Action pattern of α-amylases on modified maltooligosaccharides

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

2-Chloro-4-nitrophenyl- (CNP) and 4,6-O-benzylidene-modified 4-nitrophenyl-(Bnl-NP) β-maltooligosaccharides (DP 4-8) were synthesised from cyclodextrins using a chemical procedure. For the preparation of CNP-maltooligosides of longer chain length a new chemoenzymatic procedure was developed using rabbit skeletal muscle glycogen phosphorylase b. These substrates were used for further studies of the action pattern of porcine pancreatic α-amylase (PPA), human salivary α-amylase (HSA) and Bacillus licheniformis α-amylase (BLA). The hydrolysis products and the remaining substrates were separated and quantified by HPLC. Our results suggest at least six subsites in the binding region of HSA; four glycone (-4, -3, -2, -1) and two aglycon binding sites (+1, +2). The binding modes of the benzylidene derivatives indicated a favourable interaction between the Bnl group and subsite (-3) and an unfavourable one with subsite (-4). PPA exhibited a unique pattern of action on CNP-maltooligosaccharides by cleaving maltotriose units from the nonreducing ends and leaving CNP-glycosides, or by cleaving CNP-G2 units from the reducing ends to leave maltooligosaccharides. Modification of the nonreducing end of NP glycosides to give a 4,6-O-benzylidene-D-glucopyranosyl group indicated a favourable interaction between the Bnl group and the subsites (-3) and (-5) but an unfavourable one with subsite (-4), which resulted in a clear shift in the product pattern. The binding region is longer in BLA than in human amylases. Our results suggested the presence of at least eight subsites; five glycone binding sites and three aglycon ones. The binding modes of substrates will be discussed on the basis of the known features of the structures of α-amylases.

Original languageEnglish
Pages (from-to)171-180
Number of pages10
JournalBiologia - Section Cellular and Molecular Biology
Volume57
Issue numberSUPPL. 11
Publication statusPublished - Dec 1 2002

    Fingerprint

Keywords

  • Action patterns
  • Bacillus licheniformis α-amylase
  • Chemoenzymatic syntheses
  • Human salivary α-amylase
  • Porcine pancreatic α-amylase
  • β-maltooligosaccharide glycosides

ASJC Scopus subject areas

  • Genetics
  • Environmental Science(all)
  • Clinical Biochemistry
  • Cell Biology

Cite this