M.I. González Siso

Respirofermentative metabolism in Kluyveromyces lactis:: Insights and perspectives☆

Yeasts do not form a homogeneous group as far as energy-yielding metabolism is concerned and the fate of pyruvate, a glycolytic intermediate, determines the type of energy metabolism. Kluyveromyces lactis has become an alternative to the traditional yeast Saccharomyces cerevisiae owing to its industrial applications as well as to studies on mitochondrial respiration. In this review we summarize the current knowdeledge about the K. lactis respirofermentative metabolism, taking into account the respiratory capacity of this yeast and the molecular mechanisms controlling its regulation, giving an up-to-date picture.

Enzyme encapsulation on chitosan microbeads

Abstract The influence of two variables (cross-linking and protein concentration) on the activity shown by α-amylase and invertase immobilized on chitosan microbeads was studied by means of full factorial experimental designs. Microencapsulation on chitosan beads has shown to be an effective immobilization method for both enzymes and observed differences in their behaviour are explained mainly by the molecular weight of their substrates.

Reoxidation of the NADPH produced by the pentose phosphate pathway is necessary for the utilization of glucose by Kluyveromyces lactis rag2 mutants

Kluyveromyces lactis mutants defective in the glycolytic enzyme phosphoglucose isomerase are able to grow in glucose media and to produce ethanol, but they depend on a functional respiratory chain and do not grow in glucose‐antimycin media. We postulate that this is due to the necessity of reoxidizing, in the mitochondria, the NADPH produced by the pentose phosphate pathway, which may be highly active in these mutants in order to bypass the blockade in the phosphoglucose isomerase step. This oxidation would be mediated by a cytoplasmic‐side mitochondrial NAD(P)H dehydrogenase that would pass the electrons to ubiquinone. Data supporting this hypothesis are provided.

Lactose bioconversion by calcium-alginate immobilization of Kluyveromyces lactis cells

Abstract A new β-galactosidase based biocatalyst consisting of whole Kluyveromyces lactis cells entrapped in calcium alginate beads has been developed. Formulative parameters and their effects on the enzymatic activity were studied by a 23 full factorial experimental design. Enzymatic activity showed a bimodal trend progressing with time and appeared to be influenced by the structure and dimension of the surrounding gel. Small particles became the choice for scale-up purposes. Remarkably, it was demonstrated that β-galactosidase activity per unit of cell biomass was higher in alginate-immobilized than in free-growing cells in the same medium. Milk whey saccharification by the ethanol-permeabilized cells was studied in packed bed bioreactors. Permeabilization increased the lactose hydrolysis rate and prevented ethanol fermentation allowing 99.5% of milk whey lactose to be hydrolyzed at 30°C for 30 h.

β-Galactosidase production by Kluyveromyces lactis on milk whey : batch versus fed-batch cultures

β-Galactosidase production by the yeast Kluyveromyces lactis on milk whey as culture medium was compared in batch and fed-batch cultures. Significant prolongation of the phase of maximum β-galactosidase production can be achieved by optimizing the feeding rate in fed-batch cultures.

Metabolic engineering for direct lactose utilization by Saccharomyces cerevisiae

A recombinant strain of Saccharomyces cerevisiae, secreting β-galactosidase from Kluyveromyces lactis, grew efficiently with more than 60 g lactose l−1. The growth rate (0.23 h−1) in a cheese-whey medium was close to the highest reported hitherto for other recombinant S. cerevisiae strains that express intracellular β-galactosidase and lactose-permease genes. The conditions for growth and β-galactosidase secretion in this medium were optimized in a series of factorial experiments. Best results were obtained at 23 °C for 72 h. Since the recombinant strain produced less than 3% ethanol from the lactose, it was also assayed for the production of fructose 1,6-bisphosphate from cheese whey, and 0.06 g l−1 h−1 were obtained.

Cloning genes from a library using a clustering strategy and PCR

A new polymerase chain reaction (PCR)-based method is described for the isolation of clones of interest from a library when only part of a sequence is available. In actuality, this occurs with many genomes that have been partially sequenced using a random strategy. The method presented here, discriminating clusters by PCR (DCbyPCR), is a nonradioactive and improved alternative to colony hybridization.