Standard dimensions forcis N-methyl peptide unit and flexibility ofcis peptide units

The mean dimensions of thecis N-methyl peptide unit have been arrived at by analysing the crystal structure data on compounds containing such units. These dimensions can be used as standard in conformational studies on cyclic peptides. While the bonds meeting at C are almost coplanar, those meeting at N show a slight pyramidal disposition. A comparison of the dimensions of the normal and N-methylatedcis peptide units show that there are perceptible differences in the parameters connected with N. In addition, the flexibility of thecis peptide unit has been analysed by studying the distribution of the parameters in different classes of compounds such as cyclic di, tri and higher peptides. The salient features are: (i) The angle C^αCN in cyclic dipeptide and the angle C^δNC^α in higher peptides tend to be lower, when the peptide unit is associated with a prolyl residue; (ii) in cyclic tripeptides the internal anglesviz., C^αCN and CNC^α are significantly larger thereby increasing the intra-annular space; (iii) the bond C^α-C is distinctly shorter when it occurs in cyclic dipeptides. The results lead to the conclusion that thecis peptide unit takes up aneed-based flexibility in its dimension.     

Conformation of the peptide antibiotic – histatin 8 in aqueous and non aqueous media

Summary Histatin 8 (Lys¹-Phe-His-Glu-Lys⁵-His-His-Ser-His-Arg¹⁰-Gly-Tyr¹²) belongs to a group of related neutral and basic histidine rich peptides present in human salivary secretions that possess fungicidal and bactericidal activities. The conformation of this peptide has been examined by¹H and¹³C 2D-NMR in DMSO-d₆, water (pH 4.0) and 40% HFA solutions. MD simulations incorporating NMR data was used to generate the solution conformations. The structures were refined byMARDIGRAS employing theRANDMARDI approach. In both DMSO-d₆ and water, the peptide is seen to adopt a β-pleated sheet, while HFA induces an α-helix structure. The role of these structures in its mechanism of action has been explained. This article was presented at the Astra-Zeneca International Symposium on Progress in Drug Discovery and Development Sciences, Bangalore, India, 17–19 January 2001.     

Physiology and biochemistry of theAmaranthus cytokinin bioassay and its applications

The influence of some substances on the amaranthin biosynthesis inAmaranthus-seedlings was investigated with regard to the accuracy and sensitivity of theAmaranthus cytokinin bioassay. 1) The herbicide glyphosate inhibited plgment formation in a concentration of 10^-4 mol 1^-1 in most cases only slightly (approximately 10–25 %). 2) Shikimic acid does not seem to be a precursor, in many experiments it was slightly inhibitory. 3) Jasmonic acid promoted slightly the plgment formation with a distinct maximum at about 5 x 10^-4 mol 1^1-. 4) The oxidation product of zeatin (with KMnO₄) is not active in theAmaranthus bioassay; only weak activity was recorded at very high concentrations. 5) Glucosides, extracted fromGinkgo biloba leaves, stimulate amaranthin synthesis, as we could conclude from applications of this bioassay to senescing, abscisingGinkgo leaves. Presented at the International Symposium “Plant Growth Regulators” held on June 18–22, 1984 at Liblice, Czechoslovakia.     

A direct comparison of the performance of the seaweed biofilters, <Emphasis Type="Italic">Asparagopsis armata</Emphasis> and <Emphasis Type="Italic">Ulva rigida</Emphasis>

The tetrasporophyte of Asparagopsis armata has been previously established as a novel seaweed biofilter for integrated land-based mariculture. The species growth and biofiltration rates were much higher than the values described in the literature for Ulva spp., the most common seaweed biofilter. However, a validation of the advantage of one species over the other requires a study of the performances of these two species in the same system at the same time. In this work, we compared the biofiltration performance and biomass yield of A. armata and Ulva rigida cultivated in the effluents of a fish farm in southern Portugal. Comparisons were performed at different water renewal rates and in two seasons of the year. The maximum total ammonia nitrogen (TAN) removal rates were similar for both species in December (2.7 and 2.8 g TAN m^–2 day^–1 for U. rigida and A. armata, respectively) and higher for A. armata (6.5 g TAN m^–2 day^–1) than for U. rigida (5.1 g TAN m^–2 day^–1) in May. Higher differences were observed when estimating the nitrogen biofiltration through the organic nitrogen yield (N yield) of the biomass produced, particularly in May. This estimate is directly related with the biomass yield and the N content in the tissue which were always higher for A. armata than for U. rigida. In December, the maximum biomass yields were 71 g dry weight (DW) m^–2 day^–1 for A. armata and 44 g DW m^–2 day^–1 for U. rigida, while in May, the yield of A. armata was 125 g DW m^–2 day^–1 and of U. rigida was 73 g DW m^–2 day^–1. This study confirmed that A. armata is indeed a more efficient biofilter than U. rigida. To the best of our knowledge, the production rates reported here are the highest ever reported for macroalgae cultivated in tanks.     

Melanoma targeting with α-melanocyte stimulating hormone analogs labeled with fac-[99mTc(CO)3]+: effect of cyclization on tumor-seeking properties

Early detection of primary melanoma tumors is essential because there is no effective treatment for metastatic melanoma. Several linear and cyclic radiolabeled α-melanocyte stimulating hormone (α-MSH) analogs have been proposed to target the melanocortin type 1 receptor (MC1R) overexpressed in melanoma. The compact structure of a rhenium-cyclized α-MSH analog (Re-CCMSH) significantly enhanced its in vivo tumor uptake and retention. Melanotan II (MT-II), a cyclic lactam analog of α-MSH (Ac-Nle-cyclo[Asp-His-dPhe-Arg-Trp-Lys]-NH₂]), is a very potent and stable agonist peptide largely used in the characterization of melanocortin receptors. Taking advantage of the superior biological features associated with the MT-II cyclic peptide, we assessed the effect of lactam-based cyclization on the tumor-seeking properties of α-MSH analogs by comparing the pharmacokinetics profile of the ^99mTc-labeled cyclic peptide βAla-Nle-cyclo[Asp-His-d-Phe-Arg-Trp-Lys]-NH₂ with that of the linear analog βAla-Nle-Asp-His-dPhe-Arg-Trp-Lys-NH₂ in melanoma-bearing mice. We have synthesized and coupled the linear and cyclic peptides to a bifunctional chelator containing a pyrazolyl-diamine backbone (pz) through the amino group of βAla, and the resulting pz–peptide conjugates were reacted with the fac-[^99mTc(CO)₃]⁺ moiety. The ^99mTc(CO)₃-labeled conjugates were obtained in high yield, high specific activity, and high radiochemical purity. The cyclic ^99mTc(CO)₃-labeled conjugate presents a remarkable internalization (87.1% of receptor-bound tracer and 50.5% of total applied activity, after 6 h at 37 °C) and cellular retention (only 24.7% released from the cells after 5 h) in murine melanoma B16F1 cells. A significant tumor uptake and retention was obtained in melanoma-bearing C57BL6 mice for the cyclic radioconjugate [9.26 ± 0.83 and 11.31 ± 1.83% ID/g at 1 and 4 h after injection, respectively]. The linear ^99mTc(CO)₃-pz–peptide presented lower values for both cellular internalization and tumor uptake. Receptor blocking studies with the potent (Nle⁴,dPhe⁷)-αMSH agonist demonstrated the specificity of the radioconjugates to MC1R (74.8 and 44.5% reduction of tumor uptake at 4 h after injection for cyclic and linear radioconjugates, respectively).     

Production of phosphohydrolases by Nicotiana tabacum 1507 cell suspension culture

The time course of growth, biosynthesis and secretion of different phosphohydrolytic activities by Nicotiana tabacum 1507 cell suspension culture were investigated. It was established that the cell culture under study biosynthesised large amounts of phosphohydrolytic activities during the linear phase of growth for a relatively short period (between the 2nd and 5th days of cultivation). The highest enzyme activity was determined with bis-pNPP which is non-specific substrate for phosphodiesterases and for some nucleases (116.10³ U/L at pH 5.7 and 51.10³ U/L at pH 8.0). The different phosphohydrolytic activities were distingnished using specific substrates at pH 5.7 (20.10³ U/L – 5′-phosphodiesterases, 18,8.10³ U/L – 3′-phosphodiesterases and 15,5.10³ U/L – phosphomonoesterases) and at pH 8.0 (10,2.10³ U/L – 5′-phosphodiesterases, 9,5.10³ U/L – 3′-phosphodiesterases and 6,4.10³ U/L – phosphomonoesterases). This revised version was published online in June 2006 with corrections to the Cover Date.     

Inward and outward rectifying potassium currents inSaccharomyces cerevisiae mediated by endogenous and heterelogously expressed ion channels

Disruption of genes encoding endogenous transport proteins inSaccharomyces cerevisiae has facilitated the recent cloning, by functional expression, of cDNAs encoding K⁺ channels and amino acid transporters from the plantArabidopsis thaliana [1–4]. In the present study, we demonstrate in whole-cell patch clamp experiments that the inability oftrk1Δtrk2Δ mutants ofS. cerevisiae to grow on submillimolar K⁺ correlates with the lack of K⁺ inward currents, which are present in wild-type cells, and that transformation of thetrk1Δtrk2Δ double-deletion mutant withKAT1 fromArabidopsis thaliana restores this phenotype by encoding a plasma membrane protein that allows large K⁺ inward currents. Similar K⁺ inward currents are induced by transformation of atrk1 mutant withAKT1 fromA. thaliana. This work was supported by a grant from theForschungsgemeinschaft (A.B.), TheU.S. Department of Energy (c.L.S.), The U.S. National Science Foundation (R.F.G.) Lisboa, Portugal.     

Fusarium toxins in bread cereals of the land brandenburg — Comparison of integrated/ecological cultivation of 2000 to 2002

Over a period of three years (2000–2002), wheat and rye samples of integrated and ecological cultivation in the land Brandenburg were analyzed by HPLC for theFusarium toxins deoxynivalenol and zearalenone. In the years 2000 and 2001, the contamination generally was lower than in the year 2002. In the 3 years of testing, the contamination byFusarium toxins was significantly less frequent and lower in cereals of ecological cultivation than in cereals of integrated cultivation. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Synthesis of cyclic α-MSH peptides

Summary Cyclic lactam analogs of α-melanocyte stimulating hormone (α-MSH) have been shown to be potent agonists in the frog skin bioassay [Al-Obeidi, F. et al., J. Med. Chem., 32 (1989) 2555], demonstrating melanocortin-1 (MC1) receptor activity. We synthesized cyclic α-MSH(1–13) and α-MSH(4–10) lactam analogs. The peptides were synthesized using Fmoc chemistry. We improved the cyclization procedure: side chains of Asp⁵ and Lys¹⁰ from the deprotected peptide were coupled in DMF to form a cyclic lactam, using an excess of PyBOP reagent and DIEA as a base. The cyclization reaction was completed within 1 h and was almost quantitative. We also synthesized an α-MSH analog cyclized via a disulphide bridge. The peptides were tested for their selectivity for the rat MC4 receptor. Cyclization and substitutions at position 7 dramatically influenced the selectivity for the rMC4 receptor.     

Antimutagenic effects of caffeine during nitrosoguanidine-induced mutagenesis ofSalmonella typhimurium cells and phages

The effect of caffeine on nitrosoguanidine-induced mutagenesis ofSalmonella typhimurium & nd its P22 and L phages was studied. The detected mutations included phage “clear” mutations, reversions of phage “amber” mutation, and prototrophic reversions of thehis ⁻ auxotroph ofSalmonella typhimurium. Neither therecA mutation of the host nor theerf mutation of the phage genome were found to affect the nitrosoguanidine-induced mutagenesis of the phage during vegetative growth. Beginning with a concentration of 0.2 mg/ml, caffeine decreased the frequency of mutants by 30–60%, attaining a maximum effect at 1.5 mg/ml and retaining this effect even at higher concentrations. A similar antimutagenic effeot was observed with the mutagenesis of the host cells. The nitrosoguanidine-induced mutagenesis does not seem to be related to the function of therecA cell gene or theerf phage gene. The mechanism of mutagenesis by nitrosoguanidine probably has two components, one of them caffeine sensitive, the other caffeine-resistant.     

Conformational preferences of modified nucleoside N(2)-methylguanosine (m(2)G) and its derivative N(2), N(2)-dimethylguanosine (m(2)(2)G) occur at 26th position (hinge region) in tRNA

Conformational preferences of the modified nucleosides N²-methylguanosine (m²G) and N², N²-dimethylguanosine (m₂²G) have been studied theoretically by using quantum chemical perturbative configuration interaction with localized orbitals (PCILO) method. Automated complete geometry optimization using semiempirical quantum chemical RM1, along with ab initio molecular orbital Hartree–Fock (HF-SCF), and density functional theory (DFT) calculations has also been made to compare the salient features. Single-point energy calculation studies have been made on various models of m²G26:C/A/U44 and m₂²G26:C/A/U44. The glycosyl torsion angle prefers “syn” (χ = 286°) conformation for m²G and m₂²G molecules. These conformations are stabilized by N(3)–HC2′ and N(3)–HC3′ by replacing weak interaction between O5′–HC(8). The N²-methyl substituent of (m²G26) prefers “proximal” or s-trans conformation. It may also prefer “distal” or s-cis conformation that allows base pairing with A/U44 instead of C at the hinge region. Thus, N²-methyl group of m²G may have energetically two stable s-trans m²G:C/A/U or s-cis m²G:A/U rotamers. This could be because of free rotations around C–N bond. Similarly, N², N²-dimethyl substituent of (m₂²G) prefers “distal” conformation that may allow base pairing with A/U instead of C at 44th position. Such orientations of m²G and m₂²G could play an important role in base-stacking interactions at the hinge region of tRNA during protein biosynthesis process.     

SD-8, a novel therapeutic agent active against multidrug-resistant Gram positive cocci

Anti-bacterial drug resistance is one of the most critical concerns among the scientist worldwide. The novel antimicrobial decapeptide SD-8 is designed and its minimal inhibitory concentration and therapeutic index (TI) was found in the range of 1–8 μg/ml and 45–360, respectively, against major group of Gram positive pathogens (GPP). The peptide was also found to be least hemolytic at a concentration of 180 μg/ml, i.e., nearly 77 times higher than its average effective concentration. The kinetics assay showed that the killing time is 120 min for methicillin-sensitive Staphylococcus aureus (MSSA) and 90 min for methicillin-resistant S. aureus (MRSA). Membrane permeabilization is the cause of peptide antimicrobial activity as shown by the transmission electron microscopy studies. The peptide showed the anti-inflammatory property by inhibiting COX-2 with a K _D and K _i values of 2.36 × 10⁻⁹ and 4.8 × 10⁻⁸ M, respectively. The peptide was also found to be effective in vivo as derived from histopathological observations in a Staphylococcal skin infection rat model with MRSA as causative organism.     

Botulinum Neurotoxin Types A,B, and E: Fragmentations by Autoproteolysis and Other Mechanisms Including by <Emphasis Type="Italic">O</Emphasis>-Phenanthroline–Dithiothreitol,and Association of the Dinucleotides NAD<Superscript>+</Superscript>/NADH with the Heavy Chain of the Three Neurotoxins

The first evidence of autoproteolytic activity of the ~50-kDa light chain of the clostridial neurotoxins (NT) is traceable to the observations that the light chains of botulinum NT serotypes A and E, separated from their ~100-kDa heavy chain conjugate, were found cleaved at the amino side of Tyr250 and Arg244, respectively [DasGupta and Foley (1989). Biochimie 71: 1183–1200]. Specific cleavages of the recombinant light chain of NT type A, including at Tyr249–Tyr250, firmly established that the cleavages reported earlier were due to autoproteolysis [Ahmed et al. (2001). J. Protein Chem. 20: 221–231; Ahmed et al. (2003). Biochemistry 42:12539–12549] and not by contaminating proteases or non-enzymatic. We now report many cleavages in the NT types A, B and E and also in their separated light and heavy chains, and identification of several of the peptide bonds cleaved. None of the identified cleaved bonds (–P₁–P₁′ –) in one serotype (except Asp–Pro) was found common in other serotypes or cleaved within itself at a second site. After separation from the heavy chain self-cleavages of the light chains of type A, B and E at Tyr249–Tyr250, Gln258–Ser259 and Ile243–Arg244, respectively indicate an intriguing feature (in the aligned sequences these bonds of type A and B are 2 and type A and E are 4 peptide bonds apart) that may have some role in the NT’s structure–function relationship yet to be understood. We point out that autoproteolysis of a single peptide bond (Phe418–Thr419 or Phe422–Glu423) in NT type A reported by Ahmed et al. (2001) can potentially generate proteolytically active light chain freed of the heavy chain; this is an efficient pathway, that by-passes nicking by a trypsin-like protease(s) inside the intrachain disulfide bridge and its reductive cleavage. We offer probable explanations for the observed cleavages such as acid- and metal-mediated (non-catalytic and non-stoichiometric) reactions in addition to autoproteolysis but cannot predict which mechanism(s) of cleavage occur or prevail following NT’s entry in the body as poison or therapeutic agent. The metal chelator O-phenanthroline (above critical miceller concentration) in the presence of dithiothreitol cleaved type E NT at limited sites generating discrete 114-, 87-, 49-, 42-, and 31-kDa fragments but degraded NTs type A and B extensively. The limited cleavage of type E NT was dependent on the presence of metal ion(s) bound to the protein and its native (urea sensitive) conformation. The self-cleavage of the NTs at specific sites prompted us to search for specific binding sites on the NTs analogous to SNARE-motifs—the 9-residuelong motifs present on the NT’s natural substrates (SNAP-25, syntaxin, VAMP/synaptobrevin); such putative binding motifs (sites) noted on all clostridial NTs are reported here. Their relationship to the observed autoproteolysis remains to be determined experimentally. The dinucleotide NAD⁺/NADH associated with the NTs type A, B and E (2–3 NADH per protein molecule) via their H-chains, and a portion of the H-chain (toward the C-terminus) appears to exhibit limited amino acid sequence homology with lactate dehydrogenase—a representative NAD⁺/NADH binding protein.     

Homoserine derivatives for the preparation of base-stable nucleopeptide analogues

Summary Covalently linked peptide-oligonucleotide hybrids are good candidates for antisense or anti-gene therapeutics. The use of homoserine as the linking amino acid allows nucleopeptide analogues with a base-stable amino acid-nucleoside phosphate diester linkage to be obtained. Three N^α, O-protected homoserine derivatives (N ^α-Boc-Hse(DMT)-O⁻ HTEA⁺ (I),N ^α-Fmoc-Hse(MMT)-O⁻Hpyr⁺ (II) andN ^α-Phac-Hse(DMT)-O⁻HTEA⁺(III) were prepared after transient silylation,N ^α-acylation, desilylation and protection of the hydroxyl group. The first can be placed at any position in the peptide sequence, while the other two must be placed at theN-terminus to afford nucleopeptides with the N-terminal amine group free or permanently blocked, respectively.     

Residue Specific Ribose and Nucleobase Dynamics of the cUUCGg RNA Tetraloop Motif by MNMR 13C Relaxation

The dynamics of the nucleobase and the ribose moieties in a 14-nt RNA cUUCGg hairpin-loop uniformly labeled with ¹³C and ¹⁵N were studied by ¹³C spin relaxation experiments. R₁, R_1ρ and the ¹³C-{¹H} steady-state NOE of C₆ and C_1′ in pyrimidine and C₈ and C_1′ in purine residues were obtained at 298 K. The relaxation data were analyzed by the model-free formalism to yield dynamic information on timescales of pico-, nano- and milli-seconds. An axially symmetric diffusion tensor with an overall rotational correlation time τ_c of 2.31±0.13 ns and an axial ratio of 1.35±0.02 were determined. Both findings are in agreement with hydrodynamic calculations. For the nucleobase carbons, the validity of different reported ¹³C chemical shift anisotropy values (Stueber, D. and Grant, D. M., 2002 J. Am. Chem. Soc. 124, 10539–10551; Fiala et al., 2000 J. Biomol. NMR 16, 291–302; Sitkoff, D. and Case, D. A., 1998 Prog. NMR Spectroscopy 32, 165–190) is discussed. The resulting dynamics are in agreement with the structural features of the cUUCGg motif in that all residues are mostly rigid (0.82 < S² < 0.96) in both the nucleobase and the ribose moiety except for the nucleobase of U7, which is protruding into solution (S² = 0.76). In general, ribose mobility follows nucleobase dynamics, but is less pronounced. Nucleobase dynamics resulting from the analysis of ¹³C relaxation rates were found to be in agreement with ¹⁵N relaxation data derived dynamic information (Akke et al., 1997 RNA 3, 702–709). Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Gene cloning and expression of a new acidic family 7 endo-β-1,3-1,4-glucanase from the acidophilic fungus Bispora sp. MEY-1

Most reported microbial β-1,3-1,4-glucanases belong to the glycoside hydrolase family 16. Here, we report a new acidic family 7 endo-β-1,3-1,4-glucanase (Bgl7A) from the acidophilic fungus Bispora sp. MEY-1. The cDNA of Bgl7A was isolated and over-expressed in Pichia pastoris, with a yield of about 1,000 U ml^–1 in a 3.7-l fermentor. The purified recombinant Bgl7A had three activity peaks at pH 1.5, 3.5, and 5.0 (maximum), respectively, and a temperature optimum at 60°C. The enzyme was stable at pH 1.0–8.0 and highly resistant to both pepsin and trypsin. Belonging to the group of non-specific endoglucanase, Bgl7A can hydrolyze not only β-glucan and cellulose but also laminarin and oat spelt xylan. The specific activity of Bgl7A against barley β-glucan and lichenan (4,040 and 2,740 U mg^–1) was higher than toward carboxymethyl cellulose sodium (395 U mg^–1), which was different from other family 7 endo-β-glucanases.     

Acidification of Soil in a Dry Land Winter Wheat-sorghum/corn-fallow Rotation in the Semiarid U.S. Great Plains

Soil pH is decreasing in many soils in the semiarid Great Plains of the United States under dry land no-till (NT) cropping systems. This study was conducted to determine the rate of acidification and the causes of the acidification of a soil cropped to a winter wheat (Triticum aestivum L.)-grain sorghum [Sorghum bicolor (L.) Moench]/corn (Zea mays L.)-fallow rotation (W-S/C-F) under NT. The study was conducted from 1989 to 2003 on soil with a long-term history of either continuous NT management [NT(LT)] (1962–2003) or conventional tillage (CT) (1962–1988) then converted to NT [NT(C)] (1989–2003). Nitrogen was applied as ammonium nitrate (AN) at a rate of 23 kg N ha⁻¹ in 1989 and as urea ammonium nitrate (UAN) at an average annual rate of 50 kg N ha⁻¹ from 1990 to 2003 for both NT treatments. Soil samples were collected at depth increments of 0–5, 5–10, 10–15, and 15–30 cm in the spring of 1989 and 2003. Acidification rates for the NT(LT) and NT(C) treatments were 1.13 and 1.48 kmol H⁺ ha⁻¹ yr⁻¹ in the 0–30 cm depth, respectively. The amount of CaCO₃ needed to neutralize the acidification is 57 and 74 kg ha⁻¹ yr⁻¹ for the NT(LT) and NT(C) treatments, respectively. A proton budget estimated by the Helyar and Porter [1989, Soil Acidity and Plant Growth, Academic Press] method indicated that NO₃⁻ leaching from the 30 cm depth was a primary cause of long-term acidification in this soil. Nitrate leaching accounted for 59 and 66% of the H⁺ from the acid causing factors for NT(LT) and NT(C) treatments, respectively. The addition of crop residues to the soil neutralized 62 and 47% of the acidity produced from the leaching of NO₃⁻, and 37 and 31% of the acid resulting from NO₃⁻ leaching and the other acid-causing constituents for the NT(LT) and NT(C) treatments, respectively. These results document that surface soils in dry land W-S/C-F rotations under NT are acidifying under current management practices. Improved management to increase nitrogen uptake efficiency from applied fertilizer would help reduce the rate of acidification. The addition of lime materials to prevent negative impacts on grain yields may be necessary in the future under current management practices. A contribution of the university of Nebraska Agricultural Research Division, Lincoln, NE 68583. Journal series No. 15120     

Chemical and physical-chemical characteristics of dietary fibres from Ulva lactuca (L.) Thuret and Enteromorpha compressa (L.) Grev.

Dietary fibres from Ulva lactuca (L.) Thuret (sea lettuce) and Enteromorpha compressa (L.) Grev. (A.O. nori) were measured according to a ‘standard’ method and a ‘physiological’ protocol simulating the gastric and intestinal environments. U. lactuca contained 15.8–8.0% soluble and 24.2–32.6% insoluble fibres according to the ‘standard’ and ‘physiological’ methods, respectively. For E. compressa, these values were 14.9–15.9 and 21.6–28.7%, respectively. For both algae, the composition suggests that the soluble fibres were xylorhamnoglycuronans sulphates and insoluble fibres were essentially composed of glucans. No marked chemical compositional variation was observed between soluble fractions extracted under the simulated gastric and intestinal conditions. Fibres in both algae are hydrophilic but the water holding capacities were higher after extraction of soluble fibres (5.5–9.5 g g⁻¹ for the dry algae; 14.0–16.0 g g⁻¹ for the standard insoluble fibres). Water soluble fibres demonstrated low intrinsic viscosities at 37 °C in buffers, particularly those from E. compressa (36.0–36.5 ml g⁻¹), and was affected by pH for those of U. lactuca (147.5 ml g⁻¹ at pH 3.0 and 175.0 ml g⁻¹ at pH 7.3).     

Membrane-associated echinocandin B deacylase of Actinoplanes utahensis: purification, characterization, heterologous cloning and enzymatic deacylation reaction

Aspergillus nidulans produces echinocandin B, a neutral lipopeptide. A deacylase from Actinoplanes utahensis catalyzes cleavage of the linoleoyl group from echinocandin B, a key step in generating a potential antifungal agent. Virtually all (99.8%) deacylase activity was cell-associated. The deacylase was salt-solubilized, heat-treated and purified to apparent homogeneity by a 3-step chromatographic procedure. The enzyme was a heterodimer consisting of 63- and 18-to-20-kDa subunit, optimally active at pH 6.0, and at 60°C with salt. The K _m of the deacylase for echinocandin B was 50 μM and its V _max was 14.6 μmol cyclic hexapeptide min⁻¹ mg⁻¹protein. The substrate specificity of the enzyme was broad with respect to both acyl and cyclic peptide analogues of echinocandin B. The two deacylase subunit genes were cloned and over-expressed in Streptomyces lividans. The recombinant deacylase was purified from the culture filtrate to apparent homogeneity by a 1-step chromatographic procedure. Using the recombinant deacylase, an enzymatic deacylation of immobilized echinocandin B resulted in the generation of cyclic hexapeptide at gram-level. Journal of Industrial Microbiology & Biotechnology (2000) 24, 173–180. Received 24 August 1999/ Accepted in revised form 23 November 1999