Interaction of myoglobin with poly(methacrylic acid) at different pH in their layer-by-layer assembly films: an electrochemical study

Myoglobin (Mb), with net positive surface charges at pH 5.0, was successfully assembled into layer-by-layer films on various solid surfaces with poly(methacrylic acid) (PMAA) at different pH, designated as {PMAA(pH 5.0)/Mb}n, {PMAA(pH 6.5)/Mb}n, and {PMAA(pH 8.0)/Mb}n, respectively. As a weak polycarboxylic acid with pKa=6 - 7, PMAA carried different negative charges at different pH due to different ionization degree of its carboxylic acid groups. Quartz crystal microbalance (QCM), UV-vis spectroscopy, and cyclic voltammetry (CV) were used to monitor and confirm the assembly of {PMAA/Mb}n films. All the results showed that the adsorption amount of Mb in each bilayer had an "unexpected" sequence of {PMAA(pH 5.0)/Mb}n>{PMAA(pH 6.5)/Mb}n>{PMAA(pH 8.0)/Mb}n, which could be explained by the formation of soluble complex of PMAA-Mb at pH 8.0 and the cooperative effect of hydrogen bonding and induced electrostatic interaction between Mb and PMAA at pH 5.0. The influence of ionic strength in exposure solution and in Mb adsorbate solution was investigated, and the results supported the above explanations. The {PMAA/Mb}n films provided a suitable microenvironment for Mb to retain its near-native structure and transfer electron with underlying electrodes. The reversible CV peak pair for Mb Fe(III)/Fe(II) redox couple could be used to catalyze reduction of hydrogen peroxide electrochemically, showing the potential applicability of the films as the new type of biosensors or bioreactors based on the direct electrochemistry of Mb. The electrochemical and electrocatalytic behaviors of protein layer-by-layer films with weak polyelectrolytes could thus be controlled by adjusting the solution pH of weak polyelectrolytes.     

Production of Fab fragments of monoclonal antibodies using polyelectrolyte complexes

A new method for the production of monovalent Fab fragments of antibodies has been developed. Traditionally Fab fragments are produced by proteolytic digestion of antibodies in solution followed by isolation of Fab fragments. In the case of monoclonal antibodies against inactivated subunits of glyceraldehyde-3-phosphate dehydrogenase, digestion with papain resulted in significant damage of the binding sites of the Fab fragments. Antigen was covalently attached to the polycation, poly(N-ethyl-4-vinylpyridinium bromide). Proteolysis of monoclonal antibodies in the presence of the antigen-polycation conjugate followed by (i) precipitation induced by addition of polyanion, poly(methacrylic) acid, and pH shift from 7.3 to 6.5 and (ii) elution at pH 3.0 resulted in 90% immunologically competent Fab fragments. Moreover, the papain concentration required for proteolysis was 10 times less in the case of antibodies bound to the antigen-polycation conjugate than that of free antibodies in solution. The digestion of antibodies bound to the antigen-polyelectrolyte complex was less damaging, suggesting that binding to the antigen-polycation conjugate not only protected binding sites of monoclonal antibodies from proteolytic damage but also facilitated the proteolysis probably by exposing antibody molecules in a way convenient for proteolytic attack by papain.     

Use of TaqMan gene probe for real-time monitoring of acidophilic hydrogen-producing bacteria

Using a TaqMan gene probe targeting the 16S rDNA of acidophilic hydrogen-producing bacteria (HPB), the abundance of this HPB in the biomass was found to increase from 0.02 to 72% in a single batch treating rice slurry waste at pH 4.5 over 130 h. The corresponding abundances were 4.4% in the batch operated at pH 5.0 and 0.01–0.02% at pH 5.5–6.5. During the growth phase, the generation time for the acidophilic HPB at pH 4.5 averaged 3.5 h.     

Reactivation of glyceraldehyde-3-phosphate dehydrogenase using conjugates of monoclonal antibodies with polyelectrolyte complexes. An attempt to make an artificial chaperone

The simplified model of chaperone action when the inactive misfolded forms are removed from the reaction media preventing aggregation was developed using antibodies in combination with polyelectrolyte complexes. The antibodies, which bind specifically inactive dimers of glyceraldehyde-3-phosphate dehydrogenase but not native tetramers, were coupled covalently to poly(methacrylic acid). The treatment of inactivated GAPDH with this conjugate followed by its precipitation after equimolar addition of polycation, poly-(N-ethyl-4-vinylpyridinium bromide), resulted in a significant increase in the specific activity of the enzyme.     

Interaction of antibodies and antigens conjugated with synthetic polyanions: on the way of creating an artificial chaperone

Recently we have initiated the use of synthetic polyelectrolytes to mimic the action of chaperones in living cells [Dainiak et al., Biochim. Biophys. Acta 1381 (1998) 279-285]. The next step in this direction is done by the synthesis of conjugates of poly(methacrylic acid) (PMAA) with antigen, denatured glyceraldehyde-3-phosphate dehydrogenase (dGAPDH), and with monoclonal antibodies specific for dGAPDH (but not for the native protein). The pH-dependent properties of the conjugates have been studied using turbidimetry and light scattering. The antibody-PMAA and dGAPDH-PMAA conjugates were shown to interact with free dGAPDH and antibodies respectively as well as with each other. Insoluble aggregates of dGAPDH with antibody-PMAA and of antibodies with dGAPDH-PMAA are formed in acidic media. The same situation occurs in the mixture of antibody-PMAA and dGAPDH-PMAA: precipitation takes place in acidic media, whereas soluble associates are formed in neutral solutions. The size of the soluble associates and the number of conjugates in the associate could be regulated by pH. The competition of free dGAPDH and dGAPDH-PMAA for binding with antibody-PMAA and the dynamic release of refolded GAPDH, with no affinity to antibody-PMAA, into solution could be used for simulating chaperone action.     

Acidification of small mountain lakes in the High Tatra Mountains,Poland

A hydrochemical investigation was carried out on eight small High Tatra mountain lakes (Poland). When comparing recent data with those from the period 1935–1965, a constant process of acidification of the lakes is found. The average pH of precipitation is 4.8 in the study area, but the lakes are in two stages of acidification: weak (pH 6.0–6.5) and intermediate (pH 5.5–5.8). The differences are due to differences in water sources.     

Effect of medium-pH and MES on adventitious root formation from stem disks of apple

We have examined the effect of medium-pH on rooting using 1-mm slices cut from stems of apple microshoots. Before autoclaving, the pH of the rooting medium was set at various pH values between 4.5 and 8.0. During autoclaving, the pH drifted in particular in the alkaline region. Additional changes occurred during culture and the range set at 4.5–8.0 had shifted to 5.2–6.0 after autoclaving and 3 weeks of culture. When 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) had been added as buffering agent, the pH was stable when set at 5.0–6.5. Highest rooting was achieved at pH ~5.3 with and without MES (pH measured after autoclaving). This maximum did not correlate with highest auxin uptake. MES inhibited adventitious root formation during the initial phase of root formation when the meristemoids are being formed (ca. 30% reduction at 10 mM) but was promotive during outgrowth of the meristemoids to roots (30% increase at 10 mM). Inhibition and promotion by MES were not related to its buffering action as they were observed at all pHs.     

Polymer brush controlled bioinspired calcium phosphate mineralization and bone cell growth

Polymer brushes on thiol-modified gold surfaces were synthesized by using terminal thiol groups for the surface-initiated free radical polymerization of methacrylic acid and dimethylaminoethyl methacrylate, respectively. Atomic force microscopy shows that the resulting poly(methacrylic acid) (PMAA) and poly(dimethylaminoethyl methacrylate) (PDMAEMA) brushes are homogeneous. Contact angle measurements show that the brushes are pH-responsive and can reversibly be protonated and deprotonated. Mineralization of the brushes with calcium phosphate at different pH yields homogeneously mineralized surfaces, and preosteoblastic cells proliferate on both the nonmineralized and mineralized surfaces. The number of living cells on the mineralized hybrid surfaces is ca. 3 times (PDMAEMA) and 10 times (PMAA) higher than on the corresponding nonmineralized brushes.     

Immobilization of urease by using chitosan–alginate and poly(acrylamide-co-acrylic acid)/κ-carrageenan supports

Jack bean urease (urea aminohydrolase, E.C. 3.5.1.5) was entrapped into chitosan–alginate polyelectrolyte complexes (C-A PEC) and poly(acrylamide-co-acrylic acid)/κ-carrageenan (P(AAm-co-AA)/carrageenan) hydrogels for the potential use in immobilization of urease, not previously reported. The effects of pH, temperature, storage stability, reuse number, and thermal stability on the free and immobilized urease were examined. For the free and immobilized urease into C-A PEC and P(AAm-co-AA)/carrageenan, the optimum pH was found to be 7.5 and 8, respectively. The optimum temperature of the free and immobilized enzymes was also observed to be 55 and 60 °C, respectively. Michaelis–Menten constant (K _m) values for both immobilized urease were also observed smaller than free enzyme. The storage stability values of immobilized enzyme systems were observed as 48 and 70%, respectively, after 70 days. In addition to this, it was observed that, after 20th use in 5 days, the retained activities for immobilized enzyme into C-A PEC and P(AAm-co-AA)/carrageenan matrixes were found as 55 and 89%, respectively. Thermal stability of the free urease was also increased by a result of immobilization.     

A Novel PHB Depolymerase from a Thermophilic Streptomyces Sp.

A novel PHB depolymerase from a thermophilic Streptomyces sp. MG was purified to homogeneity by hydrophobic interaction chromatography and gel filtration. The molecular mass of the purified enzyme was 43 kDa as determined by size exclusion chromatography and 41 kDa by SDS-PAGE. The optimum pH and temperature were 8.5 and 60 °C respectively. The enzyme was stable at 50 °C and from pH 6.5–8.5. The enzyme hydrolyzed not only bacterial polyesters, i.e. poly(3-hydroxybutyric acid and poly(3-hydroxybutyrate-co-3-hydroxyvalerate), but also synthetic, aliphatic polyesters such as polypropiolactone, poly(ethylene adipate) and poly(ethylene succinate). Revisions requested 9 November 2005; Revisions received 12 December 2005     

Poly(styrene-divinylbenzene) beads surface functionalized with di-block polymer grafting and multi-modal ligand attachment: performance of reversibly immobilized lipase in ester synthesis

Fibrous poly(styrene-b-glycidylmethacrylate) brushes were grafted on poly(styrene–divinylbenzene) (P(S–DVB)) beads using surface-initiated atom transfer radical polymerization. Tetraethyldiethylenetriamine (TEDETA) ligand was incorporated on P(GMA) block. The ligand attached beads were used for reversible immobilization of lipase. The influences of pH, ionic strength, and initial lipase concentration on the immobilization capacities of the beads have been investigated. Lipase adsorption capacity of the beads was about 78.1 mg/g beads at pH 6.0. The K _m value for immobilized lipase was about 2.1-fold higher than that of free enzyme. The thermal, and storage stability of the immobilized lipase also was increased compared to the native lipase. It was observed that the same support enzyme could be repeatedly used for immobilization of lipase after regeneration without significant loss in adsorption capacity or enzyme activity. A lipase from Mucor miehei immobilized on styrene–divinylbenzene copolymer was used to catalyze the direct esterification of butyl alcohol and butyric acid.     

Biotreatment of acidic zinc- and copper-containing wastewater using ethanol-fed sulfidogenic anaerobic baffled reactor

The treatment of acidic (pH 6.5–3), sulfate- (2–3 g/L), Zn- and Cu- (total metal 0–500 mg/L) containing wastewater was studied in a four-stage anaerobic baffled reactor (ABR) at 35 °C for 250 days. Ethanol was supplemented (COD/SO₄ ²⁻ = 0.67) as carbon and electron source for sulfate reducing bacteria. Sulfate reduction, COD oxidation and metal precipitation efficiencies were 70–92, 80–94 and >99%, respectively. The alkalinity produced from sulfidogenic ethanol oxidation increased the wastewater pH from 3.0 to 7.0–8.0. The electron flow from organic oxidation to sulfate averaged 87%. Decreasing feed pH to 3 and increasing total metal concentrations to 500 mg/L did not adversely affect the performance of ABR and sufficient alkalinity was produced to increase the effluent pH to neutral values. More than 99% of metals were precipitated in the form of metal-sulfides. Accumulation of precipitated metals in the first compartment allowed metal recovery without disturbing reactor performance seriously.     

Search for catalytic properties of simple polypeptides

Simple polypeptides were used as possible supports for nucleotide polymerization, in the absence of any preformed polynucleotide template. Sequential copolymers of alanine and glycine, water soluble polypeptides based on arginine and poly(Glu-SerGlu) have been tested. No catalytic effect has been found although poly(Glu-Ser-Glu) favors the 2′–5′ internucleotide linkage. More interestingly, polypeptides containing arginine residues strongly accelerate the hydrolysis of oligoadenylic acids. The influence of pH, temperature, nature of the buffer and polypeptide sequence was investigated.     

Characterization of Water-Soluble Complexes of Polyacrylic Acid with α-Amylase from <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

Polycomplex formation of α-Amylase from Aspergillus oryzae (TAKA) with polyacrylic acid (PAA) was studied by pH titration, fluorescence, and high performance liquid chromatography (HPLC) methods in water solutions. Acording to the our results, the complex formation and its solubility were depended on nature of enzyme and the pH of solutions. Both of them correlates isoelectric points (PI). The stability of PAA–amylase complexes was negligibly weak at pH 7 [pH > pI (isoelectric pH)]. Stable water-soluble polycomplexes were formed at pH 5 (pI ~4.5) and coexisted with free protein molecules. Insoluble complexes has been observed at pH < 4.5. The frozen storage stabilities of the obtained complexes were also studied by measuring the activities at different pH.     

Growth yields and glucose metabolism of N<Subscript>2</Subscript>-fixing <Emphasis Type="Italic">Gluconacetobacter diazotrophicus</Emphasis> at different culture pH values

Gluconacetobacter diazotrophicus was grown in chemostat under N₂-fixing conditions at different culture pH values (from 2.5 to 7.5) with glucose as the C-source. Maximum glucose and oxygen utilization yields were observed at pH values between 5.0 and 6.5. Yields, although lower, were not severely affected at acidic (2.5–4.5) and moderate alkaline (7.5) pH values. But, at pH values just over 7.5, cultures became unstable and washed out. Maximum biomass yields coincided with optimal activity (and minimal synthesis) of pyrroloquinoline quinone (PQQ)-linked glucose dehydrogenase (PQQ-GDH). At external pH values of 7.0 and above, whereas PQQ-GDH was actively synthesized, a very low in situ activity could be detected. The lack of PQQ-GDH activity at moderate alkaline pH values seems to be the cause of lack of growth of this organism under these conditions.     

Production of xylanase by Aspergilli using alternative carbon sources: application of the crude extract on cellulose pulp biobleaching

The ability of xylanolytic enzymes produced by Aspergillus fumigatus RP04 and Aspergillus niveus RP05 to promote the biobleaching of cellulose pulp was investigated. Both fungi grew for 4–5 days in liquid medium at 40°C, under static conditions. Xylanase production was tested using different carbon sources, including some types of xylans. A. fumigatus produced high levels of xylanase on agricultural residues (corncob or wheat bran), whereas A. niveus produced more xylanase on birchwood xylan. The optimum temperature of the xylanases from A. fumigatus and A. niveus was around 60–70°C. The enzymes were stable for 30 min at 60°C, maintaining 95–98% of the initial activity. After 1 h at this temperature, the xylanase from A. niveus still retained 85% of initial activity, while the xylanase from A. fumigatus was only 40% active. The pH optimum of the xylanases was acidic (4.5–5.5). The pH stability for the xylanase from A. fumigatus was higher at pH 6.0–8.0, while the enzyme from A. niveus was more stable at pH 4.5–6.5. Crude enzymatic extracts were used to clarify cellulose pulp and the best result was obtained with the A. niveus preparation, showing kappa efficiency around 39.6% as compared to only 11.7% for that of A. fumigatus.     

Decolorization of Orange II dye by white-rot fungi

Agitation, temperature, inoculum size, initial pH and pH of buffered medium affected the decolorization of Orange II dye byCoriolus versicolor andFunalia trogii. The optimum temperature and initial pH value for decolorization were 30°C and 6.5–7.0, respectively; pH 4.5 was the most efficient in buffered cultures. High decolorization extents were reached at all agitation rates. At an inoculum size of more than 1 mL, the extent of decolorization changed only slightly. High extents were obtained using immobilized fungi at repeated-batch mode.     

Reversibility of structural rearrangements in the negative vesicular membrane upon electrostatic adsorption/desorption of the polycation

Interaction of small unilamellar vesicles (SUVs), composed of negative diphosphatidylglycerol (cardiolipin, CL(2-)) and neutral dipalmitoylphosphatidylcholine (DPPC), with poly(N-ethyl-4-vinylpyridinium bromide) (PEVP) was studied in water solution above and below the vesicular membrane melting point by means of differential scanning calorimetry, photon correlation spectroscopy, microelectrophoresis, conductometry, and fluorescence techniques. It has been found that CL(2-) species are homogeneously distributed within DPPC-CL(2-) SUV membrane leaflets and between them. Interaction of PEVP with DPPC-CL(2-) SUVs led to drastic structural rearrangements in the membrane if it was in the fluid state (liquid SUVs). Negative CL(2-) molecules migrated from the inner to the outer membrane leaflet and segregated in the vicinity of adsorbed PEVP chains. In addition, PEVP adsorption terminated completely the exchange of lipid molecules between the SUVs. At the same time, the integrity of liquid SUVs contacting PEVP remained unchanged. Since the interaction of PEVP with liquid SUVs was predominantly electrostatic in nature, the polycation could be completely removed from the vesicular membrane by addition of an excess of polyacrylic acid (PAA) polyanions forming a more stable electrostatic complex with PEVP. Removal of PEVP resulted in complete resumption of the original distribution of lipids in lateral and transmembrane directions as well as intervesicular lipid exchange. In contrast, PEVP interacting with DPPC-CL(2-) SUVs formed defects in the vesicular membrane if it was in the gel state (solid SUVs). Such interaction was contributed not only by electrostatic but most likely by hydrophobic interactions involving the defected membrane sites. PEVP kept contacting solid SUVs in the presence of an abundant amount of PAA. The established phenomena may be important for understanding the biological effects of polycations.     

Effect of pH on the stability of type-C toxin ofClostridium botulinum

Stability of type-C botulinum toxin at pH 1.8–12.0 and during exposure to 5 and 28°C for 20 and 16 h, respectively, was tested by titration on adult mice. The toxin was found in the samples kept at pH of 2.7–10.2, whereas, at the pH extremes of 1.8 and 12.0, it was inactivated.     

Microbial decolorization of azo dyes by Proteus mirabilis

A bacterium identified as Proteus mirabilis was isolated from acclimated sludge from a dyeing wastewater treatment plant. This strain rapidly decolorized a deep red azo dye solution (RED RBN). Features of the decolorizing process related to biodegradation and biosorption were also studied. Although P. mirabilis displayed good growth in shake culture, color removal was best in anoxic static cultures. For color removal, the optimal pH and temperature were 6.5–7.5 and 30–35°C, respectively. The organism exhibited a remarkable color removal capability, even at a high concentration of azo dye. More than 95% of azo dye was reduced within 20 h at a dye concentration of 1.0 g L⁻¹. Decolorization appears to proceed primarily by enzymatic reduction associated with a minor portion, 13–17%, of biosorption to inactivated microbial cells. Received 06 January 1999/ Accepted in revised form 22 April 1999