• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by silver stain

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to cleave the native substrate Hyaluronan (Catalog # ). The specific activity is >75,000 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  E. coli-derived Ser259-Ile1072, with an N-terminal Met and 6-His tag Accession # NP_688206

• Accession #

• N-terminal Sequence    
  Analysis

  Met

• Predicted Molecular Mass

  93 kDa

• SDS-PAGE

  86 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 500 mM Sodium Acetate, 50 mM CaCl₂, pH 6.5

• Recombinant Streptococcus agalactiae Hyaluronan Lyase (rHyluronan Lyase) (Catalog # 5150-GH)

• Substrate: Hyaluronan (Catalog # )

• 96 Well UV Transparent Plate (Costar, Catalog # 3635)

• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute Substrate to 5.0 mg/mL in Assay Buffer.

2. Dilute rHyaluronan Lyase to 2.0 µg/mL in Assay Buffer.

3. Load into a plate 50 µL of 2.0 µg/mL rHyaluronan Lyase, and start the reaction by adding 50 µL of 5.0 mg/mL Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 5.0 mg/mL Substrate.

4. Read plate at 232 nm in kinetic mode for 5 minutes.

5. Calculate specific activity:

     *Adjusted for Substrate Blank  
      **Using the extinction coefficient 3800 M  ^-1cm  ^-1  
      ***Using the path correction 0.32 cm  
     Note: the output of many spectrophotometers is in mOD Per Well:

• rHyluronan Lyase: 0.1 µg

• Substrate: 2.5 mg/mL

Background: Hyaluronan Lyase

The hyaluronan lyase from Streptococci agalactiae is highly specific towards hyaluronan (1). However, a thousand fold lower activity towards unsulfated chondroitin sulfate has also been observed (2). The enzyme activity is Ca²⁺ dependent (3). During digestion the enzyme moves processively along the hyaluronan chains continuously releasing disaccharide units as it travels (1). The recombinant enzyme can be used to digest hyaluronan and study chondroitin sulfate structure (2). Unlike hyaluronidases, hyaluronan lyase digestion of the substrate creates a double bond at the non-reducing terminus of the product; therefore, the digestion progress can be followed by UV spectrometry and the enzyme can be used for hyaluronan quantification. The recombinant enzyme can also be used for drug screening because the enzyme is a major pathogenic factor for Streptococci agalactiae that causes serious, often fatal, neonatal infections (4). The expressed protein corresponds to the mature form of the native protein (3).

• References:

1. Lin, B. et al. (1994) J. Biol. Chem. 269:30113.

2. Baker, J. et al. (1997) Biochem. J. 327:65.

3. Akhtar, M. et al. (2006) J. Biol. Chem. 281:28336.

4. Hynes, W.L. and Walton, S.L. (2000) FEMS Microbiol. Lett. 183:201.

• Entrez Gene IDs:

  3685697 (S. pombe)

• Alternate Names:

  Hyaluronan Lyase