ATP regeneration using immobilized carbamyl phosphokinase

A simple and efficient system for continuous ATP regeneration is described. The procedure is based on the enzyme-catalyzed reaction between carbamyl phosphate and ADP. The carbamyl phosphate was generated in situ by reaction between potassium cyanate and potassium phosphate. The enzyme, carbamyl phosphokinase, was isolated from extracts of Streptococcus faccalis and partially purified. Immobilization of the enzyme was achieved using glutaraldehyde-treated alkylamine glass giving 200–250 units of activity per gram of glass. A column of carbamyl phosphokinase on glass was used to form ATP continuously from ADP, phosphate, and cyanate and lost approximately 16% of the initial activity after 14 days operation at room temperature.     

Highly active immobilized hydrogenase from Desulfovibrio gigas

The hydrogenase from the sulfate reducer $\text{Desulfovibrio gigas}$ has been immobilized by covalent coupling onto a porous silica support. Two methods have been used: glutaraldehyde activation of aliphatic amino Spherosil and diazotation of aromatic amino Spherosil. The effect of cytochrome C₃ and CC₃ addition during coupling has been investigated. The highest enzymatic activity (4440 U/g support) and immobilization yield (29 %) was obtained when coupling hydrogenase in the presence of cytochrome C₃ or CC₃ with diazotized aromatic amino silica. This immobilized hydrogenase preparation which shows a very good resistance to oxygen inactivation seems suitable for hydrogen photoproduction by coupling with illuminated chloroplasts.     

An immobilized hydrogenase from Alcaligenes eutrophus H-16

Alcaligenes Eutrophus H-16 was grown in continuous culture under conditions which induced hydrogenase production. The hydrogenase enzyme was extracted, partially purified and immobilized on porous glass. This enzyme was then studied both in solution and in immobilized form as a possible candidate for a number of industrial applications. It proved to have a stability (storage and operational) which was highly temperature dependent. Temperatures near freezing caused the enzyme to retain its activity for long periods of time. Although its kinetics were more favorable at elevated temperatures of up to 40 degrees C, the loss of stability outweighed this gain substantially. The effects of buffer type and pH on enzyme activity were also studied. This enzyme has only a modest sensitivity to destruction by oxygen during storage, in contrast to hydrogenases produced by several other microorganisms.     

Application of immobilized hydrogenase for the detritiation of water

Detritiation of contaminated water is an essential part of nuclear power production. Most promising methods used for this process are based on catalyzed hydrogen isotope exchange reactions. It is proposed herein to replace the platinum catalysts which are currently used in industry with immobilized hydrogenase. Whole bacterial cells of Alcaligenes eutrophus immobilized in calcium alginate or κ-carrageenan gels were found to be efficient catalysts of the reaction of hydrogen–tritium (H–T) exchange in both a batch tank reactor and in a column. The dependence of the reaction rate on the amount of immobilized cells in the system, and on the concentration of the cells in the matrix, indicate that enzymatic H–T exchange is not controlled by diffusion. Immobilized A. eutrophus cells are enzymatically active over a wide range of pH, with a broad maximum from pH 6.0 to 8.0, and are quite resistant to inhibitors of hydrogenases such as O₂ and CO. Upon increasing the temperature from 4 to 37°C, the rate of hydrogenase-catalyzed H–T exchange increases by a factor of 5. From the standpoint of catalytic efficiency, 1 g of PtO₂ is approximately equivalent to 10 g of cells (wet weight). In contrast of platinum-based catalysts, bacterial hydrogenases (1) are potentially inexpensive; (2) can be readily available in bulk quantities; (3) are maximally active in liquid water.     

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.     

Thermal stabilities of membrane-bound,solubilized, and artificially immobilized hydrogenase from Chromatium vinosum

Thermal inactivation (at 65 °C) of both membrane-bound and solubilized hydrogenase from Chromatium vinosum has been studied. Membrane binding greatly increases thermostability relative to the solubilized enzyme. Covalent attachment (but not simple adsorption) of the solubilized enzyme to ω-NH₂-alkyl-agaroses sharply increases thermal stability, which almost attains that of membrane-bound hydrogenase. It appears that there are at least two requirements for such stabilization—the enzyme should be in a hydrophobic medium and should be firmly bound to a hydrophobic support.     

固定化酵母连续发酵生产酒精工业应用的研究

本实验以海藻酸钠为基本载体,选择性地加入适量化学物质,制备新型复合载体。该载体能较好地避免单纯海藻酸钠载体易降解、易软化［１］以及颗粒上浮的问题。经反复使用不发粘、弹性好,无毒并能保持较高的发酵水平。     

Isomaltulose production using immobilized cells

Three strains of Erwinia rhapontici especially suitable for use in the form of nongrowing immobilized cells were selected by screening strains of cells for high activity and operational stability in an immobilized form. Immobilization in calcium alginate gel pellets was easily the best method of immobilizing E. rhapontici. Much greater operational stabilities were obtained than when other immobilization methods were used. Conditions of operation which optimize the activity, stability, and yield and the ease of operation of the immobilized cell columns working in a steady state are described. These include the effects of substrate concentration, diffusional restrictions and water activity, the concentration of cells immobilized, and the type of reactor used. Thus, the immobilized cells produce about 1500 times their own weight of isomaltulose during one half-life of use (ca. 1 year). Loss of activity was most closely correlated with the volume of substrate processed and so presumably is due to the presence of low concentrations of a cummulative inhibitor in the substrate. Methods for regenerating the activity of the immobilized cells by the periodic administration of nutrients, of forming isomaltulose by continuously supplying nutrients to growing immobilized cells, and of crystallizing isomaltulose from the column eluate are also described.     

Self-Incorporation of coenzymes by ribozymes

RNA molecules that are assembled from the four standard nucleotides contain a limited number of chemical functional groups, a characteristic that is generally thought to restrict the potential for catalysis by ribozymes. Although polypeptides carry a wider range of functional groups, many contemporary protein-based enzymes employ coenzymes to augment their capabilities. The coenzymes possess additional chemical moieties that can participate directly in catalysis and thereby enhance catalytic function. In this work, we demonstrate a mechanism by which ribozymes can supplement their limited repertoire of functional groups through RNA-catalyzed incorporation of various coenzymes and coenzyme analogues. The group I ribozyme of Tetrahymena thermophila normally mediates a phosphoester transfer reaction that results in the covalent attachment of guanosine to the ribozyme. Here, a shortened version of the ribozyme is shown to catalyze the self-incorporation of coenzymes and coenzyme analogues, such as NAD⁺ and dephosphorylated CoA-SH. Similar ribozyme activities may have played an important role in the RNA world, when RNA enzymes are thought to have maintained a complex metabolism in the absence of proteins and would have benefited from the inclusion of additional functional groups.Correspondence to: G.F. Joyce     

Factors controlling initial and long-term ATP regeneration catalyzed by immobilized chromatophores

Photosynthetic ATP regeneration was measured in open reactors using immobilized Chromatophores from Rhodopseudomonas capsulata. The influence of several factors on both initial and long-term ADP photophosphorylation was studied. The effect of phosphate salts and of bovine serum albumin on the organelle activity yield was studied. Photophosphorylation was initiated either with ADP or regenerated ATP and the roles of these nucleotides were compared. Different photoreactor configurations were tested for the production of a phosphorylated compound and a flat reactor selected. The presence of inorganic pyrophosphate in the reaction medium was shown to improve the synthesis of ATP 1.4 times. Using the optimal conditions described here, the total G-6P production was 50-fold higher than in batch reactors.     

The hoxZ gene of the Azotobacter vinelandii hydrogenase operon is required for activation of hydrogenase.

The roles of the product of the hoxZ gene immediately downstream of the hydrogenase gene (hoxKG) in Azotobacter vinelandii were investigated by constructing and characterizing a mutant with the center of the hoxZ gene deleted. The strain lacking the functional hoxZ gene product exhibited a low rate of H2 oxidation with O2 as the electron acceptor relative to that of the wild-type strain. Nevertheless, when the enzyme was exogenously activated and methylene blue was used as the electron acceptor from hydrogenase, rates of H2 oxidation comparable to those in the wild-type strain were observed. These results suggest that the gene product of hoxZ plays a role in activating and maintaining hydrogenase in a reduced active state. The product of hoxZ could also be the linkage necessary for transfer of electrons from H2 to the electron transport chain.