Deamidation and glycation of a Bacillus licheniformis α-amylase during industrial fermentation can improve detergent wash performance

Connie Pontoppidan, Svend G. Kaasgaard, Carsten P. Sønksen, Carsten Andersen*, Birte Svensson*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Abstract

The industrial thermostable Bacillus licheniformis α-amylase (BLA) has wide applications, including in household detergents, and efforts to improve its performance are continuously ongoing. BLA during the industrial production is deamidated and glycated resulting in multiple forms with different isoelectric points. Forty modified positions were identified by tandem mass spectrometric peptide mapping of BLA forms separated by isoelectric focusing. These modified 12 asparagine, 9 glutamine, 8 arginine and 11 lysine residues are mostly situated on the enzyme surface and several belong to regions involved in stability, activity and carbohydrate binding. Eight residues presumed to interact with starch at the active site and surface binding sites (SBSs) were subjected to mutational analysis. Five mutants mimicking deamidation (N→D, Q→E) at the substrate binding cleft showed moderate to no effect on thermostability and kcat and KM for maltoheptaose and amylose. Notably, the mutations improved laundry wash efficiency in detergents at pH 8.5 and 10.0. Replacing three reducing sugar reactive side chains (K→M, R→L) at a distant substrate binding region and two SBSs enhanced wash performance especially in liquid detergent at pH 8.5, slightly improved enzymatic activity and maintained thermostability. Wash performance was most improved (5-fold) for the N265D mutant near substrate binding subsite +3.
Original languageEnglish
JournalAmylase
Volume5
Issue number1
Pages (from-to)38-49
Number of pages12
ISSN2450-9728
DOIs
Publication statusPublished - 2021

Keywords

  • mass spectrometric peptide mapping
  • Site-directed mutagenesis
  • Surface binding sites
  • Thermostability
  • Enzyme kinetics

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