Jean B. Hunter

The role of an NAD-independent lactate dehydrogenase and acetate in the utilization of lactate by Clostridium acetobutylicum strain P262

Clostridium acetobutylicum strain P262 utilized lactate at a rapid rate [600 nmol min−1 (mg protein)−1], but lactate could not serve as the sole energy source. When acetate was provided as a co-substrate, the growth rate was 0.05h−1. Butyrate, carbon dioxide and hydrogen were the end products of lactate and acetate utilization, and the stoichiometry was 1 lactate + 0.4 acetate →0.7 butyrate + 0.6H2 + 1CO2. Lactate-grown cells had twofold lower hydrogenase than glucose-grown cells, and the lactate-grown cells used acetate as an alternative electron acceptor. The cells had a poor affinity for lactate (Ks=1.1 mM), and there was no evidence for active transport. Lactate utilization was catabolyzed by an inducible NAD-independent lactate dehydrogenase (iLDH) that had a pH optimum of 7.5. The iLDH was fivefold more active withd-lactate thanl-lactate, and theKm ford-lactate was 3.2 mM. Lactate-grown cells had little butyraldehyde dehydrogenase activity, and this defect did not allow the conversion of lactate to butanol.

Process Options in Hepatitis B Surface Antigen Extraction from Transgenic Potato

The process conditions for recombinant hepatitis B surface antigen (HBsAg) extraction from transgenic potato were examined. The effects of temperature, the reducing agent β‐mercaptoethanol (BME), and proteinase inhibitors on the level of antigenic activity of recovered HBsAg were determined. Sedimentation profiles were performed to characterize HBsAg assembly into virus‐like particles. Increasing the temperature of the sample for about 1 min increased the measured HBsAg antigenic activity. The optimum temperature was around 50 °C. A 3‐fold enhancement of the antigenic activity was obtained in extract from transgenic potato expressing HBsAg, when monoclonal antibodies were used to assay for HBsAg. When antigenic activity was determined by polyclonal antibodies, no enhancement in the antigenic activity was obtained. Temperature may affect the conformation of the a epitope to which the monoclonal antibodies bind or alter the fluidity of surface lipid regions. BME increased the antigenic activity of HBsAg up to 4‐fold when monoclonal antibodies directed against the a determinant were used, but there was no increase with polyclonal antibodies. This observation suggests that BME affects the structure or presentation of the a epitope. In the presence of BME and leupeptin, a proteinase inhibitor, higher antigenic activity was obtained. Leupeptin might protect the antigen, which might become more susceptible to proteolytic degradation after reduction, as a result of stimulation of sulfhydryl proteases. Although both temperature and BME increased the antigenic activity of HBsAg individually, when combined their interaction was antagonistic, resulting in reduced antigenic activity. Different proteinase inhibitors, including leupeptin, aprotinin, E‐64, pefabloc, and pepstatin, had no significant effect on HBsAg from potato extract in a 2 h period in the absence of BME. The sedimentation profile of potato‐produced HBsAg was determined in 5−30% sucrose gradients. Yeast‐derived recombinant HBsAg was used as a positive control. The HBsAg from transgenic potato showed sedimentation and density properties that are very similar to the yeast‐produced antigen, indicating assembly into virus‐like particles. BME treatment did not change the sedimentation profile.

NAD-independent lactate and butyryl-CoA dehydrogenases of Clostridium acetobutylicum P262

Abstract.Clostridium acetobutylicum P262 cells that were growing on lactate and acetate had an NAD-independent lactate dehydrogenase (iLDH) activity of 200 nmol mg protein−1 min−1. Ammonium sulfate precipitation and DEAE cellulose caused a 35-fold purification. Gel filtration indicated that the iLDH had a molecular weight of approximately 55 kDa, but two bands were always observed. Phenyl sepharose could not separate the two proteins, and hydroxyapatite caused a complete loss of activity. The semi-purified iLDH had a Vmax of 13,000 nmol mg protein−1 min−1 and a Km value of 3.5 mM for D-lactate. The Vmax and Km values for L-lactate were 300 nmol mg protein−1 min−1 and 0.7 mM. The iLDH had a pH optimum of 7.5, was not activated by fructose-1,6-bisphosphate (FDP), and could be coupled to either 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or dichlorophenol-indophenol (DCPIP), but not methyl viologen (MV) or benzyl viologen (BV). The iLDH did not have strong absorbance between 500 and 300 nm, and trichloroacetic acid or acid ammonium sulfate extracts had virtually no fluorescence at 450 nm. The crude extracts also had MTT-linked butyryl-CoA dehydrogenase activity (60 nmol mg protein−1 min−1). The NAD-independent butyryl-CoA dehydrogenase eluted from DEAE-cellulose as two fractions. The yellow fraction was extremely unstable, but the green fraction could be stored for short periods of time at 5°C. The green-colored butyryl-CoA dehydrogenase had strong absorption at 450 nm, and gel filtration indicated that it had a molecular weight of 90 kDa. The NAD-independent butyryl-CoA dehydrogenase could be coupled to MTT, DCPIP, or MV, but not BV. Because the NAD-independent lactate and butyryl-CoA dehydrogenase could both be linked to low potential carriers, these two enzymes may function as oxidation-reduction system in vivo.

The use of real-time PCR to study Penicillium chrysogenum growth kinetics on solid food at different water activities.

Fungal growth on solid foods can make them unfit for human consumption, but certain specialty foods require fungi to produce their characteristic properties. In either case, a reliable way of measuring biomass is needed to study how various factors (e.g. water activity) affect fungal growth rates on these substrates. Biochemical markers such as chitin, glucosamine or ergosterol have been used to estimate fungal growth, but they cannot distinguish between individual species in mixed culture. In this study, a real-time polymerase chain reaction (rt-PCR) protocol specific for a target fungal species was used to quantify its DNA while growing on solid food. The measured amount of DNA was then related to the biomass present using an experimentally determined DNA-to-biomass ratio. The highly sensitive rt-PCR biomass assay was found to have a wide range, able to quantify the target DNA within a six orders-of-magnitude difference. The method was used to monitor germination and growth of Penicillium chrysogenum spores on a model porous food (cooked wheat flour) at 25°C and different water activities of 0.973, 0.936, and 0.843. No growth was observed at 0.843, but lag, exponential and stationary phases were identified in the growth curves for the higher water activities. The calculated specific growth rates (μ) during the exponential phase were almost identical, at 0.075/h and 0.076/h for aw=0.973 and 0.936, respectively. The specificity of the method was demonstrated by measuring the biomass of P. chrysogenum while growing together with Aspergillus niger on solid media at aw=0.973.

An Isotachophoretic Model of Counteracting Chromatographic Electrophoresis (CACE)

Abstract Counteracting chromatographic electrophoresis (CACE) is a new electrophoretic technique for preparative separation of charged macromolecules, particularly proteins. CACE combines gel chromatography of a protein sample in a packed bed of gel beads having a gradient of exclusion limit, simultaneously with electrophoresis in an electric field tending to move the protein upstream. The target proteins convective movement opposes its electrophoretic movement, focusing the protein into an accumulation zone where its net velocity is zero. A mathematical model of concentrations and electrical fields in CACE was derived from an analogy to isotachophoresis. Accumulation-zone concentrations, electrical field, and pH were calculated from the bulk flow and electrophoretic fluxes of the target protein and buffer constituents, along with expressions for charge conservation and electroneutrality. The model predicts conditions for formation of protein accumulation zones given column operating parameters and mobil...

Protein purification by counteracting chromatographic electrophoresis — The focusing window

Counteracting Chromatographic Electrophoresis (CACE) is a novel purification technique that focuses the target protein into a thin band at the interface separating two gel media which differ sharply in internal porosity. This article validates a criterion that predicts protein focusing by CACE, using the colored proteins-ferritin, hemoglobin and myoglobin. Fair comparison is shown between the theory and the experiment. Data from the past literature which were reported to exhibit nonconformity have also been shown to agree fairly well with the present model. Variations in protein focusing conditions can be partly explained by protein-ion binding.

Analysis of Joule heating in electrophoretic processes

Abstract Joule heating appears to be the most limiting factor in the scale-up of many electrophoretic processes. The heat equation is solved for the case of Counteracting Chromatographic Electrophoresis (cylindrical gel) and the importance of autothermal heating effect is emphasized.

The acetate kinase ofClostridum acetobutylicum strain P262

Clostridum acetobutylicum strain P262 fermented glucose, pyruvate, or lactate, and the butyrate production was substrate-dependent. Differences in butyrate yield could not be explained by changes in butyrate kinase activities, but the butyrate production was inversely related to acetate kinase activity. The acetate kinase had a pH optimum of 8.0, aKm for acetate of 160 mM, and akcat of 16,800 min-1. The enyzme had a native molecular mass of 78 kDa; the size of 42 kDa on SDS-PAGE indicated that the acetate kinase of strain P262 was a homodimer.

Testing of an Enhanced Brine Dewatering System

Water recovery is essential for long-duration space exploration transit and outpost missions. Primary stage wastewater recovery systems partially satisfy this need, and generate concentrated wastewater brines that are unusable without further processing. The Enhanced Brine Dewatering System (EBDS) is being developed to allow nearly complete recovery of water from wastewater brines. This paper describes the operation of the EBDS and discusses the development and testing of two EBDS full system prototypes. The alpha prototype was used to process ersatz solutions that represent the anticipated wastewater stream from a primary stage water processor, and to evaluate the effects of different additives that are mixed with the brine to improve the ability of the EBDS to generate a removable solid residue. In addition to processing ersatz formulations, the alpha prototype processed authentic brines generated by the Cascade Distillation System (CDS) with a wastewater stream made up of actual treated urine, hygiene water, and condensate water. The chemical and microbial composition of the EBDS influent solutions, dried solid residue, and condensed effluent were evaluated. The beta prototype was tested with a wastewater brine ersatz to evaluate system performance, including power requirements. The performance test results were used to evaluate the equivalent system mass of flight-like EBDS designs for a number of reference missions.

WORK MEASUREMENT FOR ESTIMATING FOOD PREPARATION TIME OF A BIOREGENERATIVE DIET

During space missions, such as the prospective Mars mission, crew labor time is a strictly limited resource. The diet for such a mission (based on crops grown in a bioregenerative life support system) will require astronauts to prepare their meals essentially from raw ingredients. Time spent on food processing and preparation is time lost for other purposes. Recipe design and diet planning for a space mission should therefore incorporate the time required to prepare the recipes as a critical factor. In this study, videotape analysis of an experienced chef was used to develop a database of recipe preparation time. The measurements were highly consistent among different measurement teams. Data analysis revealed a wide variation between the active times of different recipes, underscoring the need for optimization of diet planning. Potential uses of the database developed in this study are discussed and illustrated in this work.