A Novel Actinobacterial Cutinase Containing a Non-Catalytic Polymer-Binding Domain

The single putative cutinase-encoding gene from the genome of Kineococcus radiotolerans SRS30216 was cloned and expressed in Escherichia coli as a secreted fusion protein, designated YebF-KrCUT, where YebF is the extracellular carrier protein. The 294-amino acid sequence of KrCUT is unique among currently characterized cutinases by having a C-terminal extension that consists of a short (Pro-Thr)-rich linker and a 55-amino-acid region resembling the substrate binding domain of poly(hydroxybutyrate) (PHB) depolymerases. Phylogenetically, KrCUT takes a unique position among known cutinases and cutinase-like proteins of bacterial and fungal origin. A modeled structure of KrCUT, although displaying a typical α/ß hydrolase fold, shows some unique loops close to the catalytic site. The 39-kDa YebF-KrCUT fusion protein and a truncated variant thereof were purified to electrophoretic homogeneity and functionally characterized. The melting temperatures ( T m ) of KrCUT and its variant KrCUT206 devoid of the putative PHB-binding domain were established to be very similar at 50–51°C. Cutinase activity was confirmed by the appearance of characteristic cutin components, C 16 and C 18 hydroxyl fatty acids, in the mass chromatograms following incubation of KrCUT with apple cutin as substrate. KrCUT also efficiently degraded synthetic polyesters such as polycaprolactone and poly(1,3-propylene adipate). Although incapable of PHB depolymerization, KrCUT could efficiently bind PHB, confirming the predicted characteristic of the C-terminal region. KrCUT also potentiated the activity of pectate lyase in the degradation of pectin from hemp fibres. This synergistic effect is relevant to the enzyme retting process of natural fibres. IMPORTANCE. To date only a limited number of cutinases have been isolated and characterized from nature, the majority being sourced from phytopathogenic fungi and thermophilic bacteria. The significance of our research relates to the identification and characterization of a unique member of microbial cutinases, of name KrCUT, that was derived from the genome of the Gram-positive Kineococcus radiotolerans SRS30216, a highly radiation-resistant actinobacterium. Given the wide-ranging importance of cutinases in applications such as the degradation of natural and synthetic polymers, in the textile industry, in laundry detergents, or in biocatalysis (e.g., transesterification reactions), our results could foster new research leading to broader biotechnological impacts. This study also demonstrated that genome mining or prospecting is a viable means to discover novel biocatalysts as environmentally friendly and biotechnological tool.

Five Surfactin Isomers Produced during Cheonggukjang Fermentation by Bacillus pumilus HY1 and Their Properties

The cyclic lipopeptide produced from Bacillus pumilus strain HY1 was isolated from Korean soybean sauce cheonggukjang. The chemical structures of the surfactin isomers were analyzed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). The five potential surfactin isoforms were detected with protonated masses of m/z 994.7, 1008.7, 1022.7, 1036.7, and 1050.7 and different structures in combination with Na+, K+, and Ca2+ ions. ESI-MS/MS analysis revealed that the isolated surfactin possessed the precise amino acid sequence LLVDLL and hydroxyl fatty acids with 12 to 16 carbons. The surfactin content during cheonggukjang fermentation increased from 0.3 to 51.2 mg/kg over 60 h of fermentation. The mixture of five surfactin isoforms of cheonggukjang inhibited the growth of two cancer cell lines. The growth of both MCF-7 and Caco-2 cells was strongly inhibited with 100 μg/μL of surfactin. This study is the first-time report of five surfactin isomers of Bacillus pumilus strain HY1 during Korean soybean sauce cheonggukjang fermentation, which has cytotoxic properties.

Sophorolipid-Based Oligomers as Polyol Components for Polyurethane Systems

Due to reasons of sustainability and conservation of resources, polyurethane (PU)-based systems with preferably neutral carbon footprints are in increased focus of research and development. The proper design and development of bio-based polyols are of particular interest since such polyols may have special property profiles that allow the novel products to enter new applications. Sophorolipids (SL) represent a bio-based toolbox for polyol building blocks to yield diverse chemical products. For a reasonable evaluation of the potential for PU chemistry, however, further investigations in terms of synthesis, derivatization, reproducibility, and reactivity towards isocyanates are required. It was demonstrated that SL can act as crosslinker or as plasticizer in PU systems depending on employed stoichiometry. (ω-1)-hydroxyl fatty acids can be derived from SL and converted successively to polyester polyols and PU. Additionally, (ω-1)-hydroxyl fatty acid azides can be prepared indirectly from SL and converted to A/B type PU by Curtius rearrangement.

Comparative Fatty Acid Profiling of Klebsiella pneumoniae and Rhodococcus rhodochrous Isolated from Spoilt Paints by Gas Chromatography

The use of fatty acids to study the differences in un-related microbes is limited. This study analyzes the fatty acids produced by two unrelated microorganisms: Klebsiella pneumoniae (Gram-negative, aerobic, non-endospore forming, usually encapsulated rod-shaped bacteria of the family Enterobacteriaceae) and Rhodococcus rhodochrous (metabolically versatile, non-spore-forming, non-motile actinomycete) isolated from spoilt paints. Fatty acids produced by the organisms were analyzed using an efficient MIDI-Sherlock gas chromatography method . K. pneumoniae was characterized by a high content of straight chain, branched chain, hydroxyl and cyclo-fatty acids made up of C12: 0, C13:0, C14:0 iso, C14:0, C15:0 iso, C15:0 anteiso, C15:1 ω 8c, C15:0, C16:0 iso, C16:1w5c, C16:0, C15:03OH, C17:1 ω 8c, C17:0 cyclo, C17:0, C18:1 ω5c and C18:0. R. rhodochrous was dominated by straight chain, monounsaturated and 10-methyl fatty acids. The inability to synthesize branched, cyclo- and hydroxyl- fatty acids, was observed in R. rhodochrous which composed mainly of C14: 0, C15: 1 ω 5c, C15:0, C16:1 ω 9c, C16:0, C17:1 ω 8c, C17:0, C17:0 10-methyl, C18: 1 ω 9c, C18.0, 10 methyl-C18:0 TBSA, C20:1 ω 9c, and C20:0. Descriptive statistics reveal a mean of 2.53, 15.10 and 15.15 for retention time (RT), equivalent chain length (ECL) and Peak name, respectively. Possible implications of the variations in fatty acid distribution may include differences in their abilities to produce various secondary metabolites and potentials to degrade a variety of xenobiotics. Keywords: Fatty acids, paints, Rhodococcus rhodochrous, Klebsiella pneumoniae

Potential favourable health effects of some dietary uncommon fatty acids

In addition to the major fatty acids widely studied, our diet contains many bioactive fatty acids less frequently investigated such as n-3 docosapentaenoic acid (n-3 DPA), natural trans fatty acids, conjugated fatty acids (CLAs), furan fatty acids (FuFAs), branched chain fatty acids (BCFAs) and fatty acid esters of hydroxyl fatty acids (FAHFAs). Many of them may have beneficial health effects, particularly in the prevention of cardiovascular diseases, inflammation and metabolic disorders such as diabetes. This review aims to give a brief overview of the current knowledge on these lipids. Thus, information about biosynthesis, food and tissue content, daily intake, biological and potential health effects of these fatty acids is provided.

The Endocrine Role of Adipose Tissue and Its Management of Obesity-Related Diseases.

Adipose tissue plays a central role in regulating whole-body energy. Moreover, adipose tissue acts as an endocrine organ and produces numerous bioactive factors called adipokines which communicate with other organs and modulate a range of metabolic pathways: proteins (adiponectin, angiopoietins, chemerin, etc.), lipids (fatty acid esters of hydroxyl fatty acids, lysophosphatidic acids and sphingolipids), metabolites (uric acid and uridine) and microRNAs. However, excessive adipose tissue is associated with a chronic state of low-grade inflammation, caused by unbalanced production or secretion of these adipokines and can contribute to the development of obesity [1].

9-PAHSA Prevents Mitochondrial Dysfunction and Increases the Viability of Steatotic Hepatocytes

Nonalcoholic fatty liver disease (NAFLD) is quickly becoming the most common liver disease worldwide. Within the NAFLD spectrum, patients with nonalcoholic steatohepatitis (NASH) are at the highest risk of developing cirrhosis and disease progression to hepatocellular carcinoma. To date, therapeutic options for NASH patients have been ineffective, and therefore, new options are urgently needed. Hence, a model system to develop new therapeutic interventions is needed. Here, we introduce two new in vitro models of steatosis induction in HepG2 cells and primary murine hepatocytes. We used a recently discovered novel class of bioactive anti-inflammatory lipids called branched fatty acid esters of hydroxyl fatty acids. Among these bioactive lipids, palmitic-acid-9-hydroxy-stearic-acid (9-PAHSA) is the most promising as a representative nondrug therapy based on dietary supplements or nutritional modifications. In this study, we show a therapeutic effect of 9-PAHSA on lipotoxicity in steatotic primary hepatocytes and HepG2 cells. This could be shown be increased viability and decreased steatosis. Furthermore, we could demonstrate a preventive effect in HepG2 cells. The outcome of 9-PAHSA administration is both preventative and therapeutically effective for hepatocytes with limited damage. In conclusion, bioactive lipids like 9-PAHSA offer new hope for prevention or treatment in patients with fatty liver and steatosis.

Anticancer Effect of the Five Surfactin Isomers Produced During Cheonggukjang Fermentation by Bacillus Pumilus HY1 Isolated From Korean Traditional Fermented Soy Sauce

The cyclic lipopeptide produced from Bacillus pumilus strain HY1 was isolated from Korean soybean sauce. The chemical structures of the surfactin isomers were analyzed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). The five potential surfactin isoforms were detected with protonated masses of m/z 994.7, 1,008.7, 1022.7, 1036.7, and 1,050.7 and different structures in combination with Na+, K+, and Ca2+ ions. ESI-MS/MS analysis revealed that the isolated surfactin possessed the precise amino acid sequence LLVDLL and hydroxyl fatty acids with 12 to 16 carbons. The surfactin content during cheonggukjang fermentation increased from 0.3 to 51.2 mg/kg over 60 h of fermentation. The surfactin extraction of cheonggukjang inhibited the growth of two cancer cell lines. The growth of both MCF-7 and Caco-2 cells was strongly inhibited with 100 μg/μL surfactin. These results suggest that surfactins produced from strain HY1 have anticancer properties.

Nrp1 is Activated by Konjac Ceramide Binding-Induced Structural Rigidification of the a1a2 Domain

Konjac ceramide (kCer) is a plant-type ceramide composed of various long-chain bases and α-hydroxyl fatty acids. The presence of d4t,8t-sphingadienine is essential for semaphorin 3A (Sema3A)-like activity. Herein, we examined the three neuropilin 1 (Nrp1) domains (a1a2, b1b2, or c), and found that a1a2 binds to d4t,8t-kCer and possesses Sema3A-like activity. kCer binds to Nrp1 with a weak affinity of μM dissociation constant (Kd). We wondered whether bovine serum albumin could influence the ligand–receptor interaction that a1a2 has with a single high affinity binding site for kCer (Kd in nM range). In the present study we demonstrated the influence of bovine serum albumin. Thermal denaturation indicates that the a1a2 domain may include intrinsically disordered region (IDR)-like flexibility. A potential interaction site on the a1 module was explored by molecular docking, which revealed a possible Nrp1 activation mechanism, in which kCer binds to Site A close to the Sema3A-binding region of the a1a2 domain. The a1 module then accesses a2 as the IDR-like flexibility becomes ordered via kCer-induced protein rigidity of a1a2. This induces intramolecular interaction between a1 and a2 through a slight change in protein secondary structure.

Plasma Derived Exosomal Biomarkers of Exposure to Ionizing Radiation in Nonhuman Primates

Exposure to ionizing radiation induces a cascade of molecular events that ultimately impact endogenous metabolism. Qualitative and quantitative characterization of metabolomic profiles is a pragmatic approach to studying the risks of radiation exposure since it provides a phenotypic readout. Studies were conducted in irradiated nonhuman primates (NHP) to investigate metabolic changes in plasma and plasma-derived exosomes. Specifically, rhesus macaques (Macaca mulatta) were exposed to cobalt-60 gamma-radiation and plasma samples were collected prior to and after exposure to 5.8 Gy or 6.5 Gy radiation. Exosomes were isolated using ultracentrifugation and analyzed by untargeted profiling via ultra-performance liquid chromatography mass spectrometry (UPLC-MS) based metabolomic and lipidomic analyses, with the goal of identifying a molecular signature of irradiation. The enrichment of an exosomal fraction was confirmed using quantitative ELISA. Plasma profiling showed markers of dyslipidemia, inflammation and oxidative stress post-irradiation. Exosomal profiling, on the other hand, enabled detection and identification of low abundance metabolites that comprise exosomal cargo which would otherwise get obscured with plasma profiling. We discovered enrichment of different classes of metabolites including N-acyl-amino acids, Fatty Acid ester of Hydroxyl Fatty Acids (FAHFA’s), glycolipids and triglycerides as compared to the plasma metabolome composition with implications in mediation of systemic response to radiation induced stress signaling.