Thesis for Doctor of Philosophy

Maria Catarina Marques Dias de Almeida
“Produção de Pectinase utilizando culturas contínuas de leveduras”
Ph. D. in Chemical and Biological Engineering, UM, 2005
Supervisors: J. A. Teixeira; P. Moradas-Ferreira

Abstract

Pectin processing enzymes are used in many industrial processes dealing with fruits and vegetables, as they catalyze the degradation of pectic substances. Currently, the main source for commercial pectinase is Aspergillus niger in batch cultures. This fungus is traditionally chosen due to the wide variety of secreted proteins and metabolites and to its GRAS classification. However, to obtain pure enzymatic solutions for specific industrial needs, the broth must be subjected to high cost and time-consuming downstream separation procedures. As an alternative, in most of the cases, the enzyme can be sold as a commercial mixture including different contaminant metabolites.

A yeast strain can be chosen as a substitute for pectinase production. Wild strains can be selected from special environments where they produce the desired enzyme, and then they can be transformed in order to secrete the target enzyme or to favorably change the characteristics of the microrganism. A convenient way to increase the process productivity is to work with high cell density systems.

The main goal of this work is to establish a continuous procedure to produce endopolygalacturonase (E.C. 3.2.1.15) secreted by the yeast strain Kluyveromyces marxianus CCT 3172, selected from a cocoa fermentation in Brazil as being a good endoPG producer.

In order to retain the cells inside continuous bioreactors two different strategies were followed:

· the wild strain was transformed to over-express the GAP1 gene encoding the protein p37, and thus obtain a flocculating phenotype; to this end, an expression vector for Kluyveromyces marxianus was constructed and named pKY37; this vector is based on the commercial plasmid for gene expression in Saccharomyces cerevisiae pYES2, uses the S11 fragment as the replication origin and has the GAP1 expression under the control of the GAL1 promoter; the transformant cells were tested in a continuous reactor;

· continuous bioreactors were assayed having the wild type cells immobilized in different carriers, namely a continuous stirred tank reactor and a packed bed reactor; two carriers were tested – Siran (a porous glass) and a cellulosic support from spent grains (a by-product of brewing industries).

Keywords: flocculation, glyceraldehyde-3-phosphate dehydrogenase, continuous reactors, pectinase, immobilized yeast cells, Kluyveromyces marxianus, Siran, spent grains