Characterization of a novel α-amylase from Lipomyces kononenkoae and expression of its gene (LKA1) in Saccharomyces cerevisiae

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dc.contributor.authorSteyn A.J.C.
dc.contributor.authorPretorius I.S.
dc.date.accessioned2011-05-15T16:01:29Z
dc.date.available2011-05-15T16:01:29Z
dc.date.issued1995
dc.description.abstractA 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.
dc.description.versionArticle
dc.identifier.citationCurrent Genetics
dc.identifier.citation28
dc.identifier.citation6
dc.identifier.issn1728083
dc.identifier.other10.1007/BF00518165
dc.identifier.urihttp://hdl.handle.net/10019.1/12006
dc.subjectamylase
dc.subjectfungal protein
dc.subjectglucose polymer
dc.subjectglycosylated protein
dc.subjectmaltose
dc.subjectmaltotriose
dc.subjectsignal peptide
dc.subjectamino acid sequence
dc.subjectamino terminal sequence
dc.subjectarticle
dc.subjectcontrolled study
dc.subjectenzyme activity
dc.subjectenzyme analysis
dc.subjectenzyme glycosylation
dc.subjectenzyme purification
dc.subjectenzyme release
dc.subjectenzyme structure
dc.subjectgene expression
dc.subjecthigh performance liquid chromatography
dc.subjecthydrolysis
dc.subjectnonhuman
dc.subjectpriority journal
dc.subjectpromoter region
dc.subjectrecombinant gene
dc.subjectsaccharomyces cerevisiae
dc.subjectsouthern blotting
dc.subjectyeast
dc.subjectalpha-Amylase
dc.subjectAmino Acid Sequence
dc.subjectChromatography, High Pressure Liquid
dc.subjectElectrophoresis, Polyacrylamide Gel
dc.subjectGene Expression Regulation, Fungal
dc.subjectMetals
dc.subjectMolecular Sequence Data
dc.subjectMolecular Weight
dc.subjectRecombinant Proteins
dc.subjectSaccharomyces cerevisiae
dc.subjectSaccharomycetales
dc.subjectStarch
dc.subjectSubstrate Specificity
dc.subjectSupport, Non-U.S. Gov't
dc.subjectLipomyces kononenkoae
dc.subjectSaccharomyces cerevisiae
dc.titleCharacterization of a novel α-amylase from Lipomyces kononenkoae and expression of its gene (LKA1) in Saccharomyces cerevisiae
dc.typeArticle
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