Investigation of mechanisms of interaction between inulinase from Kluyveromyces marxianus and the matrices of ion-exchange resins and fiber

It is established that ion exchange resins AV-17-2P, KU-2, AV-16-GS, AM 21A, IMAC-HP, PUROLITE and fiber VION KN-1 can be applied as carriers for inulinase immobilization. The analysis of IR spectra for an enzyme, carriers and heterogeneous enzyme preparations showed that inulinase binding to matrices of various carriers occurs in general through electrostatic interactions. It is assumed that the mechanisms of interaction between inulinase from Kluyveromyces marxianus and the matrices of cation and anion exchange polymers differ essentially from each other: different sites of protein molecule take part in adsorption that causes various conformational reorganizations in an enzyme molecule.     

Thermal inactivation of free and immobilized inulinase

Thermal inactivation of the Kluyveromyces marxianus inulinase in a free form and immobilized on VION KN-1 cation exchange fiber was studied. Atomic force microscopy demonstrated an oligomeric structure of this enzyme, composed of two subunits differing in their size. It was assumed that the intersubunit contacts were destroyed at 60°C, and the inulinase molecule dissociated into two monomers located separately.     

Interaction of micro-dispersed forms of anionites and surface-active agents with serum albumin

The interaction of serum albumin with microdispersed forms of anion-exchange resins AV-16 GS and AV-17 I (USSR), obtained by mechanochemical destruction of polymers, and with low-molecular weight anionic (oleic acid, sulfonol) and cationic (cetazol, catamine) surfactants was being studied. Catamine and cetazol as well as microdispersions of anion-exchange resins are able to precipitate 90% of protein from the equilibrium solution, the content of protein in the initial solution being constant and equal to 6.5 mg/ml. Studies on the equilibrium in the system serum albumin--microdispersions of anion-exchange resins in the presence of ionogenic surfactants revealed that cationic surfactants are able to increase the amount of the precipitated protein, while in the presence of anionic surfactants the interactions between the protein macromolecules and microdispersions of polyelectrolytes become much weaker.     

The effect of urea, gamma- and UV-irradiation on physico-chemical characteristics of native and immobilized inulinase

The immobilization of inulinase by ionexchange AB-26 and AB-17-2P has been made by adsorbtion and glutaraldehyde methods. The effect of UV-radiation, carbamide and gamma-rays on the stability of native and immobilized enzyme has been investigated. The stability of inulinase in relation to denaturation agents has been shown to increase with the immobilization of ionexchange. The character of binding with the matrix affects greatly the stability of immobilized enzyme to physical factors.     

The interaction of inulinase molecules with a chitosan matrix: UV-induced changes in the functional properties of immobilized inulinase

We have developed a technique for the immobilization of inulinase on chitosans with different molecular weights. The acid-soluble mid-molecular-weight (200 kDa) and high-molecular-weight (350 kDa) chitosans are shown to be promising matrices for inulinase adsorption. We assumed that the formation of an inulinase-chitosan matrix complex occurs mainly due to hydrophobic interactions; electrostatic interactions also play an essential role. The enzyme complex with high-molecular-weight chitosan is more stable against the action of UV light and temperature. This allows us to recommend chitosan as a catalyst in industry for the production of fructose from inulin-containing vegetable raw mateials.     

Immobilization of Kluyveromyces marxianus cells containing inulinase activity in open pore gelatin matrix: 1. Preparation and enzymatic properties

Kluyveromyces marxianus cells with inulinase (2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7) activity have been immobilized in open pore gelatin pellets with retention of > 90% of the original activity. The open pore gelatin pellets with entrapped yeast cells were obtained by selective leaching out of calcium alginate from the composite matrix, followed by crosslinking with glutaraldehyde. Enzymatic properties of the gelatin-entrapped cells were studied and compared with those of the free cells. The immobilization procedure did not alter the optimum pH of the enzymatic preparation; the optimum for both free and immobilized cells was pH 6.0. The optimum temperature of inulin hydrolysis was 10°C higher for immobilized cells. Activation energies for the reaction with the free and immobilized cells were calculated to be 6.35 and 2.26 kcal mol^?1, respectively. K_m values were 8 mM inulin for the free cells and 9.52 mM for the immobilized cells. The thermal stability of the enzyme was improved by immobilization. Free and immobilized cells showed fairly stable activities between pH 4 and 7, but free cell inulinase was more labile at pH values below 4 and above 7 compared to the immobilized form. There was no loss of enzyme activity of the immobilized cells on storage at 4°C for 30 days. Over the same period at room temperature only 6% of the original activity was lost.     

Immobilization of inulinase obtained by solid-state fermentation using spray-drying technology

Abstract

This work focuses on the immobilization of a crude inulinase extract obtained by solid-state fermentation using spray-drying technology. Maltodextrin and arabic gum were used as immobilizing agents. The effects of inlet air temperature, maltodextrin/arabic gum ratio and mass fraction of crude enzyme extract on the activity of immobilized inulinase were assessed using a central composite rotatable design (CCRD) (2³). The optimum operational conditions for the immobilization of inulinase by spray-drying was obtained at an inlet air temperature of 200°C, mass fraction of crude enzyme extract of 0.5 wt% and using only arabic gum as immobilizing agent. The immobilized enzyme had good thermostability, comparable with other inulinases obtained from different microorganisms. The method used gave good enzyme activity after immobilization and could be applied to other enzymes which have good thermal stability.     

Biological treatment of textile dye acid violet-17 by bacterial consortium in an up-flow immobilized cell bioreactor

AIMS: To develop a cost effective and efficient biological treatment process for small scale textile processing industries (TPI) releasing untreated effluents containing intense coloured Acid violet-17 (AV-17), a triphenyl methane (TPM) group textile dye. METHODS AND RESULTS: The samples collected from effluent disposal sites of TPI were used for selective enrichment of microbial populations capable of degrading/decolourizing AV-17. A consortium of five bacterial isolates was used to develop an up-flow immobilized cell bioreactor for treatment of feed containing AV-17. The bioreactor, operating at a flow rate of 6 ml x h(-1), resulted in 91% decolourization of 30 mg AV-17/l with 94.3 and 95.7% removal of biochemical oxygen demand and chemical oxygen demand of the feed. Comparison of the input and output of the bioreactor by UV-visible, thin layer chromatography and (1)H-nuclear magnetic resonance spectroscopy indicates conversion of the parent dye into unrelated metabolic intermediates. SIGNIFICANCE: These results will form a basis for developing 'on-site' treatment system for TPI effluents to achieve decolourization and degradation of residual dyes.     

Immobilized inulinase: a new horizon of paramount importance driving the production of sweetener and prebiotics

In recent times, inulinase has emerged as one the most prominent and industrially upcoming enzymes applied to meet the ever increasing demand of d-fructose and fructooligosaccharides (FOS) as sweetener and prebiotics in the food and pharmaceutical industry, respectively. This review deals with types of inulinase and the attempts made to modify it for better thermal stability and shelf life. The ease of immobilization of inulinase has led us to the path of experimenting with different methods of enzyme immobilization since 1979. Several modes of immobilization ranging from simple cross-linking of enzymes onto a polymer support to nanoparticles have been applied over the years. The approach and concept of this review provide a yet unexplored focus on pioneering advances for the development of white biotechnology, for instance production of immobilized inulinase-based reusable biocatalysts and bioreactors designed for their use and for the continuous production of fructose and FOS.     

Inhibition of the inositol trisphosphate receptor of mouse eggs and A7r5 cells by KN-93 via a mechanism unrelated to Ca2+/calmodulin-dependent protein kinase II antagonism

KN-93, a Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor, concentration-dependently and reversibly inhibited inositol 1,4,5-trisphosphate receptor (IP(3)R)-mediated [Ca(2+)](i) signaling in mouse eggs and permeabilized A7r5 smooth muscle cells, two cell types predominantly expressing type-1 IP(3)R (IP(3)R-1). KN-92, an inactive analog, was ineffective. The inhibitory action of KN-93 on Ca(2+) signaling depended neither on effects on IP(3) metabolism nor on the filling grade of Ca(2+) stores, suggesting a direct action on the IP(3)R. Inhibition was independent of CaMKII, since in identical conditions other CaMKII inhibitors (KN-62, peptide 281-309, and autocamtide-related inhibitory peptide) were ineffective and since CaMKII activation was precluded in permeabilized cells. Moreover, KN-93 was most effective in the absence of Ca(2+). Analysis of Ca(2+) release in A7r5 cells at varying [IP(3)], of IP(3)R-1 degradation in eggs, and of [(3)H]IP(3) binding in Sf9 microsomes all indicated that KN-93 did not affect IP(3) binding. Comparison of the inhibition of Ca(2+) release and of [(3)H]IP(3) binding by KN-93 and calmodulin (CaM), either separately or combined, was compatible with a specific interaction of KN-93 with a CaM-binding site on IP(3)R-1. This was also consistent with the much smaller effect of KN-93 in permeabilized 16HBE14o(-) cells that predominantly express type 3 IP(3)R, which lacks the high affinity CaM-binding site. These findings indicate that KN-93 inhibits IP(3)R-1 directly and may therefore be a useful tool in the study of IP(3)R functional regulation.     

Free energy simulations and MM-PBSA analyses on the affinity and specificity of steroid binding to antiestradiol antibody

Antiestradiol antibody 57-2 binds 17beta-estradiol (E2) with moderately high affinity (K(a) = 5 x 10(8) M(-1)). The structurally related natural estrogens estrone and estriol as well synthetic 17-deoxy-estradiol and 17alpha-estradiol are bound to the antibody with 3.7-4.9 kcal mol(-1) lower binding free energies than E2. Free energy perturbation (FEP) simulations and the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method were applied to investigate the factors responsible for the relatively low cross-reactivity of the antibody with these four steroids, differing from E2 by the substituents of the steroid D-ring. In addition, computational alanine scanning of the binding site residues was carried out with the MM-PBSA method. Both the FEP and MM-PBSA methods reproduced the experimental relative affinities of the five steroids in good agreement with experiment. On the basis of FEP simulations, the number of hydrogen bonds formed between the antibody and steroids, which varied from 0 to 3 in the steroids studied, determined directly the magnitude of the steroid-antibody interaction free energies. One hydrogen bond was calculated to contribute about 3 kcal mol(-1) to the interaction energy. Because the relative binding free energies of estrone (two antibody-steroid hydrogen bonds), estriol (three hydrogen bonds), 17-deoxy-estradiol (no hydrogen bonds), and 17alpha-estradiol (two hydrogen bonds) are close to each other and clearly lower than that of E2 (three hydrogen bonds), the water-steroid interactions lost upon binding to the antibody make an important contribution to the binding free energies. The MM-PBSA calculations showed that the binding of steroids to the antiestradiol antibody is driven by van der Waals interactions, whereas specificity is solely due to electrostatic interactions. In addition, binding of steroids to the antiestradiol antibody 57-2 was compared to the binding to the antiprogesterone antibody DB3 and antitestosterone antibody 3-C4F5, studied earlier with the MM-PBSA method.     

Hydrolysis of inulin: a kinetic study of the reaction catalyzed by an inulinase from Aspergillus ficuum

A kinetic study of the hydrolysis of inulin was performed by using as catalyst a commercial inulinase from Aspergillus ficuum. The reaction was studied carrying out initial rate as well as time course measurements. Both inulinase and invertase activities of the enzyme were taken into account, and the corresponding kinetic parameters were determined in the temperature range 30-50 degrees C. The activation energies of the turnover constant for inulinase and invertase activities were found to be similar (56-57 kJ . mol(-1)). The ratio S/I of invertase to inulinase activity was 1.6 regardless of temperature. The thermal degradation of the enzyme was also investigated up to 70 degrees C, and an activation energy of 350-370 kJ . mol(-1) was evaluated.     

Synthesis and evaluation of a ring-constrained Hsp90 C-terminal inhibitor that exhibits neuroprotective activity

KU-596 is a second-generation C-terminal heat shock protein 90 KDa (Hsp90) modulator based on the natural product, novobiocin. KU-596 has been shown to induce Hsp70 levels and manifest neuroprotective activity through induction of the heat shock response. A ring-constrained analog of KU-596 was designed and synthesized to probe its binding orientation and ability to induce Hsp70 levels. Compound 2 was found to exhibit comparable or increased activity compared to KU-596, which is under clinical investigation for the treatment of neuropathy.     

Characterization of molecular-sieve-bound inulinase

The enzyme inulinase (2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7), prepared from Kluyveromyces marxianus has been immobilized using an inorganic solid support, molecular sieve 4A via the metal link method. The immobilized enzyme had around 22 units of inulinase activity per g of the support with retention of 72% of the original activity. The optimum protein to molecular sieve ratio for the maximum retention of inulinase activity was 9 mg/g molecular sieve. The properties of soluble and immobilized enzyme differed in many respects. The optimum pH of the enzyme shifted from 6 to 5 and the optimum temperature of enzyme activity changed from 50 to 55°C. K_m values were 6.7 mM for soluble enzyme and 10 mM for immobilized enzyme. The heat stability of the enzyme was improved by immobilization. Immobilized enzyme retained about 76% of the original activity after 40 days of storage at room temperature (30±2°C).     

Inhibiting heat-shock protein 90 reverses sensory hypoalgesia in diabetic mice

Increasing the expression of Hsp70 (heat-shock protein 70) can inhibit sensory neuron degeneration after axotomy. Since the onset of DPN (diabetic peripheral neuropathy) is associated with the gradual decline of sensory neuron function, we evaluated whether increasing Hsp70 was sufficient to improve several indices of neuronal function. Hsp90 is the master regulator of the heat-shock response and its inhibition can up-regulate Hsp70. KU-32 (N-{7-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyl-tetrahydro-2H-pyran-2-yloxy]-8-methyl-2-oxo-2H-chromen-3-yl}acetamide) was developed as a novel, novobiocin-based, C-terminal inhibitor of Hsp90 whose ability to increase Hsp70 expression is linked to the presence of an acetamide substitution of the prenylated benzamide moiety of novobiocin. KU-32 protected against glucose-induced death of embryonic DRG (dorsal root ganglia) neurons cultured for 3 days in vitro. Similarly, KU-32 significantly decreased neuregulin 1-induced degeneration of myelinated Schwann cell DRG neuron co-cultures prepared from WT (wild-type) mice. This protection was lost if the co-cultures were prepared from Hsp70.1 and Hsp70.3 KO (knockout) mice. KU-32 is readily bioavailable and was administered once a week for 6 weeks at a dose of 20 mg/kg to WT and Hsp70 KO mice that had been rendered diabetic with streptozotocin for 12 weeks. After 12 weeks of diabetes, both WT and Hsp70 KO mice developed deficits in NCV (nerve conduction velocity) and a sensory hypoalgesia. Although KU-32 did not improve glucose levels, HbA1c (glycated haemoglobin) or insulin levels, it reversed the NCV and sensory deficits in WT but not Hsp70 KO mice. These studies provide the first evidence that targeting molecular chaperones reverses the sensory hypoalgesia associated with DPN.     

KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II

1-[N,O-Bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpipera zine (KN-62), a selective inhibitor of rat brain Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) was synthesized and its inhibitory properties in vitro and in vivo were investigated. KN-62 inhibited phosphorylation of exogenous substrate (chicken gizzard myosin 20-kDa light chain) by Ca2+/CaM kinase II with Ki value of 0.9 microM, but no significant effect up to 100 microM on activities of chicken gizzard myosin light chain kinase, rabbit brain protein kinase C, and bovine heart cAMP-dependent protein kinase type II. KN-62 also inhibited the Ca2+/calmodulin-dependent autophosphorylation of both alpha (50 kDa) and beta (60 kDa) subunits of Ca2+/CaM kinase II dose dependently in the presence or absence of exogenous substrate. Kinetic analysis indicated that this inhibitory effect of KN-62 was competitive with respect to calmodulin. However, KN-62 did not inhibit the activity of autophosphorylated Ca2+/CaM kinase II. Moreover, Ca2+/CaM kinase II bound to a KN-62-coupled Sepharose 4B column, but calmodulin did not. These results suggest that KN-62 affects the interaction between calmodulin and Ca2+/CaM kinase II following inhibition of this kinase activity by directly binding to the calmodulin binding site of the enzyme but does not affect the calmodulin-independent activity of already autophosphorylated (activated) enzyme. We examined the effect of KN-62 on cultured PC12 D pheochromocytoma cells. KN-62 suppressed the A23187 (0.5 microM)-induced autophosphorylation of the 53-kDa subunit of Ca2+/CaM kinase in PC12 D cells, which was immunoprecipitated with anti-rat forebrain Ca2+/CaM kinase II polypeptides antibodies coupled to Sepharose 4B, thereby suggesting that KN-62 could inhibit the Ca2+/CaM kinase II activity in vivo.     

Inulinase-producing Marine Yeasts: Evaluation of their Diversity and Inulin Hydrolysis by Their Crude Enzymes

Total 427 yeast strains from seawater, sediments, mud of salterns, guts of the marine fish, and marine algae were obtained. After inulinase activity of the yeast cultures was estimated, we found that four strains (OUC1, G7a, OUC2, and G7a1) of the marine yeasts grown in the medium with inulin could secrete a large amount of inulinase into the medium. The results of routine identification and molecular methods show that they belong to Pichia guilliermondii OUC1, Cryptococcus aureus G7a, Yarrowia lipolytica OUC2, and Debaryomyces hansenii G7a1, respectively. The optimal pHs of inulinase activity produced by them were 6.0, 5.0, 5.0, and 5.0, respectively, while the optimal temperatures of inulinase activity produced by them were 60°, 50°, 60°, and 50°C, respectively. A large amount of monosaccharides and a trace amount of oligosaccharides were detected after the hydrolysis by the crude inulinase produced by P. guilliermondii OUC1, indicating that the crude inulinase had a high exoinulinase activity while a large amount of monosaccharides and oligosaccharides were detected after inulin hydrolysis by the crude inulinase produced both by C. aureus G7a and D. hansenii G7a1. However, no monosaccharides and disaccharides were detected after inulin hydrolysis by the crude inulinase produced by Y. lipolytica OUC2, suggesting that the crude inulinase had no exoinulinase activity.     

Temporal and spatial expression of a position specific antigen, AV-1, in chick limb buds

In a previous study, we demonstrated the presence of a position-specific antigen (AV-1) in chick limb buds at an early developmental stage. Here, we reported the temporal and spatial expressions and the biochemical characterization of the AV-1 antigen. Indirect immunofluorescence staining and immunoblot analysis clearly showed that the AV-1 antigen is a glycoprotein that is localized on the plasma membrane and that it is expressed from stage 19 and highly expressed at stages 22-26 in some middle-distal to anterior-distal region of limb buds. In the wing bud, at stage 28, the AV-1 antigen was faintly detected in the restricted space between the precartilaginous regions of the radius and the ulna, and those of the metacarpals 2 and 3, but not those of the metacarpals 3 and 4. Such stage-specific and "position-specific" expressions of the AV-1 antigen in limb buds strongly suggest that the AV-1 antigen or cells containing it are involved in determination of the limb pattern formation.     

高产菊粉酶酵母筛选、发酵和酶学性质研究

筛选到1株菊粉酶高产克鲁维酵母菌株,采用酵母高密度细胞发酵方法,最高菊粉酶产量达到288.78u/mL,比80～90年代国际上报道的克鲁维酵母菊粉酶最高产量高6.8倍。该酶的菊粉酶/转化酶活性比为1/24.72;菊糖m=13.3mmol/L,蔗糖Km=62.6mmol/L;最适反应pH值为4.4,但在pH3.8～5.6的范围内均保持了较高的活性,相当于最适pH值下活性的90%;最适反应温度为55℃,在50～575℃范围内能够保持较高活性,50℃下酶的半衰期约为16h;外加Mg2+提高酶活性11.28%。