Characterization of a novel α-amylase from Lipomyces kononenkoae and expression of its gene (LKA1) in Saccharomyces cerevisiae
Date
1995
Authors
Steyn A.J.C.
Pretorius I.S.
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Abstract
A highly active α-amylase (76 250 Da) secreted by the raw starch-degrading yeast Lipomyces kononenkoae strain IGC4052B was purified and characterized. Using high performance liquid chromatography (HPLC), end-product analysis indicated that the L. kononenkoae α-amylase acted by endo-hydrolysis on glucose polymers containing α-1,4 and α-1,6 bonds, producing mainly maltose, maltotriose and maltotetraose. The following NH2-terminal amino acids were determined for the purified enzyme: Asp-Cys-Thr-Thr-Val-Thr-Val-Leu-Ser-Ser-Pro-Glu-Ser-Val-Thr-Gly. The L. kononenkoae α-amylase-encoding gene (LKA1), previously cloned as a cDNA fragment, was expressed in Saccharomyces cerevisiae under the control of the PGK1 promoter. The native signal sequence efficiently directed the secretion of the glycosylated protein in S. cerevisiae. De-glycosylation of the enzyme indicated that post-translational glycosylation is different in S. cerevisiae from that in L. kononenkoae. Zymogram analysis indicated that glycosylation of the protein in S. cerevisiae had a negative effect on enzyme activity. Southern-blot analysis revealed that there is only a single LKA1 gene present in the genome of L. kononenkoae.
Description
Keywords
amylase, fungal protein, glucose polymer, glycosylated protein, maltose, maltotriose, signal peptide, amino acid sequence, amino terminal sequence, article, controlled study, enzyme activity, enzyme analysis, enzyme glycosylation, enzyme purification, enzyme release, enzyme structure, gene expression, high performance liquid chromatography, hydrolysis, nonhuman, priority journal, promoter region, recombinant gene, saccharomyces cerevisiae, southern blotting, yeast, alpha-Amylase, Amino Acid Sequence, Chromatography, High Pressure Liquid, Electrophoresis, Polyacrylamide Gel, Gene Expression Regulation, Fungal, Metals, Molecular Sequence Data, Molecular Weight, Recombinant Proteins, Saccharomyces cerevisiae, Saccharomycetales, Starch, Substrate Specificity, Support, Non-U.S. Gov't, Lipomyces kononenkoae, Saccharomyces cerevisiae
Citation
Current Genetics
28
6
28
6