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Production and partial characterization of an endopolygalacturonase from Saccharomyces cerevisiae

1994; Canadian Science Publishing; Volume: 40; Issue: 11 Linguagem: Inglês

10.1139/m94-155

1480-3275

Pilar Blanco, Carmen Sieiro, Ana Díaz, Tomás G. Villa,

Biofuel production and bioconversion

Saccharomyces cerevisiae CECT1389 secreted an extracellular endopolygalacturonase (EC 3.2.1.15) when grown in shake flasks in medium containing galactose alone, or either galactose and polygalacturonic acid or galactose and galacturonic acid as the carbon sources. The synthesis of the enzyme was repressed by glucose and by high oxygen tensions. The enzyme was partially purified by gel exclusion chromatography over Sephacryl S-200, where it showed an apparent molecular mass of 39 kDa; the value determined by high-performance liquid chromatography (HPLC) was 65 kDa. The optimal temperature and pH for enzyme activity were45 °C and 5.5, respectively. The K m and V max values for polygalacturonic acid were 4.7 mg∙mL −1 and 6.4 nmol∙mL −1 ∙min −1 . The K i for HgCl 2 was 6.8 × 10 −5 M. The enzyme exhibited an endo-splitting mechanism as deduced from viscosimetry experiments as well as from an HPLC study of the end products.Key words: characterization, endopolygalacturonase, Saccharomyces cerevisiae.