Insights into hydrogen bonding and stacking interactions in cellulose.

In this quantum chemical study, we explore hydrogen bonding (H-bonding) and stacking interactions in different crystalline cellulose allomorphs; namely, cellulose I(ß) and cellulose III(I). We consider a model system representing a cellulose crystalline core made from six cellobiose units arranged in three layers with two chains per layer. We calculate the contributions of intrasheet and intersheet interactions to the structure and stability in both cellulose I(ß) and cellulose III(I) crystalline cores. Reference structures for this study were generated from molecular dynamics simulations of water-solvated cellulose I(ß) and III(I) fibrils. A systematic analysis of various conformations describing different mutual orientations of cellobiose units is performed using the hybrid density functional theory with the M06-2X with 6-31+G(d,p) basis sets. We dissect the nature of the forces that stabilize the cellulose I(ß) and cellulose III(I) crystalline cores and quantify the relative strength of H-bonding and stacking interactions. Our calculations demonstrate that individual H-bonding interactions are stronger in cellulose I(ß) than in cellulose III(I); however, the total H-bonding contribution to stabilization is larger in cellulose III(I) because of the highly cooperative nature of the H-bonding network. In addition, we observe a significant contribution from cooperative stacking interactions to the stabilization of cellulose I(ß). The theory of atoms-in-molecules (AIM) has been employed to characterize and quantify these intermolecular interactions. AIM analyses highlight the role of nonconventional CH···O H-bonding in the cellulose assemblies. Finally, we calculate molecular electrostatic potential maps for the cellulose allomorphs that capture the differences in chemical reactivity of the systems considered in our study.

MALDI-MS as a monitor of the purification and folding of synthetic eclosion hormone.

Analogues of the small protein Manduca sexta eclosion hormone (62 amino acids) were synthesized by Fmoc solid-phase methodology. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was used to analyze the products of the syntheses and this information was used to design an efficient purification scheme. MALDI-MS was used to monitor the target products through purification and it was also used to monitor folding of the purified materials. The folded EH analogues were shown to be biologically active proteins with an in vivo bioassay using pharate adult moths, Heliothis virescens.

Experimental and theoretical evidence for convective nutrient transport in an immobilized cell support.

Even though immobilized-cell reactors possess several engineering advantages over free-cell reactors, their full potential has not been realized because mass transfer often limits the rate of nutrient supply and product removal from immobilized cell supports. We studied the interaction between mass transfer and reaction kinetics in the anaerobic conversion of glucose to CO2 and ethanol by yeast immobilized in a porous rotating disk on the agitator shaft of a conventional CSTR. A Sherwood number correlation was used to show that external mass-transfer resistances were negligible under typical operating conditions. The modulus of Weisz based on observable reaction parameters was used to gauge the importance of pore diffusion limitations. Under conditions for which significant pore diffusion effects and hence low effectiveness factors (eta = ca. 0.1) would be predicted, the observed reaction rates were much higher than expected (eta = ca. 1), suggesting that pore diffusion limitations were at least partially relieved by convective transport of glucose into the support. Two possible mechanisms of convective transport are discussed. We hypothesize that gas evolution was responsible for the convective enhancement of glucose supply.

Heterologous protein expression affects the death kinetics of baculovirus-infected insect cell cultures: a quantitative study by use of n-target theory.

The death of cultured insect cells after baculovirus infection is a time-dependent event. Without a quantitative model, it is difficult to characterize its kinetics. Our group has shown that the cell survival rate can be characterized by use of the n-target theory, which involves only two parameters: the number of hypothetical inactivation targets (n) and the first-order death rate (k). In this study, we used different recombinant viruses to examine the effect of heterologous protein expression on the cell survival rate. The proteins expressed were beta-galactosidase, human T-cell leukemia virus type I p40x, human interleukin-2, and human tissue plasminogen activator (tPA). The survival rate was affected by protein expression, but the n value remained constant if the protein expression level was high (above 30 mg/L). Low-level expression of secreted, glycosylated tPA resulted in a reduced n value, which was restored to the normal value when the tPA signal peptide and prosequence were deleted. In addition, if the n value was normal (10-11), the level of protein expression correlated negatively with the death rate. However, if the n value was reduced by unfavorable culture conditions or foreign protein expression, the expression level correlated positively with the death rate. A dimensionless plot with kt as the dimensionless time shows that alteration of the k value while retaining constant n is equivalent to a rescaling of time. Therefore, the survival curves with constant n reduce to a single curve on the dimensionless plot.(ABSTRACT TRUNCATED AT 250 WORDS)

Alteration of glucose consumption kinetics with progression of baculovirus infection in Spodoptera frugiperda cells.

We have used the initial-rate approach to characterize changes in the glucose consumption kinetics of baculovirus-infected Spodoptera frugiperda clone 9 (Sf9) cells with the progression of the infection process. The specific glucose consumption rate (qG) of cultured baculovirus-infected Sf9 cells was measured at 4, 8, 12, 16, and 24 h postinfection (h.p.i.) in media containing 4-35 mM glucose. Higher medium glucose concentrations resulted in higher final extracellular virus and recombinant beta-galactosidase yields. qG was related to the extracellular glucose concentration by means of a Michaelis-Menten relationship. The apparent Michaelis-Menten constant (K(m)) for glucose consumption was found not to change significantly during the progression of the infection process, and remained between 6.2 and 7.2 mM. However, the maximal specific glucose consumption rate (qGmax) was found to rapidly increase after infection, peaking at 16 h.p.i. at a value four times that for uninfected Sf9 cells. The kinetic analysis of glucose consumption rates in baculovirus-infected Sf9 cells presented here will aid in the optimal design and operation of bioreactor systems for the large-scale production of recombinant products from the baculovirus/insect cell system.

Enzymatic hydrolysis of ammonia-treated sugar beet pulp.

Sugar beet pulp is a carbohydrate-rich coproduct generated by the table sugar industry. Beet pulp has shown promise as a feedstock for ethanol production using enzymes to hydrolyze polymeric carbohydrates and engineered bacteria to ferment sugars to ethanol. In this study, sugar beet pulp underwent an ammonia pressurization depressurization (APD) pretreatment in which the pulp was exploded by the sudden evaporation of ammonia in a reactor vessel. APD was found to substantially increase hydrolysis efficiency of the cellulose component, but when hemicellulose- and pectin-degrading enzymes were added, treated pulp hydrolysis was no better than the untreated control.

Optimizing ammonia processing conditions to enhance susceptibility of legumes to fiber hydrolysis: alfalfa.

An ammonia process was applied at several ammonia loadings, moisture contents, temperatures, and dwell times. A cellulase loading of 5 FPU/g dry matter and a 24 h incubation time were used to produce the sugars, which were measured as reducing sugars and by HPLC. Optimal processing conditions caused a 76% of theoretical yield (2.9-fold above untreated). Cellulose and hemicellulose conversions were 68 and 85% (vs 38 and 34% in untreated, respectively). The short hydrolysis time and relatively low enzyme loading suggests great potential to produce sugars from alfalfa.

Optimizing ammonia processing conditions to enhance susceptibility of legumes to fiber hydrolysis: Florigraze rhizoma peanut.

A warm-season legume, Florigraze rhizoma peanut (FRP), was used as the source of fiber to produce sugars. FRP was subjected to several ammonia-processing conditions using temperature, biomass moisture content, and ammonia loading as process variables during a 5-min treatment. A cellulase loading of 2 FPU/g DM and 24 h incubation were used to produce the sugars. Total sugar yield was 3.34-fold higher in the optimal treatment (1.5 g ammonia/g DM-60%-90 degrees C) compared to untreated and was 65.3% of theoretical. Cellulose and hemicellulose conversions increased from 30 and 15.5% in untreated FRP to 78 and 34% in treated FRP.

Optimizing ammonia pressurization/depressurization processing conditions to enhance enzymatic susceptibility of dwarf elephant grass.

An ammonia pressurization/depressurization process was investigated to evaluate the potential of producing reducing sugars from dwarf elephant grass, a warm-season forage. Moisture, temperature, and ammonia loading affected sugar yield (p < 0.0001). At optimal conditions, ammonia processing solubilized 50.9% of the hemicellulose and raised the sugar yield (percentage of theoretical) from 18 to 83%. Glucose and xylose production were increased 3.2- and 8.2-fold, respectively. The mild processing conditions of the ammonia treatment (90-100 degrees C, 5 min), the low enzyme loading (2 international filter paper units/g), and the short hydrolysis time (24 h), greatly enhance the potential of using forages to produce sugars valuable for several applications.

An attempt towards simultaneous biobased solvent based extraction of proteins and enzymatic saccharification of cellulosic materials from distiller's grains and solubles.

Distiller's grains and solubles (DGS) is the major co-product of corn dry mill ethanol production, and is composed of 30% protein and 30-40% polysaccharides. We report a strategy for simultaneous extraction of protein with food-grade biobased solvents (ethyl lactate, d-limonene, and distilled methyl esters) and enzymatic saccharification of glucan in DGS. This approach would produce a high-value animal feed while simultaneously producing additional sugars for ethanol production. Preliminary experiments on protein extraction resulted in recovery of 15-45% of the protein, with hydrophobic biobased solvents obtaining the best results. The integrated hydrolysis and extraction experiments showed that biobased solvent addition did not inhibit hydrolysis of the cellulose. However, only 25-33% of the total protein was extracted from DGS, and the extracted protein largely resided in the aqueous phase, not the solvent phase. We hypothesize that the hydrophobic solvent could not access the proteins surrounded by the aqueous phase inside the fibrous structure of DGS due to poor mass transfer. Further process improvements are needed to overcome this obstacle.

Nuclear magnetic resonance spectroscopy of dense cell populations for metabolic studies and bioreactor engineering: a synergistic partnership.

Commercial exploitation of the fruits of recombinant DNA and cell fusion technologies is significantly limited by the lack of fundamental metabolic information on the cell lines of interest, whether these are plant, animal, insect, or microbial cells. NMR can help to provide this information and thereby improve bioreactor design and operation. However, in the case of on-line NMR of dense cell culture devices for metabolic studies, these devices are inherently heterogeneous bioreactors. To ensure that the metabolic information generated is reliable, a number of precautions should be taken. These are the same precautions that should be taken to ensure that commercial bioreactors operate in a reaction-controlled regime. Therefore, reactor engineering methodologies, particularly diffusion and reaction analyses and reaction monitoring by whole-cell NMR must go hand in hand, each extending, complementing, and validating the other.

Enzymatic hydrolysis and recrystallization behavior of initially amorphous cellulose.

Cellulose samples from cotton and wood pulps with varying low degrees of crystallinity (mechanically decrystallized) were studied. The influence of initial cellulose crystallinity on sugar yield after enzymatic hydrolysis was determined by two different methods. As expected, samples with low crystallinity were much more accessible to enzymatic attack and glucose yields were higher than were samples of high initial crystallinity. Hydrolysis of cellulose seems more dependent on cellulose crystallinity than on the source of cellulose. It is known that decrystallized or amorphous cellulose can recrystallize under proper conditions, e.g., during acid hydrolysis. The data reported here also reveal some recrystallization during enzymatic hydrolysis which probably occurs simulataneously with a selective enzymatic attack on the amorphous regions of cellulose. In all cases, the amorphous celluloses recrystallized in the original lattice form, that of native cellulose.

Degradation of ribonucleic acid by immobilized ribonuclease.

An immobilized enzyme (pancreatic ribonuclease bound to porous titania) was investigated for the degradation of purified yeast ribonucleic acid as a substrate. The immobilized enzyme is active and stable in the pH range 4--8. Dependence of enzymatic activity on ionic strength, pH, temperature, fluid flow rate, and substrate concentration were investigated. A cumulative fluid residence time of 6 sec is sufficient for 50% substrate conversion at 25 degrees C and pH 7.0. The critical flow rate (i.e., the fluid flow rate necessary to remove film diffusion resistance) approximately doubles with each 10 degree C rise in reaction temperature. The critical flow rates obtained in this study are about 40 times greater than those obtained for a similar study on immobilized glucose oxidase. Arrhenius plots gave activation energies of -9.6 and -7.1 kcal/g mol at pH 4.6 and 7.0, respectively. The work reported herein is a bench-scale investigation of an immobilized enzyme with primary emphasis on the mass transfer and kinetic characteristics of the system. The rapid reaction rates obtainable at relatively low temperatures offer a potential alternative method of purifying yeast single cell protein (SCP) with miminum loss of desired protein. The key questions are how such a system would react in a yeast homogenate, what conditions in such a system must be controlled, and what type of immobilized reactor should be utilized, if such further work continued to show promise.

Oxygen transfer properties of a bioreactor for use within a nuclear magnetic resonance spectrometer.

Nuclear magnetic resonance (NMR) spectroscopic analysis of whole cells is an important emerging technique for noninvasive and nondestructive monitoring of cell physiology. However, this technique requires extremely high cell densities. Attempts to maintain densities above the carrying capacity of a maintenance system result in the demise of the entire culture. To define conditions for maintaining mammalian cells at high densities for NMR studies, we have designed a bioreactor to operate under defined, oxygen-limited conditions within an NMR spectrometer. The bioreactor utilizes hollow fibers to deliver nutrients and remove wastes from an agitated cell suspension. The mass transfer properties of the fibers with respect to oxygen were determined. Ehrlich Ascites Tumor (EAT) cells were supplied with glutamine as the respiratory carbon source. The maximum viable cell density supported by a given oxygen concentration in the fluid flowing through the fiber lumen was predicted and then confirmed experimentally on the bench and in the spectrometer.

Optimum fiber spacing in a hollow fiber bioreactor.

A high surface area hollow fiber reactor was developed for mammalian cell culture. The reactor employs an interfiber gel matrix of agar or collagen for cell support. A model was developed to predict cell density as a function of fiber spacing. Optimum spacings are calculated for two sizes of Celgard hollow fibers. Ehrlich Ascites Tumor (EAT) cells were grown to an estimated density of 1.1 x 10(8) viable cells/mL in the extracapillary space-corresponding to an overall reactor density of 7 x 10(7) cells/mL. On the basis of available kinetic and diffusivity data, the model predicts that lactate accumulation may limit cell growth in the early stage of medium utilization, while oxygen delivery becomes limiting at later stages.

Kinetic characterization of baculovirus-induced cell death in insect cell cultures.

The death process of baculovirus-infected insect cells was divided into two phases: a constant viability (or delay) phase characterized by a delay time (t(d)) and a first-order death phase characterized by a half-life (t(1/2)). These two parameters were used in conjunction with the n-target theory to classify the kinetics of cell death under various conditions, including different multiplicity of infection (MOI), host cell lines, virus types, incubation volumes, cell density and extracellular L(+)-lactate and ammonium concentrations. Two groups of kinetic effects were found: one characterized by a constant number of hypothetical targets and the other by decreased numbers of hypothetical targets. The first group includes effects such as MOI, virus types, and host cell lines. The second includes the effects of environmental perturbations, such as incubation volume, cell density, and extracellular concentrations of L(+)-lactate and ammonium. Although the underlying mechanisms of these effects are as yet unknown, the death kinetics of infected cells significantly affects the recombinant protein production. In general, foreign protein production does not correlate with the cell life after infection.

FTIR evidence for alcohol binding and dehydration in phospholipid and ganglioside micelles.

We theorize that intoxicants and modern anesthetics bind at the membrane-water interface and displace (dehydrate) bound water molecules by breaking the hydrogen bonds. We tested this hypothesis by examining the effect of butanol on the binding of water to the polar regions of lipids in reversed micelles. Understanding the mechanisms of intoxication requires studies in physiologically relevant systems such as systems containing sialoglycoconjugates, especially gangliosides, which concentrate in the synapses of neural tissue. Therefore, we compared butanol effects on phospholipid with effects on ganglioside. Hydrogen-bond breaking activity of 1-butanol was studied in reversed micelles made of dipalmitoylphosphotidylcholine (DPPC), ganglioside (GM1 and GT1b) or the lipid mixture in a D2O-CCl4 medium. Fourier transform infrared spectroscopy (FTIR) data indicated that 1-butanol binds to DPPC and to gangliosides. Adding GM1 to the DPPC micelles introduces a new binding site for the alcohol. GT1b binds more butanol than GM1, because of more binding sites provided by extra sialic acid moieties. Spectral red shifts indicate that both water and butanol bind to the C = O group of sialic acid. Butanol partially releases the surface-bound water by disrupting hydrogen bonds, as indicated by an appearance of a sharp new free OD stretching band of the released D2O molecules. However, control studies with lipid-free systems in CCl4 revealed that a free OD peak could occur from a deuterium exchange reaction between D2O and 1-butanol(ol-h).(ABSTRACT TRUNCATED AT 250 WORDS)

A stirred bath technique for diffusivity measurements in cell matrices.

A stirred bath technique was developed for determining effective diffusivities in cell matrices. The technique involves cell immobilization in a dilute gel which has negligible effect on solute diffusion. Agar and collagen were tested as immobilizing gels. Agar gel was shown to have minor interactions with the diffusion of various biological molecules, and was used for immobilization of Ehrlich Ascites Tumor (EAT) cells. Diffusivities of glucose and lactic acid were measured in EAT matrices for cell loadings between 20 and 45 vol %. Treatment with glutaraldehyde was effective in quenching the metabolic activity of the cells while preserving their physical properties and diffusive resistance. The measured data agree favorably with predictions based on Maxwell's equation for effective diffusion in a periodic composite material. The stirred bath technique is useful for diffusivity determinations in immobilized matrices or free slurries, and is applicable to both microbial and mammalian cell systems.