Vincenzo Scardi

Improved method for immobilizing invertase-active whole cells of Saccharomyces cerevisiae in gelatin

Abstract Conditions are presented for the simple, rapid and reproducible immobilization of invertase [β- d -fructosidase, β- d -fructofuranoside fructohydrolase, EC 3.2.1.26] -active whole cells of Saccharomyces cerevisiae with gelatin. The efficiency of the resulting immobilized-cell preparation is compared with a preparation obtained by a previously described method1 as well as with yeast whole-cells entrapped in alginate and κ-carrageenan gels.

Enzyme inactivation and stabilization studies in an ultrafiltration reactor

SummaryAn ultrafiltration membrane enzymatic reactor is used in connection with different reacting systems.The experimental conditions are such that the enzyme, which operates at fairly high concentration levels because of the concentration polarization phenomena taking place in the reactor, is still in soluble form.The analysis of the system unsteady-state response enables the identification of the mechanism of enzyme deactivation and the extraction of the kinetic parameters of both the deactivation and the main reaction.The stabilizing effect observed in connection with enzyme entrapment within an inert gel deposited onto the U.F. membrane active surface is also discussed.

A new method of whole microbial cell immobilization

SummaryA simple method for producing gelatin-immobilized microbial cells is described. The microorganism used as an example was bakers yeast (Saccharomyces cerevisiae). The gel particles containing these cells were utilized as an immobilized enzyme (invertase) both in stirred batch and packed bed systems.

Gelatin-entrapped whole-cell invertase

SummaryThe investigated biocatalyst consists of gelatin-entrapped cells of Saccharomyces cerevisiae retaining invertase activity. Comparative examination of pH profile, apparent Km, saturation velocity and activation energy indicates that the entrapment procedure did not influence invertase affinity with sucrose but lead to some loss of activity probably due to either enzyme inactivation or cell-wall impairment as well as to substrate diffusion limitation in the gel matrix.

Kinetic behaviour of immobilized enzyme membrane reactors

Abstract Enzyme immobilization techniques which are founded on protein gelification onto the active surface of an ultrafiltration membrane are discussed in connection with p -nitro-phenyl-phosphate hydrolysis by acid phosphatase (E.C.3.1.3.2.) Two different immobilized enzyme membrane reactor layouts have been considered. In both the enzyme has been confined within a gel layer onto the active surface of the U.F. membrane. The gel formation has been obtained by means of a concentration polarization technique. In the first reactor the enzyme has been cogelified with albumin, in the second it has been previously co-cross-linked with the same albumin and subsequently ultrafiltered. The reduction in activation energy which is shown by both immobilized enzyme configurations as compared to the corresponding soluble enzyme tests, clearly indicates that a combined substrate mass transfer/reaction step is rate controlling.

Ultrafiltration processing with enzyme-gel composite membranes☆

Abstract The so-called gel polarization model describes, in the ultrafiltration of proteins, the formation of an adherent gel layer of concentrated protein at the pressurized surface of an ultrafiltration membrane. This formation occurs in a wide variety of fluid-dynamic regimes. The question arose as to whether a gelled enzyme, associated with an ultrafiltration merebrane in this way can be used as a type of immobilized enzyme. p]The kinetic behaviour of gelled acid phosphatase overlying cellulose acetate membranes is reported. Experiments have been carried out in an unstirred batch system and in a recirculating system with various substrate concentrations and trans-membrane pressure drops. The results are discussed and an attempt at modelling is presented.

A rapid colorimetric method for the determination of isonicotinic acid hydrazide in blood serum

Abstract A rapid and convenient method for the determination of isoniazid in blood serum or plasma has been described. This method is based on the reaction between INH and sodium pentacyanoammineferroate, which results in a yellowish chromogen whose maximum absorption is at 430 mμ. Factors that influence the reaction are discussed in order to attain the maximal sensitivity. In comparison with other methods for INH estimation, the present method is superior, since it is specific and simple. It can be carried out in not more than half an hour with ordinary laboratory equipment.

Invertase and acid phosphatase in free and gel-immobilized cells of Saccharomyces cerevisiae grown under different cultural conditions

Abstract A systematic investigation of the growth mode and appearance of two cell wall enzymes (invertase E.C. 3.1.1.26, and acid phosphatase E.C.3.1.3.2) in free and immobilized cells of Saccharomyces cerevisiae was carried out. Gel-entrapped yeast cells were stimulated into growth by incubation in media differing from one another in composition, under different experimental conditions. In immobilized cells, lower growth rates were observed which were mainly due to the resistance of the matrix to nutrient diffusion. Invertase activity reached levels higher than those of corresponding free cells. This result was independent of the cultural system adopted for growth and of the nature of the gel matrix chosen for immobilization, and could not be explained merely in terms of diffusional resistance. A possible explanation could be some physiological change related to the state of immobilization of the yeast cells. What was observed with invertase seems to be peculiar to this enzyme, as the extent of repression, in the case of acid phosphatase, was almost the same for both free and gel-immobilized cells.

Mechanical stability and diffusional resistance of a polymeric gel used for biocatalyst immobilization

The mechanical strength of gelatin gels insolubilized by crosslinking with formaldehyde was measured at various gelatin percentages and formaldehyde-to-gelatin ratios. This property was shown to be related to the characteristic sponge-like structure of the insolubilized gelatin gel, a structure that unexpectedly is also responsible for the resistance to substrate and product diffusion. A comparison between immobilizates of invertase and invertase-active yeast cells prepared with different gelatin concentrations showed that the enzyme, in contrast to cells, is deeply involved in the gel insolubilization process. The catalytic behavior of agar, kappa-carrageenan, alginate, and gelatin immobilizates was compared under the same conditions of cell loading.

Immobilization of yeast cells by adhesion on tuff granules

SummaryA method for immobilizing yeast cells (Saccharomyces cerevisiae) possessing invertase activity by direct adhesion on tuff granules coated with insolubilized gelatin is described. The immobilized cells, firmly fixed as a monolayer onto the surface of the support granules display catalytic properties (in terms of apparent Km) close to free cells and are particularly suitable for continuous sucrose hydrolysis in a fixed-bed reactor. From an industrial point of view, the immobilization method described here has two advantages over other immobilization methods, i.e. the immobilized yeast cells have a fairly good operational stability and their proliferation on tuff granules can be controlled.