Isolation and Molecular Characterization of Biofouling Bacteria and Profiling of Quorum Sensing Signal Molecules from Membrane Bioreactor Activated Sludge

The formation of biofilm in a membrane bioreactor depends on the production of various signaling molecules like N-acyl homoserine lactones (AHLs). In the present study, a total of 200 bacterial strains were isolated from membrane bioreactor activated sludge and screened for AHLs production using two biosensor systems, Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136. A correlation between AHLs production and biofilm formation has been made among screened AHLs producing strains. The 16S rRNA gene sequence analysis revealed the dominance of Aeromonas and Enterobacter sp. in AHLs production; however few a species of Serratia, Leclercia, Pseudomonas, Klebsiella, Raoultella and Citrobacter were also identified. The chromatographic characterization of sludge extract showed the presence of a broad range of quorum sensing signal molecules. Further identification of sludge AHLs by thin layer chromatography bioassay and high performance liquid chromatography confirms the presence of C4-HSL, C6-HSL, C8-HSL, 3-oxo-C8-HSL, C10-HSL, C12-HSL, 3-oxo-C12-HSL and C14-HSL. The occurrence of AHLs in sludge extract and dominance of Aeromonas and Enterobacter sp. in activated sludge suggests the key role of these bacterial strains in AHLs production and thereby membrane fouling.     

Production of the Quorum-Sensing Molecules N-Acylhomoserine Lactones by Endobacteria Associated with Mortierella alpina A-178

Gram‐negative bacteria communicate with one another using N‐acylhomoserine lactones (AHLs) as signaling molecules. This mechanism, known as quorum sensing (QS), is needed to develop pathogenicity, as well as symbiotic interactions with eukaryotic hosts, such as animals and plants. Increasing evidence indicates that certain bacteria, namely endobacteria, also inhabit fungal cells and establish symbiotic relationships with their hosts. However, it has not been clear whether bacterial QS acts in developing the relationships. Here we describe the isolation and identification of N‐heptanoylhomoserine lactone and N‐octanoylhomoserine lactone from the culture broth of the zygomycete fungus Mortierella alpina A‐178. This suggested the presence of endobacteria in the fungus, as was confirmed by PCR, fluorescence in situ hybridization, and transmission electron microscopy. Two major bands obtained by PCR‐denaturing gradient gel electrophoresis showed sequence identity to genes in the β‐proteobacterium Castellaniella defragrans (100 %) and the Gram‐positive bacterium Cryobacterium sp. (99.8 %). The production of AHLs depended on the presence of endobacteria and was induced in response to the increase in the concentration of AHLs, suggesting that the bacterium conducts AHL‐mediated QS in the fungus. This paper is the first to report the production of AHLs by endofungal bacteria and raises the possibility that QS plays roles in the development of fungus–endobacterium symbiosis.     

Quorum Quenching in Culturable Phyllosphere Bacteria from Tobacco

Many Gram-negative plant pathogenic bacteria employ a N-acylhomoserine lactone (AHL)-based quorum sensing (QS) system to regulate their virulence traits. A sustainable biocontrol strategy has been developed using quorum quenching (QQ) bacteria to interfere with QS and protect plants from pathogens. Here, the prevalence and the diversity of QQ strains inhabiting tobacco leaf surfaces were explored. A total of 1177 leaf-associated isolates were screened for their ability to disrupt AHL-mediated QS, using the biosensor Chromobacterium violaceum CV026. One hundred and sixty-eight strains (14%) are capable of interfering with AHL activity. Among these, 106 strains (63%) of the culturable quenchers can enzymatically degrade AHL molecules, while the remaining strains might use other QS inhibitors to interrupt the chemical communication. Moreover, almost 79% of the QQ strains capable of inactivating AHLs enzymatically have lactonase activity. Further phylogenetic analysis based on 16S rDNA revealed that the leaf-associated QQ bacteria can be classified as Bacillus sp., Acinetobacter sp., Lysinibacillus sp., Serratia sp., Pseudomonas sp., and Myroides sp. The naturally occurring diversity of bacterial quenchers might provide opportunities to use them as effective biocontrol reagents for suppressing plant pathogen in situ.     

Novel brominated phospholipid fatty acids from the Caribbean spongeAgelas sp.

The very long-chain fatty acids, (5E,9Z)-6-bromo-5,9-tetracosadienoic, (5E,9Z)-6-bromo-23-methyl-5,9-tetracosadienoic, (5E,9Z)-6-bromo-5,9-pentacosadienoic and (5E,9Z)-6-bromo-24-methyl-5,9-pentacosadienoic acids, were identified in the phospholipids (mainly phosphatidylethanolamine) of the spongeAgelas sp. Structure elucidation was accomplished by means of mass spectrometry and chemical transformations, including deuteration with Wilkinson's catalyst. All of the sterols from the sponge had the Δ5,7 nucleus, with 24-methylcholesta-5,7,22-trien-3β-ol (ergosterol) and 24-ethylcholesta-5,7,22-trien-3β-ol being the most abundant.     

Homoserine Lactones,Methyl Oligohydroxybutyrates,and Other Extracellular Metabolites of Macroalgae‐Associated Bacteria of the Roseobacter Clade: Identification and Functions

Twenty‐four strains of marine Roseobacter clade bacteria were isolated from macroalgae and investigated for the production of quorum‐sensing autoinducers, N‐acylhomoserine lactones (AHLs). GC/MS analysis of the extracellular metabolites allowed us to evaluate the release of other small molecules as well. Nineteen strains produced AHLs, ranging from 3‐OH‐C10:0‐HSL (homoserine lactone) to (2E,11Z)‐C18:2‐HSL, but no specific phylogenetic or ecological pattern of individual AHL occurrence was observed when cluster analysis was performed. Other identified compounds included indole, tropone, methyl esters of oligomers of 3‐hydroxybutyric acid, and various amides, such as N‐9‐hexadecenoylalanine methyl ester (9‐C16:1‐NAME), a structural analogue of AHLs. Several compounds were tested for their antibacterial and antialgal activity on marine isolates likely to occur in the habitat of the macroalgae. Both AHLs and 9‐C16:1‐NAME showed high antialgal activity against Skeletonema costatum, whereas their antibacterial activity was low.     

Active Efflux Influences the Potency of Quorum Sensing Inhibitors in Pseudomonas aeruginosa

Many bacteria regulate gene expression through a cell–cell signaling process called quorum sensing (QS). In proteobacteria, QS is largely mediated by signaling molecules known as N‐acylated L ‐homoserine lactones (AHLs) and their associated intracellular LuxR‐type receptors. The design of non‐native small molecules capable of inhibiting LuxR‐type receptors (and thereby QS) in proteobacteria is an active area of research, and numerous lead compounds are AHL derivatives that mimic native AHL molecules. Much of this previous work has focused on the pathogen Pseudomonas aeruginosa, which controls an arsenal of virulence factors and biofilm formation through QS. The MexAB‐OprM efflux pump has been shown to play a role in the secretion of the major AHL signal in P. aeruginosa, N‐(3‐oxododecanoyl) L ‐homoserine lactone. In the current study, we show that a variety of non‐native AHLs and related derivatives capable of inhibiting LuxR‐type receptors in P. aeruginosa display significantly higher potency in a P. aeruginosa Δ(mexAB‐oprM) mutant, suggesting that MexAB‐OprM also recognizes these compounds as substrates. We also demonstrate that the potency of 5,6‐dimethyl‐2‐aminobenzimidazole, recently shown to be a QS and biofilm inhibitor in P. aeruginosa, is not affected by the presence/absence of the MexAB‐OprM pump. These results have implications for the use of non‐native AHLs and related derivatives as QS modulators in P. aeruginosa and other bacteria, and provide a potential design strategy for the development of new QS modulators that are resistant to active efflux.     

β-Cyclodextrin Encapsulation of Synthetic AHLs: Drug Delivery Implications and Quorum-Quenching Exploits

Many bacteria, such as Pseudomonas aeruginosa, regulate phenotypic switching in a population density-dependent manner through a phenomenon known as quorum sensing (QS). For Gram-negative bacteria, QS relies on the synthesis, transmission, and perception of low-molecular-weight signal molecules that are predominantly N-acyl-l -homoserine lactones (AHLs). Efforts to disrupt AHL-mediated QS have largely focused on the development of synthetic AHL analogues (SAHLAs) that are structurally similar to native AHLs. However, like AHLs, these molecules tend to be hydrophobic and are poorly soluble under aqueous conditions. Water-soluble macrocycles, such as cyclodextrins (CDs), that encapsulate hydrophobic guests have long been used by both the agricultural and pharmaceutical industries to overcome the solubility issues associated with hydrophobic compounds of interest. Conveniently, CDs have also demonstrated anti-AHL-mediated QS effects. Here, using fluorescence spectroscopy, NMR spectrometry, and mass spectrometry, we evaluate the affinity of SAHLAs, as well as their hydrolysis products, for β-CD inclusion. We also evaluated the ability of these complexes to inhibit wild-type P. aeruginosa virulence in a Caenorhabditis elegans host infection study, for the first time. Our efforts confirm the potential of β-CDs for the improved delivery of SAHLAs at the host/microbial interface, expanding the utility of this approach as a strategy for probing and controlling QS.     

Interkingdom Responses to Bacterial Quorum Sensing Signals Regulate Frequency and Rate of Nodulation in Legume–Rhizobia Symbiosis

Density‐dependent phenotypic switching in bacteria, the phenomenon of quorum sensing (QS), is instrumental in many pathogenic and mutualistic behaviors. In many Gram‐negative bacteria, QS is regulated by N‐acylated‐l ‐homoserine lactones (AHLs). Synthetic analogues of these AHLs hold significant promise for regulating QS at the host–symbiont interface. Regulation depends on refined temporal and spatial models of quorums under native conditions. Critical to this is an understanding of how the presence of these signals may affect a prospective host. We screened a library of AHL analogues for their ability to regulate the legume–rhizobia mutualistic symbiosis (nodulation) between Medicago truncatula and Sinorhizobium meliloti. Using an established QS‐reporter line of S. meliloti and nodulation assays with wild‐type bacteria, we identified compounds capable of increasing either the rate of nodule formation or total nodule number. Most importantly, we identified compounds with activity exclusive to either host or pathogen, underscoring the potential to generate QS modulators selective to bacteria with limited effects on a prospective host.     

Cytotoxic Petrosiacetylenes from the Marine Sponge Petrosia sp.

A novel petrosiacetylene analog (petrosiacetylene E) has been isolated from the Korean marine sponge Petrosia sp., along with petrosiacetylene A, B and C. Their structures were elucidated on the basis of spectroscopic methods and the stereochemistry of the new compound was determined by using the modified Mosher’s method. Petrosiacetylene E showed higher cytotoxicity against five human cancer cell lines than petrosiacetylene A and B, presumably due to the additional hydroxy group located at C-16.     

Non‐native N‐Aroyl L‐Homoserine Lactones Are Potent Modulators of the Quorum Sensing Receptor RpaR in Rhodopseudomonas palustris

Quorum sensing (QS) is a process by which bacteria use low‐molecular‐weight signaling molecules (or autoinducers) to assess their local population densities and alter gene expression levels at high cell numbers. Many Gram‐negative bacteria use N‐acyl L ‐homoserine lactones (AHLs) with aliphatic acyl groups as signaling molecules for QS. However, bacteria that utilize AHLs with aroyl acyl groups have been recently discovered; they include the metabolically versatile soil bacterium Rhodopseudomonas palustris, which uses p‐coumaroyl HL (p‐cAHL) as its QS signal. This autoinducer is especially unusual because its acyl group is believed to originate from a monolignol (i.e., p‐coumarate) produced exogenously by plants in the R. palustris environment, rather than through the endogenous fatty acid biosynthesis pathway like other native AHLs. As such, p‐cAHL could signal not only bacterial density, but also the availability of an exogenous plant‐derived substrate and might even constitute an interkingdom signal. Like other Gram‐negative bacteria, QS in R. palustris is controlled by the p‐cAHL signal binding its cognate LuxR‐type receptor, RpaR. We sought to determine if non‐native aroyl HLs (ArHLs) could potentially activate or inhibit RpaR in R. palustris, and thereby modulate QS in this bacterium. Herein, we report the testing of a set of synthetic ArHLs for RpaR agonism and antagonism by using a R. palustris reporter strain. Several potent non‐native RpaR agonists and antagonists were identified. Additionally, the screening data revealed that lower concentrations of ArHL are required to strongly agonize RpaR than to antagonize it. Structure–activity relationship analyses of the active ArHLs indicated that potent RpaR agonists tend to have sterically small substituents on their aryl groups, most notably in the ortho position. In turn, the most potent RpaR antagonists were based on either the phenylpropionyl HL (PPHL) or the phenoxyacetyl HL (POHL) scaffold, and many contained an electron‐withdrawing group at either the meta or para positions of the aryl ring. To our knowledge, the compounds reported herein represent the first abiotic chemical modulators of RpaR, and more generally, the first abiotic ligands capable of intercepting QS in bacteria that utilize native ArHL signals. In view of the origins of the p‐cAHL signal in R. palustris, the largely unknown role of QS in this bacterium, and R. palustris' unique environmental lifestyles, we anticipate that these compounds could be valuable as chemical probes to study QS in R. palustris in a range of fundamental and applied contexts.     

Cerebrosides from a Far‐Eastern Glass Sponge Aulosaccus sp.

Nine new cerebrosides 1a–d , 2a , 2b , 3a–c were found in the extract of a Far‐Eastern glass sponge Aulosaccus sp. (class Hexactinellida). These β‐d ‐glucopyranosyl‐(1 → 1)‐ceramides contain sphingoid bases (2S,3S,4R,11Z)‐2‐aminoeicos‐11‐ene‐1,3,4‐triol (in 1a – d ), (2S,3S,4R,13Z)‐2‐aminoeicos‐13‐ene‐1,3,4‐triol (in 2a , b ) and (2S,3S,4R,13S*,14R*)‐2‐amino‐13,14‐methylene‐eicosane‐1,3,4‐triol (in 3a – c ), which are N‐acylated by (2R,15Z)‐2‐hydroxydocos‐15‐enoic (in 1a , 2a , 3a ), (2R,16Z)‐2‐hydroxytricos‐16‐enoic (in 1b , 2b , 3b ), (2R,17Z)‐2‐hydroxytetracos‐17‐enoic (in 1d ) and (2R)‐2‐hydroxydocosanoic (in 1c , 3c ) acids. The monoenoic and cyclopropane‐containing sphingoid bases of compounds 1a–d , 2a , 2b , 3a–c have not been found previously in any sphingolipids. The structures of the cerebrosides were elucidated on the basis of ¹H‐, ¹³C‐NMR spectroscopy, mass spectrometry, optical rotation data and chemical transformations. A simplified method for the assignment of the absolute configuration of 2‐hydroxy fatty acids by GC analysis of their (2R)‐ and (2S)‐oct‐2‐yl esters was proposed.     

Structural Analysis of the Minor Cerebrosides from a Glass Sponge Aulosaccus sp.

The minor cerebrosides from a Far‐Eastern glass sponge Aulosaccus sp. were analyzed as constituents of some multi‐component RP‐HPLC fractions. The structures of eighteen new and one known cerebrosides were elucidated on the basis of NMR spectroscopy, mass spectrometry, optical rotation data and chemical transformations. These β‐D‐glucopyranosyl‐(1→1)‐ceramides contain sphingoid bases N‐acylated with straight‐chain (2R)‐2‐hydroxy fatty acids, namely, (2S,3S,4R,11Z)‐2‐aminoeicos‐11‐ene‐1,3,4‐triol, acylated with 15E‐22:1, 16Z‐21:1, 15Z‐21:1, 15Z‐20:1, 15E‐20:1, 19:0, 18:0 acids, (2S,3S,4R)‐2‐amino‐13‐methyltetradecane‐1,3,4‐triol—with 19Z‐26:1, 16Z‐23:1, 23:0, 22:0 acids, (2S,3S,4R)‐2‐amino‐14‐methylpentadecane‐1,3,4‐triol—with 16Z‐23:1, 16E‐23:1, 15Z‐22:1, 22:0 acids, (2S,3S,4R)‐2‐amino‐14‐methylhexadecane‐1,3,4‐triol, linked to 16Z‐23:1, 15Z‐22:1 acids, (2S,3S,4R)‐2‐amino‐9‐methylhexadecane‐1,3,4‐triol—to 16Z‐23:1 acid, and (2S,3S,4R)‐2‐aminohexadecane‐1,3,4‐triol, attached to 15Z‐22:1 acid. The 13‐methyl and 9‐methyl‐branched trihydroxy sphingoid base backbones (C₁₅ and C₁₇, respectively) have not been found previously in sphingolipids. The ceramide parts, containing other backbones, present new variants of N‐acylation of the marine sphingoid bases with the 2‐hydroxy fatty acids. The combination of the instrumental and chemical methods used in this study improved the efficiency of the structural analysis of such complex cerebroside mixtures that gave more detailed information on glycosphingolipid metabolism of the organism.     

Lipids and ultrastructure of Thraustochytrium sp. ATCC 26185

As a representative of a genus with species considered to be potential commercial producers of the nutritionally important polyunsaturated fatty acid docosahexaenoic acid (DHA), Thraustochytrium sp. ATCC 26185 was investigated to determine its potential for DHA production and lipid composition. Cells from liquid shake cultures contained 32% (w/w) lipid, 18% of which was nonsaponifiable lipid. The major saturated fatty acids (14∶0 and 16∶0) comprised up to 59% of the total fatty acids, and DHA was up to 25% after 6 d incubation. Squalene represented 63% of the nonsaponifiable lipid, and cholesterol composed 41% of the total sterols. The phospholipids expected for eucaryotic microbes were detected with phosphatidylcholine as the major phospholipid at 76% of the total. The ultrastructure of this species was similar to other Thraustochytrium species except that the cells did not have surface scales and they contained unusual membrane-like structures that appeared to be associated with oil formation.     

Identification of New N‐Acylhomoserine Lactone Signalling Compounds of Dinoroseobacter shibae DFL‐12T by Overexpression of luxI Genes

Bacteria of the Roseobacter clade are widespread in the ocean and occur in many different habitats. In the genome of Dinoroseobacter shibae DFL‐12, luxI homologous genes that encode synthases responsible for the formation of N‐acylhomoserine lactones (AHLs) have been described. These compounds are known autoinducers that regulate several biological traits—namely, flagella formation and cell differentiation—in D. shibae through quorum sensing. The AHLs produced by D. shibae mainly consisted of N‐octadecadienoylhomoserine lactone (C18:2‐AHL) and N‐octadecenoylhomoserine lactone (C18:1‐HSL). In the wild type these AHLs are synthesized only in low abundance. The luxI genes were therefore expressed in Escherichia coli; this resulted in the formation of AHLs mostly different from those found in the D. shibae wild type. A luxI₁‐deficient mutant of D. shibae was then reprovided with an overexpressed luxI₁ gene. This strain produced large amounts of C18:2‐AHL and C18:1‐AHL, allowing full characterization of these compounds by mass spectrometric techniques and derivatization. Synthesis of the proposed structures confirmed that the major compound is (2E,11Z)‐N‐octadeca‐2,11‐dienoylhomoserine lactone ( 6 , C18:2‐HSL), accompanied by (Z)‐N‐octadec‐11‐enoylhomoserine lactone ( 5 , C18:1‐HSL). AHL 6 has not been reported before from other organisms and contains an unusual 2E double bond.     

The Lipid A Structure from the Marine Sponge Symbiont Endozoicomonas sp. HEX 311

Endozoicomonas sp. HEX311 is a Gram-negative bacterium known to establish a commensal interaction with the marine demosponge Suberites domuncula. The molecular bases of the sponge–microbe interaction events are still poorly defined. Nevertheless, it has been proved that S. domuncula possesses an innate immune system with similarities to the mammalian one and is able to recognize the main component of the Gram-negative bacteria cell wall: the lipopolysaccharide. Whether this recognition occurs in a structure-dependent manner, which is typical for mammalian immune system receptors, is still under investigation. Herein, we report the Endozoicomonas sp. HEX311 lipid A structure obtained by a combination of data attained from chemical, MALDI MS, and MS² approaches. The lipid A is a complex family of species decorated by pyrophosphate and phosphate units and carrying (R)-3-hydroxydodecanoic acid, (R)-3-hydroxytetradecanonic acid, iso-2-hydroxyundecanoic acid, iso-(R)-3-hydroxyundecanoic acid, and iso-nonanoic acid as acyl chains.     

Terpenes in sponge cell membranes: Cell separation and membrane fractionation studies with the tropical marine spongeAmphimedon sp.

The differing sponge and symbiotic microbial cell types in the tropical marine spongeAmphimedon sp. were fractionated according to density, investigated by electron microscopy, and analyzed by high-performance liquid chromatography and nuclear magnetic resonance for the presence of the terpene metabolite diisocyanoadociane (1) and Δ^5,7-sterols (2–7). A sample of whole sponge was dissected into superficial ectosome and deeper choanosome. The superficial tissue was found to be enriched in sterol relative to choanosome; however, extracts from both tissues contained terpene. Dissociation of whole sponge followed by Ficoll density gradient fractionation showed that there are two chemically distinct types of sponge cells inAmphimedon sp.—small non-nucleolated cells of low density contain terpene 1 together with sterols, while larger nucleolated cells contain significant levels of terpene, but only traces of sterol. Membrane fractionation studies were undertaken to establish whether the terpene components were located specifically in the cell membranes of these two cell types. A membrane vesicle pellet spun down at 100,000×g from small sponge cells contained sterols, but only traces of terpene, whereas the membrane vesicle preparation from heavier cells contained both terpenes and sterols. Subsequently, the presence of terpenes together with sterols was demonstrated in a membrane vesicle preparation of purity >90% prepared from bacteria-free sponge cells. These results provide the first experimental evidence that terpenes are associated with sponge cell membranes, where they may function as structural components. Part of this work has been published in preliminary form (ref. 1)     

Enzymatic Incorporation of Selected Long-Chain Fatty Acids into Triolein

Acidolysis of triolein (tri C18:1) with selected long-chain fatty acids (LCFA) was carried out using Candida antarctica (Novozym 435), Rhizomucor miehei (Lipozyme RM IM), Pseudomonas sp. (PS-30), Aspergillus niger (AP-12), and Candida rugosa (AY-30). A better incorporation of stearic acid (SA), α-linolenic acid (ALA), γ-linolenic acid (GLA), arachidonic acid (AA), and docosapentaenoic acid (DPA) was achieved using lipase from Rhizomucor miehei. Lipase from Pseudomonas sp. catalyzed a better incorporation of linoleic acid (LA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) into triolein. Thus, Rhizomucor miehei and to a lesser extent Pseudomonas sp. might be considered as providing the most effective enzymes for acidolysis of triolein with selected LCFA. In general, incorporation of LCFA into triolein (tri C18:1) may be affected by chain length, number of double bonds, and the location and geometry of the double bonds as well as reaction conditions and reactivity and specificity of lipases used. As the ratio of the number of moles of a mixture of equimole quantities of C18 FA to triolein changed from 1 to 3, incorporation of C18 FA into triolein increased accordingly with Rhizomucor miehei lipase. Similarly, incorporation of n-3 FA into triolein increased when ALA, DPA, DHA, and EPA were used. The same trend was noticed for a mixture of n-6 FA (LA + GLA + AA) and triolein.     

A new lipid from an australian marine sponge, Callyspongia sp

A specimen of the sponge Callyspongia sp. collected off the coast of New South Wales, Australia, has yielded the novel lipid (6Z,9Z,12Z,15Z)-1,6,9,12,15-octadecapenten-3-one, together with (4Z,7Z,10Z,13Z)-4,7,10,13-hexadecatetraenoic acid.     

Rational Biocatalyst Design for a Cyanide-Free Synthesis of Long-Chain Fatty Nitriles from Their Aldoximes

Recently, the capability of the aldoxime dehydratase from Bacillus sp. (OxdB) for the transformation of fatty aldoximes into fatty nitriles with impressive substrate loadings is reported. However, the substrate scope of this biocatalyst turned out to be limited in terms of the chain length with decanal oxime being the substrate with the longest well tolerated n-alkyl chain. Besides the increased bulkiness of the long-chain aldoximes, their strongly decreased water solubility represents a further hurdle for an efficient biotransformation. Addressing this challenge of an expanded substrate spectrum comprising long-chain fatty aldoximes, this work investigates the substrate solubility and enzyme kinetics in combination with molecular modeling in order to find an enzyme mutant being suitable for C12- to C16-aldoximes. Both, fatty aldoxime solubility in water and the active site of the wild-type enzyme OxdB are identified as critical issues for an efficient biotransformation of these substrates. The activity issue is addressed by a rational design of a mutant using a homology modeling as well as a molecular modeling software suitable for enzymes. With the resulting double mutant OxdB-F289A/L293A, this report can achieve successful biotransformations with the C12- to C16-aldoximes at substrate concentrations of 250 × 10⁻³ to 1000 × 10⁻³ m . For example, an excellent conversion of >99% is obtained with tetradecanal oxime. Practical applications: Fatty nitriles with a prolonged chain length of C12 or more are of high interest in industry due to their use for the production of fatty amines on large technical scale. As an alternative route, fatty nitriles can be generated from their aldoximes by means of an aldoxime dehydratase (Oxd) as biocatalyst. The conversion of long-chain fatty aldoximes, however, remained a challenge up to now. This work describes the optimization of the aldoxime dehydratase OxdB from Bacillus sp. for the dehydration of nonsoluble bulky fatty aldoximes. The created variant can convert long chain fatty aldoximes toward the corresponding nitrile as demonstrated for C12- to C16-nitriles. In addition, high conversion (of up to >99%) is achieved when operating at high substrate concentrations of up to 1000 × 10⁻³ m , thus making this approach interesting for industrial applications.     

Identification of Novel Acetylenic Alcohols and a New Dihydrothiopyranone from the Tropical Sponge <Emphasis Type="Italic">Reniochalina</Emphasis> sp.

Two new acetylenic alcohols (1–2) and a new dihydrothiopyranone (3) were isolated from the tropical sponge Reniochalina sp. Their structures were determined by spectroscopic and chemical methods to be (3R)-hydroxyoctatriacont-(4E)-en-1-yne (1), 5-hydroxyheptatriacont-(3Z)-en-1-yne (2) and 2-hexadecyl-2,3-dihydrothiopyran-4-one (3). The acetylenic alcohol (1) exhibited significant growth inhibitory effect against human tumor cell lines.