Estudio sobre enzimas amilolíticas de las levaduras no convencionales. Phaffia rhodozyma y Schwanniomyces occidentalis
Author
Marín Alberdi, DoloresAdvisor
Fernández Lobato, MaríaEntity
UAM. Departamento de Biología Molecular; Centro de Biología Molecular Severo Ochoa (CBM)Date
2005-02-25Subjects
Enzimas-Aspectos genéticos-Tesis doctorales; Amilasas-Tesis doctorales; Levaduras-Tesis doctorales; Biología y Biomedicina / BiologíaNote
Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular, Fecha de lectura 25-02-2005Abstract
One of the most interesting objectives in biotechnology is the generation of
industrial yeast strains with amylolytic activity, which can be used for food production.
In this work, an amylolytic industrial baker strain of Saccharomyces cerevisiae
containing the Schwanniomyces occidentalis SWA2 a-amylase gene was generated. The
new strain contains DNA exclusively from yeast and expresses a high starch
hydrolysing activity. Yeast transformation was canied out by an integrative process
targeted to a dispensable upstream region of a mutated ILV2 locus that determines
sulfometuron resistance. The SWA2 gene was constitutively expressed under the ADHl
promoter. We studied the growth, starch utilization and fermentative capacity of this
novel strain. However, the results of the bakery tests were not fuliy satisfactory in terms
of bread volume. We look for new yeast activities that could be expressed with the
previously used. So, we decided to study the yeast Phafla rhodozyma as a donor of
amylolytic genes.
The yeast Phafia rhodozyma has been widely used in the food industry and has
been approved as a dietary ingredient for humans. In this work, we studied the growth
of this yeast in different substrates and describe its amylolytic system. We purified an
extracellular a-glucosidase from this yeast by using ion exchange chromatography. The
molecular weight was 115 f 7% kDa according to molecular filtration in non
denaturising conditions. SDS gel electrophoresis determines that the protein is formed
by two subunits of approximately 60 kDa each. The maximal activity was achieved at
pH 5.5 and 45 "C. The enzyme presented high activity towards maltose, maltotriose and
oligosaccharides, low rates of hydrolysis of starch and no activity with a-(1,6)-
glycosidic bonds substrates. The novel a-giucosidase presents transglicosidase activity
and could be used for synthesis of oligosaccharides with prebiotic capacities. The
kinetic constants of the enzyme for different substrates were determined. In order to
obtain the gene encoding for this a-glycosidase, we generated a cDNA library of P.
rhodozyma using the yeast expression vector pYES2 S'I. We also designed degenerated
oligonucleotides targeted to consewed regions for different a-glucosidase genes, which
were used as primers in a PCR reaction using genomic DNA of Phafla rhodozyma as a
template. So far, this appmach has shown no positive results.
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