making DLS measurements and for a support in the optical microscopy. The authors sincerely thank the reviewers (especially referee #1) for their helpful advice and comments, which, in our humble opinion, significantly improved the manuscript.

Additional information

Funding

This article was supported by the Croatian Science Foundation, Project Nos. UIP-2020-02-7669, IP-2018-01-1754 and IP-2018-01-5475.