Recombinant, truncated B. circulans keratanase-II: Description and characterisation of a novel enzyme for use in measuring urinary keratan sulphate levels via LC-MS/MS in Morquio A syndrome

Clin Biochem. 2015 Aug;48(12):796-802. doi: 10.1016/j.clinbiochem.2015.03.024. Epub 2015 Apr 10.


Objective: Morquio A syndrome (mucopolysaccharidosis IVA; MPS IVA) is an autosomal recessive lysosomal storage disorder caused by deficient N-acetylgalactosamine-6-sulphatase (GALNS) activity. Early and accurate diagnosis of this condition is critical for improved patient outcomes, particularly as enzyme replacement therapy has recently become available. An LC-MS/MS assay utilising keratan sulphate (KS) disaccharides derived from keratanase-II digestion provides a sensitive and specific means for quantitation of urinary KS, a screening biomarker for Morquio A (Oguma et al., 2007; Martell et al., 2011). To ensure a reliable supply of keratanase-II, we sought to produce a Bacillus circulans-derived enzyme via a recombinant approach in Escherichia coli.

Design and methods: Bioinformatics analysis of the B. circulans keratanase-II enzyme identified likely dispensable C-terminal domains amenable to enhancement via protein engineering. A truncated form of the enzyme was designed to remove the domains predicted to be unnecessary for catalytic activity and detrimental to recombinant expression in E. coli.

Results: C-terminally truncated, recombinant B. circulans keratanase-II was purified to >98% homogeneity and extensively characterised, demonstrating desired activity, specificity and utility in LC-MS-based quantitation of urinary KS from Morquio A and control samples, and is functionally indistinguishable from full-length, native B. circulans-derived keratanase-II.

Conclusions: This novel, recombinant keratanase-II meets all performance requirements and can be produced in a rapid and reproducible manner. We speculate that other related bacterial enzymes of biomedical or industrial interest may be amenable to similar engineered enhancements.

Keywords: Keratanase; LC/MS–MS; Morquio syndrome; Mucopolysaccharidosis IV; Recombinant enzyme.

MeSH terms

  • Acetylglucosaminidase / biosynthesis
  • Acetylglucosaminidase / chemistry*
  • Acetylglucosaminidase / genetics
  • Acetylglucosaminidase / metabolism
  • Adolescent
  • Adult
  • Animals
  • Bacillus / enzymology
  • Bacillus / genetics
  • Bioengineering / methods
  • Biomarkers / urine
  • Case-Control Studies
  • Catalysis
  • Cattle
  • Child
  • Child, Preschool
  • Chromatography, Liquid / methods
  • Cloning, Molecular
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Humans
  • Keratan Sulfate / urine*
  • Mucopolysaccharidosis IV / urine*
  • Protein Structure, Tertiary
  • Tandem Mass Spectrometry / methods
  • Young Adult


  • Biomarkers
  • Keratan Sulfate
  • keratanase II
  • Acetylglucosaminidase