The β-fructofuranosidase activities of a strain of Clostridium acetobutylicum grown on inulin

Summary The -fructofuranosidase activities of a strain of Clostridium acetobutylicum, selected for its capacity to grow on inulinic substrates, were investigated. When grown on inulin, this strain produced extracellular and intracellular -fructofuranosidases, both of which hydrolysed inulin (inulinase activity) and sucrose (invertase activity). Inulinase activity was higher than invertase activity in the extracellular preparation, the opposite being observed for the cellular preparation. The effects of pH and temperature, substrate specificity and the kinetic constants for inulin and sucrose were studied on both preparations, as well as induction by inulin and repression by glucose and fructose of inulinase and invertase activities. The overall results were consistent with the existence of a least one inulinase, (EC 3.2.1.7), mainly but not entirely released in the extracellular medium, and an invertase (3.2.1.26) localized within the cell.Time course hydrolysis experiments of dalhia inulin and Jerusalem artichoke inulofructans by extracellular inulinase showed that this preparation had a remarkably high specificity for hydrolysis of long chain inulofructans.     

Influence of sugars and light on rhizosecretion of α-glucosidase and acid phosphatase during micropropagation of potato

The effects of carbohydrate supply and light on rhizosecretion during micropropagation of potato plantlets (Solanum tuberosum L., cv. ‘Iwa’) in liquid medium were investigated. Soluble protein content was higher in the spent medium for plantlets grown under light conditions than in the dark. For those plantlets grown under light conditions and on different sugar-supplemented media, they rhizosecreted the highest amount of soluble protein when grown in the presence of maltose, while they rhizosecreted the lowest amount of soluble protein when grown on medium containing glucose. Moreover, plantlets grown under light and on a medium containing sucrose were the most vigorous, and exhibited the highest levels of rhizosecreted acid phosphatase activity. However, there was no direct relationship between plantlet growth and rhizosecretion. When plantlets were grown in the dark and on medium containing maltose, a higher α-glucosidase activity was detected than those grown on medium containing sucrose. These results suggested that rhizosecretion of certain proteins from plantlets grown in vitro might not require exposure to light conditions.     

Simultaneous production of high activities of thermostable endoglucanase and β-glucosidase by the wild thermophilic fungus Thermoascus aurantiacus

The culture-medium composition was optimised, on a shake-flask scale, for simultaneous production of high activities of endoglucanase and β-glucosidase by Thermoascus aurantiacus using statistical factorial designs. The optimised medium containing 40.2 g l⁻¹ Solka Floc as the carbon source and 9 g l⁻¹ soymeal as the organic nitrogen source yielded 1130 nkat ml⁻¹ endoglucanase and 116 nkat ml⁻¹β-glucosidase activities after 264 h as shake cultures. In addition, good levels of β-xylanase (3479 nkat ml⁻¹) and low levels of filter-paper cellulase, β-xylosidase, α-l-arabinofuranosidase, β-mannanase, β-mannosidase, α-galactosidase and β-galactosidase were detected. Batch fermentation in a 5-l laboratory fermentor using the optimised medium allowed the production of 940 nkat ml⁻¹ endoglucanase and 102 nkat ml⁻¹β-glucosidase in 192 h. Endoglucanase and β-glucosidase showed optimum activity at pH 4.5 and pH 5, respectively, and they displayed optimum activity at 75 °C. Endoglucanase and β-glucosidase showed good stability at pH values 4–8 and 4–7, respectively, after a prolonged incubation (48 h at 50 °C). Endoglucanase had half-lives of 98 h at 70 °C and 4.1 h at 75 °C, while β-glucosidase had half-lives of 23.5 h at 70 °C and 1.7 h at 75 °C. Alkali-treated bagasse, steam-treated wheat straw, Solka floc and Sigmacell 50 were 66, 48.5, 33.5 and 14.4% hydrolysed by a crude enzyme complex of T. aurantiacus in 50 h. Received: 12 November 1999 / Accepted: 14 November 1999     

Influence of β-endorphin on the proliferative and phagocytic activities of peripheral blood cells of men and women from different age groups

It has been found that β-endorphin modulation of lymphocyte proliferative activity in male donors is mainly observed at a relatively young age (in groups aged 20–29 and 30–39 years), it gradually becomes lower with age, and disappears in donors at aged 50–60 years. At the same time, women have a prolonged modulating effect of peptide on proliferation. In women aged 50–59 years, the peptide has a marked promotional effect on spontaneous proliferation at concentrations of 10^?7, 10^?8, and 10^?10 M induced by a suboptimal concentration of phytohemagglutinin (PHA) at 10^?10 M, while in women aged 30–39 years, β-endorphin suppresses PHA-induced proliferative response. In men aged 20–29 years, β-endorphin stimulates the uptake capacity of neutrophils, whereas in those aged 50–59 years, this capacity is suppressed by β-endorphin. In female donors from any age groups, β-endorphin was not found to influence the activity of neurophils.     

Influence of proline and β-turn mimetics on the cyclization of penta- and hexapeptides

Summary Proline and Pro-derived peptidomimetics, such as meoxPro-Oic (4-methoxy-proline-octahydro indolic acid), and DBF (2-aminoethyl-6-dibenzofuran propionic acid) were introduced into thymopentin-derived penta-[SP5-] and hexa-[SP6-] peptides and penta-, hexa- and hepta-alanine. Surprisingly, we found that cyclomonomer formation in the investigated penta- and hexapeptides was drastically hindered by the presence of proline regardless of position.     

Simultaneous production of endoglucanase and β-glucosidase using synthetic two cistron genes

Summary Simultaneous production of endoglucanase and -glucosidase by using a synthetic two cistron system inEscherichia coli was attempted as a possible way of reducing production cost. The first cistron in this system we constructed is an endoglucanase gene fused to a tac promoter that provides for efficient expression. The second cistron is a -glucosidase structural gene. A ribosome binding site sequence of 33-base was inserted between the two cistron genes.E. coli cells transformed with the system produced 12.4 units/mg protein of endoglucanase and 327 units/mg protein of -glucosidase, which represent 15% and 22% of total cellular protein, respectively, in L medium within three hours after induction with IPTG.     

Influence of Oil Type on Characteristics of β-Sitosterol and Stearic Acid Based Oleogel

Oleogels were prepared from extra virgin olive oil, corn oil, sunflower oil, and flaxseed oil with a mixture of β-sitosterol and stearic acid (Sit1:SA4, w/w) at concentrations of 15 and 20 g/100 g oil. The prepared oleogels were characterized by different methods to study the influence of oil type on the oleogel properties. The oil type influenced the colour and appearance of the oleogel. The flaxseed oil based oleogel showed lower oil loss and higher firmness than those of other oils based oleogels. The increase of gelator mixture from 15 to 20 g/100 g oil reduced the oil loss and improved the firmness of oleogel samples. The microscopy and small-angle x-ray scattering analyses showed different microstructures and crystallographic reflections for oleogels prepared from different oil types. Also, the oil type and concentration of gelator mixture influenced the melting and crystallization enthalpies of oleogel. Furthermore, different oils based oleogels showed varying values of viscosity, storage modulus (G’), and loss modulus (G”). Therefore, it can be concluded that the oil type and concentration of gelator influence the functional properties of oleogel and the flaxseed oil resulted in oleogel with good properties compared with other oils used in the study.     

Influence of truncation of avian β-defensin-4 on biological activity and peptide-membrane interaction

Defensins are important components in host defense systems. The therapeutic use of β-defensins is limited by their innate toxicity and high cost due to the size and complex disulfide pairing. In this study, we used linear avian β- defensin-4 (RL38) without disulfide bonds as model peptide to derive two peptides by the truncation. GL23 is the C-terminal truncated sequence of RL38, and GLI23 is the derivative of GL23 by the replacement of cysteines with isoleucines. Results showed that these peptides exhibited strong antibacterial activity against gram-negative and gram-positive bacteria. An exception was that GL23 showed weak antimicrobial activity against gallinaceous pathogenic bacteria Salmonella Pullorum C79-13. Two truncated peptides GL23 and GLI23 displayed no or weak hemolysis, which was in accordance with little blue shifts of the peptides in the presence of synthetic eukaryotic membranes. CD spectroscopy demonstrated that these peptides appeared to be unfolded in aqueous solution but acquire structure in the presence of membrane- mimicking phospholipids. GLI23 kept the antibacterial activity at high concentrations of NaCl or low concentration of divalent cations (Mg2+ and Ca2+). The peptides preferentially bound to negatively charged phospholipids over zwitterionic phospholipids, which led to greater cell selectivity. The outer and inner membranes assay displayed that GLI23 killed bacteria by targeting the cell membrane. These results suggest the peptides derived by truncation of linear β-defensins may be a promising candidate for future antibacterial agent.     

Influence of lactose and lactate on growth and β-galactosidase activity of potential probiotic Propionibacterium acidipropionici

Dairy propionibacteria are microorganisms of interest for their role as starters in cheese technology and as well as their functions as probiotics. Previous studies have demonstrated that Propionibacterium acidipropionici metabolize lactose by a β-galactosidase that resists the gastrointestinal transit and the manufacture of a Swiss-type cheese, so that could be considered for their inclusion in a probiotic product assigned to intolerant individuals. In the present work we studied the effect of the sequential addition of lactose and lactate as first or second energy sources on the growth and β-galactosidase activity of P. acidipropionici Q4. The highest β-galactosidase activity was observed in a medium containing only lactate whereas higher final biomass was obtained in a medium with lactose. When lactate was used by this strain as a second energy source, a marked increase of the intracellular pyruvate level was observed, followed by lactate consumption and increase of specific β-galactosidase activity whereas lactose consumption became negligible. On the contrary, when lactose was provided as second energy source, lactic acid stopped to be metabolized, a decrease of the intracellular pyruvate concentration was observed and β-galactosidase activity sharply returned to a value that resembled the observed during the growth on lactose alone. Results suggest that the relative concentration of each substrate in the culture medium and the intracellular pyruvate level were decisive for both the choice of the energetic substrate and the β-galactosidase activity in propionibacteria. This information should be useful to decide the most appropriate vehicle to deliver propionibacteria to the host in order to obtain the highest β-galactosidase activity.     

Simultaneous expression of genes encoding endoglucanase and β-glucosidase in Zymomonas mobilis

Summary As a step towards constructing strains of Z. mobilis capable of converting cellulose to ethanol, DNA fragments encoding endoglucanase (from Xanthomonas albilineans) and -glucosidase (from either X.albilineans or Pseudomonas sp.) were linked on the same vector and transferred to Z. mobilis. All clones expressed endoglucanase. -Glucosidase was only produced by clones containing the Xanthomonas gene, and when two copies of this gene were present the -glucosidase activity was higher.     

Influence of cultivation and induction conditions on β-lactamase production in Acinetobacter calcoaceticus

Summary The formation and localization of the -lactamase of Acinetobacter calcoaceticus CCM 5593 is strongly affected by cultivation and induction conditions. Optimal parameters for enzyme yield are cultivation on minimal salts medium with acetate (10 g·1^–1) as carbon source and addition of yeast extract (5–10 g·l^–1), induction by cefotaxime (50g·ml^–1) immediately after inoculation and growth for 24 h at 25° C. The strain forms a basal level of -lactamase constitutively [70 units (U)·g^–1]. Nearly all of this was found to be cell-bound. However, -lactamase activity additionally produced after induction (up to 500 U·g^–1 wet bacteria) was located in the culture medium (up to 96%). This unusual localization is a special feature of A. calcoaceticus and is not attributed to cell lysis. Offprint requests to: P. Borneleit     

Specificity of cellulase and β-xylanase induction in Trichoderma reesei QM 9414

Cellulose- and xylan-degrading enzymes of Trichoderma reesei QM 9414 induced by, sophorose, xylobiose, cellulose and xylan were analyzed by isoelectric focusing. The sophorose-induced enzyme system contained two types of endo-1,4--glucanases (EC 3.2.1.4), one specific for cellulose and the other non-specific, hydrolyzing both cellulose and xylan, and exo-1,4--glucanases (cellobiohydrolases I, EC 3.2.1.91), i.e. all types of glucanases that are produced during growth on cellulose. Specific endo-1,4--xylanases (EC 3.2.1.8) present in the cellulose-containing medium were less abundant in the sophorose-induced enzyme system. Xylobiose and xylan induced only specific endo-1,4--xylanases. It is concluded that syntheses of cellulases and -xylanases in T. reesei QM 9414 are under separate control and that the non-specific endo-1,4--glucanases are constituents of the cellulose-degrading enzyme system.     

Influence of amylose,amylopectin and pullulan on β-glucuronidase activity of starch-degrading Escherichia coli

Synthesis of -glucuronidase in starch-degrading Escherichia coli (S1) was induced by amylose, amylopectin and pullulan supplied in mineral medium as the sole carbon source (1%, w/v). The maximum activity occurred after 4 days when cultures reached the stationary phase of growth, but induction was also evident during log-phase. The effects obtained with amylose, amylopectin and pullulan were higher than that obtained with maize starch.     

Influence of β-alanine on ultrastructure,tanning, and melanization of Drosophila melanogaster cuticles

In Drosophila melanogaster, the chitinous microfibrils arising from the tips of the epidermal villi in adult cuticles remain irregular and loose in the mutant ebony (which fails in cuticular incorporation of -alanine) but closely knit and regular in normal flies. Addition of -alanine to cuticles from which nonchitinous materials have been removed with alkali converts the loose arrangement of the microfibrils to a compact and sharply delineated arrangement. -alanine also accelerates tyrosinase-catalyzed oxidation of N-acetyldopamine by reacting with the oxidized product of the reaction to produce an orange-red complex. Similarly, -alanine accelerates oxidation of N-acetyldopamine when these two substances are added to fluids from the hemocoel, to lead to tanning instead of normal blackening. These findings may help explain why -alanine induces tanning while inhibiting melanization in insects.This study was supported by Grant GM-18680 from the National Institutes of Health.     

Production of cellulase and β-glucosidase activities following growth of Streptomyces hygroscopicus on cellulose containing media

The actinomycete, Streptomyces hygroscopicus was shown to be capable of producing extracellular cellulase and cell associated -glucosidase activity during growth on cellulose containing media. Cell homogenates were shown to contain a -glucosidase fraction which was stable for up to 24h. at 30°C and had half-lives of 480min. and 220min. at 40 and 50°C, respectively. The enzyme fraction was also shown to be optimally active at pH 4.0 suggesting that it might represent a suitable supplement for fungal cellulase systems, deficient in -glucosidase activity.     

Phylogenetic variation in glycosidases and glycanases acting on plant cell wall polysaccharides, and the detection of transglycosidase and trans-β-xylanase activities

Wall polysaccharide chemistry varies phylogenetically, suggesting a need for variation in wall enzymes. Although plants possess the genes for numerous putative enzymes acting on wall carbohydrates, the activities of the encoded proteins often remain conjectural. To explore phylogenetic differences in demonstrable enzyme activities, we extracted proteins from 57 rapidly growing plant organs with three extractants, and assayed their ability to act on six oligosaccharides ‘modelling’ selected cell‐wall polysaccharides. Based on reaction products, we successfully distinguished exo‐ and endo‐hydrolases and found high taxonomic variation in all hydrolases screened: β‐d ‐xylosidase, endo‐(1→4)‐β‐d ‐xylanase, β‐d ‐mannosidase, endo‐(1→4)‐β‐d ‐mannanase, α‐d ‐xylosidase, β‐d ‐galactosidase, α‐l ‐arabinosidase and α‐l ‐fucosidase. The results, as GHATAbase, a searchable compendium in Excel format, also provide a compilation for selecting rich sources of enzymes acting on wall carbohydrates. Four of the hydrolases were accompanied, sometimes exceeded, by transglycosylase activities, generating products larger than the substrate. For example, during β‐xylosidase assays on (1→4)‐β‐d ‐xylohexaose (Xyl₆), Marchantia, Selaginella and Equisetum extracts gave negligible free xylose but approximately equimolar Xyl₅ and Xyl₇, indicating trans‐β‐xylosidase activity, also found in onion, cereals, legumes and rape. The yield of Xyl₉ often exceeded that of Xyl_7–8, indicating that β‐xylanase was accompanied by an endotransglycosylase activity, here called trans‐β‐xylanase, catalysing the reaction 2Xyl₆→ Xyl₃ + Xyl₉. Similar evidence also revealed trans‐α‐xylosidase, trans‐α‐arabinosidase and trans‐α‐arabinanase activities acting on xyloglucan oligosaccharides and (1→5)‐α‐l ‐arabino‐oligosaccharides. In conclusion, diverse plants differ dramatically in extractable enzymes acting on wall carbohydrate, reflecting differences in wall polysaccharide composition. Besides glycosidase and glycanase activities, five new transglycosylase activities were detected. We propose that such activities function in the assembly and re‐structuring of the wall matrix.     

Influence of β-cyclodextrin on the Properties of Norfloxacin Form A

Cyclodextrins are able to form host–guest complexes with hydrophobic molecules to result in the formation of inclusion complexes. The complex formation between norfloxacin form A and β-cyclodextrin was studied by exploring its structure affinity relationship in an aqueous solution and in the solid state. Kneading, freeze-drying, and physical mixture methods were employed to prepare solid complexes of norfloxacin and β-cyclodextrin. The solubility of norfloxacin significantly increased upon complexation with β-cyclodextrin as demonstrated by a solubility isotherm of the A_L type along with the results of an intrinsic dissolution study. The complexes were also characterized in the solid stated by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), Fourier-transform infrared (FT-IR) spectroscopy, X-ray diffractometry, scanning electron microscopy (SEM), and solid-state nuclear magnetic resonance (ssNMR) spectrometry. The thermal analysis showed that the thermal stability of the drug is enhanced in the presence of β-cyclodextrin. Finally, the microbiological studies showed that the complexes have better potency when compared with pure drug.KEY WORDS: bioassay, complexation, intrinsic dissolution, norfloxacin, β-cyclodextrin     

Influence of lignin-derived phenolic compounds on the Clostridium thermocellum endo-β-1,4-xylanase XynA

Phenolic compounds released during pretreatment of lignocellulosic biomass influence its enzymatic hydrolysis. To understand the effects of these compounds on the kinetic properties of xylan-degrading enzymes, the present study employed the recombinant cellulosomal endo-β-1,4-xylanase, thermostable GH11 XynA protein from Clostridium thermocellum, as an enzyme model to evaluate the effects of 4-hydroxybenzoic acid, gallic acid, vanillin, tannic acid, p-coumaric acid, ferulic acid, syringaldehyde, and cinnamic acid. XynA was deactivated by the assayed phenols at 60 °C, presenting the strongest deactivation in the presence of tannic acid, with an activity reduction of about 80 %. Thermal stability of XynA was influenced by ferulic acid, syringaldehyde, cinnamic acid, 4-hydroxybenzoic acid, and p-coumaric acid. The hydrolysis rate of oat-spelt xylan by XynA was influenced by temperature, being unable to hydrolyze at 40 °C in the presence of tannic acid. On hydrolysis at 60 °C, the presence of gallic and tannic acid caused a major reduction in reducing sugar production, generating 3.74 and 2.15 g.L^-1 of reducing sugar, respectively, whereas the reaction in the absence of phenols generated 4.41 g.L^-1. When XynA was pre-deactivated by phenols it could recover most of its activity at 40 °C, however, at 60 °C activity could not be reestablished.     

Influence of 4′-6-diamidino-2-phenylindole on the secondary structure and template activities of DNA and polydeoxynucleotides

Summary The interaction between 4-6-Diamidino-2-Phenylindole-hydrochloride (DAPI) and a variety of DNAs and synthetic polydeoxynucleotides was investigated in order to delineate the nucleic acid structural features necessary for binding. The spectra of DAPI-DNA complexes, measured at various DAPI-DNA molar ratios (r), are hypochromic relative to DNA in the region of its maximum absorption. All the curves pass through an isosbestic point at 268 nm. A new maxima appears in the region of 380–392 nm for DAPI-DNA complexes. The magnitude of the peaks in the region are directly proportional to the amount of drug present in the complex.Studies with various DNA types and synthetic polydeoxynucleotides indicate that the drug preferentially binds to dAT-rich regions of DNA. This was also confirmed by enzymatic studies. The inhibition of template action by DAPI in a purified DNA-polymerase reaction was dependent on the dAT-content of the template. The implication of these data to explain a selective binding of DAPI to mitochondrial DNA have been discussed.