Genetic and Functional Characterization of the Hyaluronate Lyase HylB and the Beta-N-Acetylglucosaminidase HylZ in Streptococcus zooepidemicus

Abstract

Hyaluronic acid (hyaluronan) is a high molecular weight glycosaminoglycan composed of repeating disaccharides of glucuronic acid and N-acetylglucosamine. The molecular weight of hyaluronan is important for its rheological property, biological function, and application. However, genes important for regulation of hyaluronan production or molecular weight remain poorly understood. Here, we address the roles of two predicated hyaluronate lyase-encoding genes, hylB and hylZ in Streptococcus zooepidemicus. Semi-quantitative RT-PCR assays showed that hylZ was constitutively expressed while the expression level of hylB was growth-phase dependent. Using recombinantly expressed 6His-HylB and -HylZ protein, enzyme assays revealed that HylB was a hyaluronate lyase, and its K(m) and V(max) were 0.57 μM and 1.43 mM min(-1), respectively. 6His-HylZ showed no activity against hyaluronan while it hydrolyzed pNp-β-GlcNAc suggesting that HylZ was a beta-N-acetylglucosaminidase. Under the optimal conditions (pH 6.0 and 50 °C), the K(m) and V(max) for 6His-HylZ to degrade pNp-β-GlcNA were 1.16 mM and 26.18 μmol min(-1) mg(-1), respectively. Characterization of ΔhylB and ΔhylZ demonstrated that loss of hylB or/and hylZ had no significant effects on cell growth, lactic acid, and hyaluronan yields. Significantly, as compared to the wild type, ΔhylB produced hyaluronan with an 18 % increase in molecular weight. Our results strongly suggest that hylB encodes a hyaluronate lyase while hylZ encodes a β-N-acetylglucosaminidase. hylB-deficient strain has the potential to produce high molecular weight hyaluronan.

Zinc Oxide Quantum Dots May Provide a Novel Potential Treatment for Antibiotic-Resistant Streptococcus agalactiae in Lama glama

“…ZnO is a material with excellent antibacterial activity in many drug-resistant pathogens, including S. aureus , Escherichia coli ( E. coli ) [ 36 ], and Candida albicans [ 37 ]. Compared to traditional metal bacteriostatic materials, when the size of ZnO is reduced to the nanometer range, ZnO QDs typically exhibit additional morphologies and significant antibacterial activity against various bacterial species, e.g., E. coli , S. aureus , and Salmonella pullorum , which have been explored by numerous researchers [ 18 , 38 , 39 , 40 ], prompting us to explore whether they also have inhibitory effects on drug-resistant S. agalactiae in our study. Our experimental results showed that the inhibition ability of ZnO QDs for S. agalactiae WJYT1 was concentration dependent.…”

Cloning and Biochemical Characterization of a Hyaluronate Lyase from Bacillus sp. CQMU-D

“…Microbial HAases are recognized as good sources for HAase development with potential clinical applications. They are found in Bacillus [12,13], Streptococcus [14,15], Streptomyces [16], and other bacteria strains [17][18][19]. Most microbial HAases are hyaluronate lyases, which are convenient for operation and cost-effective for fermentation [17].…”