Comparative Studies on the Susceptibility of (R)-2,3-Dipalmitoyloxypropylphosphonocholine (DPPnC) and Its Phospholipid Analogues to the Hydrolysis or Ethanolysis Catalyzed by Selected Lipases and Phospholipases

Susceptibility of soybean phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and its phosphono analogue (R)-2,3-dipalmitoyloxypropylphosphonocholine (DPPnC) towards selected lipases and phospholipases was compared. The ethanolysis of substrates at sn-1 position was carried out by lipase from Mucor miehei (Lipozyme®) and lipase B from Candida antarctica (Novozym 435) in 95% ethanol at 30 °C, and the hydrolysis with LecitaseTM Ultra was carried out in hexane/water at 50 °C. Hydrolysis at sn-2 position was carried out in isooctane/Tris-HCl/AOT system at 40 °C using phospholipase A2 (PLA2) from porcine pancreas and PLA2 from bovine pancreas or 25 °C using PLA2 from bee venom. Hydrolysis in the polar part of the studied compounds was carried out at 30 °C in acetate buffer/ethyl acetate system using phospholipase D (PLD) from Streptococcus sp. and PLD from white cabbage or in Tris-HCl buffer/methylene chloride system at 35 °C using PLD from Streptomyces chromofuscus. The results showed that the presence of C-P bond between glycerol and phosphoric acid residue in DPPnC increases the rate of enzymatic hydrolysis or ethanolysis of ester bonds at the sn-1 and sn-2 position and decreases the rate of hydrolysis in the polar head of the molecule. The most significant changes in the reaction rates were observed for reaction with PLD from Streptococcus sp. and PLD from Streptomyces chromofuscus that hydrolyzed DPPnC approximately two times slower than DPPC and soybean PC. The lower susceptibility of DPPnC towards enzymatic hydrolysis by phospholipases D gives hope for the possibility of using DPPnC-like phosphonolipids as the carriers of bioactive molecules that, instead of choline, can be bounded with diacylpropylphosphonic acids (DPPnA).

Streptomyces gossypiisoli sp. nov., isolated from cotton soil in Xinjiang, PR China

A novel actinobacterium, designated TRM 44567T, was isolated from cotton soil in Xinjiang Uygur Autonomous Region, northwest PR China. Growth occurred at 16–45 °C, pH 5.0–9.0, and 0–7 % (w/v) NaCl, with optimum growth at 37 °C, pH 7.0–8.0 and 1 % (w/v) NaCl, respectively. Comparative 16S rRNA gene sequence analysis indicated that strain TRM 44567T was phylogenetically most closely related to Streptomyces chromofuscus NBRC 12851T (98.48 % sequence similarity); however, the average nucleotide identity between strain TRM 44567T and S. chromofuscus NBRC 12851T was only 83.77 %. Strain TRM 44567T possessed ll-diaminopimelic acid as the diagnostic cell-wall diamino acid. The predominant menaquinones were MK-9(H10), MK-9(H6) and MK-9(H4). The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol and phosphatidylinositol mannoside. The major fatty acids were iso-C16 : 0, C16 : 0, anteiso-C15 : 0, anteiso-C17 : 0, iso-C14 : 0 and iso-C15 : 0. The genomic DNA G+C content was 70.8 mol%. Strain TRM 44567T represents a novel species of the genus Streptomyces , for which the name Streptomyces gossypiisoli sp. nov. is proposed. The type strain is TRM 44567T (=KCTC 39957 T=CCTCC AA 2017011T).

Symmetrically substituted dichlorophenes inhibit N-acyl-phosphatidylethanolamine phospholipase D

N-Acyl-phosphatidylethanolamine phospholipase D (NAPE-PLD) (EC 3.1.4.4) catalyzes the final step in the biosynthesis of N-acyl-ethanolamides. Reduced NAPE-PLD expression and activity may contribute to obesity and inflammation, but a lack of effective NAPE-PLD inhibitors has been a major obstacle to elucidating the role of NAPE-PLD and N-acyl-ethanolamide biosynthesis in these processes. The endogenous bile acid lithocholic acid (LCA) inhibits NAPE-PLD activity (with an IC50 of 68 μm), but LCA is also a highly potent ligand for TGR5 (EC50 0.52 μm). Recently, the first selective small-molecule inhibitor of NAPE-PLD, ARN19874, has been reported (having an IC50 of 34 μm). To identify more potent inhibitors of NAPE-PLD, here we used a quenched fluorescent NAPE analog, PED-A1, as a substrate for recombinant mouse Nape-pld to screen a panel of bile acids and a library of experimental compounds (the Spectrum Collection). Muricholic acids and several other bile acids inhibited Nape-pld with potency similar to that of LCA. We identified 14 potent Nape-pld inhibitors in the Spectrum Collection, with the two most potent (IC50 = ∼2 μm) being symmetrically substituted dichlorophenes, i.e. hexachlorophene and bithionol. Structure–activity relationship assays using additional substituted dichlorophenes identified key moieties needed for Nape-pld inhibition. Both hexachlorophene and bithionol exhibited significant selectivity for Nape-pld compared with nontarget lipase activities such as Streptomyces chromofuscus PLD or serum lipase. Both also effectively inhibited NAPE-PLD activity in cultured HEK293 cells. We conclude that symmetrically substituted dichlorophenes potently inhibit NAPE-PLD in cultured cells and have significant selectivity for NAPE-PLD versus other tissue-associated lipases.

Analysis of Carotenoids and the Identification of Mangrove Sediment Bacteria of Segara Anakan, Cilacap

The excessive consumption of artificial dyes can lead to negative effects on human body. Thus, the invention of natural dyes, such as carotenoids, is needed in order to reduce the negative impacts. Carotenoids are yellow, orange, and reddish yellow pigments produced by plants, animals, algae, and microorganisms. This study was aimed to analyze the carotenoid pigments of mangrove sediment bacteria in Segara Anakan, Cilacap, and to identify species of bacteria that can produce carotenoids. Carotenoids were analysed by using Thin Layer Chromatography (TLC) and UV-Vis spectrophotometer. Meanwhile molecular identification of bacteria was carried by 16S rDNA PCR and DNA sequence was analysed through a program called Basic Local Alignment Search Tool (BLAST). The kinship of bacteria was shown in Phylogenetic tree by using Bioedit and MEGA 5 software. Qualitative analysis by using TLC produced several pigments like: β-carotene, β-isorenieraten, lycopene, flavonoids, chlorophyll a, chlorophyll b and a feofitin with Rf value of 0.36-0.95. Quantitative analysis showed that bacteria KH (greenish yellow), KT (dark yellow) and KM (light yellow) produced carotenoids at 95.30 mg.g-1, 110.34 mg.g-1 and 25.349 mg.g-1. KH, KT and KM were suspected to be bacteria known as Streptomyces chartreusis, Bacillus megaterium and Streptomyces chromofuscus with similarity of 99%. Mangrove sediment bacteria had the potential of producing carotenoids as an alternative of eco-friendly natural dyes.