Palma Parascandola

Hydrolysis of Lactose in a Fluidized Bed of Zeolite Pellets Supporting Adsorbed ß - Galactosidase

The purpose of this work is to verify the possibility of using a simple physical adsorption technique to improve enzyme efficiency in performing lactose hydrolysis. Immobilization experiments of ß -galactosidase on zeolite pellets showed that this process is fairly slow and can be accomplished with contact times shorter than few hours. Comparison between homogeneous and heterogeneous hydrolysis experiments in mixed batch reactors indicates that the enzyme distribution within the zeolite pellets is concentrated in a thin shell under the pellet surface. Continuous conversion experiments were carried out on a purposely-built fluidization column. Results were fitted with a mathematical model for fluidized bed conversion without the use of any further adjustable parameter than those used in the heterogeneous batch conversion. Furthermore, no change of the parameter values was necessary. Scale-up predictions given by the model showed that fluidized columns few meters high of pellets supporting b-galactosidase are sufficient for an industrially suitable process of lactose hydrolysis. which can be performed on columns of few meters and, therefore, these are applicable to relatively small process scales as those found in traditional Italian cheese making factories.

Lactose hydrolysis by immobilized β-galactosidase: the effect of the supports and the kinetics

Abstract The kinetic behaviour of β-galactosidase from Kluyveromices marxianus (Saccharomyces) lactis, immobilized on different oxides supports, such as alumina, silica, and silicated alumina has been studied. We observed a strong dependence of the immobilized enzyme activities on the chemical nature and physical structure of the supports. In particular, when the particle sizes of the supports are increased, the enzymatic activity strongly decreases. The hydrolysis of lactose, promoted by the mentioned enzyme immobilized on small commercial silica spheres, has been studied in different operative conditions, by changing: feed rate, reagent and products concentration and temperature, while pH has been kept constant (7.0). A depressing effect originated by both the reaction products, has been observed. Therefore, a kinetic model based on the Michaelis–Menten mechanism, in which the depressing effects of both the hydrolysis products (galactose and glucose) are also considered, have been developed and the related parameters determined.

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.

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.

Performance of the auxotrophic Saccharomyces cerevisiae BY4741 as host for the production of IL-1β in aerated fed-batch reactor: role of ACA supplementation, strain viability, and maintenance energy

BackgroundSaccharomyces cerevisiae BY4741 is an auxotrophic commonly used strain. In this work it has been used as host for the expression and secretion of human interleukin-1β (IL1β), using the cell wall protein Pir4 as fusion partner. To achieve high cell density and, consequently, high product yield, BY4741 [PIR4-IL1β] was cultured in an aerated fed-batch reactor, using a defined mineral medium supplemented with casamino acids as ACA (auxotrophy-complementing amino acid) source. Also the S. cerevisiae mutant BY4741 Δyca1 [PIR4-IL1β], carrying the deletion of the YCA1 gene coding for a caspase-like protein involved in the apoptotic response, was cultured in aerated fed-batch reactor and compared to the parental strain, to test the effect of this mutation on strain robustness. Viability of the producer strains was examined during the runs and a mathematical model, which took into consideration the viable biomass present in the reactor and the glucose consumption for both growth and maintenance, was developed to describe and explain the time-course evolution of the process for both, the BY4741 parental and the BY4741 Δyca1 mutant strain.ResultsOur results show that the concentrations of ACA in the feeding solution, corresponding to those routinely used in the literature, are limiting for the growth of S. cerevisiae BY4741 [PIR4-IL1β] in fed-batch reactor. Even in the presence of a proper ACA supplementation, S. cerevisiae BY4741 [PIR4-IL1β] did not achieve a high cell density. The Δyca1 deletion did not have a beneficial effect on the overall performance of the strain, but it had a clear effect on its viability, which was not impaired during fed-batch operations, as shown by the kdvalue (0.0045 h-1), negligible if compared to that of the parental strain (0.028 h-1). However, independently of their robustness, both the parental and the Δyca1 mutant ceased to grow early during fed-batch runs, both strains using most of the available carbon source for maintenance, rather than for further proliferation. The mathematical model used evidenced that the energy demand for maintenance was even higher in the case of the Δyca1 mutant, accounting for the growth arrest observed despite the fact that cell viability remained comparatively high.ConclusionsThe paper points out the relevance of a proper ACA formulation for the outcome of a fed-batch reactor growth carried out with S. cerevisiae BY4741 [PIR4-IL1β] strain and shows the sensitivity of this commonly used auxotrophic strain to aerated fed-batch operations. A Δyca1 disruption was able to reduce the loss of viability, but not to improve the overall performance of the process. A mathematical model has been developed that is able to describe the behaviour of both the parental and mutant producer strain during fed-batch runs, and evidence the role played by the energy demand for maintenance in the outcome of the process.

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.

Ethanolic fermentation by yeast cells immobilized in polyaldehyde-hardened gelatin beads

Abstract Viable cells of Saccharomyces cerevisiae were entrapped within gelatin beads by means of a novel technique using oxystarch as a harmless insolubilizing agent instead of formaldehyde or glutaraldehyde. The beads, after incubation in a nutrient medium, were examined in a packed-bed reactor for the continuous production of ethanol under different experimental conditions. Compared with Ca-alginate and κ-carrageenan, oxystarch-hardened gelatin showed the highest cell loading and mechanical stability.

A novel process-based model of microbial growth: self-inhibition in Saccharomyces cerevisiae aerobic fed-batch cultures

AbstractBackgroundMicrobial population dynamics in bioreactors depend on both nutrients availability and changes in the growth environment. Research is still ongoing on the optimization of bioreactor yields focusing on the increase of the maximum achievable cell density.ResultsA new process-based model is proposed to describe the aerobic growth of Saccharomyces cerevisiae cultured on glucose as carbon and energy source. The model considers the main metabolic routes of glucose assimilation (fermentation to ethanol and respiration) and the occurrence of inhibition due to the accumulation of both ethanol and other self-produced toxic compounds in the medium. Model simulations reproduced data from classic and new experiments of yeast growth in batch and fed-batch cultures. Model and experimental results showed that the growth decline observed in prolonged fed-batch cultures had to be ascribed to self-produced inhibitory compounds other than ethanol.ConclusionsThe presented results clarify the dynamics of microbial growth under different feeding conditions and highlight the relevance of the negative feedback by self-produced inhibitory compounds on the maximum cell densities achieved in a bioreactor.