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Three different yeast strains, namely Saccharomyces cerevisiae mnn1mnn9, Kluyveromyces lactis JA6/GAA and Zygosaccharomyces bailii [pZ 3klIL-1β], were entrapped in polyvinyl alcohol (PVA) gel particles, obtained following the commercially available immobilization kit named Lentikat®. After immobilization in the PVA-gel particles, yeast cells remained viable: colonization of the gel matrix reached up 100 mg d.w. of cells cm-3 gel for all the strains examined. Lentikats® of K. lactis JA6/GAA and Z. bailii [pZ3klIL-1β] showed to be suitable for the continuous production of glucoamylase and interleukin 1β, respectively, when employed under non-selective conditions. They were of easy handiness and showed excellent mechanical properties during prolonged operation in stirred tank reactors. © 2005 Elsevier Inc. All rights reserved.

To circumvent cell wall retention commonly associated to Saccharomyces cerevisiae when used as a host for heterologous protein production, we have created a translational fusion of human interleukin-1β (IL-1β) to the Pir4 cell wall protein, so as to drive the secretion of the recombinant product to the growth medium. The auxotrophic S. cerevisiae BY4741 was used as host to express the Pir4-IL1β fusion protein. Once it was ascertained that the fusion protein was secreted to the culture medium and behaved as a growth-linked product, S. cerevisiae BY4741 [PIR4-IL1β] was cultured in an aerated fed-batch reactor to achieve high cell density and, consequently, high product concentration in the medium. Two cultivation media were employed, a rich complex and a defined mineral medium, the latter suitably supplemented with bacto-casamino acids as ACA (auxotrophy-complementing amino acid) source. The rich complex medium allowed a good performance of the producer strain only during batch growth, but was revealed to be inadequate for long-term fed-batch operations. The defined mineral medium ensured a better performance, even though not yet satisfactory in spite of a proper ACA supplementation. The behaviour of BY4741 was attributed to an intrinsic sensitivity of the producer strain to long-term aerated fed-batch operations.

A systematic investigation on the effects of auxotrophies on the performance of yeast in aerated fed-batch reactor was carried out. Six isogenic strains from the CEN.PK family of Saccharomyces cerevisiae, one prototroph and five auxotrophs, were grown in aerated fed-batch reactor using the same operative conditions and a proper nutritional supplementation. The performance of the strains, in terms of final biomass decreased with increasing the number of auxotrophies. Auxotrophy for leucine exerted a profound negative effect on the performance of the strains. Accumulation of reactive oxygen species (ROS) in the cells of the strain carrying four auxotrophies and its significant viability loss, were indicative of an oxidative stress response induced by exposure of cells to the environmental conditions. The mathematical model was fundamental to highlight how the carbon flux, depending on the number and type of auxotrophies, was diverted towards the production of increasingly large quantities of energy for maintenance. © 2011 Elsevier Inc.

Viable cells of Kluyveromyces lactis, transformed with the glucoamylase gene from Arxula adeninivorans, were entrapped in beads of Ca-alginate and employed on a lab scale in a continuous stirred and a fluidised bed reactor (FBR), both fed with a rich medium (YEP) containing lactose as carbon source. Experiments with freely suspended cells in batch and chemostat had demonstrated that glucoamylase production was favoured in the presence of lactose and YEP medium. Employing controlled-sized beads having a 2.13 mm diameter, specific glucoamylase productivity was higher in the stirred reactor (CSTR) than in the FBR; in the latter a higher volumetric productivity was achieved, due to the lower void degree. The performance of the immobilised cell systems, in terms of specific glucoamylase productivity, was strongly affected by mass transfer limitations occurring throughout the gel due to the high molecular weight of the product. In the perspective to improve and scale-up the immobilised cell system proposed, a mathematical model, which takes into account substrate transfer limitations throughout the gel, has been developed. The effective lactose diffusivity was related to the bead reactive efficiency by means of the Thiele modulus. The regression of the model parameters on the experimental data of substrate consumption obtained both in the CSTR and in the FBR allowed to estimate lactose diffusivity and the kinetic parameters of the immobilised yeast.

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. © 2003 Elsevier Science B.V. All rights reserved.

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, κ-carrageenan, alginate, and gelatin immobilizates was compared under the same conditions of cell loading. 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, k-carrageenan, alginate, and gelatin immobilizations was compared under the same conditions of cell loading.