Stereospecific hydrogenations with immobilized microbial cells or enzymes

Stereospecific hydrogenations according to the general scheme [formula: see text] are of interest from a preparative and mechanistic point of view. Proteus mirabilis is suitable for the hydrogenation of a-keto-acids to R-hydroxy-acids, and a Clostridium strain for the reduction of enoates. Both have been immobilized in formaldehyde crosslinked gelatin and the latter also in polyacrylamide. Immobilized as well as free cells showed usually half lives of 100-200 h. The immobilized cells could be separated from the products and reused. In order to hydrogenate enoates stereospecifically, formate dehydrogenase and enoate reductase have been separately immobilized and coimmobilized on controlled pore glass. The yields for the separately immobilized enzymes were about 30 per cent and 70-80 per cent, respectively. The measured rate of the coupled system with immobilized enzymes was compared with the calculated rate, taking into account effects of pore diffusion for the pyridine nucleotide. Under operational conditions the half-life of the immobilized formate dehydrogenase was 36 h versus 45 h for the free enzyme. The corresponding values for the enoate reductase turned out to be about 17 h versus about 15 h. So far the immobilized as well as the free enzymes seem to be less stable than immobilized or free cells.     

Effect of irradiation on immobilized enzymes compared with that on enzymes in solution

Summary Glucose oxidase and catalase were immobilized by attaching them to nylon fibers that had been treated with triethyloxonium-tetrafluoroborate, diaminohexane and glutardialdehyde according to Morris, Campbell and Hornby (1975). This method assures that the enzymes are bound to a side chain of the polyamide structure. Enzyme activity (as measured by the O₂-uptake and by microcalorimetry) was found to be unchanged after 2 years. The apparent K_m-constants of the immobilized enzymes with glucose were the same as those for enzymes in solution. GOD and catalase immobilized in poly(acrylamide) gel had the same K_m-value.Despite the high stability during storage, the radiation induced inactivation of enzymes immobilized on gel or chromosorb, an inorganic carrier, was of the same order of magnitude as that of the dissolved enzymes. The enzymes bound to nylon fibers showed a higher radiation sensitivity. This might have been caused by an additional attack on the binding site of the carrier.Dedicated to Professor Dr. Dr. U. Hagen on the occasion of his 60^th birthdayDAAD-Fellow from AustriaDAAD-Fellow from South-Korea     

Mathematical modeling of flow-tube extracorporeal shunts with immobilized enzymes

A complex of computer programs "IF-reactor" for model calculations and processing of experimental data related to flow tube extracorporeal shunts with immobilized enzymes has been developed. Qualitative and quantitative comparison of theory with experiment has been made.     

Increasing the activity of immobilized enzymes with nanoparticle conjugation

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Bacteriolysis by immobilized enzymes.

Bacteriolytic enzymes produced by Achromobacter lunatus were immobilized in collagen membrane. Intact bacteria such as Pseudomonas solanacearum, Xanthomonas oryzae, Staphylococcus aureus, and Pseudomonas aeruginosa were lyzed with the bacteriolytic enzyme-collagen membrane. Relative activity of the bacteriolytic enzyme-collagen membrane against Pseu. solanacearum was about 2% of that of native bacteriolytic enzymes. No difference in the optimum pH was observed between immobilized enzymes and native enzymes. The bacteriolytic enzymes in the collagen membrane were stable against sodium chloride which was an inhibitor of the native bacteriolytic enzymes. Xanthomonas oryzae and Pseu. aeruginosa were continuously lyzed by a reactor containing the rolled bacteriolytic enzyme-collagen membrane.     

Carrier-free immobilized enzymes for biocatalysis

Methods for the preparation of carrier-free insoluble enzymes are reviewed. The technology of cross-linked enzyme aggregates has now been applied to a range of synthetically useful activities. Fusion proteins are also gaining momentum because they allow a relatively selective aggregation or even a specific self-assembly of the desired enzyme activity into insoluble particles in the absence of potentially denaturing chemicals required for precipitation and cross-linking. Recycling of insoluble protein particles for multiple rounds of batchwise reaction has been demonstrated in selected biotransformations. However, for application in a fully continuous biocatalytic process, low resistance to mechanical stress and high compressibility are issues for consideration on carrier-free enzyme particles.     

Glucose analysis utilizing immobilized enzymes

The four commercial instruments that measure glucose by incorporation of immobilized reagents are described and compared. The design of the immobilized enzyme or enzymes is shown to be related to the type of instrument. Three of the instruments are of the partitioned enzyme-sensor type requiring an immobilized enzyme capable of rapid, constant flow rate when inserted in a flowing stream. Moderately high enzyme loading is required if the instrument is designed to operate in the equilibrium mode while lower enzyme loading can be tolerated in kinetic mode. Only one instrument is an enzyme electrode in which the immobilized glucose oxidase is in the immediate vicinity of the electrochemical detector. In that case the immobilized enzyme must have very high enzyme activity per unit volume, but need not have high physical durability. The design of the instrument and immobilized enzyme(s) is also affected by whether the instrument is to be used in an industrial or a clinical laboratory.     

Comments on diffusive and electrostatic effects with immobilized enzymes

Shuler, Aris &; Tsuchiya (1972) have recently considered the combined effects of diffusive resistance and electrostatic field on the rate of reaction catalyzed by an enzyme immobilized on a non-porous surface. They employed a potential distribution for the electrical double layer which is asymptotically valid when surface potential is small.The complete Gouy-Chapman solution, which is valid for higher surface potential, is employed here. Numerical values of the effectiveness factor calculated with this potential distribution agree very closely with the results of Shuler et al. for most cases. It is shown that the effectiveness factor can (i) attain magnitudes much greater than unity in physically realizable systems, (ii) approach the solution for “infinite” surface potential at reasonable values of surface charge density, and (iii) pass through a maximum as bulk substrate concentration is varied. This behavior leads to the existence of an optimum surface concentration for enzyme immobilized on a highly charged non-porous support such that the most effective catalytic action on a charged substrate is ensured. Finally, it is established that significant electrical and/or diffusive effects result in non-linear Lineweaver-Burk plots of reciprocal observed reaction velocity against reciprocal bulk substrate concentration. These non-linear plots cannot be interpreted in the same way as linear plots obtained when enzyme is unbound.     

Sunflower oil transesterification with methanol using immobilized lipase enzymes

Bioprocess and Biosystems Engineering - The transesterification of sunflower oil with methanol, using immobilized lipase enzymes as catalysts, was studied. The process was carried out in a...     

Distribution of immobilized enzymes on porous membranes

In this article, the results from a theoretical and experimental investigation of enzyme immobilization in porous membranes are reported. A theoretical model of the immobilization process, which accounts for restricted diffusion of enzyme in the pores of the membrane, has been developed. The model predicts the effect of immobilization kinetics and time of immobilization on the enzyme distribution in the pores of the membrane. The immobilization of glucose oxidase and glucose oxidase-biotin conjugate on porous alumina membranes was experimentally investigated. Enzyme uptake data was correlated to the theory to determine the rate constant of imobilization and the distribution of the enzyme in the pore. Immobilization studies were carried out for enzyme adsorption and for enzyme attachment by covalent coupling. The distribution of enzyme was experimentally studied by assembling five membranes in the diffusion cell. Following immobilization, the membranes were separated and each was assayed for activity. The amount of active enzyme present in each membrane yielded a discrete distribution that compared well with that predicted by theory. (c) 1992 John Wiley & Sons, Inc.     

A cross-flow reactor with immobilized pectolytic enzymes for juice clarification

Summary A new system for continuous juices clarification is presented. The bioreactor combines microporous plates commercially available and industrial pectinases immobilized on nylon membranes in a cross-flow configuration. The kinetic behaviour of the reactor for different recirculation flow rates has been determined. Fresh apricot juice has been continuously clarified in the bioreactor with excellent results.     

The coagulation of milk with immobilized enzymes: A critical review

The milk coagulation process is described including current understanding of the mechanism and kinetics involved with emphasis on aspects which impact on coagulation with immobilized enzymes. From the studies of coagulation with immobilized enzymes reported to date there is little evidence to suggest that immobilized enzymes can be used to coagulate milk.