A Novel Hyaluronidase Produced by Bacillus sp. A50

Abstract

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 1.5×104 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 1.02×106 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 Ca2+, Mg2+, or Ni2+, and inhibited by Zn2+, Cu2+, 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.

Purification of hyaluronidase as an anticancer agent inhibiting CD44

“…However, all of the gas atoms occupy axial positions with less sterically beneficial interactions. Thus, the oligosaccharide has an energetically stable structure (Bansal, Kedige, & Anand, 2010, Guo et al, 2014, Necas et al, 2008. HA acts as a significant component in inflammation and inflammation-related tumorigenesis (Huang, Zhao, & Radi, 2014, Ward, Vasquez, Tolg, Telmer, & Turley, 2011.…”

Constructing a recombinant hyaluronic acid biosynthesis operon and producing food-grade hyaluronic acid in Lactococcus lactis

“…Then, three volumes of ethanol were added to one volume of the solution to precipitate HA and after centrifugation, the precipitate was dissolved in distilled water. HA polysaccharides (100 mg) were degraded by hyaluronate lyase as previously described [12] and desalted on a Bio-Gel P-2 column (0.5 × 200 cm) in 0.1 M NH 4 HCO 3 . The eluent was monitored at a UV wavelength of 232 nm.…”

“…The polysaccharide was purified from the culture and degraded by hyaluronate lyase as previously described [12,26]. It was then desalted on a Bio-Gel P-2 column and analyzed on a C18 reversed-phase column (Fig.…”

Determination of modification degree in BDDE-modified hyaluronic acid hydrogel by SEC/MS

“…Table 5 Crosslinked and pendent modified fragments in Fraction 1, 2, 3 confirmed by 1 H NMR and MS. MS analysis give the accurate MW and structure information of all fractions ( Table 5) 6 Hz) and ␦ 6.084 (d, 1H, J = 21.6 Hz), chemical shift of the proton in CH C group also increase (␦ 5.930). The HAase-B, as a hyaluronate lyase, can hydrolyze (1 → 4) bonds between GlcA and GlcNAc by a ␤-elimination reaction to form unsaturated bonds like other microbial hyaluronidase (Guo et al, 2014;Meyer, Hobby, Chaffee, & Dawson, 1940;Meyer, 1971;Wiegrebe, Buschauer, Bernhardt, & Göpferich, 2005). It is very unusual to hydrolyze (1 → 3) bonds between GlcA and GlcNAc to generate unsaturated monosaccharide.…”

“…The HAase-B, which has high activity and mild reaction condition, can hydrolyze (1 → 4) bonds between GlcA and GlcNAc by a ␤-elimination reaction and digest HA into unsaturated disaccharide ( DiHA) completely with UV absorbance at 232 nm wavelength ( Fig. 3) (Guo, Shi, Sheng, & Wang, 2014). When a BDDE-modified HA hydrogel is treated with this enzyme, DiHA and modified fragments with special MW are obtained.…”