Franco Morisi

Substrate and steric specificity of hydropyrimidine hydrase

Eadie et al. [ 1 ] partially purified animal and plant hydantoinases which catalyze the hydrolysis of hydantoin to hydantoic acid. These authors have also found that dimethylhydantoin is a competitive inhibitor of the enzymes. Later Wallach and Grisolia [2] prepared from calf liver an enzyme, termed hydropyrimidine hydrase (EC 3.5.2.2.), which plays an important role in the metabolism of pyrimidines. The same enzyme rapidly hydra, lyzes hydantoin to hydantoic acid and is inactive with disubstituted hydantoins and most probably is identical with the rat liver hydantoinase of Eadie et al. [ I] . Dudley et al. [3] studied the metabolism of the racemic forms of N-substituted S-phenylhydantoins which undergo N-dealkylation as an initial reaction and then are hydrolyzed giving the R-form of phenyl hydantoic acid as a major metabolic product. On the basis of these findings Dudley and Bius [4] formulated the hypothesis that only the R-isomer of S-phenylhydantoin could undergo the ring-opening reaction and that the residual S-isomer undergoes spontaneous and/or enzymatic in vivo racemization. In this communication, we report results of a study designed to gain insight into the specificity of the enzyme hydropyrimidine hydrase. It will be shown that this enzyme rapidly hydrolyzes the R-isomer ot several hydantoins including 5-phenyl-hydantoin and is inactive with the S-isomer. The latter can undergo spontaneous racemization under the conditions of the hydrolysis.

[57] Lactose reduction of milk by fiber-entrapped β-galactosidase. Pilot-plant experiments

Publisher Summary Milk treated by enzymatic methods retains its original nutritional value because glucose and galactose, the products of lactose hydrolysis, are not removed. Moreover, the enzymatic process is particularly well adapted for immobilized lactase preparations. Because milk is a food, it is of the utmost importance not to transform any of its components, in this case lactose, into harmful substances, nor to add anything to the processed milk, nor to change the properties of milk. Furthermore, because of widespread lactose intolerance a very inexpensive industrial process is needed to increase milk consumption. Otherwise, the low lactose dairy products will be available for only a limited few. Now, the only inexpensive process involves using immobilized enzymes. This chapter discusses problems involved in the utilization of fiber-entrapped lactase for the industrial production of milk having low lactose content. The properties of enzymes from two different sources, E. coli and yeast, were studied and compared; then the behavior of some fiber-entrapped preparations and the problems relating to industrial application were investigated. Finally, pilot-plant experiments were carried out to establish optimal operating conditions.

Properties of free and immobilized glucose isomerase

In recent years, a considerable amount of work has been carried out on immobilized enzymes [l-4] , but relatively few studies are concerned with the immobilization of glucose isomerase (D-xylose ketol-isomerase EC 5.3.1.5) [5,6] . Glucose isomerase (GI) was entrapped in polyacrylamide gel [5] and covalently bound to porous glass [6], but poor results were obtained for the instability and the low activity of these preparations. The present communication describes the properties of the glucose isomerase from Sfrepfomyces griseolus as free and immobilized form. A new method of immobilization [7], consisting in the physical entrapment of proteins in filamentous structures, was used to prepare insoluble derivatives of glucose isomerase. Optimal conditions for enzyme stability and activity in free and immobilized form are reported.

[18] Fiber-entrapped enzymes

Publisher Summary This chapter discusses the analytical determination, properties, and application of fiber-entrapped enzymes. Entrapment of invertase is reported in the chapter. It is stressed that any other enzyme can be entrapped using the same procedure. The activity of a fiber-entrapped enzyme is determined by incubating the enzyme fibers and the substrate solution while stirring. The physical properties of enzyme fibers depend on the chemical nature of the polymer and are generally different from those of fibers normally obtained in the textile industry. These differences arise chiefly from the fact that, while in the normal wet-spinning procedure a homogeneous solution of the polymer is spun, in the technology of enzyme entrapment an emulsion is extruded. The catalytic properties of fiber-entrapped enzymes are generally different from those of the corresponding free enzymes. The principal differences are ascribed to, the restricted diffusion of substrates and products inside the porous enzyme support. Because of their excellent activity and stability, fiber-entrapped enzymes have potential technological application in industry, medicine, and analytical chemistry. The easy and inexpensive preparation procedures for enzyme fibers show a clear economic advantage in a large number of applications, which include both replacement of currently used free enzymes and the use of sophisticated enzymes considered too expensive in the soluble form.

Fiber-entrapped enzymes.

Fixed enzymes, regarded as very selective heterogeneous catalysts working at room temperature in aqueous solution, appear very attractive for any industrial activity connected with chemical transformations. However, their development is determined by the economy realized in their applications. We shall not consider here the problems related to enzyme production and purification, but only problems related to enzyme fixation. In particular we shall describe the desirable characteristics of an enzyme fixation technique developed in our laboratories and based on entrapment of enzymes in fibers (1,2).

Synthesis of l-Tryptophan from Indole and dl-Serine by Tryptophan Synthetase Entrapped in Fibres: I. Preparation and Properties of Free and Entrapped EnzymeII. Reactor Studies

Optimal culture conditions for microbial production of tryptophan synthetase were studied. It was found that on cultivation of Escherichia coli 476, a tryptophan auxotroph, in a medium containing 5g/liter glycerol as C source, supplemented with 1 g/liter of acid-treated peptone, cells with high tryptophan synthetase activity could be obtained.The enzyme was extracted from cells and 3-fold purified by heat treatment and ammonium sulfate precipitation. The overall yield of the isolation procedure was 60%.The partially purified tryptophan synthetase was entrapped in cellulose triacetate fibres. Under storage conditions, in refrigerator, the entrapped enzyme was stable at least for 6 months. The activity of the entrapped enzyme was about 75% with respect to the free enzyme.Similar behaviour for the free and entrapped enzyme was observed as to the effect of temperature and pH on the enzymic activity. The operational stability of the entrapped tryptophan synthetase was very good (activity unchanged after 50 day...