Enzyme recirculation in saccharification of lignocellulosic materials

Steam-exploded aspen wood and wheat straw were enzymically hydrolysed for 2 days when sugar yields of 53% and 49% were obtained. Removal of hydrolysate after 1 day and continued hydrolysis for a further 24 h increased the yields to 67 and 56%, respectively. After hydrolysis, 50% or more of the enzymes was adsorbed on the solid residue with the remainder in solution along with the hydrolysate. Enzymes in the hydrolysate were easily recovered by a few minutes contact with a plug of new substrate. A small quantity of sugar is also adsorbed, but ≈90% passes through the substrate plug. We propose here a simple technique for recirculating the enzymes attached to the solid residue, thereby improving significantly the total enzyme recovery and sugar yield per enzyme unit. An enzyme recovery factor, ERF, was calculated on the basis of sugar yields obtained with recovered enzyme and was compared with the initial amount of enzyme. ERF values of 0.79 and 0.73 were obtained with steam-exploded aspen wood and wheat straw, respectively. Various aspects associated with the adsorption of enzymes in the hydrolysate onto new substrate and the extent to which sugars are bound to the substrate and residue are discussed.     

Re-sorption of organic compounds by roots of Zea mays L. and its consequences in the rhizosphere

The influx and efflux of sugar-C and the cycling of C within intact maize roots (Zea mays L.) was studied in sterile solution culture. Using metabolic inhibitors it was shown that roots could take up sugars against the concentration gradient probably via H⁺-ATPase dependent plasmalemma proton cotransporters. In contrast to this, no evidence was found for an ATPase mediated efflux of sugars from the root. All parts of the root were capable of taking up exogenous sugars. Examination of sugar exudation sites along the root slowed efflux at all locations, with the amount of efflux linearly correlated with internal cellular concentration. The results clearly indicated that the influxefflux mechanisms are linked both spatially, temporally and with respect to the sugars capable of transportation. The turnover of C within the root was found to be extremely rapid with turnover of the soluble sugar pool being 0.8 to 15 times daily depending on root spatial location. The results strongly suggest that the recapture of sugars from outside the root plays an important role in regulating the amount of C lost to the soil which in turn will reduce both pathogen attraction and the size of the rhizosphere microbial population and will also increase the plant's C efficiency.     

小分子有机酸对恒电荷土壤胶体Pb2＋吸附-解吸的影响

供试土壤胶体对Pb²⁺吸附及吸附态Pb²⁺的解吸等温线均符合Freundlich和Langmuir等温式,吸附常数Ka值大小为塿土>黄绵土>黑垆土>黄褐土,其大小次序与表面总电荷密度σ0大小一致,表明了各土壤胶体对Pb²⁺吸附强度的大小,在小分子有机酸作用下,吸附量降低,吸附亲和力增加,柠檬酸的影响大于草酸的影响;解吸后残留Pb^2+吸附常数杨值的大小基本为塿土>黄褐土>黑垆土>黄绵土,反映了解吸残留Pb²⁺吸附强度的大小,与各土壤胶体有机质和游离氧化铁含量有关,在NaNO₃和草酸溶液中,吸附-解吸等温线相距较远,吸附-解吸之间存在着滞后性;在柠檬酸作用下,吸附-解吸等温线基本接近,二者之间具有一定的可逆性。     

Effect of solution conditions on protein damage in foam

In this study we investigated the conditions under which protein damage during foaming could be reduced. We used bovine serum albumin (BSA), immunoglobulin G (IgG), pepsin, catalase and lysozyme. The parameters examined were ionic strength, pH, protein concentration and the addition of sugars (trehalose and sucrose). Results showed that protein damage can be reduced by operating at optimal ionic strength and pH, and to a lesser extent, by the addition of sugars. Solution conditions under which the native structure of the protein was stabilised in solution favoured a reduction in the amount of damage, due to lower surface adsorption. The actual quantity of protein damaged in foaming was found to be relatively insensitive to changes in the bulk protein concentration, provided that the concentration was near to, or greater than, the apparent CMC value.     

Adsorption of fibrinogen on silicon oxide with controlled hydrophobic/hydrophilic properties

The amount of fibrinogen irreversibly adsorbed on silicon dioxide does not exceed 3.6 pmol/cm2 and depends on the protein concentration, solution pH and surface hydrophobic/hydrophilic properties. Electrostatic interactions determine the fibrinogen adsorption rate. Partial denaturation of fibrinogen takes place in its adsorption form diluted solutions with the pH value lower than the protein isoelectric point.     

Interaction between collagen and adenosine 5'-triphosphate.

A quantitative study was made of the adsorption of adenosine 5′-triphosphate (ATP) on collagen by following the change in the absorbance at 258 nm of ATP in the soaking solution. The amount of ATP adsorbed decreased exponentially with the increase of pH up to pH 8 and fell off more rapidly at higher pH values. At a given pH, when the concentration of ATP was increased, the amount of ATP adsorbed increased following the pattern of a Langmuir isotherm. The adsorption was independent of the cation present. The adsorption of adenosine 5′-diphosphate was essentially the same as that for ATP. For tendons deposited with calcium phosphate, the amount of ATP adsorbed decreased compared to natural tendons. The adsorption of ATP on collagen fibers inhibited the contraction caused by calcium chloride, calcium bromide, and lithium bromide. In solution, ATP had very little effect on the denaturation of acid-soluble collagen caused by calcium chloride.     

The control of the proventriculus in the honeybee (Apis mellifera carnica L.) I. A dynamic process influenced by food quality and quantity?

The control of crop emptying in foraging honeybees was investigated in individuals trained to collect defined amounts of sugar solutions. Following feeding, they were dissected after fixed periods of time in order to measure crop content and haemolymph sugar titers. Between feeding and dissection, the metabolic rate of every investigated forager was measured using open-flow respirometry, so as to assess the effects of both food quality (concentration, molarity and viscosity of the fed sugar solution) and food quantity on the transport rate through the proventriculus. The sugar transport rate through the proventriculus was observed to be mainly dependent on the metabolic expenditure of the individual. Bee foragers were able to precisely adjust the sugar transport rate to their metabolic rates, but under certain conditions, an excess of sugars was transported through the proventriculus, more than needed to cover the bee’s energetic demands. This excess depended on the nutritive value and quantity of the fed sugar solution, and on the time after feeding. It did not depend on the metabolic rate of the bee, the molarity, or the viscosity of the fed sugar solution. As long as the bees did not exhaust their crop contents, the haemolymph sugar titers were unaffected by this excess amount transported, by the time after feeding, the concentration and the viscosity of the fed sugar solution. For all feeding conditions assayed, the haemolymph trehalose titer remained constant, while the titers of other haemolymph sugars varied. It is suggested that the trehalose concentration in the haemolymph is regulated in honeybees, and that it represents the controlled variable in the feedback loop responsible for the transport rate through the proventriculus.     

PROTEIN FILMS ON COLLODION MEMBRANES

1. Collodion membranes of high permeability were found to adsorb weighable amounts of gelatin and egg albumin from solution at 37°C. 2. The effect of protein concentration could be expressed fairly well by a hyperbolic equation proposed by Langmuir for the adsorption of gases by a plane surface, while the usual parabolic adsorption equation of Freundlich did not fit the results. 3. In comparing this effect with solutions of varying pH, it was found there was a decided maximum of adsorption in solutions of isoelectric protein. The effects of acids and salts on the amount of gelatin adsorbed were like those observed by Loeb on the viscosity of gelatin solutions, but opposite in direction. The effects of pH on the amount of adsorbed gelatin and on the fluidity of the gelatin solutions were nearly parallel. 4. Membranes made impermeable by long drying took up very little or no gelatin from solution. 5. In the case of membranes of varying permeability the maximum amount of adherent gelatin increased with the permeability and thickness of the membranes, and appeared to be, within limits, a linear function of the relative pore surface of the membranes as calculated from Poiseuille''s law. 6. The film of gelatin greatly decreased the permeability of the membranes, as measured by the flow of water through them. The relative cross-section of the pore openings, as calculated from the permeability measurements, was a linear function of the amount of adherent gelatin. These results led to the conclusion that the gelatin formed a film inside the pores.     

Adsorption of BSA on QAE-dextran: equilibria

Equilibrium isotherms for adsorption of bovine serum albumin (BSA) on a strong-base (QAE) dextran-type ion exchanger have been determined experimentally. They were not affected by the initial concentration of BSA but were affected by pH considerably. They were correlated by the Langmuir equation when pH >/= 5.05 and by the Freundlich equation of pH 4.8, which is close to pl approximately 4.8 of BSA. The contribution of ion exchange to adsorption of BSA on the ion exchanger was determined experimentally. The maximum amounts of inorganic anion exchanged for BSA were 1% and 0.4% of the exchange capacity of the ion exchanger at pH 6.9, respectively. Since the effect of the ion exchange on the adsorption appeared small, BSA may be adsorbed mainly by electrostatic attraction when pH >/= 5.05 and by hydrophobic interaction or hydrogen bonding at pH 4.8. When NaCl coexisted in the solution, the shape of the isotherm was similar to the Langmuir isotherm, but it is shifted to the right. When the concentration of NaCl was 0.2 mol/dm(3), BsA was not adsorbed on the resin. When BSA was dissolved in pure water, the saturation capacity of BSA on HPO(4) (2-),-orm resin was about 2 times larger than that for adsorption from the solution with buffer (pH 6.9 and 8.79). The saturation capacity for adsorption of BSA in pure water on HPO(4) (2-) + H(2)O(4) (-)-from resin was much smaller than that from the solution with buffer. The isotherms for univalent Cl(-)-and H(2)PO(4) (-)-form resin was peculiar; that is, the amount of BSA adsorbed decreased with increasing the liquid-phase equilibrium concentration of BSA. (c) 1993 John Wiley & Sons, Inc.     

Adsorption, desorption, and activity of glucose oxidase on selected clay species.

The adsorption of the enzyme glucose oxidase (EC 1.1.3.4) to clays followed the pattern described for other proteins as being pH dependent. Maximum adsorption occurred at or below the isoelectric point of the enzyme. The amount of enzyme adsorbed to clay was influenced by the type of clay used, and also the saturating cations. Initially adsorbed enzyme showed low specific activities, and as amounts of enzyme adsorbed approached maximum stauration of clay, specific activities increased approaching that determined for free enzyme. The adsorption of glucose oxidase involved a temperature-independent cation-exchange mechanism, and enzyme adsorbed to surfaces of clay could be desorbed in active form by elevation of pH of suspending solution. This was followed by a slower temperature-dependent fixation, probably by hydrogen bonding, which resulted in protein being irreversibly adsorbed to clay surfaces. It is proposed that on adsorption of glucose oxidase to clay surfaces unravelling of the protein structure occurred, which allowed penetration of protein into the interlamellar spaces of montmorillonite. This proposal was based on the observed expansion of montmorillonite to 23 A, and the decreases in amount of a second-protein lysozyme adsorbed with extended incubation times of glucose oxidase - clay complexes at pH 4.5.     

Adsorption of β-Lactoglobulin onto the Surface of Stainless Steel Particles

The adsorption of β-Lactoglobulin (β-Lg), one of the main constituents of fouling deposits in milk processing, onto the surface of stainless steel particles was studied under various conditions. The adsorption isotherm of β-Lg at 25°C was of the Langmuir type, and the plateau suggested that the surface was covered by a monolayer of β-Lg. The amount of β-Lg adsorbed steeply increased above 65°C. At 75°C, it increased almost linearly with the protein concentration in the bulk solution. Heating and chemical modification of the SH-group caused a much smaller amount of β-Lg to be adsorbed at 75°C. These findings indicate that the thermal aggregation of denatured β-Lg at the surface is important in the adsorption. More β-Lg was adsorbed at pH 4 than at pH 6.85. This suggests that the electrostatic interaction between β-Lg and the surface contributes to the adsorption behavior.     

The control of the proventriculus in the honeybee (Apis mellifera carnica L.) II. Feedback mechanisms

The mechanisms underlying the control of solution transport rates through the proventriculus in foraging honeybees were investigated in individuals trained to collect defined amounts of sugar solutions. Following feeding, bees were injected either with metabolisable (glucose, fructose, trehalose), or non-metabolisable (sorbose) sugars, in order to distinguish between haemolymph osmolarity and haemolymph sugar levels as factors controlling the solution transport rates through the proventriculus. After a fixed period, workers were dissected in order to measure crop content and haemolymph sugar titers. Between feeding and dissection, the metabolic rate of every investigated forager was measured using open-flow respirometry. Bees injected with metabolisable sugars 15 min after feeding were observed to reduce their solution transport rates through the proventriculus, but injection of non-metabolisable sugars had no influence on them. This suggests that the solution transport rate through the proventriculus is controlled by the concentration of metabolisable compounds in the haemolymph, and not by the haemolymph osmolarity. A period of 10 min after injection of metabolisable sugars was enough to observe reduced solution transport rates. However, if bees were injected only 5 min after feeding, no reduced solution transport rates were observed 10 min after injection.     

Enzymic saccharification of pretreated wheat straw

Studies of pretreatment of wheat and its subsequent saccharification by Trichoderma reesei cellulases are reported. Steam explosion was found to be the most effective of the pretreatment methods tested. Data are presented describing the effect of enzyme and substrate concentration on the rate and degree of hydrolysis. Significant inhibition of the cellulases was observed when sugar concentrations were 6% or higher. This inhibition increased when glucose and ethanol were present simultaneously. Adsorption of enzymes to the substrate was followed during a 24-h hydrolysis period. An initial rapid and extensive adsorption occurred, followed by a short desorption period that was followed in turn by a further increased adsorption peaking after 3 h. Intermediate removal of hydrolysate, particularly in combination with a second addition of enzyme, clearly improved the yield of saccharification compared to an uninterrupted hydrolysis over a 24-h period. Thus, a 74% yield of reducing sugars was obtained. Furthermore, an increase in the amount of recoverable enzymes was observed under these conditions. Evidence is presented that suggests that a countercurrent technique, whereby free enzymes in recovered hydrolysate are adsorbed onto new substrate, may provide a means of recirculating dissolved enzymes.     

Effects of sulphate and pH on the plant-availability of phosphate adsorbed on goethite

The adsorption of phosphate on metal (hydr)oxides may be influenced by the pH and by the adsorption of other ions. In this study, the influence of sulphate and pH on phosphate adsorption on goethite and the availability to plants of adsorbed phosphate was examined. Maize plants were grown on suspensions of goethite with adsorbed phosphate, containing the same total amount of phosphate and either 0.11 mM or 2.01 mM sulphate at pH 3.7, 4.6 or 5.5. The uptake of phosphorus by the plants increased with the larger sulphate concentration and decreasing pH. Mean P uptake in the treatment with 2.01 mM sulphate and pH 3.7 was 55 µmol plant^-1, whereas in the treatment with 0.11 mM sulphate and pH 5.5 it was 2 µmol plant^-1. Batch adsorption experiments using³² P and speciation modelling of ion adsorption showed that in the presence of sulphate, the phosphate concentration in solution strongly increased with decreasing pH, due to competitive adsorption between sulphate and phosphate on goethite. Modelled phosphate concentrations in solution in the uptake experiment were all below 0.6 µM and correlated well with the observed P uptake. This correlation indicates that the strong influence of the sulphate concentration and pH on the plant-availability of adsorbed phosphate results from the competition between sulphate and phosphate for adsorption on goethite.     

Adsorption of thermomonospora fusca E(5) cellulase on silanized silica

The adsorption kinetics and dodeceyltrimethylammonium-bromide-mediated elution of Thermomonospora fusca E(5) cellulase were recorded in situ, at hydrophobic, silanized silica. Experiments were performed at different solution concentrations, ranging from 0.001 to 0.70 mg/mL. Plateau values of adsorbed mass generally increased with increasing solution concentration, with the adsorbed layer being only partially eluted by buffer. Treatment with surfactant removed more of the adsorbed enzyme in each case, with the remaining adsorbed mass varying little among experiments. Adsorption of E(5) into this nonremovable state was suggested to occur early in the adsorption process and continue until some critical surface concentration was reached. Beyond this critical value of adsorbed mass, adsorption progressed with the protein adopting more loosely bound states. Adsorption kinetic data were interpreted with reference to an adsorption mechanism allowing for irreversible adsorption into two dissimilar states. These states were distinguished by differences in occupied interfacial area, and binding strength, presumably a result of differences in structure. Comparison of the data to the kinetic model based on this mechanism showed that the fraction of adsorbed molecules present in the more tightly bound state decreased as adsorption occurred from solutions of increasing concentration. However, the absolute values of more tightly bound molecules were less dependent on adsorption conditions.     

Reduction of albumin adsorption onto silicon surfaces by Tween 20

The ability of Tween 20 to reduce the adsorption of albumin on silicon surfaces of different hydrophobicity was investigated by ellipsometry. As expected, protein adsorption was found to depend on the degree of hydrophobicity of the surfaces and on the concentration of the surfactant. A reduction of 90% in albumin adsorption on hydrophobic methylated surfaces by 0.05% Tween 20 was achieved, whereas a reduction of only 15% on hydrophilic surfaces was observed. Experiments of time-dependent protein adsorption in both pure protein and protein-surfactant mixtures were conducted to ascertain the stability of physically adsorbed Tween 20 films on intermediate silicon surfaces. It was found that the adsorbed Tween 20 film was robust and there was no evidence of exchange of the Tween molecules with albumin for up to 240 min exposure. Adsorption minima were confirmed to correlate with minima in contact angle and critical micelle concentration (CMC). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 618-625, 1997.     

Pysicochemical studies of taste reception. V. Suppressive effect of salts on sugar response of the frog.

The tast responses of frog to various kinds of sugars were measured quantitatively by use of the glossopharyngeal nerve activity under an appropriate condition where the water response was completely suppressed. The concentration dependences of response of frog tongue to D-fructose, D-glucose, and sucrose were almost the same, D-galactose, however, elicited a much larger response in comparison with the other sugars in the whole range of concentrations examined. The sugar response was suppressed extensively by the presence of small amount of salts in the stimulating sugar solution. The suppressive effects of NaCl, KCl, MgCl2, MgSO4, and K4Fe(CN)6 were examined with a fixed concentration of sugar. The results obtained with these salts, added in various concentrations, fell on a single curve when the data were plotted against the ionic strength in the stimulating solution. The present results were consistent with the notion that the taste receptor potential for salts or acids is attributable to a change in the phase boundary potential at the membrane-solution interface as proposed in the previous papers of this series.     

草酸对土壤胶体与矿物表面酶的吸附及活性影响

采用平衡批处理法,研究了模拟根系分泌物--草酸溶液的浓度、pH对酸性磷酸酶在针铁矿、高岭石及黄棕壤和砖红壤胶体(＜2μm)上的吸附及比活的影响.结果表明,针铁矿对磷酸酶的吸附量受草酸浓度的影响较小,其它供试胶体对蛋白的吸附量随草酸浓度的升高,一般表现为先急剧降低(0～5mmol·L^-1),之后逐渐升高到与对照相当或略低.这与草酸在土壤胶体和矿物表面的配位形态及其对载体表面的电荷改变、溶解有关.草酸体系中,供试胶体对磷酸酶的吸附顺序为针铁矿>黄棕壤＞高岭石＞砖红壤.酶在草酸体系中的最大吸附点位一般出现在蛋白的等电点(IEP)和供试胶体的PZC之间,而酶在草酸体系中被固定到供试胶体上之后,其最适比活点随胶体类型的不同而没有变化或有所高移.     

乙酸对土壤胶体矿物吸附酸性磷酸酶的影响

研究了不同pH值、不同浓度乙酸对酸性磷酸酶在土壤胶体和矿物表面吸附的影响,结果表明,在pH2～8的乙酸体系中,酶在胶体矿物表面的最大吸附pH一般出现在蛋白的等电点和矿物的零电荷点(PZC)之间,各土壤胶体和粘粒矿物对酶的吸附量大小顺序为针铁矿》黄棕壤>砖红壤>高岭石>二氧化锰,乙酸浓度对酶在胶体矿物表面的吸附量和吸附结合能具有较显著影响,在0～200mmol·L^-1范围内,随着乙酸浓度的增加,酶吸附量呈现先升高、后降低、再稳定的趋势,而吸附结合能的变化与此相反,并就乙酸对酶在胶体矿物表面吸附影响的可能机理进行了初步探讨。     

De-icers derived from corn steep water

Corn steep water (CSW) and other byproducts derived from fermentations and sugar productions are presently forming the base of compositions for de-icing and anti-icing materials. Since the de-icing and anti-icing values are in part a colligative property, increase in the molar concentration of ionic species has been frequently necessary to decrease further the freezing point of this byproducts stream. In the present study this has been achieved by the generation of biodegradable organic acid salts in situ, without the use of chloride or other inorganic salts, by the alkaline degradation of reducing sugars added to corn steep water, which alone is not an efficient de-icer. Reducing sugars, such as glucose, react with alkali metal hydroxides to produce principally hydroxy carboxylic acids that react with the alkali metal hydroxide to form a mixture of organic acid salts. The ionic strength of the resulting solution is increased since each sugar molecule produces nearly two acid molecules upon degradation. The ionic strength necessary to achieve the desired freezing point depression is determined by the amount and concentration of the alkali metal hydroxide used, with the necessary counter anions being derived from the degradation of the reducing sugar. The amount of the sugar used is that required to result in a near to neutral final solution. The well-known anti-corrosive property of CSW is used in the de-icer preparations, either by conducting the alkaline degradation of the sugar in this medium, or by using water for the degradation of the sugar followed by dilution of the resulting solution with CSW to adjust the viscosity of the final solution to meet the requirements for spraying. The monovalent metal hydroxides are more efficient in producing de-icer solutions than the divalent metal hydroxides.