O conhecimento ao nível molecular dos mecanismos envolvidos nas interacções entre leveduras que originam a formação de flocos, tem necessariamente de se iniciar pela identificação dos factores influentes e/ou participantes nos próprios mecanismos. A caracterização fisiológica da floculação da Kluyveromyces marxianus foi alvo de estudo por outros autores (Teixeira, 1989; Sousa et ai, 1992), sendo objectivo do trabalho apresentado nesta tese o estudo da floculação da Kluyveromyces marxianus numa perspectiva essencialmente bioquímica/biologia molecular. Através de uma análise dos dados obtidos experimentalmente constatamos que:
( i ) a acumulação de uma proteína com 37 kDa (p37) na parede celular de Kluyveromyces
marxianus é concomitante com a indução da sua floculação, por qualquer dos métodos
mencionados, sendo a sua distribuição na parede aparentemente aleatória, tal como é observado por imunolocalização com anticorpos conjugados com partículas de ouro coloidal e microscopia electrónica.
( ii ) a referida proteína de 37 kDa foi purificada e parcialmente caracterizada. Possui características hidrofóbicas, um ponto isoeléctrico aproximado de 7 e é uma glicoproteína com cerca de 5% de carbohidratos ligados a um resíduo de asparagina. A sua microsequenciação mostrou a existência de uma elevada homologia da estrutura primária desta proteína com a enzima glicolítica gliceraldeído-3-fosfato desidrogenase (GAPDH). ( iii ) a floculação da Kluyveromyces marxianus pode ser induzida numa cultura a 40°C em presença de 200 mM de CaCl2.
( iv ) a clonagem do gene GAP1, codifiante para a proteína p37, mostrou a ausência de um péptido sinal característico das proteínas translocadas para o retículo endoplasmático, pelo que a p37 é secretada para a parede por uma via ou mecanismo possivelmente diferente do classicamente descrito.
( v ) ao contrário do gene GAP2 codificante para a enzima glicolítica GAPDH, o gene GAP1 é constitutivamente expresso, estando os seus níveis de transcrição aumentados a 40°C. No entanto, o transporte e acumulação da p37 na parede celular da Kluy\>eromyces
marxiatms só é verificado em condições de indução da floculação.
( vi ) a família multigénica de GAPDH está representada no genoma de Kluyveromyces
marxianus por 3 membros, GAP 1-3. Não foram detectados transcriptos do gene GAP3 e
a expressão do gene GAP2 é profundamente afectada pela presença de glucose. Utilizando etanol como fonte de carbono, os níveis de expressão do gene GAP2 são extremamente reduzidos.
( vii ) a utilização de PCR/SSCP, técnica normalmente usada na detecção de mutações, pode ser efectuada na identificação e estudos de expressão (através de RT-PCR/SSCP) de famílias multigénicas.
Os resultados obtidos neste estudo permitem a colocação de várias questões, cujo esclarecimento em muito contribuiria para o conhecimento do mecanismo de floculação da
Kluyveromyces marxianus. A expressão do gene GAP 1 em S. cerevisiae e K. marxianus
utilizando para o efeito vectores específicos, reveste-se de particular interesse, dado que vai permitir verificar em que condições se dá a exportação da p37. A retenção na parede celular desta proteína, bem como o seu envolvimento nas interacções que resultam na floculação destas células, serão questões mais facilmente esclarecidas a partir do momento em que o controlo da expressão da proteína pode ser efectuado de maneira simples, na presença de um promotor apropriado.
De elevado interesse, e não apenas para o estudo da floculação, seria o esclarecimento do mecanismo de secreção da p37, já que a sua translocação para o retículo endoplasmático não é aparentemente efectuada pela via classicamente descrita, devido à inexistência de um péptido sinal associado com a p37. Até à presente data, as
evidências que sugerem a existência de um mecanismo de transporte alternativo, envolvendo nomeadamente "chaperones", são resultantes de experiências in vitro ou de manipulação genética, pelo que a existência in vivo, de proteínas glicosiladas e exportadas por um mecanismo alternativo, como é sugerido para a p37, seria de todo o interesse.
A GAPDH é uma das proteínas mais abundantes a nível celular, chegando a atingir em levedura cerca de 5% do seu peso seco. Este facto, torna particularmente importante o estudo das regiões promotoras, já clonadas parcialmente, dos genes GAP 1-3. Poucas regiões promotoras de genes da levedura Kluyveromyces estão já caracterizadas, pelo que a importância industrial que esta levedura poderá assumir pelas suas características já discutidas na presente tese, justifica também um estudo pormenorizado de regiões específicas a montante das regiões codificantes, em particular de elementos para ligação de factores de transactivação.
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