Solubility properties and diffusional extraction behavior of natamycin from streptomyces gilvosporeus biomass

Natamycin is a type of polyene macrolide antibiotic and has been produced in submerged microbial cultures of some natural Streptomyces strains. Natamycin extraction from cellular biomass is greatly affected by the molecular and solubilization characteristics of the extraction solvent, and this is a major reason for the routine attainment of low volumetric titers, resulting from sparing natamycin solubility. In this work, a series of experiments were conducted to investigate the solubility of natamycin in some selected organic solvents in order to assess the influence on natamycin extraction yield. Natamycin showed the highest solubility in 75% aqueous methanol under the conditions of pH 2, 30°C and 1 atm. Furthermore, the extraction of natamycin using 75% aqueous methanol was performed and the highest extraction yield of 45.7% was obtained under pH 2. A mathematical model derived from Fick's law of the biomolecular diffusion process was developed to fit the experimental kinetic data of natamycin extraction. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Characteristics of hexadecane partition by the growth medium of Acinetobacter sp.

The partition of n-hexadecane in the spent growth medium of Acinetobacter sp. HOI-N was determined by measuring the increase in the relative aqueous solubility of ³H-hexadecane as compared to controls. The amount of hexadecane partitioned was proportional to the protein concentration. The specific solubility of hexadecane (nmol/mg protein) was analyzed by least-squares fitting yielding an average slope of 0.6 with a standard deviation of 0.3, indicating either nonequilibrium of hexadecane or physical aggregation of protein. The amount of hexadecane partitioned was concentration dependent yielding optically clear microemulsions at hexadecane concentrations of less than 1.4mM and macroemulsions at hexadecane concentrations of 1.4mM or greater. Preliminary results indicated that hexadecane and partitioned by a lipoprotein complex.     

Engineered biosynthesis and characterisation of disaccharide-modified 8-deoxyamphoteronolides

Several polyene macrolides are potent antifungal agents that have severe side effects. Increased glycosylation of these compounds can improve water solubility and reduce toxicity. Three extending glycosyltransferases are known to add hexoses to the mycosaminyl sugar residues of polyenes. The Actinoplanes caeruleus PegA enzyme catalyses attachment of a D-mannosyl residue in a β-1,4 linkage to the mycosamine of the aromatic heptaene 67-121A to form 67-121C. NppY from Pseudonocardia autotrophica adds an N-acetyl-D-glucosamine to the mycosamine of 10-deoxynystatin. NypY from Pseudonocardia sp. P1 adds an extra hexose to a nystatin, but the identity of the sugar is unknown. Here, we express the nypY gene in Streptomyces nodosus amphL and show that NypY modifies 8-deoxyamphotericins more efficiently than C-8 hydroxylated forms. The modified heptaene was purified and shown to be mannosyl-8-deoxyamphotericin B. This had the same antifungal activity as amphotericin B but was slightly less haemolytic. Chemical modification of this new disaccharide polyene could give better antifungal antibiotics.

     

Polymeric nanoparticle-encapsulated curcumin ("nanocurcumin"): a novel strategy for human cancer therapy

Background  

Curcumin, a yellow polyphenol extracted from the rhizome of turmeric (Curcuma longa), has potent anti-cancer properties as demonstrated in a plethora of human cancer cell line and animal carcinogenesis models. Nevertheless, widespread clinical application of this relatively efficacious agent in cancer and other diseases has been limited due to poor aqueous solubility, and consequently, minimal systemic bioavailability. Nanoparticle-based drug delivery approaches have the potential for rendering hydrophobic agents like curcumin dispersible in aqueous media, thus circumventing the pitfalls of poor solubility.     

Monitoring protein stability <Emphasis Type="Italic">in vivo</Emphasis>

Reduced protein stability in vivo is a prerequisite to aggregation. While this is merely a nuisance factor in recombinant protein production, it holds a serious impact for man. This review focuses on specific approaches to selectively determine the solubility and/or stability of a target protein within the complex cellular environment using different detection techniques. Noninvasive techniques mapping folding/misfolding events on a fast time scale can be used to unravel the complexity and dynamics of the protein aggregation process and factors altering protein solubility in vivo. The development of approaches to screen for folding and solubility in vivo should facilitate the identification of potential components that improve protein solubility and/or modulate misfolding and aggregation and may provide a therapeutic benefit.     

Borate complexes of amphotericin B: Polymeric species and aggregates in aqueous solutions

Amphotericin B, a polyene macrolide antibiotic, exists in aqueous solution as a poorly soluble, high-molecular-weight aggregate. A borate complex of this polyene was prepared that has greater solubility and is less aggregated. In aqueous solution this borate complex exists as a mixture of several molecular species differing in borate content, molecular weight, and molecular conformation. The solubility varied with pH and was minimal at neutrality. Throughout the pH range it was one to two orders of magnitude higher than that of the parent compound. The molecular size distribution, as determined by differential ultrafiltration, showed a progressive increase in the weight fraction of aggregates going from acid to alkaline solutions. The sizes of aggregates ranged from under 25 to over 100 molecules. The borate content of the complexes increased with increasing pH. No borate was complexed in acid solutions. This indicated that amphotericin B and borate ions can complex to form copolymer chains of varying length in which these species alternate, since both are bifunctional. The complexation equilibrium is favored by high pH. Absorption and CD spectra indicated that the polyene molecules can stack reversibly to form dimers. Dimerization constants calculated from the spectra were highest in neutral solution and declined with increasing acidity or alkalinity. In alkaline solutions the polymer chains are long and extended, with minimal stacking. In neutral solution the chains are shorter and extensively stacked. In acid solutions no borate complexes are formed, and the polyenes are stacked to an intermediate degree. The very different effects of pH and concentration on the degree of complexation with borate and on the degree of dimerization of the polyenes shows that these equilibria are independent of each other.     

β-Naphthoflavone analogs as potent and soluble aryl hydrocarbon receptor agonists: Improvement of solubility by disruption of molecular planarity

The physiological role of aryl hydrocarbon receptor (AhR) is not yet fully understood, and investigation is hampered by the limited solubility of reported AhR ligands in aqueous media. To achieve improved solubility, we focused on our previous finding that planarity-disruption of molecules leads to less efficient crystal packing and greater aqueous solubility. Here, we describe chemical modification of an AhR agonist, β-naphthoflavone, focusing on planarity-disruption. As expected, introduction of substituents at the ortho-positions of the phenyl group resulted in greater solubility. Among the compounds prepared, the fluoro analog showed more potent AhR agonistic activity and greater solubility than did β-naphthoflavone. Our results indicate that this strategy to improve aqueous solubility, that is, introduction of substituent(s) that disrupt planarity, may be generally applicable to bicyclic molecules.     

Exonuclease III action on microarrays: Observation of DNA degradation by fluorescence correlation spectroscopy

DNA with all cytosines, thymines, or all pyrimidines of one strand substituted by fluorescently labeled analogs shows diminished solubility in aqueous media and a strong tendency to aggregation that hampers enzymatic downstream processing. In this study, immobilization of fully fluorescently labeled DNA on microarrays was shown to resolve the named problems and to enable successive DNA degradation by exonuclease III. Fluorescence correlation spectroscopy and single-molecule counting for monitoring the course of DNA hydrolysis in real time revealed the virtually processive degradation of labeled DNA that occurred at an average rate of approximately 4 nt/s.     

The Influence of Cosolvent on the Complexation of HP-β-cyclodextrins with Oleanolic Acid and Ursolic Acid

The present work was aimed at the influence of ethanol on the complex formation of hydroxypropyl-β-cyclodextrin (HP-β-CD) with oleanolic acid (OA) and ursolic acid (UA), two insoluble isomeric triterpenic acids. Phase solubility studies were carried out to evaluate the solubilizing power of HP-β-CD, in association with ethanol, toward OA and UA. A mathematical model was applied to explain and predict the solubility of OA and UA influenced by HP-β-CD and ethanol. The solid complexes were prepared by evaporating the filtrate of samples which was prepared in different complexing media. The solubility of OA is much higher than that of UA in all the tested aqueous solutions. The solubility of OA and UA can be increased over 900 and 200 times, respectively, by forming complex with HP-β-CD. Ethanol (0.5%, v/v) can help the formation of OA-HP-β-CD complex, but is harmful to the formation of UA-HP-β-CD complex. Increasing solubility in water can be achieved by adding ethanol into the complexing media, but the concentration of ethanol should be optimized. The ring E of the chemical compounds has a great influence on the complexing process.     

Peptide purification using the chemoselective reaction between N‐(methoxy)glycine and isothiocyanato‐functionalized resin

An efficient peptide purification strategy is established, comprising the selective reaction of an N‐terminal N‐(methoxy)glycine residue of the peptide and isothiocyanato‐functionalized resins, and subsequent Edman degradation. These reactions take place in acidic media; in particular, the Edman degradation proceeds smoothly in media containing more than 50% trifluoroacetic acid (v/v). These acidic conditions offer increased solubility, making them advantageous for the purification of hydrophobic and aggregation‐prone peptides. The effectiveness of this method, together with scope and limitations, is demonstrated using model peptides and the practical purification of the loop region of the human dopamine D2 receptor long isoform (residues 240–272). Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Modulation of polyene antibiotics self-association by ions from the Hofmeister series

The toxicity of the antifungal polyene antibiotic amphotericin B (AMB) has been related to its low solubility, more specifically to a self-associated form termed toxic aggregate. In addition, AMB in aqueous medium gives rise to concentration, ionic strength, and time-dependent polydisperse systems. For this reason different approaches, including the use of several lipid aggregates, have been used in attempts to improve the drug's solubility and increase its therapeutic index. In this context, understanding AMB's self-association properties should help in the preparation of less toxic formulations. Ions from the Hofmeister series alter water properties: while kosmotropes (water structure makers-sulfate, citrate, phosphate) decrease solute solubility, chaotropes (water structure breakers-perchlorate, thiocyanate, trichloroacetate, and the neutral molecule urea) have opposite effects. This work reports a study of the effect of Hofmeister ions and urea on the self-aggregation of AMB and some of its derivatives. Optical absorption and circular dichroism spectra were used to monitor monomeric and aggregated antibiotic. While kosmotropes increased aggregation in a concentration-dependent manner, the opposite was observed for chaotropes. It is shown, for the first time, that thiocyanate and trichloroacetate can induce complete AMB monomerization. The understanding of these processes at the physicochemical and molecular levels and the possibility of modulating the aggregation state of AMB and its derivatives should contribute to elucidate the mechanisms of action and toxicity of this widely used antibiotic and to develop more efficient and less toxic preparations.     

l-Arginine hydrochloride increases the solubility of folded and unfolded recombinant plasminogen activator rPA

L ‐Arginine hydrochloride (L ‐ArgHCl) was found to be an effective enhancer for in vitro protein refolding more than two decades ago. A detailed understanding of the mechanism of action, by which L ‐ArgHCl as co‐solvent is capable to effectively suppress protein aggregation, while protein stability is preserved, has remained elusive. Concepts for the effects of co‐solvents, which have been established over the last decades, were found to be insufficient to completely explain the effects of L ‐ArgHCl on protein refolding. In this article, we present data, which clearly establish that L ‐ArgHCl acts on the equilibrium solubility of the native model protein recombinant plasminogen activator (rPA), while for S‐carboxymethylated rPA (IAA‐rPA) that served as a model protein for denatured protein states, equilibrium solubilities could not be obtained. Solid to solute free transfer energies for native rPA were lowered by up to 14 kJ mol^‐1 under the tested conditions. This finding is in marked contrast to a previously proposed model in which L ‐ArgHCl acts as a neutral crowder which exclusively has an influence on the stability of the transition state of aggregation. The effects on the apparent solubility of IAA‐rPA, as well as on the aggregation kinetics of all studied protein species, that were observed in the present work could tentatively be explained within the framework of a nucleation‐aggregation scheme, in which L ‐ArgHCl exerts a strong effect on the pre‐equilibria leading to formation of the aggregation seed.     

Silica‐modified luminescent LaPO4:Eu@LaPO4@SiO2 core/shell nanorods: Synthesis,structural and luminescent properties

Monoclinic‐type tetragonal LaPO₄:Eu (core) and LaPO₄:Eu@LaPO₄ (core/shell) nanorods (NRs) were successfully prepared using a urea‐based co‐precipitation process under ambient conditions. An amorphous silica layer was coated around the luminescent core/shell NRs via the sol–gel process to improve their solubility and colloidal stability in aqueous and non‐aqueous media. The prepared nano‐products were systematically characterized by X‐ray diffraction pattern, transmission electron microscopy, energy dispersive X‐ray analysis, and FTIR, UV/Vis, and photoluminescence spectroscopy to examine their phase purity, crystal phase, surface chemistry, solubility and luminescence characteristics. The length and diameter of the nano‐products were in the range 80–120 nm and 10–15 nm, respectively. High solubility of the silica‐modified core/shell/Si NRs was found for the aqueous medium. The luminescent core NRs exhibited characteristic excitation and emission transitions in the visible region that were greatly affected by surface growth of insulating LaPO₄ and silica layers due to the multiphonon relaxation rate. Our luminescence spectral results clearly show a distinct difference in intensities for core, core/shell, and core/shell/Si NRs. Highly luminescent NRs with good solubility could be useful candidates for a variety of photonic‐based biomedical applications.     

Halophilic β-lactamase as a new solubility- and folding-enhancing tag protein: production of native human interleukin 1α and human neutrophil α-defensin

The amino acid composition of halophilic enzymes is characterized by an abundant content of acidic amino acid, which confers to the halophilic enzymes extensive negative charges at neutral pH and high aqueous solubility. This negative charge prevents protein aggregation when denatured and thereby leads to highly efficient protein refolding. β-Lactamase from periplasmic space of moderate halophile (BLA), a typical halophilic enzyme, can be readily expressed as a native, active form in Escherichia coli cytoplasm. Similar to other halophilic enzymes, BLA is soluble upon denaturation by heat or urea treatments and, hence, can be efficiently refolded. Such high solubility and refolding efficiency make BLA a potential fusion partner for expression of aggregation-prone heterologous proteins to be expressed in E. coli. Here, we succeeded in the soluble expression of several “difficult-to-express” proteins as a BLA fusion protein and verified biological activities of human interleukin 1α and human neutrophil α-defensin, HNP-1.     

Carboxyl-terminal domain characterization of polyene-specific P450 hydroxylase in <Emphasis Type="Italic">Pseudonocardia autotrophica</Emphasis>

A polyene compound NPP identified in Pseudonocardia autotrophica was shown to contain an aglycone identical to nystatin, but to harbor a unique disaccharide moiety that led to higher solubility and reduced hemolytic activity. Recently, it was revealed that the final step of NPP (nystatin-like polyene) biosynthesis is C10 regio-specific hydroxylation by the cytochrome P450 hydroxylase (CYP) NppL (Kim et al. [7]). Through mutation and cross-complementation, here we found that NppL preferred a polyene substrate containing a disaccharide moiety for C10 hydroxylation, while its orthologue NysL involved in nystatin biosynthesis showed no substrate preference toward mono- and disaccharide moieties, suggesting that two homologous polyene CYPs, NppL and NysL might possess a unique domain recognizing a sugar moiety. Two hybrid NppL constructs containing the C-terminal domain of NysL exhibited no substrate preference toward 10-deoxy NPP and 10-deoxy nystatin-like NysL, implying that the C-terminal domain plays a major role in differentiating the sugar moiety responsible for substrate specificity. Further C-terminal domain dissection of NppL revealed that the last fifty amino acids play a critical role in determining substrate specificity of polyene-specific hydroxylation, setting the stage for the biotechnological application of hydroxyl diversification for novel polyene biosynthesis in actinomycetes.     

Effects of osmolytes on solvent features of water in aqueous solutions

The solvatochromic solvent features of water (dipolarity/polarizability, π*, hydrogen bond donor acidity, α, and hydrogen bond acceptor basicity, β) of water have been determined in aqueous solutions of erythritol, glucose, inositol, sarcosine, xylitol and urea with concentrations from 0 to ~3 M and higher. The concentration effects of the osmolytes on the solvent features of water were characterized and compared with those reported previously for sorbitol, sucrose, trimethylamine N-oxide (TMAO), and trehalose. The solvent features of water in solutions of all osmolytes except TMAO and sarcosine were established to be linearly interrelated. It is shown that the concentration effects of essentially all nonionic osmolytes depend on osmolytes’ lipophilicity, molecular polarizability, and polar surface area. It is demonstrated that solubility of various compounds in aqueous solutions of glucose, sucrose, sorbitol, and urea of varied concentrations may be described in terms of solvent dipolarity/polarizability of water in these solutions. Surface tension of aqueous solutions of sucrose and sorbitol may also be described in the same terms. The relative permittivity of aqueous solutions of glucose and sucrose may be described in terms of the solvent hydrogen bond donor acidity of water. It is suggested that the effects of nonionic osmolytes on behavior of proteins and nucleic acids in aqueous media may be considered in terms of the altered solvent features of water instead of “nano-molecular crowding” effect.     

Performance of Luffa cylindrica as an immobilization matrix for the biotransformation of cholesterol by Mycobacterium species

Abstract

The microbial cleavage of the side chain of cholesterol is a slow process due to the low solubility of the substrate in aqueous media (< 1 μM). Cell immobilization has been shown to be an efficient technology for enhancing the yield of cholesterol biotransformation. In these experiments, living cells of Mycobacterium sp. DSM 2966 and Mycobacterium sp. DSM 2967 were immobilized by passive adsorption on different types of solid carriers. As compared to the control and other solid supports, Luffa cylindrica resulted in a 3–4-fold increase of the specific side chain cleavage activity after 7 days of incubation. Luffa cylindrica had no significant negative influence on cell growth. Furthermore, it is a natural, inexpensive, non-toxic and mechanically strong material and therefore suitable for follow-up experiments.     

Anomalously high aggregation level of the polyene antibiotic amphotericin B in acidic medium: implications for the biological action

Amphotericin B (AmB) is a polyene antibiotic used to treat deep-seated mycoses. Both the therapeutic action and the toxic side effects of this drug are dependent on its molecular organization. AmB appears as a zwitterion at neutral pH owing to NH(3)(+) and COO(-) groups. The results obtained with electronic absorption, fluorescence, resonance light scattering and infrared absorption spectroscopic analyses show that in the aqueous medium at pH above 10 AmB appears in the monomeric form owing to the negative net electric charge of the molecule. On the contrary, anomalously high aggregation level has been observed at pH below 2, despite the positive net electric charge. The effect is interpreted in terms of the permanent polarization of the polyene chain at low pH, associated with relative rotational freedom of the charged mycosamine fragment of the molecule. The pH-dependent aggregation of AmB is discussed in aspect of pharmacological action of the drug.     

Oral formulation of a novel antiviral agent,PG301029, in a mixture of Gelucire 44/14 and DMA (2∶1, wt/wt)

To develop an oral formulation for PG301029, a novel potent agent for the treatment of Hepatitis C virus infection, that not only has very low aqueous solubility but also degrades rapidly in water. The solubility of PG301029 was determined in water, various aqueous media, and several neat organic solvents. The stability of PG301029 was monitored at room temperature in buffess for 4 days, and in several neat organic solvents for up to 8 mo. Drug concentrations were measured by high-performance liquid chromatography (HPLC). Based on solubility and stability data, Gelucire 44/14 and DMA (N,N-dimethylacetamide) at a weight ratio of 2 to 1 were chosen as the formulation vehicle. After the vehicle was prepared, it was maintained in liquid form at ∼40°C until the PG301029 was dissolved. The final formulation product was a semisolid at room temperature. The bioavailability of the formulation was tested on 4 female BALB/c mice. PG301029 is insoluble in all tested aqueous media, while its solubility is promising in DMA. This compound is unstable in aqueous media and some organic solvents; however, it is stable in DMA. This proposed formulation is able to hold up to 10 mg/mL of drug and is stable at 4°C. The shelf life for this formulation stored at 4°C is extrapolated to be greater than 4 years. This formulation dramatically increases the bioavailability of PG301029. This nonaqueous formulation solves the stability, solubility, and bioavailability problems for PG301029. This semisolid formulation can easily be incorporated into soft elastic capsules.     

Effects of pisatin on Dictyostelium discoideum: its relationship to inducible resistance to nystatin and extension to other isoflavonoid phytoalexins

Dictyostelium discoideum amoebae can acquire resistance to otherwise inhibitory concentrations of pisatin, an isoflavonoid phytoalexin of pea, and nystatin, a polyene antibiotic, following pretreatment with sublethal concentrations of these compounds. Additionally, growth on medium containing pisatin can induce nystatin resistance. We show here that distinct mechanisms mediate the inducible resistance to these two compounds because it is possible to isolate mutations that specifically block the induction of nystatin resistance but do not affect the induction of pisatin resistance. Pisatin did not affect wild-type sterol biosynthesis; therefore, the induction of nystatin resistance by pisatin is probably not via an alteration of membrane sterols. The inducible pisatin resistance phenotype was shown to extend to the isoflavonoid phytoalexins maackiain and biochanin A, and all three compounds inhibited the aggregation of amoebae that is normally triggered by starvation. Received: 23 February 1998 / Accepted: 26 June 1998