Brave new surfactant world revisited by thermoalkalophilic lipases: computational insights into the role of SDS as a substrate analog.

Non-ionic surfactants were shown to stabilize the active conformation of thermoalkalophilic lipases by mimicking the lipid substrate while the catalytic interactions formed by anionic surfactants have not been well characterized. In this study, we combined µs-scale molecular dynamics (MD) simulations and lipase activity assays to analyze the effect of ionic surfactant, sodium dodecyl sulfate (SDS), on the structure and activity of thermoalkalophilic lipases. Both the open and closed lipase conformations that differ in geometry were recruited to the MD analysis to provide a broader understanding of the molecular effect of SDS on the lipase structure. Simulations at 298 K showed the potential of SDS for maintaining the active lipase through binding to the sn-1 acyl-chain binding pocket in the open conformation or transforming the closed conformation to an open-like state. Consistent with MD findings, experimental analysis showed increased lipase activity upon SDS incubation at ambient temperature. Notably, the lipase cores stayed intact throughout 2 µs regardless of an increase in the simulation temperature or SDS concentration. However, the surface structures were unfolded in the presence of SDS and at elevated temperature for both conformations. Simulations of the dimeric lipase were also carried out and showed reduced flexibility of the surface structures which were unfolded in the monomer, indicating the insulating role of dimer interactions against SDS. Taken together, this study provides insights into the possible substrate mimicry by the ionic surfactant SDS for the thermoalkalophilic lipases without temperature elevation, underscoring SDS's potential for interfacial activation at ambient temperatures.

Assessing the concentration of conjugated fatty acids within pomegranate seed oil using quantitative nuclear magnetic resonance (qNMR).

INTRODUCTION: Pomegranate seed is rich in oil, and seed oil of pomegranate consists of conjugated fatty acids with different percentages. OBJECTIVES: The current contribution covers how to determine percentages of different isomeric conjugated fatty acids. METHODS: The percentages of these isomers are analysed by quantitative nuclear magnetic resonance (qNMR) using benzoic acid as an internal reference chemical with a well-defined amount. Linear mathematical equations are developed for the quantitative analysis of fatty acids found in pomegranate seed oil. RESULTS: The developed approach is utilised for the pomegranate seed oils prepared in the laboratory and tested for commercial samples. Among the oils derived at the laboratory, the Yeni Hicaz pomegranate cultivar seeds yielded the highest fraction of punicic acid. Among the acids, punicic acid was the one with the highest fraction, while linolenic acid was the one with the lowest percentage. CONCLUSIONS: These results are important in identifying pomegranate seed oils. Among the commercial samples tested with the current approach, only one of them showed similar content analysis as in the laboratory-derived oils.

Enzymes for Efficient CO2 Conversion.

The accumulation of carbon dioxide in the atmosphere as a result of human activities has caused a number of adverse circumstances in the world. For this reason, the proposed solutions lie within the aim of reducing carbon dioxide emissions have been quite valuable. However, as the human activity continues to increase on this planet, the possibility of reducing carbon dioxide emissions decreases with the use of conventional methods. The emergence of compounds than can be used in different fields by converting the released carbon dioxide into different chemicals will construct a fundamental solution to the problem. Although electro-catalysis or photolithography methods have emerged for this purpose, they have not been able to achieve successful results. Alternatively, another proposed solution are enzyme based systems. Among the enzyme-based systems, pyruvate decarboxylase, carbonic anhydrase and dehydrogenases have been the most studied enzymes. Pyruvate dehydrogenase and carbonic anhydrase have either been an expensive method or were incapable of producing the desired result due to the reaction cascade they catalyze. However, the studies reporting the production of industrial chemicals from carbon dioxide using dehydrogenases and in particular, the formate dehydrogenase enzyme, have been remarkable. Moreover, reported studies have shown the existence of more active and stable enzymes, especially the dehydrogenase family that can be identified from the biome. In addition to this, their redesign through protein engineering can have an immense contribution to the increased use of enzyme-based methods in CO2 reduction, resulting in an enormous expansion of the industrial capacity.

High-yield production of active recombinant S. simulans lysostaphin expressed in E. coli in a laboratory bioreactor.

Staphylococcus aureus (S. aureus), which has developed multidrug resistance, leads to many healthcare-associated infections resulting in significant medical and economic losses. Therefore, the development of new efficient strategies to deal with these bacteria has been gaining importance. Lysostaphin is a peptidoglycan hydrolase that has considerable potential as a bacteriocin. However, there have been few reported optimization and scale-up studies of the lysostaphin bioproduction process. Our preliminary results have revealed that the composition of auto-induction media at 30 °C increases the produced lysostaphin around 10-fold in shake flasks. In this study, achieving higher yields for recombinant lysostaphin in E. coli at a laboratory scale has been the aim, through the use of auto-induction media. Optimized medium composition and fermentation parameters were transferred to a laboratory-scale bioreactor. The tested conditions improved protein yields up to 184 mg/L in a 3 L stirred bioreactor and the productivity was improved 2-fold in comparison to previously published reports. Furthermore, this study also showed that lysostaphin is an effective bacteriocin on both commercially available and isolated S. aureus strains. These results will contribute to future larger-scale production of lysostaphin via the proposed fermentation conditions.

Lipase and Water in a Deep Eutectic Solvent: Molecular Dynamics and Experimental Studies of the Effects of Water-In-Deep Eutectic Solvents on Lipase Stability.

Given the accumulated evidence on the effects of water-in-deep eutectic solvents (DESs) on the solvent nanostructure and the yield of lipase reactions, here we have used molecular dynamics (MD) simulations to delineate the structure and dynamics of thermoalkalophilic lipases in choline chloride/urea-based DES (reline) with varying hydration levels. Results indicated that pure reline almost froze the lipase backbone, while hydrated reline that showed a less ordered nanostructure than the pure form introduced some fluctuations to lipase structures, particularly to the lid domain. Although none of the solvents led to unfolding, solvation by 8 M urea or water when accompanied with elevated temperature caused the most significant loss of secondary structure. Experimental results indicated that lipase incubation in slightly hydrated reline [5% (v/v)] led to the highest level of residual activity, implying interfacial activation. Overall, we report that slightly hydrated reline activates thermoalkalophilic lipases, consistent with the particular MD observation showing enhanced mobility of the lid domain. These insights provided by this study contribute to designing efficient lipase applications in DES-based reaction media, giving cues for customizing water-in-DESs for optimal enzyme stability and activity.

Enhanced production of recombinant Staphylococcus simulans lysostaphin using medium engineering.

Staphylococcus aureus, among other staphylococcal species, developed multidrug resistance and causes serious health risks that require complex treatments. Therefore, the development of novel and effective strategies to combat these bacteria has been gaining importance. Since Staphylococcus simulans lysostaphin is a peptidoglycan hydrolase effective against staphylococcal species, the enzyme has a significant potential for biotechnological applications. Despite promising results of lysostaphin as a bacteriocin capable of killing staphylococcal pathogens, it is still not widely used in healthcare settings due to its high production cost. In this study, medium engineering techniques were applied to improve the expression yield of recombinant lysostaphin in E. coli. A new effective inducible araBAD promoter system and different mediums were used to enhance lysostaphin production. Our results showed that the composition of autoinduction media enhanced the amount of lysostaphin production 5-fold with the highest level of active lysostaphin at 30 °C. The production cost of 1000 U of lysostaphin was determined as 4-fold lower than the previously proposed technologies. Therefore, the currently developed bench scale study has a great potential as a large-scale fermentation procedure to produce lysostaphin efficiently.

A new lipase as a pharmaceutical target for battling infections caused by Staphylococcus aureus: Gene cloning and biochemical characterization.

Staphylococcus aureus lipases along with other cell-wall-associated proteins and enzymes (i.e., catalase, coagulase, protease, hyaluronidase, and ß-lactamase) play important roles in the pathogenesis of S. aureus and are important subject of drug targeting. The appearance of antibiotic-resistant types of pathogenic S. aureus (e.g., methicillin-resistant S. aureus, MRSA) is a worldwide medical problem. In the present work, a novel lipase from a newly isolated MRSA strain from a cow with subclinical mastitis was cloned and biochemically characterized. The mature part of the lipase was expressed in Escherichia coli and purified by nickel affinity chromatography. It displays a high lipase activity at pH 8.0 and 25 °C using p-nitrophenyl palmitate and has a preference for medium-long-chain substrates of p-nitrophenyl esters (pNPC10-C16). Furthermore, in search of inhibitors, the effect of farnesol on the growth of S. aureus and the lipase activity was also studied. Farnesol inhibits the growth of S. aureus and is a mixed-type inhibitor with Ki and Ki (') values of 0.2 and 1.2 mmol L(-1), respectively. A lipase with known properties could not only serve as a template for developing inhibitors for S. aureus but also a valuable addition to enzyme toolbox of biocatalysis. The discovery of this lipase can be potentially important and could provide a new target for pharmaceutical intervention against S. aureus infection.