A Novel Hyaluronidase Produced by Bacillus sp. A50

Hyaluronidases are a family of enzymes that degrade hyaluronic acid (hyaluronan, HA) and widely used in many fields. A hyaluronidase producing bacteria strain was screened from the air. 16S ribosomal DNA (16S rDNA) analysis indicated that the strain belonged to the genus Bacillus , and the strain was named as Bacillus sp. A50. This is the first report of a hyaluronidase from Bacillus , which yields unsaturated oligosaccharides as product like other microbial hyaluronate lyases. Under optimized conditions, the yield of hyaluronidase from Bacillus sp. A50 could reach up to 1.5×10 4 U/mL, suggesting that strain A50 is a good producer of hyaluronidase. The hyaluronidase (HAase- B ) was isolated and purified from the bacterial culture, with a specific activity of 1.02×10 6 U/mg protein and a yield of 25.38%. The optimal temperature and pH of HAase- B were 44°C and pH 6.5, respectively. It was stable at pH 5–6 and at a temperature lower than 45°C. The enzymatic activity could be enhanced by Ca 2+ , Mg 2+ , or Ni 2+ , and inhibited by Zn 2+ , Cu 2+ , EDTA, ethylene glycol tetraacetic acid (EGTA), deferoxamine mesylate salt (DFO), triton X-100, Tween 80, or SDS at different levels. Kinetic measurements of HAase- B towards HA gave a Michaelis constant ( K m ) of 0.02 mg/mL, and a maximum velocity ( V max ) of 0.27 A 232 /min. HAase- B also showed activity towards chondroitin sulfate A (CSA) with the kinetic parameters, K m and V max , 12.30 mg/mL and 0.20 A 232 /min respectively. Meanwhile, according to the sequences of genomic DNA and HAase- B ’s part peptides, a 3,324-bp gene encoding HAase- B was obtained.