Urokinase bound to fibrocollagenous tubes: an in vitro kinetic study

Urokinase (UK) has been immobilized to the inner surfaces of fibrocollagenous tubes (FCT) in an attempt to develop a fibrinolytic biomaterial which may be suitable for use as a small diameter vascular prosthesis. The enzyme was bound by adsorption followed by glutaraldehyde crosslinking. An in virto kinetic study of immobilized urokinase was conducted by employing the tubular material as a flow through reactor operated in a batch recycle mode in which the esterolysis of the model substrate, N-alpha-acetyl-L-lysine methyl ester (ALME), was monitored as a function of substrate concentration, recycle flow rate, and temperature. Results were compared with data from the soluble enzyme reaction, which was conducted in the presence and absence of 10% swine skin gelatin, in order to identify the specific effects of a collagenous microenvironment. Observed rates for the UK-FCT catalyzed reaction were observed to be dependent on recycle flow rates below 12 mL/min (Re = 107). Apparent Michaelis-Menten rate parameters were determined by a nonlinear search technique for two flow rates: one above the critical point for external diffusion effects (Re = 282) and one within the mass-transfer-limited region (Re = 71). When the latter data were corrected for external diffusion by applying the Graetz correlation for laminar flow in tubes to estimate themass transfer coefficient, the corrected K(m) of 6.45 +/- 0.38 mM agreed very closely with the diffusion free parameter (i.e. 6.13 +/- 0.63). Furthermore, this value was observed to be an order of magnitude higher than that of the soluble enzyme but approximately equal to the K(m) of the soluble enzyme in a 10% gelatin environment (8.13 +/- 1.53 mM). It is postulated that the difference in kinetic parameters between soluble and collagen immobilized UK is due to an inherent interaction between collagen and enzyme rather than to mass transfer effects. Such aninteraction is supported by the effects of collagen on thermal stability and energy of activation.     

The production of cellulase in a spouted bed fermentor using cells immobilized in biomass support particles

Continuous cellulase production by Trichoderma viride QM 9123, immobilized in 6 mm diameter, spherical, stainless steel biomass support particles, has been achieved using a medium containing glucose as the main carbon source. Experiments were carried out in a 10-L spouted bed fermentor. In this type of reactor-recycled broth is used to create a jet at the base of a bed of particles, causing the particles to spout and circulate. During the circulation, particles pass through a region of high shear near the jet inlet. This effectively prevents a buildup of excess biomass and thus enables steady-state conditions to be achieved during continuous operation. Continuous production of cellulase was achieved at significantly higher yield and productivity than in conventional systems. At a dilution rate of 0.15 h(-1) (nominal washout rate for freely suspended cells is 0.012 h(-1)), the yield of cellulase on glucose was 31% higher than that measured during batch operation, while the volumetric productivity (31.5 FPA U/L. h) was 53% greater than in the batch system. The specific cellulase productivity of the immobilized cells was more than 3 times that of freely suspended cells, showing that diffusional limitations can be beneficial. This offers significant opportunity for the further development of biomass support particles and associated bioreactors.     

Mode of action and properties of xylanase and beta-Xylosidase from Neurospora crassa

Extracellular beta-xylosidase (1,4-beta-D-xylan xylohydrolase, EC 3.2.1.37) from culture filtrates of Neurospora crassa was purified to homogeneity by preparative isoelectric focusing followed by gel electrophoresis. The molecular weight of the purified xylosidase was 83,000 D and the K(m) on p-nitrophenyl-beta-D-xyloside was 0.047mM. The homogeneous xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) and beta-xylosidase showed differences in their mode of action towards xylooligosaccharides. The degree of hydrolysis of D-xylan by xylanase of N. crassa was 18%. Supplementation of beta-xylosidase from the same organism resulted in 48% hydrolysis. The synergistic effect was more pronounced, with the hydrolysis of 68%, when a homogeneous preparation of beta-xylosidase from Sclerotium rolfsii was added to the saccharification system.     

Effect of temperature and pH on immobilized Zymomonas mobilis for continuous production of ethanol

The effects of temperature and inlet pH of the medium on the ethanol productivity and activity of the immobilized Z. mobilis cells during continuous fermentation of glucose have been studied at various temperatures and pH. On changing the temperature from one steady state level to a new one, 6-8 h were required in order to fully experience the effect of a change in temperature; whereas 8-20 h were required on changing the pH. The optimum temperature of 37 degrees C and a broad pH range of 4.4-6.0 were observed for maximum ethanol productivity and ethanol yield.     

A kinetic model for anaerobic digestion of biological sludge

The principal objective of this study was the development and evaluation of a comprehensive kinetic model capable of predicting digester performance when fed biological sludge, preliminary conversion mechanisms such as cell death, lysis, and hydrolysis responsible for rendering viable biological sludge organisms to available substrate were studied in depth. The results of this study indicate that hydrolysis of the dead, particulate biomass-primarily consisting of protein-is the slowest step, and therefore kinetically controls the overall process of anaerobic digestion of biological sludge. A kinetic model was developed which could accurately describe digester performance and predict effluent quality.     

Operation and pressure distribution of immobilized cell hollow fiber bioreactors

It has been cited in the literature on hollow fiber systems that pressure gradients persist, and the transmembrane flux of the hollow fiber system is dependent on the pattern of the pressure gradients. The pattern can be used to its advantage in immobilized enzyme systems. However, with immobilized living cell systems, the pressure gradients lead to a nonuniform environment within the hollow fiber cartridge and not necessarily favorable results. This article provides pertinent pressure-drop data on hollow fiber cartridges which are in flow configurations typical of immobilized cell culture work. The results illuminate operational problems that may arise in the culture of either anchorage dependent or independent cells. Possible solutions with crossflow systems are suggested.     

Immobilization of BSA, enzymes and cells of Bacillus stearothermophilus onto cellulose, polygalacturonic acid and starch based graft copolymers containing maleic anhydride

Poly(maleic anhydride styrene) graft copolymers of cellulose, pectin polygalacturonic acid salt, calcium polygalacturonate, and starch were prepared and used to immobilize proteins. The cellulose grafts coupled quite appreciable quantities of acid phosphatase, glucose oxidase, and trypsin. However, the general retention of activity was somewhat disappointing. Further investigation with acid phosphatase showed that the amount of enzyme immobilized increased as the amount of anhydride in the graft copolymer increased but no such relationship existed for the enzymic activity. The cellulose graft copolymers were hydrolyzed and it appeared that the carboxyl group aided adsorption of the enzyme. Attempts to couple acid phosphatase using CMC through the free carboxyl groups, created by hydrolysis, gave only a small increase in the extent of protein coupling. However, the unhydrolyzed system gave a useful degree of immobilization of cells of Bacillus stearothermophilus, as did a poly(maleic anhydride/styrene)-cocellulose system. Attempts to improve the activity by using grafts based on other polysaccharide supports met with mixed success. Pectin products were soluble. Polygalacturonic acid products were partially soluble and extremely high levels of enzymic activity were obtained. This was probably due in part to the hydrophilic nature of the system, which also encouraged absorption of the enzyme. Attempts were made to reduce the solubility by using the calcium pectinate salt. Immobilization of acid phosphatase and trypsin resulted in inceased protein coupling but relatively poor activities were attained. A starch based system gave similar results. Calcium polygalacturonate was used to prepare an insoluble graft copolymeric system containing acrylonitrile-comaleic anhydride. The resulting gels gave excellent coupling with acid phosphatase which had a very good retention of activity.     

Effects of solute hydrophobicity and phospholipid composition on permeability of composite membranes containing phospholipids

Permeabilities of several solutes through the composite membranes containing phospholipids have been measured. They were inversely proportional to the content of the phospholipids in the membrane. Both the permeability of solutes and the degree of permeability change around the phase transition temperature of the phospholipids for the hydrophobic solutes such as n-butanol and salicylamide were larger than those for the hydrophilic solutes such as amino acids and pyridoxine. These results suggest thatthe permeation path of hydrophobic solutes is different from that of hydrophilic ones. The addition of phosphatidyl ethanolamine, phosphatidyl serine, or phosphatidic acid to the composite membrane influenced the solute permeability due to the introduced negative charge and/or the change in the molecular packing of phospholipid.     

Methane recovery from water hyacinth through anaerobic activated sludge process

The concepts of phase separation, anaerobic activated sludge process, and alkali pretreatment have been incorporated in this investigation with the objective of developing rational and cost-effective designs of diphasic anaerobic activated sludge systems, with and without alkali treatment, for methane recovery from water hyacinth (WH). Evaluation of process kinetics and optimization analyses of laboratory data reveal that a diphasic system with alkali treatment could be designed with an alkali pretreatment step (3.6% Na(2)CO(3) + 2.5% Ca(OH)(2) (w/w) of WH, 24 h duration) followed by an open acid phase (2.1 days HRT) and closed methane reactor with sludge recycle (5.7 days HRT, 7.7 days MCRT) for gas yield of 50 L/kg WH/d at 35-37 degrees C. Likewise, a diphasic system without alkali treatment could be designed with an open acid phase (2 days HRT) followed by closed methane reactor with sludge recycle (3.2 days HRT, 6 days MCRT) for gas yield of 32.5 L/kg WH/d at 35-37 degrees C. Detailed economic analyses bring forth greater cost-efficacy of the diphasic system without alkali treatment and reveal that the advantage accrued in terms of higher gas yield is overshadowed by the cost of chemicals in the diphasic system with alkali treatment.