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J A Prieto

Publications and source records attributed to J A Prieto.

41 records · Page 3Linked to original sources
PubMedDate not supplied

Stable high-copy-number integration of Aspergillus oryzae alpha-AMYLASE cDNA in an industrial baker's yeast strain.

The Aspergillus oryzae alpha-amylase cDNA was placed under the control of the Saccharomyces cerevisiae actin promoter (pACT1) and introduced into the ribosomal DNA locus of an industrial baker's yeast strain. To obtain a strain eligible for commercial use, we constructed an integrative cassette lacking bacterial DNA sequences but containing the alpha-amylase cDNA and ribosomal DNA sequences to target the integration to this locus. High-copy-number integrants were obtained including a defective TRP1d promoter in the integrative cassette. We selected one transformant, Rib-AMY (CECT10872), in which the multi-integrated sequences were stable even after 200 generations of growth in nonselective medium. This transformant also expressed and secreted high levels of alpha-amylase. Bread made with this strain had a higher volume, lower density, and softer crumbs than bread made with a control strain. The Rib-AMY transformant also was useful in retarding bread firming. This new strain fulfills all the requirements for commercial utilization and should reduce or eliminate the requirement for addition of exogenous alpha-amylase to the flour, reducing allergenic work-related symptoms due to this enzyme.

Aspergillus oryzae↗
PubMedDate not supplied

Purification and characterization of a new alpha-amylase of intermediate thermal stability from the yeast Lipomyces kononenkoae.

A new alpha-amylase from the extracellular culture of the yeast Lipomyces kononenkoae CBS 5608 has been purified to homogeneity by ammonium sulphate treatment, affinity binding on cross-linked starch, and DEAE-Biogel A chromatography. The enzyme was monomeric, with an apparent M(r) of 76 kilodaltons, pI < 3.5, and optimum pH 4.5-5.0, and exhibited intermediate thermal stability. The temperature for optimal enzyme activity was 70 degrees C. It is a glycoprotein with both N- and O-linked sugars. Kinetic analyses indicate that the enzyme has an endoamylolytic mechanism. The kM for soluble starch was 0.80 g.L-1 and the kcat was 622.s-1.

Enzyme Stability↗