A halotolerant hyaluronidase from newly isolated Brevibacterium halotolerans DC1: Purification and characterization

An enzyme hyaluronidase (hyase) producing halotolerant bacterium was isolated from dental caries and identified as Brevibacterium halotolerans DC1. Higher growth and hyase production were observed in nutrient broth fortified with hyaluronic acid at pH 7.0, temperature 37 °C, 120 rpm upon 48 h of incubation. Hyase was purified using salt precipitation, DEAE cellulose ion exchange, and Sephadex G-100 gel filtration chromatography. The enzyme was purified to 13-fold with 67.19% recovery of activity and 26.37 U/mg of specific activity. SDS-PAGE and zymography revealed it to be near to homogeneity showing a relative molecular weight of about 43 kDa that was confirmed by MALDI-TOF MS. This hyase was very active and stable at pH 7.0 and temperature 40 °C. The presence of metal ions Ca²⁺ and Mg²⁺ increased its activity while Zn²⁺ and Cu²⁺ severely inhibited it. Being stable at 2 M NaCl, hyase exhibited its halotolerant nature. This enzyme showed wide substrate specificity where hyaluronic acid (HA) was the best substrate. The kinetic studies revealed that K_m and V_max were 91.3 μg/mL and 306.2 μg/mL/min respectively. This is the first report of hyaluronidase from a halotolerant Brevibacterium spp. which can find applications under high salinity.