Introducing transglycosylation activity in a liquefying α-amylase

• Published in: FEBS letters Vol. 453; no. 1; pp. 100 - 106
• Main Authors: Saab-Rincón, Gloria, del-Rı́o, Gabriel, Santamarı́a, Rosa I, López-Munguı́a, Agustı́n, Soberón, Xavier
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 18-06-1999
• Subjects: ABST, α-amylase from Bacillus stearothermophilus; alpha-Amylases - genetics; alpha-Amylases - metabolism; Amino Acid Sequence; Amino Acid Substitution; Catalytic Domain; CGTase, cyclodextrin glycosyltransferase from Bacillus circulans; DNS, 3,5-dinitrosalicylic acid; G1, glucose; G2, maltose; G3, maltotriose; G4, maltotetraose; G5, maltopentaose; G6, maltohexaose; G7, maltoheptaose; Geobacillus stearothermophilus - enzymology; Glycoside Hydrolases - metabolism; Glycosylation; Glycosyltransferases - genetics; Glycosyltransferases - metabolism; HPLC, high-pressure liquid chromatography; Hydrogen-Ion Concentration; Hydrolysis; MES, 2-(N-morpholino) ethanesulfonic acid; Models, Molecular; Molecular Sequence Data; MOPS, 3′-(N-porpholino) propanesulfonic acid; Mutagenesis, Site-Directed; PCR, polymerase chain reaction; PDB, Protein Databank; Reaction mechanism; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Sequence Alignment; Sequence Analysis; Site-directed mutagenesis; TAA, taka amylase or alpha-amylase from Aspergillus oryzae; TLC, thin-layer chromatography; Transglycosylation; α-Amylase; CGTase, cyclodextrin glycosyltransferase from Bacillus circulans; G1, glucose; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; G2, maltose; G5, maltopentaose; MES, 2-( N-morpholino) ethanesulfonic acid; Transglycosylation; Hydrolysis; HPLC, high-pressure liquid chromatography; TAA, taka amylase or alpha-amylase from Aspergillus oryzae; DNS, 3,5-dinitrosalicylic acid; G3, maltotriose; Site-directed mutagenesis; MOPS, 3′-( N-porpholino) propanesulfonic acid; Reaction mechanism; ABST, α-amylase from Bacillus stearothermophilus; G4, maltotetraose; G6, maltohexaose; G7, maltoheptaose; PCR, polymerase chain reaction; PDB, Protein Databank; α-Amylase; TLC, thin-layer chromatography
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(99)00671-7

By mutating Ala-289 by Phe or Tyr in the
Bacillus stearothermophilus α-amylase, we induced this enzyme to perform alcoholytic reactions, a function not present in the wild-type enzyme. This residue was selected from homology analysis with neopullulanase, where the residue has been implicated in the control of transglycosylation [Kuriki et al. (1996) J. Biol. Chem. 271, 17321–17329]. We made some inferences about the importance of electrostatic and geometrical modifications in the active site environment of the amylase to explain the behavior of the modified enzyme.