Phenylalanine ammonia-lyase immobilized in semipermeable microcapsules for enzyme replacement in phenylketonuria

Phenylalanine ammonia-lyase immobilized within semipermeable microcapsules has an assayed enzyme activity which is 20% +/- 4% of the enzyme in free solution. The Km for the immobilized enzyme remained the same as that of the free enzyme. The pH optimum also remained unchanged at pH 8.5 +/- 1.0. At the lower pH range, enzyme activity is higher for the immobilized enzyme. Daily oral administration of microencapsulated phenylalanine ammonia-lyase to phenylketonuric rats decreased the systemic phenylalanine level by 35 +/- 8% in 2 days (P less than 0.05) and by 75 +/- 8% in 7 days (P less than 0.001).     

Biodegradable semipermeable microcapsules containing enzymes, hormones, vaccines, and other biologicals.

Semipermeable microcapsules were prepared using biodegradable material as the enclosing membranes. For instance, polylactic acid was used as membrane material to microencapsulate biologically active materials. Asparaginase microencapsulated within polylactic acids functions effectively in converting external asparagine into aspartic acid and ammonium. By variations in permeability characteristics, insulin microencapsulated within polylactic acid can be released at pre-adjusted rates. Thus, release rates of 50% in 5 hours, 50% in 20 hours, and 2.5% in 24 hours have been demonstrated. Drugs and vaccines have also been similarily microencapsulated. The advantage of the biodegradable microcapsules is the ability of the body to convert the injected polymer material to normal body metabolites (e.g., CO2 and H2O in the case of polylactic acid) after completion of its function.     

Epnzymatric recycling of coenzymes by a multi-enzyme system immobilized within semipermeable collodion microcapsules.

Hexokinase (ATP:D-glucose 6-phosphotransferase EC 2.7.1.2) and pyruvate kinase (ATP:pyruvate 2-0-phosphotransferase EC 2.7.1.40) were co-immobilized within semipermeable collodion microcapsules. The resulting microcapsules displayed excellent hexokinase and pyruvate kinase activities, with the measured pyruvate kinase activity considerably greater than that measured for hexokinase. The co-immobilized enzymes, when used sequentially were capable of recycling both ATP and ADP when exposed to the appropriate conditions. Furthermore, when exposed to limiting amounts of coenzyme, the cycles were capable of reusing the total amount of coenzyme supplied at least three times in 90 min. The use of microencapsulation to produce partially "self sufficient" enzyme systems is discussed.     

The recycling of NAD+ (free and immobilized) within semipermeable aqueous microcapsules containing a multi-enzyme system.

Semipermeable aqueous collodion microcapsules were prepared containing both yeast alcohol dehydrogenase (EC 1.1.1.1) and malic dehydrogenase (EC 1.1.1.37). These microcapsules exhibited both enzymic activities in good amount in the ratio 3:1 with respect to malic dehydrogenase:alcohol dehydrogenase.Both NAD⁺ and NADH were successfully cycled within the microcapsules by employing the included enzyme activities acting sequentially. A soluble, immobilized NAD⁺ derivative was also recycled within the semipermeable microcapsules.     

High cell density cultivation of human leukemia T cells (Jurkat cells) in semipermeable polyelectrolyte microcapsules

The ex vivo expansion of human T cells is of considerable scientific and medical interest. Currently, this requires the addition of massive amounts of stimuli. Here, human leukemia T cells (Jurkat cells) were used as model cells to demonstrate the in vitro expansion of T cells in the absence of added stimuli after encapsulation in semipermeable sodium cellulose sulfate/poly(diallyldimethyl) ammonium chloride polyelectrolyte membrane capsules (molecular weight cutoff <10 kDa, average diameter ca. 800 μm). For comparison, free and encapsulated cells were cultivated in standard T‐flasks and spinner bottles (both 50 mL culture medium) as well as in hanging drops (35 μL, only nonencapsulated cells). Encapsulation led to a significantly higher specific growth rate, a prolonged exponential growth phase together with a reduced tendency for apoptosis, as evidenced by shifts in the cell cycle distribution toward the S and G2/M phases together with a reduced percentage of cells in the sub‐G0/G1 phase. As a consequence, very high cell densities (>140×10⁶ cells/mL_capsule) were obtained in the capsules, particularly for the spinner cultivations. No evidence for nonspecific activation/stimulation, that is IL‐2 and CD25 expression, was found, while specific stimulation by phorbol‐12‐myristate‐13‐acetate/ionomycin was still possible. Since Jurkat cells commonly serve as model cells for primary T lymphocytes, the proposed method may present a strategy for high‐density proliferation of primary human T lymphocytes.     

Integration of mixing, heat transfer, and biochemical reaction kinetics in anaerobic methane fermentation

An extensive investigation of anaerobic methane fermentation requires identifying the relationship between the physical environment and biological process. In this study, a computational fluid dynamics (CFD) technique was used to characterize bacterial fermentation mechanisms intertwined with mixing and heat transfer in anaerobic digesters. The results demonstrate that the methane yield remains almost unchanged while the energy efficiency decreases with increasing mixing power in a complete‐mix digester, and that the energy output increases nonlinearly with the increase in heating energy in a plug‐flow digester. The CFD method can be applied to other bioreactors to gain valuable insights into their behavior as well. Integrating flow and temperature with kinetic behavior for anaerobic digestion not only solves the controversy about how mixing influences the digestive process, but also assists in optimizing the digester design and increasing the efficiency of energy conversion, and additionally, provides a reference for improving the mixing guidelines recommended by the U.S. Environmental Protection Agency. Biotechnol. Bioeng. 2012; 109: 2864–2874. © 2012 Wiley Periodicals, Inc.     

Characteristic reaction paths of biochemical reaction systems with time scale separation

A technique has been developed for characterizing the in vivo behavior of key enzymes from intermediate measurements. The technique is based on the identification of characteristic reaction paths, and it depends on the time scale separation characteristics of the systems. It is shown that useful information can be obtained from the phase plots of properly selected intermediate pairs or combinations which typically show process insensitive algebraic relations approached on time scales short compared to those of most practical interest. These characteristic reaction paths provide useful global measures of enzyme activity. The mathematical basis of reaction path analysis is investigated using linear transformation techniques. General theorems are developed predicting the existence of characteristic reaction paths as asymptotic limits whenever there is effective time scale separation. These limits are reached when fast reactions are relaxed, and available evidence suggests that these conditions will occur for the majority of reaction networks.     

Mass transfer effects on the reaction rate for heterogeneously distributed immobilized yeast cells

Here we examine the efficiency of different immobilized cell gradients applied to immobilized Saccharomyces cerevisiae fermenting glucose to ethanol. We developed a simulation model to fully study the competing effects of mass transfer hindrance and kinetics. It is based on a diffusion-reaction model and can be used to analyze the different cell concentration profiles inside an immobilized gel bead, in terms of effectiveness factors, productivity, and mass flux. The internal diffusion coefficient, which varies with the local cell concentration, as well as the external mass transfer, is taken into account when describing the efficiency. Although the diffusion hindrance is greater at higher cell concentrations, high cell concentration is still advantageous in the present case because the increase in reaction rate outweighs the diffusion hindrance. Thus, high cell concentrations contribute to increased productivity. The influence of the cell concentration gradient on the efficiency of the beads is negligible. Within the range of cell profiles studied it has been established that the location of the cells within the bead is of lesser importance. However, a steep cell gradient increases the importance of the external mass transfer.     

Evaluation of histochemical observations of activity of acid hydrolases obtained with semipermeable membrane techniques: A combined histochemical and biochemical investigation

Summary The reliability of enzyme histochemical observations of activities of acid hydrolases was investigated with a combined histochemical and biochemical study. Specimens of m. soleus, m. plantaris, m. gastrocnemius and diaphragm of normal and of vitamin E deficient rabbits were used. For the histochemical investigation, activity and localization of acid phosphatase, -glucuronidase, leucine aminopeptidase and E₆₀₀ resistant non-specific aryl-esterase were examined with semipermeable membrane techniques. For the biochemical investigation, activity of acid phosphatase, -glucuronidase, cathepsin D, acid maltase and neutral maltase was determined.By means of statistical calculations the enzyme activities demonstrated with histochemical techniques were compared with the enzyme activities determined with biochemical techniques.In the present communication the histochemical findings are reported and discussed. From the histochemical findings it appeared that activity of the acid hydrolases investigated is strongly increased in both a granular and a diffuse pattern in skeletal muscle of vitamin E deficient rabbits. The statistical calculations of the histochemical findings clearly reveal that the increased activity of one acid hydrolase was highly significantly paralleled by an increased activity of a second acid hydrolase. Moreover the probability that the activity of all other histochemically studied acid hydrolases was significantly increased was rather high.The increase in activity of the acid hydrolases studied was the same in muscles with an aerobic or an anaerobic metabolism. Moreover there was no difference in activity and localization of the acid hydrolases in aerobic type I and anaerobic type II fibres.The localization of acid phosphatase and -glucuronidase activity in muscle fibres mostly coincided. In cases where these enzymes were localized both centrally and in the subsarcolemnal areas of the muscle fibres, the activity of E₆₀₀ resistant naphtholesterase was usually, and the activity of leucine aminopeptidase was exclusively located in the subsarcolemnal areas. All of the examined acid hydrolases were found to be present in the inflammatory exudate and in the connective tissue.This study was partly extracted from the Ph. D. thesis of D.E. Israël (1977).     

Models of growth, self-reproduction and autoregulation based on consideration of reaction vessels with distensible, semipermeable walls]

We constructed non-equilibrium thermodynamics of the open physical-chemical irreversible processes in reactors with the strain semipermeable walls. This thermodynamics does not use the reciprocal relations of Onsager, so it may be applied when the stability stationary state is far from equilibrium. One of a general consequences of this thermodynamics is the statement: coordinated growth and self-reproduction are possible near the absolute equilibrium of the dissolvent and near the absolute stability stationary state of all chemicals with the absolute conservation of the differential equations of chemical kinetics. The supposition of ideal mixing is unnecessary; this condition is fulfilled automatically with diffusion. Growth and self-reproduction are not connected with positive eigenvalue of the differential equation of chemical kinetics. It is possible to construct a model of autoregulation and differentiation with this thermodynamics. The uniquness of such autoregulation follows from the mathematical theory [1]. The mathematical foundation of this thermodynamics is given in [1].     

Mass transfer in fermentation

An essential consideration in the design and operation of commercial fermenters is to ensure adequate mass transfer. The complex composition of fermentation liquids makes it difficult to predict accurately the mass transfer characteristics in large vessels. Here various aspects of mass transfer are discussed and their relationships examined. Strategies for predicting the most important type of mass transfer — between gases and liquids — in large scale fermentations are presented.     

Evaluation of histochemical observations of activity of acid hydrolases obtained with semipermeable membrane techniques: a combined histochemical and biochemical investigation 2. The biochemical investigation and comparison with the histochemical observations.

The reliability of enzyme histochemical semipermeable membrane techniques for the demonstration of acid hydrolases was investigated with a combined histochemical and biochemical study. In part 1 the histochemical findings were presented. In this communication the biochemical findings are reported and compared with the histochemical findings. In m. soleus, m. plantaris, m. gastrocnemius and diaphragm of vitamin E deficient rabbits the activity of the lysosomal acid hydrolases, cathepsin D, acid maltase, acid phosphatase and beta-glucuronidase is significantly increased. This increase in activity of the investigated acid hydrolases was equal for muscles with an aerobic or an anaerobic metabolism. By means of statistical calculations the activity of the enzymes demonstrated with histochemical techniques was compared with the enzyme activity determined with biochemical techniques. From the results of this investigation it can be concluded that the histochemical semipermeable membrane techniques for the demonstration of activity of acid hydrolases are very reliable. Considering the fact that these techniques are also tissue-saving, they are therefore extremely suitable for the study of catabolic wasting processes in skeletal muscle tissues of patients with inherited or acquired muscular diseases.     

Lumping analysis of biochemical reaction systems with time scale separation

Due to the complexity of the systems, successful modelling of intracellular reaction networks must rely on lumping techniques which systematically reduce the number of variables and parameters. Fortunately, the time scale separation characteristics of biochemical systems provide opportunities for eliminating unnecessary details. Through the proper interpretation of eigenvalues and eigenvectors, this article presents a theoretical basis for systematic model reduction. Results are generalized as a semiheuristic basis for lumping systems without complete kinetic information. It is also illustrated that the simplified system can yield new insight which is otherwise unavailable.     

Genipin cross-linked alginate-chitosan microcapsules: membrane characterization and optimization of cross-linking reaction

The genipin cross-linked alginate-chitosan (GCAC) microcapsule, composed of an alginate core and a genipin cross-linked chitosan membrane, was recently proposed for live cell encapsulation and other delivery applications. This article for the first time describes the details of the microcapsule membrane characterization using a noninvasive and in situ method without any physical or chemical modifications on the samples. Results showed that the cross-linking reaction generated the fluorescent chitosan-genipin conjugates. The cross-linked chitosan membrane was clearly visualized by confocal laser scanning microscopy (CLSM). A straightforward assessment on the membrane thickness and relative intensity was successfully achieved. CLSM studies showed that the shell-like cross-linked chitosan membranes of approximately 37 microm in thickness were formed surrounding the microcapsule. The reaction variables, including cross-linking temperature and time significantly affected the fluorescence intensity of the membranes. Elevating the cross-linking temperature from 4 to 37 degrees C drastically intensified the membrane fluorescence, suggesting the attainment of a high degree of cross-linking on the chitosan membrane. Extended cross-linking time altered the cross-linked membranes in modulation. Although genipin concentration and cross-linking time had little effects on the membrane thickness, cross-linking at higher temperatures tended to form relatively thinner membranes.     

Optimal identification of biochemical reaction networks

Advances in biotechnology and computer science are providing the possibility to construct mathematical models for complex biological networks and systematically understand their properties. Traditional network identification approaches, however, cannot accurately recover the model parameters from the noisy laboratory measurements. This article introduces the concept of optimal identification (OI), which utilizes a global inversion algorithm to extract the full distribution of parameters consistent with the laboratory data. In addition, OI integrates suitable computational algorithms with experimental capabilities in a closed loop fashion to maximally reduce the breadth of the extracted parameter distribution. The closed loop OI procedure seeks out the optimal set of control chemical fluxes and data observations that actively filter out experimental noise and enhance the sensitivity to the desired parameters. In this fashion, the highest quality network parameters can be attained from inverting the tailored laboratory data. The operation of OI is illustrated by identifying a simulated tRNA proofreading mechanism, in which OI provides superior solutions for all the rate constants compared with suboptimal and nonoptimal methods.