Study On The Hyaluronidases From Bacillus Sp. A50

Hyaluronan (HA), a naturally occurring linear high-molar-mass polysaccharide, is widely distributed in all the tissues and fluids of animals, with the highest concentrations in soft connective tissues. As an important component in the extracellular matrix (ECM) of vertebrate tissues, HA contributes significantly to cell adhension, migration, proliferation and differentiation. Despite HA from different sources has the same primary structures, their molecules are thought to be very different, ranging from lOkDato5000kDa. HA fragments with different sizes often show different and even opposing biological functions. For example, HA oligosaccharides of4-25disaccharides (1600-10000Da) can promote angiogenesis and induce expression of inflammatory mediators, whereas HA of high molecular weight is anti-angiogenic and has no effect on inflammatory mediators.Currently, HA is mainly produced from microbial fermentation and a little is extracted from animal tissues. The molecular weight of HA is mostly in the range from from1000kDa to3000kDa. Traditionally, Low molecular weight HA (LMW-HA), ranging from10kDa to500kDa, is prepared through physical or chemical degradation. However, the depolymerization will be accompanied by destruction of constituent sugar residues under harsh degradation conditions like strong acidic or alkaline and in the presence of oxidants. When the molecular weight of HA is degraded down to lower than100kDa, the primary structure of HA macromolecular chains will be significantly changed.Thus, HA of low molecular weight (lower than100kDa), especially HA oligosaccharides (oligo-HA), can be prepared by degradationwith hyaluronidase. As a result of its high efficiency and mildness of reaction conditions, HA degradation by hyaluronidase is attracting more interest in recent years. Hyaluronidases are a family of enzymes that degrade hyaluronic acid. HA is the key component of the extracellular matrix(ECM) that maintainswater. The degradation of HA by hyaluronidases can temperally decrease the viscosity of ECM of local tissue and increase the diffusion of drugs, so hyaluronidases are important drug diffusion promoting agents.However, hyaluronidases from animals are limited, but from microbes are unlimited. At present, the microbial hyaluronidases are reported to be obtained from many microorganisms of various genera, including C. perfringens, S. aureus, Peptostreptococcus,and Streptomyces. But, the productivities of microbial hyaluronidases are far from the requirements of industry, even the highest activity reported by Mahesh was only591U/mL. So it is necessary to screen and isolate a new and more promising strain with higher yield of hyaluronidase.A hyaluronate lyase producing bacterial strain, Bacillus sp.A50, was screened from the air around, and the medium constituents and fermentation conditions were optimized to improve the production of the hyaluronate lyase. Then the lyase was purified and characterized, and applied to prepare oligo-HA which performed outstanding effects as cosmetic ingredients. The main results are summerized as follows.1. Screening of strains producing high-level hyaluronidase and identification of strain A50A high-level hyaluronidaseproducing bacteria screened from the air around was named A50. It was identified as gram-positive and selected for further study. The16S rDNA of strain A50was sequenced and submitted to GenBank with accession number of KC522837. Homologous analysis based on the known16S rDNA sequences in NCBI revealed that the strain A50had99%identity with many Bacillus strains. Therefore, the strain A50was one kind of Bacillus, and we named it as Bacillus sp. A50.2. Optimizing medium constituents and fermentation conditions for hyaluronate lyase production from Bacillus sp. A50 (1) The medium components for hyaluronate lyase production from Bacillus sp. A50, including carbon, nitrogen and other factors were optimized. The medium that yielded maximum average hyluronate lyase comprised10g/L peptone,10g/L hydrolyzed milk protein,2g/L K2HPO4·3H2O,0.5g/L MgSO4·7H2O, and5g/L glucose (pH7.0).(2) On the basis of the optimization of the fermentation medium, the effects of incubation temperature and time, agitation rate, and aeration rate were studied on a fermenter of10L. The maximum enzymatic activity was obtained with agitation rate of150r/min, aeration rate of9L/min, incubation temperature35℃andincubation time of8h, the activity of the supernatant was1.5×105U/mL.3. Purification of the hyaluronidase produced by Bacillus sp. A50After salting out, ion-exchange and gel filtration chromatography, the purity of hyaluronidase produced by Bacillus sp. A50was21-fold of the starting culture medium, with a specific activity of1.02×107U/mg protein and a final yield of25.38%. On a DEAE Sepharose Fast Flow column, the protein was eluted with0-0.5mol/L NaCl. The fraction of each peak was collected to analyze the hyaluronidase activity. It was found that only one of the protein peaks had hyaluronidase activity. After further purification on a Superdex200column, a single protein band on the SDS-PAGE gel was obtained with the MW of approximately120kDa, which was named as HAase-B.4. Characterization of HAase-B(1)Effects of temperature and pH on HAase-BWith HA as the substrate, HAase-B displayed an optimal temperature of44℃. The activity of HAase-B remained high and relatively stable between40℃and44℃, but only25%of maximum activity at46℃. When the temperature reached60℃or higher, HAase-B would lose its activity completely. After incubation at40℃or45℃for120min, HAase-B showed little change with its activity, but its stability was very low at55℃. The activity reduced by99%after incubation at55℃for10min. The optimum pH for HAase-B was6.5, and there was no detectable activity at pH4.0and pH9.0. HAase-B was most stable at pH5.0-6.0and retained more than90%activity after incubation at this pH range for2h.(2) Effects of metal ions, metal ion chelators and surfactants on HAase-BMost of the investigated metal ions, including Ca2+, Mg2+, Ni2+, Co2+and Ba2+, had positive effects on the activity of HAase-B. HAase-B showed the highest activity in100mmol/L CaCl2solution, with activity increased to154%. Zn+(10mmol/L) or Cu+(10mmol/L) severely inhibited the activity of HAase-B by more than70%and completely inhibited the enzyme activity at the concentrations of50mmol/L and100mmol/L. Although the activity of HAase-B was decreased by metal ion chelators (EDTA, EGTA and DFO), it was not inhibited completely even when the concentrations of metal ion chelators were raised to100mmol/L. Non-ionic surfactants (Tween80and Triton X-100) moderately inhibit HAase-B activity by3%-30%at different concentrations. However, ionic detergent (SDS) completely inhibited the enzyme activity at any concentrations.(3) Substrate specificity of HAase-BHAase-B was found to degrade HA, chondroitin sulfate A (CSA), chondroitin sulfate A (CSC) and CSD, and release unsaturated products which showed a strong absorption peak at232nm.When using other polysaccharides as substrate, it did not show any activity. Kinetic measurement of HAase-B towards HA gave a Michaelis constant (Km) of0.02mg/mL, and a maximum velocity (Vmax) of0.27A232/min. HAase-B also showed activity towards CSA with the kinetic parameters Km and Vmax12.30mg/mL and0.20A232/min, respectively.(4) Gene sequencing of HAase-B and sequence analysisThe complete nucleotide sequence encoding HAase-B,composed of3324bp, was sequenced and determined to be part of the bacterial genome. The DNA sequence of HAase-B obtained in our work was submitted to GeneBank with KC522838as its accession number. According to the deduced amino acid sequence of HAase-B, the MW and pⅠ of HAase-B were predicted to be123242Da and5.05, respectively.The deduced amino acid sequence of HAase-B had relatively high identity to those of other members of glycosaminoglycan (GAG) lyase family. The alignment result showed the highest similarity with HAase-B sequence occurred in a precursor enzyme of Xanthan gum lyase XalB from Paenibacillus, up to46%similarity, and31%to46%similarities with all the other proteins of GAG lyase family. Compared with the conserved regions of all the GAG lyases above, the active sites of HAase-B might be N513, H563and Y572. By alignment of the deduced Bacillus sp. A50HAase-B amino acid sequence with the GAG lyase family enzymes, we also found the substrate binding sites of HAase-B might be R406, T409, K413, N453, W455, H456, G460, N463, S467, N513, K519, H563, Y572, V575, E578, D582, R626, A627, R630, M741, and N742.5. Preparation and Characterization of oligo-HA by degradation of HAase-BIR spectroscopy of oligo-HA prepared by degradation with HAase-B consistented with European Pharmacopoeia reference spectrum of HA. The content determination by HPLC showed that the content of the oligo-HA was obviously higher than that by HCl degradation. This indicated that the chemical structure of oligo-HAprepared by degradation with HAase-B was intact while the structure of HA oligosaccharides degraded by HCl changed.Oligo-HAprepared by degradation with HAase-B has significant antioxidant activity. When mixed with DPPH, oligo-HA degraded by HAase-B could remove DPPH effectively, suggesting high DPPH radical scavenging ability. When HaCaT human epidermal keratinocytes were damaged by UVA and UVB radiation, the keratinocytes incubated with the oligo-HA could be repaired and the cell proliferation rate return to normal. This is because that the oligo-HAprepared by degradation with HAase-B reduced the generation of intracellular reactive oxygen free radicals.Additionally, the oligo-HA has angiogenesis capability.