Purification and properties of the β-fructofuranosidase from Kluyveromyces fragilis

The β-fructofuranosidase from Kluyveromyces fragilis was purified to one band on electrophoresis by 3 different methods. Two of the preparations were found to be impure by isoelectric focusing. This demonstrates the need for more than one criteria of homogeneity when purifying this enzyme. The enzyme was found to be a glycoprotein, stable at 50°C, with a pH optimum of 4.5. The cations Hg²⁺, Ag⁺, Cu²⁺ and Cd²⁺ exhibited a marked inhibition of the enzyme. Competitive inhibition was observed with the fructose analog 2,5-anhydro-D-mannitol suggesting that the enzyme is inhibited by the furanose form of fructose.     

Modulation of immobilized enzyme activity by altering the hydrophobicity of nylon-grafted membranes: Part 2: Non-isothermal conditions

Lactose hydrolysis by β-galactosidase immobilized on two nylon membranes, differently grafted, has been studied in a bioreactor operating under isothermal and non-isothermal conditions. One membrane (M₁) was obtained by chemical grafting of methylmethacrylate (MAA); the other one (M₂) by a double chemical grafting: styrene (Sty) and MAA. Hexamethylenediamine was used as a spacer between the grafted membranes and the enzyme. Both membranes have been physically characterized studying their permeabilities in presence of pressure or temperature gradients. Under non-isothermal conditions, the increase in activity of membrane M₂ was higher than that of membrane M₁. The and β coefficients, giving the percentage of activity increase when a temperature difference of 1°C is applied across the catalytic membranes, have been calculated. Results have been discussed with reference to the greater hydrophobicity of membrane M₂ with respect to membrane M₁, the hydrophobicity being a prerequisite for the occurrence of the process of thermodialysis.     

Optimization of β-galactosidase extraction from Kluyveromyces marxianus

The influence of several parameters, such as temperature, pH, and concentration of buffer and solvent, on the release of β-galactosidase from Kluyveromyces marxianus cells was studied. In optimal conditions (37°C, pH 9.5–10.5) greater than 90% of the intracellular β-galactosidase activity was released into 0.1-0.5 phosphate buffer after 1.5-2.0 h treatment with 1% chloroform. The described method is simple, effective, relatively fast, and selective.     

Influence of the microenvironment on the activity of enzymes immobilized on Teflon membranes grafted by γ-radiation

The effect of the microenvironment and immobilization method on the activity of immobilized β-galactosidase was investigated. Immobilization was done on Teflon membranes grafted with different acrylic monomers by γ-radiation and activated by two different coupling agents through the functional groups of the grafted monomers. 2-Hydroxyethyl methacrylate (HEMA) and methacrylic acid (MAA) were grafted on the membrane, and 1,6-hexamethylenediamine (HMDA) was used as a spacer. Glutaraldehyde or cyanuric chloride were used as coupling agents to bind the enzyme to the membrane. Four different catalytic membranes were obtained using the same solid support. Direct comparison between the isothermal behaviour of the biocatalyst in its free and immobilized form was carried out. In particular the dependence of the isothermal activity on the temperature and pH was studied and the kinetic parameters determined. The influence of the microenvironment on the observed activity of the four membranes was evidenced and discussed. The way of improving the yield of these catalytic membranes is discussed also.     

Modulation of immobilized enzyme activity by altering the hydrophobicity of nylon-grafted membranes: Part 1. Isothermal conditions

The catalytic behaviour under isothermal conditions of two different membranes loaded with β-galactosidase was investigated. One membrane (M₁) was constituted by a nylon sheet grafted with methylmethacrylate by means of chemical grafting. The other, (M₂), was prepared by a double chemical grafting: the first one with styrene (Sty) and the second one with methylmethacrylate. Membrane activity was characterized as a function of temperature, pH and substrate concentration. The role of Sty in increasing membrane hydrophobicity has been discussed. Membrane M₂ was found to be better suited for employment in non-isothermal bioreactors.     

Reversible β-hydride elimination of amine ligands forming stable cis-η-iminium hydride Os complexes

Octahedral tetraammineosmium(II) species are generated from their Os^III precursors containing an amine ligand cis to a labile alcohol or triflate. These compounds undergo reversible β-hydride eliminations resulting in the formation of cis-η²-iminium hydride complexes. Judging from NMR data, the η²-iminium group in these products lies parallel to the osmium-hydride bond with the iminium carbon eclipsing the hydride. Attempts to form η²-arene complexes of an Os^II ammine system bearing a stereogenic carbon are also described.     

Immobilization of thermostable β-glucosidase from Sulfolobus shibatae by cross-linking with transglutaminase

Thermostable β-glucosidase from Sulfolobus shibatae was immobilized on silica gel modified or not modified with 3-aminopropyl-triethoxysilane using transglutaminase as a cross-linking factor. Obtained preparations had specific activity of 3883 U/g of the support, when measured at 70 °C using o-nitrophenyl β-d-galactopyranoside (GalβoNp) as substrate. The highest immobilization yield of the enzyme was achieved at pH 5.0 in reaction media. The most active preparations of immobilized β-glucosidase were obtained at a transglutaminase concentration of 40 mg/ml at 50 °C. The immobilization was almost completely terminated after 100 min of the reaction and prolonged time of this process did not cause considerable changes of the activity of the preparations. The immobilization did not influence considerably on optimum pH and temperature of GalβoNp hydrolysis catalyzed by the investigated enzyme (98 °C, pH 5.5). The broad substrate specifity and properties of the thermostable β-glucosidase from S. shibatae immobilized on silica-gel indicate its suitability for hydrolysis of lactose during whey processing.     

Production of alkyl glucoside from cellooligosaccharides using yeast strains displaying Aspergillus aculeatus β-glucosidase 1

For efficient alkyl glucoside production from cellooligosaccharides, we constructed a yeast strain for alkyl glucoside synthesis by genetically inducing the display of β-glucosidase 1 (BGL1) from the filamentous fungus Aspergillus aculeatus No. F-50 on the cell surface. The localization of BGL1 on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying BGL1 catalyzed alkyl glucoside synthesis from p-nitrophenyl β-d-glucoside and primary alcohols. The highest yield of alkyl glucoside was 27.3% of the total sugar. The substrate specificities of the BGL1-displaying yeast strain and almond β-glucosidase were compared using different-chain-length cellooligosaccharides. The BGL1-displaying yeast showed efficient alkyl glucoside production from not only glucose but also cellohexaose. This yeast is applicable as a whole-cell biocatalyst for alkyl glucoside production from cellulose hydrolysates.     

Catalytic function and affinity purification of site-directed mutant β-cyclodextrin glucanotransferase from alkalophilic Bacillus firmus var. alkalophilus

Subsites −3 and −7 in the active site of β-cyclodextrin glucanotransferase (β-CGTase) from alkalophilic Bacillus firmus var. alkalophilus were modified through site-directed mutagenesis to obtain novel mutant CGTases. Four mutant CGTases, H59Q, Y96M, 90-PPI-92, and Δ(154–160) were constructed and produced using a recombinant E. coli with a secretive expression system extracellularly. The secreted mutant β-CGTases were purified by one-step affinity adsorption chromatography using a β-cyclodextrin (CD) polymer as an adsorbent to nearly homogeneous purity. The catalytic activities were modified significantly compared to the wild-type. In particular, the Y96M and Δ(154–160) mutants increased cyclization activity around 1.5 times without any significant reduction of coupling and hydrolyzing activities. Meanwhile, the Y96M and Δ(154–160) mutants exhibited a much higher conversion yield into CDs from 28.6 to 39% without any recognizable change in the CD ratio. The conversion yield into linear maltooligosaccharides was also significantly reduced. The catalytic functions of subsites −3 and −7 in the active site of β-CGTase would appear to be related to the overall productivity of CDs rather than the product specificity.     

Thermostability of β-xylosidase from Aspergillus sydowii MG49

Heating of Aspergillus β-xylosidase at 85°C ± 1°C and pH 5.5–6.0 (optimum for activity), causes irreversible, covalent thermoinactivation of the enzyme, involving oxidation of the thiol groups that are required for catalysis. Exogenous addition of cysteine, DTT, GSH and mercaptoethanol stabilizes the enzyme by extending its half-life. A similar effect is also exhibited by bivalent cations like Mg²⁺, Mn²⁺, Co²⁺, Ca²⁺and Zn²⁺ while, on the other hand Cu²⁺ accelerates thermoinactivation. Chemical modification of crude β-xylosidase with cross-linking agents like glutaraldehyde or covalent immobilization to a nonspecific protein like gelatin and BSA also enhances enzyme thermostability. These results suggest that addition of thiols and bivalent metal ions to a crude β-xylosidase preparation or immobilization/chemical modification enhances its thermal stability, thus preventing loss of catalytic activity at elevated temperatures.     

Antinoceptive effect of triterpenoid α,β-amyrin in rats on orofacial pain induced by formalin and capsaicin

The effects of α,β-amyrin, a pentacyclic triterpene isolated from Protium heptaphylum was investigated on rat model of orofacial pain induced by formalin or capsaicin. Rats were pretreated with α,β-amyrin (10, 30, and 100 mg/kg, i.p.), morphine (5 mg/kg, s.c.) or vehicle (3% Tween 80), before formalin (20 μl, 1.5%) or capsaicin (20 μl, 1.5 μg) injection into the right vibrissa. In vehicle-treated controls, formalin induced a biphasic nociceptive face-rubbing behavioral response with an early first phase (0–5 min) and a late second phase (10–20 min) appearance, whereas capsaicin produced an immediate face-rubbing (grooming) behavior that was maximal at 10–20 min. Treatment with α,β-amyrin or morphine significantly inhibited the face-rubbing response in both test models. While morphine produced significant antinociception in both phases of formalin test, α,β-amyrin inhibited only the second phase response, more prominently at 30 mg/kg, in a naloxone-sensitive manner. In contrast, α,β-amyrin produced much greater antinociceptive effect at 100 mg/kg in the capsaicin test, which was also naloxone-sensitive. These results provide first time evidence to show that α,β-amyrin attenuates orofacial pain atleast, in part, through a peripheral opioid mechanism but warrants further detailed study for its utility in painful orofacial pathologies.     

Stress-induced morphological and physiological changes in γ-linolenic acid production by Mucor fragilis in batch and continuous cultures

Batch and continuous cultures conditions were studied in order to increase γ-linolenic acid production by Mucor fragilis CCMI 142, in response to the presence of ethanol. Continuous cultures were used to add ethanol pulses to steady state pellet cultures. It was demonstrated that pellet size, which allowed homogeneous fungal cultures, can be obtained by means of pH adjustment, thus enabling steady state continuous growth at 2.90±0.05.

The 5 and 2% (v/v) ethanol pulses induced hyphal morphological changes with arthrospore formation. A 1% (v/v) pulse of ethanol did not immediately affect growth, but induced morphological changes, which led to autolysis at the pellet core. A 0.5% (v/v) pulse of ethanol did not affect neither the morphology nor the physiology of the microorganism to any significant extent. The 0.5 and 1% (v/v) ethanol pulses resulted in an increase in the proportion of γ-linolenic acid production up to 11%. Data from batch cultures showed a higher enhancement of ethanol, attaining 30% of γ-linolenic acid.

The increase of γ-linolenic acid content observed in batch and continuous conditions appears to be a response associated with stress induced by the ethanol which seems to be of value as an industrial medium component.     

Enzymic properties of β-mannanase from Polyporus versicolor

Substrate specificities and the kinetic parameters, K_m and V_max, of the four multiple enzyme forms of extracellular β-mannanase activity purified from Polyporus versicolor were determined. Although K_m values were significantly greater than those encountered in other β-mannanase systems V_max values were equivalent or much greater, rendering the physiological efficiencies of the β-mannanase comparable to those of other β-mannanases. All enzymes preferred glucomannan as substrate, were highly refractory at low concentrations to n-octylglucopyranoside, sodium deoxylcholate, and sodium dodecylsulfate, and were largely insensitive to methanol, ethanol, acetonitrile, and dimethylsulfoxide.     

Development of a continuous solid phase process for reduction and thiol-dependent immobilization of yeast β-galactosidase

Reduction and covalent immobilization of Kluyveromyces lactis β-galactosidase through disulfide bonds onto thiolsulfinate-agarose was performed using two fixed-bed mini-reactors connected in series, one packed with thiopropyl-agarose (a solid phase reducing agent) and the other with thiolsulfinate-agarose (a thiol-reactive support). With the aim of optimizing the whole process, two reactor systems were assessed. In System I, the percolate from thiopropyl-agarose containing the reduced enzyme was re-circulated through the thiolsulfinate-agarose reactor alone. In System II, re-circulation was performed through both the reactors, improving the immobilization yield from 17% (System I) to 42% and the expressed activity from 25% (System I) to 56%. When the bio-reactor achieved with System II was fed with skimmed milk at 22 °C at a flow rate of 48 ml/h, steady state lactose hydrolysis reached 80%. In addition, it could be reused four times without losing its lactose hydrolysis capacity.     

α3β1 integrin promotes cell survival via multiple interactions between 14-3-3 isoforms and proapoptotic proteins

Laminin-5 and α3β1 integrin promote keratinocyte survival; however, the downstream signaling pathways for laminin-5/α3β1 integrin-mediated cell survival had not been fully established. We report the unexpected finding of multiple interactions between 14-3-3 isoforms and proapoptotic proteins in the survival signaling pathway. Ln5-P4 motif within human laminin-5 α3 chain promotes cell survival and anti-apoptosis by inactivating Bad and YAP. This effect is achieved through the formation of 14-3-3ζ/p-Bad and 14-3-3σ/p-YAP complexes, which is initiated by α3β1 integrin and FAK/PI3K/Akt signaling. These complexes result in cytoplasmic sequestration of Bad and YAP and their subsequent inactivation. An increase in Akt1 activity in cells induces 14-3-3ζ and σ, p-Bad, and p-YAP, promoting cell survival, whereas decreasing Akt activity suppresses the same proteins and inhibits cell survival. Suppression of 14-3-3ζ with RNA-interference inhibits cell viability and promotes apoptosis. These results reveal a new mechanism of cell survival whereby the formation of 14-3-3ζ/p-Bad and 14-3-3σ/p-YAP complexes is initiated by laminin-5 stimulation via the α3β1 integrin and FAK/PI3K/Akt signaling pathways, thereby resulting in cell survival and anti-apoptosis.     

Fungus β-glycosidases: immobilization and use in alkyl-β-glycoside synthesis

Production of β-glycosidases: β-xylosidase and β-glucosidase by the fungus Sclerotinia sclerotiorum was optimized in the presence of different carbon sources. Immobilization supports with different physico-chemical characteristics were evaluated for use in continuous reactors. Immobilization and activity yields were calculated. Among the adsorption on Duolite, Amberlite, Celite and DEAE-sepharose, and entrapment in polyacrylamide gel or reticulation using glutaraldehyde, highest yields were obtained when β-xylosidase was adsorbed on Duolite A 7 and when β-glucosidase was adsorbed on DEAE-sepharose.

Enzyme preparations from S. sclerotiorum cultures were used in a biphasic (alcohol/aqueous) medium for the synthesis of alkyl-glycosides by trans-glycosylation of sugars and long-chain alcohols. The synthesis was studied under different conditions with primary and secondary alcohols as substrates, in the presence of free or immobilized enzyme. Xylan and cellobiose were used for the synthesis of alkyl-xylosides and alkyl-glucosides, respectively. The majority of the immobilized preparations were unable to catalyze the synthesis of alkyl-glycosides.

Highest yields were obtained when using xylan and C₄–C₆-alcohols. The reaction produced alkyl-β-xyloside and alkyl-β-xylobioside, as confirmed by MS/MS. Up to 22 mM iso-amyl-xyloside and 14 mM iso-amyl-xylobioside were produced from iso-amyl alcohol and xylan.     

β-Adrenoreceptor participation in the formation of the thermoregulatory and immune responses under the effect of rapid deep cooling

The effect of β-adrenoantagonist (obzidan) iontophoresis to skin on the thermoregulatory response and immune response to antigen was analyzed to elucidate the significance of β-adrenoceptors in formation of these responses at deep rapid cooling in rats.

On the background of β-adrenoceptors blockade in thermoneutral conditions the skin and core temperatures decreases; at rapid cooling non-shivering thermogenesis is attenuated and shivering thermogenesis is considerably enhanced.

Administration of β-adrenoantagonist affect the modulating influence of cold exposure on the immune response—the immunosuppressive effect of deep cooling on the immune response is abolished. This concerns both antigen binding function of spleen and peritoneal cells and antibody formation.

The results support the idea that β-adrenoceptors participates in the processes of the stimulation of thermogenesis and suppression of the immune response to antigen at rapid deep cooling.     

Molecular cloning and expression of a cDNA for human kidney cysteine conjugate β-lyase

Kidney cysteine conjugate β-lyase (glutamine transaminase K, kyneurenine aminotransferase, EC 2.6.1.64) metabolises the cysteine conjugates of certain halogenated alkenes and alkanes to form reactive metabolites which can produce nephrotoxicicity and neurotoxicicity in experimental animals and man. Using a combination of hybridisation screening and PCR techniques we have isolated a full-length cDNA for human kidney cysteine conjugate β-lyase. Comparison of the deduced amino acid sequence with that of the rat enzyme indicated an 82% overall similarity, with 90% similarity around the pyridoxal phosphate binding site, many of the changes being conservative in nature. Expression of the cDNA in Cos-1 cells resulted in the production of a cytosolic enzyme which showed both cysteine conjugate β-lyase and glutamine transaminase K activity. Preliminary mapping of the gene for human cysteine conjugate β-lyase by PCR analysis of genomic DNA from human-rodent hybrid cells indicated that it is located on human chromosome 9.