Differences in the chitinolytic activity of mammalian chitinases on soluble and insoluble substrates

Benjamin A. Barad, Lin Liu, Roberto E. Diaz, Ralp Basilio, Steven J. Van Dyken, Richard M. Locksley, James S. Fraser

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

Chitin is an abundant polysaccharide used by many organisms for structural rigidity and water repulsion. As such, the insoluble crystalline structure of chitin poses significant challenges for enzymatic degradation. Acidic mammalian chitinase, a processive glycosyl hydrolase, is the primary enzyme involved in the degradation of environmental chitin in mammalian lungs. Mutations to acidic mammalian chitinase have been associated with asthma, and genetic deletion in mice increases morbidity and mortality with age. We initially set out to reverse this phenotype by engineering hyperactive acidic mammalian chitinase variants. Using a screening approach with commercial fluorogenic substrates, we identified mutations with consistent increases in activity. To determine whether the activity increases observed were consistent with more biologically relevant chitin substrates, we developed new assays to quantify chitinase activity with insoluble chitin, and identified a one-pot fluorogenic assay that is sufficiently sensitive to quantify changes to activity due to the addition or removal of a carbohydrate-binding domain. We show that the activity increases from our directed evolution screen were lost when insoluble substrates were used. In contrast, naturally occurring gain-of-function mutations gave similar results with oligomeric and insoluble substrates. We also show that activity differences between acidic mammalian chitinase and chitotriosidase are reduced with insoluble substrate, suggesting that previously reported activity differences with oligomeric substrates may have been driven by differential substrate specificity. These results highlight the need for assays against physiological substrates when engineering metabolic enzymes, and provide a new one-pot assay that may prove to be broadly applicable to engineering glycosyl hydrolases.

Original languageEnglish
Pages (from-to)966-977
Number of pages12
JournalProtein Science
Volume29
Issue number4
DOIs
StatePublished - Apr 1 2020

Keywords

  • chitin
  • directed evolution
  • enzymes
  • glycobiology

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