Thermochemical pretreatment of lignocellulose to enhance methane fermentation: II. Evaluation and application of pretreatment model

A model was developed and evaluated as a tool for predicting the formation of soluble products from staged thermochemical treatment of lignocellulosic materials under acidic conditions typical of autohydrolysis. The model was used to predict the general trend of hemi-cellulose and cellulose hydrolysis between pH 2 and 4 and temperatures of 170-230 degrees C, and results were compared with experimental data. When the model was evaluated for this range of temperatures and pH values, results indicated: (1) a relatively low temperature (175 degrees C) during the first stage allows hydrolysis of the hemi-cellulose polysaccharides without significant mono-saccharide decomposition, (2) subsequent stages at higher temperatures (equal or greater than 200 degrees C) are needed for significant cellulose hydrolysis, but glucose decomposition will also occur, and, (3) a pH in the range of 2-2.5 will enhance polysaccharide hydrolysis while limiting monosaccharide decomposition. The model's predictions, indicating that the formation of biodegradable products could be optimized using Pretreatments at pH 2-2.5 for the pH range evaluated, were confirmed in experiments with white fir as a representative lig nocellulose.     

Physicochemical Parameters for Growth of the Sea Ice Bacteria Glaciecola punicea ACAM 611T and Gelidibacter sp. Strain IC158

The water activity and pH ranges for growth of Glaciecola punicea (a psychrophile) were extended when this organism was grown at suboptimal rather than optimal temperatures. No such extension was observed for Gelidibacter sp. strain IC158 (a psychrotolerant bacterium) at analogous temperatures. Salinity and pH may be primary physicochemical parameters controlling bacterial community development in sea ice.     

Enzyme immobilization using a cellulose-binding domain: properties of a beta-glucosidase fusion protein.

Using molecular genetic techniques, a fusion protein has been produced which contains the cellulose-binding domain (CBD) of an exoglucanase (Cex) from Cellulomonas fimi fused to a beta-glucosidase (Abg) from Agrobacterium sp. The CBD functions as an affinity tag for the simultaneous purification and immobilization of the enzyme on cellulose. Binding to cellulose was stable for prolonged periods at temperatures from 4 degrees C to at least 50 degrees C, at ionic strengths from 10 mM to greater than 1 M, and at pH values below 8. The fusion protein can be desorbed from cellulose with distilled water or at pH greater than 8. Immobilized enzyme columns of the fusion protein bound to cotton fibers exhibited stable beta-glucosidase activity for at least 10 days of continuous operation at temperatures up to 37 degrees C. At higher temperatures, the bound enzyme lost activity. The thermal stability of the fusion protein was greatly improved by immobilization. Immobilization did not alter the pH stability. Except for its ability to bind to cellulose, the properties of the fusion protein were virtually the same as those of the native enzyme.     

B.K. virus haemagglutinin.

Among the widely applied buffered media, the HSAG (hepes-salt-albumin-gelatin) medium at pH 5.75--6.25 was found to be the most favourable for B.K. virus haemagglutinin titration. The optimum temperature was at 4 degrees C. The haemagglutinin was not affected by temperatures up to 37 degrees C, pHs between 5.5 and 9.5, and NaCl concentrations between 0.063 M and 2.56 M. When incubated at 56 degrees C, the haemagglutinin shows a time and pH dependent decline in titre. No significant time dependent titre fall occurred at 56 degrees C if NaCl molarity was varied between 1.31 and 2.56.     

Reassessment of the cold-labile nature of phosphofructokinase from a hibernating ground squirrel.

This study reassesses the proposal that cellular conditions of low temperature and relative acidosis during hibernation contribute to a suppression of phosphofructokinase (PFK) activity which, in turn, contributes to glycolytic rate suppression during torpor. To test the proposal that a dilution effect during in vitro assay of PFK was the main reason for activity loss (tetramer dissociation) at lower pH values, the influence of the macromolecular crowding agent, polyethylene glycol 8000 (PEG), on purified skeletal muscle PFK from Spermophilus lateralis was evaluated at different pH values (6.5, 7.2 and 7.5) and assay temperatures (5, 25 and 37degrees C). A 78 +/- 2.5% loss of PFK activity during 1 h incubation at 5 degrees C and pH 6.5 was virtually eliminated when 10% PEG was present (only 7.0 +/- 1.5% activity lost). The presence of PEG also largely reversed PFK inactivation at pH 6.5 at warmer assay temperatures and reversed inhibitory effects by high urea (50 or 400 mM). Analysis of pH curves at 5 degrees C also indicated that approximately 70% of activity would remain at intracellular pH values in hibernator muscle. The data suggest that under high protein concentrations in intact cells that the conditions of relative acidosis, low temperature or elevated urea during hibernation would not have substantial regulatory effects on PFK.     

Electron paramagnetic resonance studies on Pseudomonas nitrosyl nitrite reductase. Evidence for multiple species in the electron paramagnetic resonance spectra of nitrosyl haemoproteins.

The e.p.r. spectra of reduced 14NO- and 15NO-bound Pseudomonas nitrite reductase have been investigated at pH 5.8 and 8.0 in four buffer systems. At pH 8.0, absorption spectra indicated that only the haem d1 was NO-bound, but, although quantification of the e.p.r. signals in all cases accounted for NO bound the the haem d1 in both subunits of the enzyme, the precise form of the signals varied with buffer and temperature. A rhombic species, with gx = 2.07, gz = 2.01 and gy = 1.96, represented in the low-temperature spectra seen in all the buffers was converted at high temperatures (approx. 200K) into a form showing a reduced anisotropy. Hyperfine splitting on the gz component of this rhombic signal indicated a nitrogen atom trans to NO and it is proposed that histidine provides the endogenous axial ligand for haem d1. At pH 5.8, absorption spectra indicated NO binding to both haems c and d1 and e.p.r. quantifications accounted for NO-bound haems c and d1 in both enzyme subunits. The e.p.r. spectra at pH 5.8 were generally similar to those at pH 8.0 with respect to g-values and hyperfine coupling constants, but were broader with less well defined hyperfine splittings. As at pH 8, rhombic signals present in spectra at low temperatures were converted to less anisotropic forms at high temperatures. The results are discussed in relation to work on model nitrosyl-protohaem complexes [Yoshimura, Ozaki, Shintani & Watanabe (1979) Arch. Biochem, Biophys. 193, 301-313]. No. e.p.r. signal was observed from oxidized NO-bound Pseudomonas nitrite reductase at pH 6.0, over the temperature range 6-100K.     

Comparative study of energy-transducing properties of cytoplasmic membranes from mesophilic and thermophilic Bacillus species.

The properties of enzymes involved in energy transduction from a mesophilic (Bacillus subtilis) and a thermophilic (B. stearothermophilus) bacterium were compared. Membrane preparations of the two organisms contained dehydrogenases for NADH, succinate, L-alpha-glycerophosphate, and L-lactate. Maximum NADH and cytochrome c oxidation rates were obtained at the respective growth temperatures of the two bacteria. The enzymes involved in the oxidation reactions in membranes of the thermophilic species were more thermostable than those of the mesophilic species. The apparent microviscosities of the two membrane preparations were studied at different temperatures. At the respective optimal growth temperatures, the apparent microviscosities of the membranes of the two organisms were remarkably similar. The transition from the gel to the liquid-crystalline state occurred at different temperatures in the two species. In the two species, the oxidation of physiological (NADH) and nonphysiological (N,N,N',N'-tetramethyl-p-phenylenediamine or phenazine methosulfate) electron donors led to generation of a proton motive force which varied strongly with temperature. At increasing temperatures, the efficiency of energy transduction declined because of increasing H+ permeability. At the growth temperature, the efficiency of energy transduction was lower in B. stearothermophilus than in the mesophilic species. Extremely high respiratory activities enabled B. stearothermophilus to maintain a high proton motive force at elevated temperatures. The pH dependence of proton motive force generation appeared to be similar in the two membrane preparations. The highest proton motive forces were generated at low external pH, mainly because of a high pH gradient. At increasing external pH, the proton motive force declined.     

The self-association of chicken-erythrocyte histones.

The self-association of the separate histone fractions isolated from chicken erythrocytes has been studied in solution at a number of different pH values and ionic strengths. The apparent molecular weights of the histones were determined over a range of macromolecular concentrations using the techniques of osmotic pressure and sedimentation equilibrium. Histone F2c (H5) did not associate under any of the conditions investigated whereas the other histone fractions all appeared to undergo self-association forming dimers, dimers of dimers, etc. The degree of association increased with the pH and ionic strength of the medium. The tendency to aggregate increased in the order; histone F2c (H5) (non-aggregating), histone F2b (H2B), histone F2a2 (H2A), histone F3 (H3), histone F2a1 (H4) (highly aggregating). In the case of histone F2a2 (H2A) at pH 3.0 and ionic strength 0.1, the apparent weight-average molecular weight was determined at a number of macromolecular concentrations at five different temperatures. The self-association was analysed according to the method of Adams (published by Beckman Instruments Inc. in 1967) and shown to be a monomer-dimer-tetramer equilibrium. The association constants were evaluated at each of the temperatures studied and from their variation with temperature the values of the enthalpy and entropy of association were calculated. The intermolecular association was characterised by only a small change in enthalpy but a large, positive, change in entropy. This suggests that the association of histones at acid pH is due to hydrophobic interactions between the relatively uncharged segments of like polypeptide chains.     

Thermal denaturation and regeneration of japanese-radish peroxidase.

Thermal denaturation of Japanese-radish peroxidase [EC 1.11.1.7] was investigated with respect to its spectrophotometric properties and effect on the enzymatic activity. Inactivation of the peroxidase occurred at temperatures higher than 60degrees and involved three processes, i.e., dissociation of protohemin from the holoperoxidase, a conformation change in the apperoxidase, and the modification or degradation of protohemin. The splitting process of protohemin from holoperoxidase as followed by the change in the absorption spectrum at high temperatures coincided with the degrease in the activity, and it was found to be at least biphasic. The regeneration of peroxidase on cooling to room temperature was essentially reversible at neutral pH, while at pH 5 and pH 9 these processes were irreversible. The irreversibility at acidic pH was mainly due to an irreversible change in the conformation of the apoenzyme. The difference spectrum of heat-treated apoperoxidase exhibited a denaturation blueshift with negative maxima at 287 and 294 nm, and the total protein fluorescence quantum yield. qprotein, increased by 20% compared to that of the untreated apoenzyme. On the other hand, the irreversibility at alkaline pH was largely attributable to the modification of protohemin. Apoperoxidase was more resistnat to heat denaturation but the modification or degradation of protohemin in heated enzyme was greater at alkaline pH than at acidic pH. The pyridine-ferrohemochrome spectrum of peroxidase exhibited slight shifts of the maxima of the alpha-band to shorter wavelength on heat treatment, and the paper chromatogram showed the presence of a new derivative other than protohemin. The modified product is probably (2(4)-vinyl-4(2)-hydroxyethyldeuterohemin.     

The oxygen uptake of Sarotherodon niloticus L. and the oxygen binding properties of its blood and hemolysate (Pisces: Cichlidae)

The oxygen consumption of Sarotherodon niloticus L. was found to decline below a critical oxygen concentration of about 2 mg O2/l. An important influence of CO2 on the oxygen affinity of whole blood was observed at all temperatures between 20 and 35 degrees C for gas mixtures containing 5.6% CO2. Purified hemolysate showed extremely high oxygen affinities (p50 = 1.08 mmHg at pH 8.2 and 20 degrees C). Low cooperativity was observed at all temperatures from 20 to 35 degrees C, and pH values between 6.5 and 8.2. The Bohr effect proved to be important at pH values lower than pH 7.5 (phi = delta log P50/delta pH = -0.58 between pH 6.5 and 7.0 at 35 degrees C). The oxygen affinities show high thermal sensitivity without a marked pH influence (delta H value for overall oxygenation at pH was -71.7 kJ/mol). The obtained results are interpreted as adaptations to diurnal variations in ambient temperature and oxygen availability.     

Conformational analysis of the melanin-concentrating hormone core by circular dichroic spectroscopy. Disulphide bridge and tyrosine contributions.

A detailed circular dichroic (CD) study of the conformational flexibility of the melanin-concentrating hormone core [MCH(5-14)] is reported. Variable pH (2-10) and temperature (-80 degrees to +80 degrees C) in aqueous media reveal that CD contributions from tyrosine, disulphide bridge and the amide backbone can be discriminated. Only below -10 degrees C does a preferred -S-S-conformation (P chirality, dihedral angle phi = 90 +/- 10 degrees) dominate. The amide backbone CD contribution varies over all temperatures (-80 degrees to +80 degrees C) providing evidence for a type-II beta-turn at low temperatures, with the emergence of a type-I beta-turn at higher temperatures. Tyrosine exhibits a special behaviour at pH 7. These conclusions are in broad agreement with published NMR studies. Nevertheless, the MCH(5-14) core is seen to be conformationally flexible in aqueous solution at ambient temperatures. Conformation differences are observed in a non-aqueous environment.     

The influence of temperature and organic matter on the bactericidal activity of short-chain organic acids on salmonellas.

Acetic and lactic acids and BioAdd, a commercial preparation of formic and propionic acid, were tested at a concentration of 0.1% (w/w) at 20, 30, 40 and 50 degrees C and in the presence of organic material for bactericidal activity against Salmonella serotype Kedougou. BioAdd was the most active of the solutions at all temperatures, followed by lactic acid and acetic acid. The presence of horse blood at all four temperatures, and milk and serum at 50 degrees C, did not greatly affect the antibacterial activity of the acids although yeast extract (50 degrees C) provided some protection for the salmonella. Acid activity was related to low pH values although the bactericidal activity of acetic acid with blood and milk was greater than the unadulterated acid even though the pH was 0.4 units higher.     

Some catalytic and molecular properties of threonine deaminase from Bacillus stearothermophilus.

Threonine deaminase [EC 4.2.1.16] was highly purified from Bacillus stearothermophilus. The enzyme exhibited maximum activity at 65 degrees and at pH 9.2--9.6. It was inactivated on dilution and on storage at 4 degrees, but was protected by egg albumin. The enzyme was labile at 65 degrees, but became stable in the presence of egg albumin and isoleucine at pH 7.0. The substrate saturation curve for the enzyme reaction at 40 or 65 degrees was hyperbolic, but in the presence of isoleucine, the curve became sigmoidal (n = 2). The enzyme was more sensitive to isoleucine at 40 degrees than at 65 degrees, while valine slightly inhibited the enzyme at both 40 and 65 degrees. Inhibition of the enzyme by isoleucine was antagonized by valine at 40 and 65 degrees. These properties were essentially similar to those of the enzymes from mesophilic and thermophilic bacteria. The enzyme existed in two forms with different molecular sizes, 1.5-5 X 10(6) and 2 X 10(5) daltons, at pH 7.0 and at temperatures below 40 degrees. The larger component disaggregated into the small one at pH 8.5 or above, at temperatures above 50 degrees or in the presence of isoleucine and valine.     

Precipitation of egg white proteins below their isoelectric points by sodium dodecyl sulphate and temperature.

The precipitating of effect of sodium dodecyl sulphate (SDS) on the egg white proteins ovalbumin, conalbumin and lysozyme was studied at 25 degrees C and at different pH values. The proteins precipitated below their respective isolectric points, provided the detergent to protein ratio was appropriate. The pH profile of precipitation was different for the three proteins reflecting net charge differences. The binding of SDS to the proteins was studied with [35S]-labelled SDS and for ovalbumin a ratio of 21--28 SDS molecules/protein molecule, dependent on the concentration of SDS initially used, seem to be required for precipitation at pH 4.5. This number, however, is dependent on pH and increases with an increased positive net charge of the protein. The precipitating effect of SDS was identical for ovalbumin, conalbumin and lysozyme when compared on a gram to gram basis (0.1--0.15 g SDS/g precipitated protein). The precipitated protein was denatured as measured by differential scanning calorimetry, but was also completely redissolved if pH was increased to above the isoelectric point. The precipitating effecto f SDS was also examined at elevated temperatures. The two-phase systems of the proteins induced by SDS at 25 degrees C were heated from 25 degrees C to 90 degrees C at a rate of 1.25 degrees C/min. The precipitation behaviour was similar for the three proteins upon heating. When the SDS concentration was increased the precipitation curves were transferred towards lower temperatures and the courses of precipitation became less sharp. The synergistic effect of SDS and heat on protein precipitation was differentiated by denaturation measurements and radioactive labelling. The ratio SDS to precipitated protein gradually diminished towards higher temperatures but no purely thermal precipitation was found.     

Temperature dependence of the decay of the UV absorption difference spectrum of heavy meromyosin induced by adenosine triphosphate and inosine triphosphate.

The UV absorption difference spectrum of heavy meromyosin induced by ATP was measured at various temperatures. At higher temperatures, the difference spectrum formed rapidly after adding ATP and continued steadily during the steady state which we have called the ATP-form of difference spectrum. At lower temperatures, the ATP-form of difference spectrum decayed into the other form before the steady state was attained. This was identical to the difference spectrum obtained by adding ADP and has been called the ADP-form of difference spectrum. At intermediate temperatures, biphasic decay was observed. The results indicate that the dominant intermediate at the steady state is altered from the one showing the ATP-form of difference spectrum at higher temperatures to that showing the ADP-form at lower temperatures. The population of the two intermediates depends on the temperature between the two extremes. This temperature-induced transition was observed in the presence of any divalent cation such as Mg2+, Mn2+, or Ca2+. A similar transition was observed with the difference spectrum induced by ITP in the presence of MgCl2. The pH dependence of the single early decay of the ATP-induced difference spectrum was measured in the presence of MnCl2 at 1 degree. The apparent rate constant of the decay showed a biphasic pH dependence, having the same shape as the pH activity curve of ATPase [EC 3.6.1.3] observed at higher temperatures. The rate determining step for the steady state ATPase at higher temperatures is thought to be the step of changing from the intermediate complex showing the ATP-form of difference spectrum to that showing the ADP-form. This is inconsistent with our previous mechanism (Yazawa, M. et al. (1973) J. Biochem. 74, 1107-1117). The rate determining step at lower temperatures was assigned as a step of ADP dissociation.     

The influence of charge on phosphatidic acid bilayer membranes.

A complete titration of phosphatidic acid bilayer membranes was possible for the first time by the introduction of a new anaologue, 1,2-dihexadecyl-sn-glycerol-3-phosphoric acid, which has the advantage of a high chemical stability at extreme pH values. The synthesis of the phosphatidic acid is described and the phase transition behaviour in aqueous dispersions is compared with that of three ester phosphatidic acids; 1,2-dimyristoyl-sn-glycerol-3-phosphoric acid, 1,3-dimyristoylglycerol-2-phosphoric acid and 1,2-dipalmitoyl-sn-glycerol-3-phosphoric acid. The phase transition temperatures (Tt) of aqueous phosphatidic acid dispersions at different degrees of dissociation were measured using fluorescence spectroscopy and 90 degrees light scattering. The Tt values are comparable to the melting points of the solid phosphatidic acids in the fully protonated states, but large differences exist for the charged states. The Tt vs. pH diagrams of the four phosphatidic acids are quite similar and of a characteristic shape. Increasing ionisation results in a maximum value for the transition temperatures at pH 3.5 (pK1). The regions between the first and the second pK of the phosphatidic acids are characterised by only small variations in the transition temperatures (extended plateau) in spite of the large changes occurring in the surface charge of the membranes. The slope of the plateau is very shallow with increasing ionisation. A further decrease in the H+ concentration results in an abrupt change of the transition temperature. The slope of the Tt vs. pH diagram beyond pK2 becomes very steep. This is the result of reduced hydrocarbon interaction energy, which was demonstrated by differential scanning calorimetry (Blume, A. and Eibl, H., unpublished data).     

Phase behaviour of skin barrier model membranes at pH 7.4.

The main function of the skin is to protect the body against exogenous substances. The skin barrier is located in the outermost layer of the skin, the stratum corneum (SC). This layer consists of keratin enriched cells embedded in lipid lamellae that form the main barrier for diffusion of substances through the skin. The main lipid classes in this barrier are ceramides, cholesterol and free fatty acids. Cholesterol sulfate and calcium are also present in SC. Furthermore it has been suggested that a pH gradient exists. In a previous paper the effect of cholesterol sulfate and calcium on the lipid phase behaviour of mixtures prepared from cholesterol, ceramides and free fatty acids at pH 5 was reported (approximate pH at the skin surface). In the present study the phase behaviour of mixtures prepared from cholesterol, ceramides and free fatty acids prepared at pH 7.4 (the pH of viable cells) has been examined between 25 and 95 degrees C. Our studies reveal that a reversed hexagonal phase has been formed at elevated temperatures. Addition of calcium inhibits the formation of the reversed hexagonal phase, while cholesterol sulfate promotes the presence of the reversed hexagonal phase at increased temperatures. From our results we can conclude that the lipid mixtures prepared at pH 5 resemble more closely the lipid phase behaviour in intact SC than the lipid mixtures prepared at pH 7.4.     

Comparison of the inactivation of IgM and IgG complement fixation sites by acid and base.

Rabbit IgM antibodies to denatured mammalian or T6 bacteriophage DNA or poly(A)-poly(U) irreversibly lost complement-(C) fixation reactivity on exposure to low pH and reneutralization, with a halving of the complement-fixation titer occurring after treatment at about pH 3. The titers of IgG antibodies to denatured phage DNA, to poly(A)-poly(U), or to hemocyanin were halved only after exposure to pH 2. Inactivation by acid was enhanced by low protein concentrations, incubation at higher temperatures, and by slow reneutralization; under all these conditions it was more extensive with IgM than with IgG. Inactivation of IgM C-fixation activity at pH 2.5 and room temperature was a first order reaction, with a half-time of about 20 min. Both classes retained antigen-binding activity after exposure to pH 2. In the alkaline range, full C-fixation reactivity was retained by both classes after reneutralization from pH 11.5, some loss occurred at pH 12, and total irreversible inactivation occurred by pH 12.5. In the latter case, antigen-binding activity was also lost. The C-fixation inactivation curves in the alkaline range were similar for IgG and IgM antibodies.     

Calorimetric measurements of thermal denaturation of stefins A and B. Comparison to predicted thermodynamics of stefin-B unfolding.

Thermal denaturation of two homologous proteins, low-M(r) cysteine-proteinase inhibitors stefins A and B, has been investigated by microcalorimetry. Calorimetric enthalpies, as well as the temperatures at maximum heat capacity, were determined as a function of pH for each protein. Transitions were found reversible at all pH values examined (5.0, 6.5, 8.1) for the thermally more stable stefin A, in contrast to stefin B. Stefin B shows a sharp irreversible transition around 65 degrees C at pH 6.5 and 8.1, probably due to unfolding of a dimeric state followed by oligomerisation. At pH 5.0, both proteins exhibit a reversible transition with temperatures of half-denaturation at 50.2 degrees C and 90.8 degrees C for stefins B and A, respectively. The calorimetric enthalpies, which equal the van't Hoff enthalpies to within 10%, are 293 kJ/mol and 490 kJ/mol for stefins B and A, respectively. Using the predictive method of Ooi and Oobatake (1991) [Proc. Natl Acad. Sci. USA 88, 2859] the thermodynamic functions of unfolding were calculated for stefin B, whose three-dimensional structure has been determined. The calculated enthalpy, heat-capacity change on unfolding and the temperature of half denaturation compare well to the microcalorimetric data.     

5'-methylthioadenosine phosphorylase from Caldariella acidophila. 1. Purification and partial characterization]

5'-Methylthioadenosine phosphorylase has been isolated from C.acidophila, a thermophilic bacterium living in acid hot springs at temperatures ranging from 63 to 89 degrees C. The enzyme has been purified to homogeneity in 32% yield. The enzyme shows a high degree of thermophilicity, its temperature optimum being 93 degrees C in the in vitro assay. The enzyme is exceptionally stable; no loss of activity was observable after exposure for 1 h at 100 degrees C. The optimum pH is about 7,2, with one-half of the maximal activity occurring at pH 6 and 9. The apparent Km for the substrates are: 8,3 x 10(-5) M for MTA and 4,3 x 10(-4) M for phosphate ions.