A whole-cell process for the production of ε-caprolactone in aqueous media

ε-Caprolactone is an industrially important intermediate produced in multi-10,000 ton scale annually with broad applications. We report on a whole-cell biocatalytic conversion of cyclohexanol to ε-caprolactone using the combination of alcohol dehydrogenase (ADH) with two stability-improved variants (QM and M15) of the Baeyer-Villiger monooxygenase CHMO with a special focus on process development at the 200 mM scale. Influence of parameters such as volumetric mass transfer co-efficient, stirrer speed and catalytic loading (amount of E. coli whole-cells expressing ADH and CHMO) on the process efficiency were studied and optimised. This resulted in over 98% conversion, a product titer of 20 g L^–1 and an isolated product amount of 9.1 g (80%). This corresponds to a space-time yield of 1.1 g L^–1 h⁻¹ and a reaction yield (mole of product per mole substrate) of 0.9. Comparing the two CHMO variants a significant difference in catalytic yield (weight of product to weight of catalyst; 0.6 vs 0.3) was observed without any inherent changes in the process. Hence, the reported process can accommodate in the future improved variants of the CHMO.     

A comparative study of the regioselectivity of the β-galactosidases from Kluyveromyces lactis and Bacillus circulans in the enzymatic synthesis of N-Acetyl-lactosamine in aqueous media

The enzymatic synthesis of N-acetyl-lactosamine (LacNAc) was studied in aqueous media with high substrate concentrations using the transgalactosylation of N-acetyl-D-glucosamine (GlcNAc), starting from lactose as a galactosyl donor. The efficiency and regioselectivity of the β-galactosidases from Kluyveromyces lactis (KlβGal) and Bacillus circulans (BcβGal) were compared. The reaction was optimized by varying the experimental conditions (pH, catalytic activity concentration, and mass concentration ratio of the substrates), which enhanced the synthesis yields with both enzymes and especially with BcβGal. BcβGal catalyzed the formation of the maximal LacNAc concentration obtained (101 mM or 39 g L(-1), corresponding to a yield of 11% on the basis of GlcNAc conversion), after 5 h at pH 6.5 and for a substrate mass concentration ratio of 1. This enzyme also gave an optimal synthesis yield of about 17.5%. No change in regioselectivity was observed when using KlβGal, whereas the regioselectivity of BcβGal proved to be subject to variations, the 1-4 and 1-6 linkages being favored under kinetic and thermodynamic control conditions, respectively. Finally, it was demonstrated that the N-acetyl-allolactosamine synthesized during the GlcNAc transgalactosylation catalyzed by BcβGal was a thermodynamic product and did not result from a chemical and/or enzymatic isomerization of LacNAc.     

Evidence for organic synthesis in high temperature aqueous media — Facts and prognosis

Hydrothermal systems are common along the active tectonic areas of the earth. Potential sites being studied for organic matter alteration and possible organic synthesis are spreading ridges, off-axis systems, back-arc activity, hot spots, volcanism, and subduction. Organic matter alteration, primarily reductive and generally from immature organic detritus, occurs in these high temperature and rapid fluid flow hydrothermal regimes. Hot circulating water (temperature range — warm to >400 °C) is responsible for these molecular alterations, expuslion and migration. Compounds that are obviously synthesized are minor components because they are generally masked by the pyrolysis products formed from contemporary natural organic precursors. Heterocyclic sulfur compounds have been identified in high temperature zones and hydrothermal petroleums of the Guaymas Basin vent systems. They can be interpreted as being synthesized from formaldehyde and sulfur or HS _x ^– in the hydrothermal fluids.Other products from potential synthesis reactions have not yet been found in the natural systems but are expected based on known industrial processes and inferences from experimental simulation data. Various industrial processes have been reviewed and are of relevance to hydrothermal synthesis of organic compounds. The reactivity of organic compounds in hot water (200–350 °C) has been studied in autoclaves, and supercritical water as a medium for chemistry has also been evaluated. This high temperature aqueous organic chemistry and the strong reducing conditions of the natural systems suggest this as an important route to produce organic compounds on the primitive earth. Thus a better understanding of the potential syntheses of organic compounds in hydrothermal systems will require investigations of the chemistry of condensation, autocatalysis, catalysis and hydrolysis reactions in aqueous mineral buffered systems over a range of temperatures from warm to >400 °C.Presented in part at the International Society for the Study of the Origin of Life Meeting, Barcelona, Spain, July 1993.     

Conformation and cooperative order-disorder transition in aqueous solutions of β-1,3-d-glucan with different degree of branching varied by the Smith degradation

β-1,3-d -glucan with different degrees of branching were obtained by selectively and gradually removing side chains from schizophyllan, a water-soluble triple helical polysaccharide, using the Smith degradation. Size exclusion chromatography combined with a multi-angle light scattering detection was performed in aqueous 0.1 M NaCl. The degree of branching decreased after the Smith degradation, while the molar mass distributions were almost unchanged. The molecular conformation of the Smith-degraded β-1,3-d -glucan was analyzed on the basis of the molar mass dependency of the radius gyration, and found to be comparable to the original triple helix of schizophyllan. Differential scanning calorimetry in deuterium oxide–hexadeuterodimethylsulfoxide mixtures was performed to investigate the effects of the degree of branching on the cooperative order-disorder transition. Removal of side chains affects both the transition temperature and transition enthalpy. The ordered structure is formed by the residual side chains in the triplex unit, so that the linear cooperative system of the triplex is maintained after the Smith degradation.     

Plural media ethics? Reformist Islam in India and the limits of global media ethics

Dialectical Anthropology - The transatlantic field of global media ethics is premised on a search for the conceptual foundations of plurality. This article is a critique of this very endeavor. I...     

Self-assembly of β-lactoglobulin and the soluble fraction of gum tragacanth in aqueous medium

Spectrophotometric and light scattering measurements, along with optical microscopy, were used to follow the complexation and coacervation process that occur when β-lactoglobulin (BLG)/tragacanthin (T) mixed dispersions (0.3 wt.% total concentration; BLG:T ratio of 2:1) were brought from pH 6 to pH 2. In addition, the coupling of slow in situ acidification of the mixture and rheometry was utilised to gain deeper insights into pH-induced structural transitions during the assembly process. The results obtained by this multi-methodological approach allowed the associative phase separation process to be parameterised in terms of a set of characteristic pH values (~5.3, ~4.8, ~4.5, ~4.15, ~4, ~3.8, ~2.5) at which critical structural changes took place. Investigation of the absorbance profiles of complexed/coacervated systems as a function of time revealed that several transitions could occur at different time scales. Morphological changes in the assemblies and the subsequent formation of some flocculant substances during the late stage of process were clearly visualised using microscopy.     

γ-Glutamyl and d- or l-peptide linkages in mycobacillin, a cyclic peptide antibiotic

Mycobacillin lacks amino groups but contains two free alpha-carboxyl groups, indicating the presence of two side-chain peptide linkages. The five aspartic acid residues of mycobacillin are all in alpha-peptide linkage whereas the two glutamic acid residues are in gamma-linkage. Mycobacillin does not react with hydroxylamine to give hydroxamate, indicating the absence of anhydride, lactone and ester linkages. This is also confirmed by i.r. spectroscopy and titration of the molecule. Of the 15 peptides obtained from partial hydrolysates of mycobacillin, 12 contain aspartic acid. Results obtained by treatment of hydrolysates of aspartic acid-containing peptides with d-amino acid oxidase and l-glutamate decarboxylase (containing l-aspartate decarboxylase activity) indicate that residue 5 is l-aspartic acid and residues 2, 8, 11 and 13 are d-aspartic acid. The d- or l-peptide sequence and nature of peptide linkages in mycobacillin are proposed on the basis of these findings and the amino acid sequence reported earlier.     

Design and synthesis of a photocleavable biotin-linker for the photoisolation of ligand–receptor complexes based on the photolysis of 8-quinolinyl sulfonates in aqueous solution

The ability of avidin (Avn) to form strong complex with biotin (Btn) is frequently used in the detection and isolation of biomolecules in biochemical, analytical, and medicinal research. The fact that the binding is nealy irreversible, however, constitutes a drawback in term of the isolation and purification of intact biomolecules. We recently found that 8-quinolinyl esters of aromatic or aliphatic sulfonic acids undergo photolysis when irradiated at 300–330 nm in aqueous solution at neutral pH. In this work, a biotin–dopamine (BD) conjugate containing a photocleavable 8-quinolinyl benzenesulfonate (QB) linker, BDQB, was designed and synthesized for use in the efficient recovery of dopamine–protein (e.g., antibody) complexes from an Avn–Btn system. The complexation of BDQB with a primary anti-dopamine antibody (anti-dopamine IgG₁ from mouse) on an Avn-coated plate was confirmed by an enzyme-linked immunosorbent assay (ELISA) utilizing a secondary antibody (anti-IgG₁ antibody) conjugated with horseradish peroxidase (HRP). Upon the photoirradiation (at 313 nm) of the BDQB–IgG₁ complex, the release of dopamine–IgG₁ complex was confirmed by ELISA. Characterization of the resulting photoreleased dopamine–anti-dopamine IgG₁ complex was performed by SDS–PAGE and Western blot.     

Conformation of αs-Casein in Various Concentrations

Conformation of α_s-casein and its association were investigated from behaviors of tyrosyl and tryptophyl residues and hydrophobic sites. The chromophoric residues and ANS binding sites were buried into a region inaccessible to solvent with increasing concentration of α_s-casein. It is considered that the association of α_s-casein with concentration is proceeded by the hydrophobic sites to be able to bind ANS and the hydrophobic segments in which tyrosyl and tryptophyl residues exist. Below 0.04% of α_s-casein, α_s-casein exists in the monomer state and 80% of tyrosyl and tryptophyl residues are accessible to aqueous solvent. The hydro-phobic sites of α_s-casein may be exposed to solvent in the monomer state.     

Speciation of dimethyltin(IV) – and trimethyltin(IV) – carbocysteinate and – glutamate systems in aqueous media

Abstract

The formation of complex species in the dimethyltin(IV) and trimethyltin(IV)-carboxymethyl-L-cysteinate (carbocysteinate) systems in NaCl_aq, at different ionic strengths, and in a multicomponent Na⁺, K⁺, Ca²⁺ ,Mg²⁺, Cl^? and SO₄^2-? medium representative of the seawater major composition, is discussed. Experimental results give evidence for the formation of the following species (L = carbocysteinate): [(CH₃)₂Sn(L)]⁰, [(CH₃)₂Sn(HL)]⁺, [(CH₃)₂Sn(OH)(L)]^?, [(CH₃)₂Sn(OH)₂(L)]^2? in the DMT–CCYS system, and [(CH₃)₃Sn(HL)]⁰, [(CH₃)₃Sn(L)]^? and [(CH₃)₃Sn(OH)(L)]^2? in the TMT-CCYS system. The ionic strength dependence of formation constants was taken into account by an extended Debye Hückel type equation and by the SIT (Specific ion Interaction Theory). Measurements were carried out also on the dimethyltin(IV)-glutamate and trimethyltin(IV)-glutamate systems in NaCl_aq, owing the strict similarity of glutamate and carbocysteinate. Results obtained show the formation of complex species having the same stoichiometry as those formed in the DMT- and TMT-carbocysteinate systems, with very similar stability, confirming that carbocysteinate behaves as a dicarboxylic amino acid without involving the sulfur-bridge potential binding site in metal coordination.     

1,8-Naphthalimide–Cu(ІІ) ensemble based turn-on fluorescent probe for the detection of thiols in organic aqueous media

A 1,8-naphthalimide–Cu(II) ensemble was rationally designed and synthesized as a new turn-on fluorescent probe utilizing the ‘chemosensing ensemble’ method for detections of thiols (Cys, Hcy and GSH) with high selectivity over other α-amino acids at pH 7.4 in organic aqueous media (EtOH/HEPES, v/v = 9:1). The recognition mechanism was attributed to the remove Cu(II) from the 1,8-naphthalimide–Cu(II) ensemble by thiols and the release of flurescence of ligand 1. Remarkable fluorescence enhancements were therefore observed in the sensing process of thiols by the 1,8-naphthalimide–Cu(II) ensemble. Furthermore, the 1,8-naphthalimide–Cu(II) ensemble was successfully applied to the fluorescence imaging of thiols in CHO cells with high sensitivity and selectivity.     

Synthesis of (R)- or (S)-valinol using ω-transaminases in aqueous and organic media

Valinol is part of numerous pharmaceuticals and has various other important applications. Optically pure valinol (ee >99%) was prepared employing different ω-transaminases from the corresponding prochiral hydroxy ketone. By the choice of the enzyme the (R)- as well as the (S)-enantiomer were accessible. Reductive amination was performed in organic solvent (MTBE) using 2-propyl amine as amine donor whereas alanine was applied in or in aqueous medium. Transformations in phosphate buffer were successfully performed even at 200 mM substrate concentration (20.4 g/L) leading to 99% (R) and 94% (S) conversion with perfect optical purity (>99% ee).     

Structure and Conformation of the Antiviral Agent 5-Methoxymethyl-2′-deoxyuridine

Abstract

The three dimensional structure of the activiral agent, 5-methoxymethyl-2′-deoxyuridine (MmdUrd) was determined by x-ray diffraction methods. MMdUrd crystallized in space group P2₁2₁2₁ of the orthorhombic system with a = 9.166(1)A, b, = 25.348(1)Amm c = 5.270(1)A and Z = 4. The conformation of the glycosyl bond is anti (χ = 233.30), the deoxyribose ring has the C(2′)-endo envelope conformation (²E), the CH₂OH side chain has the g⁺ conformation and the methoxy group at the C(5) position is on the same side of pyrimidine plane as the 0(4′) oxygen. NMR spectroscopy was used to determine the conformation in solution. The spectra indicate that the sugar ring exists in a 60:40 equilibrium of the S- and N-states. The population of the three rotamers about the exocyclic c(4′)–C(5′) bond were estimated to be g⁺:t:g::61%:31%:8%. The correlaiton of molecular conforation with antiviral activity is discussed.     

Liquid–liquid extraction of antimicrobial peptide P34 by aqueous two-phase and micellar systems

Antimicrobial peptide P34 is a promising biopreservative for utilization in the food industry. In this work, aqueous biphasic systems (ABS) and aqueous biphasic micellar systems (ABMS) were studied as prestep for purification of peptide P34. The ABS was prepared with polyethylene glycol (PEG) and inorganic salts and the ABMS with Triton X-114 was chosen as the phase-forming surfactant. Results indicate that peptide P34 partitions preferentially to PEG-rich phase and extraction with ammonium sulfate [(NH₄)₂SO₄], yielding a 75% recovery of the antimicrobial activity, specific activity of 1,530 antimicrobial units per mg of protein, and purification fold of 2.48. Protein partition coefficient and partition coefficient for the biological activity with (NH₄)₂SO₄ system were 0.48 and 64, respectively. Addition of sodium chloride did not affect recovery, but decreased protein amount in the PEG-rich phase, indicating a higher partition of biomolecules. ABMS did not yield good recovery of antimicrobial activity. Purification fold using PEG–(NH₄)₂SO₄ and 1.0?mol l^?1 sodium chloride was twice higher than that obtained by conventional protocol, indicating a successful utilization of ABS as a step for purification of peptide P34.     

Conformation of the C-terminal pentapeptide—the ‘message sequence’—of tachykinins as determined by consensus dynamic

The tachykinins are a family of gastrointestinal peptides comprising eight members: substance P, neurokinin A, neurokinin B, eledoisin, physalemin, uperolein, kassinin and phyllomedusin. Consensus dynamics was carried out on an ensemble of seven tachykinins to determine the binding conformation of the common C-terminal fragment: Phe-X-Gly-Leu-Met-NH ₂ ,the ’message sequence’ of tachykinins. Three binding modes for the C-terminal pentapeptide were determined. The first binding conformation is folded due to an intramolecular H-bond between the NH of the variable residue (X) and CO of Met. Other features include γ-bends at both the variable amino acid (X) and at Gly. The global minimum of the simulation has this conformation for the C-terminal pentapeptide. The other two binding modes have slightly higher energies. The second is chiefly characterized by a β-turn around the segment X-Gly-Leu-Met, with additional β-bends at the variable amino acid (X) and Met. The final binding conformation is composed of β-bends around the variable amino acid (X) and Leu, and a ’pseudo’ γ-bend at the terminal Met. This paper was presented at the MBU Silver Jubilee Symposium on Structural Biology and 24th Annual Meeting of the Indian Biophysical Society held at Indian Institute of Science, Bangalore, Dec. 9–12, 1996.     

Differentiation of subnucleus-sized oligomers and nucleation-competent assemblies of the Aβ peptide

A significant feature of Alzheimer’s disease is the formation of amyloid deposits in the brain consisting mainly of misfolded derivatives of proteolytic cleavage products of the amyloid precursor protein amyloid-β (Aβ) peptide. While high-resolution structures already exist for both the monomer and the amyloid fibril of the Aβ peptide, the mechanism of amyloid formation itself still defies precise characterization. In this study, low and high molecular weight oligomers (LMWOs and HMWOs) were identified by sedimentation velocity analysis, and for the first time, the temporal evolution of oligomer size distributions was correlated with the kinetics of amyloid formation as determined by thioflavin T-binding studies. LMWOs of subnucleus size contain fewer than seven monomer units and exist alongside a heterogeneous group of HMWOs with 20–160 monomer units that represent potential centers of nucleus formation due to high local monomer concentrations. These HMWOs already have slightly increased β-strand content and appear structurally similar regardless of size, as shown by examination with a range of fluorescent dyes. Once fibril nuclei are formed, the monomer concentration begins to decrease, followed by a decrease in oligomer concentration, starting with LMWOs, which are the least stable species. The observed behavior classifies the two LMWOs as off pathway. In contrast, we consider HMWOs to be on-pathway, prefibrillar intermediates, representing structures in which nucleated conformational conversion is facilitated by high local concentrations. Aβ40 and Aβ42 M35^ox take much longer to form nuclei and enter the growth phase than Aβ42 under identical reaction conditions, presumably because both the size and the concentration of HMWOs formed are much smaller.