title: Molecular mutagenesis at Tyr-101 of the amylomaltase transcribed from a gene isolated from soil DNA.
abstract: The wild-type (WT) amylomaltase gene was directly isolated from soil DNA and cloned into a pET19b vector to express in E. coli BL21(DE3). The ORF of this gene consisted of 1,572 bp, encoding an enzyme of 523 amino acids. Though showing 99% sequence identity to amylomaltse from Thermus thermophilus ATCC 33923, this enzyme is unique in its alkaline optimum pH. In order to alter amylomaltase with less coupling or hydrolytic activity to enhance cycloamylose (CA) formation through cyclization reaction, site-directed mutagenesis of the second glucan binding site involving in CA production was performed at Tyr-101. The result revealed that the mutated Y101S enzyme showed a small increase in cyclization activity while significantly decreased disproportionation, coupling and hydrolytic activities. Mutation also resulted in the change in substrate specificity for disproportionation reaction. The WT enzyme preferred maltotriose, while the activity of mutated enzyme was the highest with maltopentaose substrate. Product analysis by HPAEC-PAD demonstrated that the main CAs of the WT amylomaltase were CA29-CA37. Y101S mutation did not change the product pattern, however, the amount of CAs formed by the mutated enzyme tended to increase especially at long incubation time.
authors.forename: S
authors.initials: S
authors.lastname: Watanasatitarpa
authors.forename: P
authors.initials: P
authors.lastname: Rudeekulthamrong
authors.forename: K
authors.initials: K
authors.lastname: Krusong
authors.forename: W
authors.initials: W
authors.lastname: Srisimarat
authors.forename: W
authors.initials: W
authors.lastname: Zimmermann
authors.forename: P
authors.initials: P
authors.lastname: Pongsawasdi
authors.forename: J
authors.initials: J
authors.lastname: Kaulpiboon
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D003500:Cyclization; D003505:Cyclodextrins / Q000096:biosynthesis*; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006004:Glycogen Debranching Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012987:Soil / Q000737:chemistry; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000201:enzymology / Q000235:genetics; D014158:Transcription, Genetic; D014312:Trisaccharides / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003505:Cyclodextrins; D004269:DNA, Bacterial; D006004:Glycogen Debranching Enzyme System; D009844:Oligosaccharides; D011994:Recombinant Proteins; D012987:Soil; D014312:Trisaccharides; C019193:maltopentaose; C008317:maltotriose; C020569:4 alpha-glucanotransferase