Purification of recombinant hyaluronan lyase of Streptococcus pyogenes bacteriophage H4489A expressed in Escherichia coli and its application for the specific determination of hyaluronan concentration

Hyaluronan (HA) lyase of Streptococcus pyogenes bacteriophage was expressed in Escherichia coli and purified to homogeneity by immobilized metal affinity chromatography (IMAC). Unlike most bacterial HA lyases, the phage enzyme specifically cleaved HA to unsaturated oligosaccharides which has an optimum absorption at 232 nm. The absorbance of the digestion product reached a limiting value as reaction time increased. The limiting absorbance showed linearity in the range of concentrations 0.05–0.5 mg/mL. Based on this fact, a specific, simple, easy to apply, low cost, and fast enough method was developed for routine determination of HA concentration of a microbial HA production process. This phage HA lyase-based limiting absorbance method has same accuracy and sensitivity as conventional carbazole method for the determination of HA produced from Streptococcus zooepidemicus cultivation.