Preparation Of Unsaturated Hyaluronic Acid Disaccharide By A Streptomyces Hyaluronate Lyase

Hyaluronic acid(HA)is an acidic mucopolysaccharide polymerized from D-glucuronic acid(GlcUAc)and N-acetyl-D-glucosamine(GlcNAc).It shows different biological activities according to its molecular weight.The molecular weight of hyaluronan oligosaccharides(o-HAs)is less than 10 kDa.O-HAs have important functions on inhibition of oxidative damage of skin cells,repairation of UV damaged cells,reduction of intracellular reactive oxygen species and treatment of cancer.Enzymatic preparation of o-HAs takes advantage in mild reaction conditions,high yield and strong specificity,which is the most desired method for industrial large-scale production.Hyaluronidase is the kind of enzymes that decomposes HA.Hyaluronidases from microorganisms mostly belong to hyaluronate lyases,which degrade HA and produce unsaturated o-HAs by β-elimination mechanism.In this study,a hyaluronic acid lyase SaHyaL,belonging to polysaccharide lyase(PL)family 8,was found by Carbohydrate-Active enZyme(CAZy)analysis on the whole genome sequence of Streptomyces alfalae ACCC40021.Multiple alignment of amino acid sequences indicated that it was a novel hyaluronate lyase with low similarity to any known hyaluronate lyase.The gene fragment of 2520 bp was amplified by PCR using the genome DNA of S.alfalae ACCC40021 as template.The recombinant plasmid pET28a-SUMO-Saryal was constructed and transformed into Escherichia coli BL21(DE3)for heterologous expression.The soluble expression of SaHyaLwas succeeded with co-expression of ubiquitin like specific protease(ULP)to remove the SUMO tag in vivo.The SDS-PAGE analysis showed that the molecular weight of recombinant SaHyaL was consistent with the predicted size of 89.5 kDa.The optimum temperature and pH of SaHyaL were 50℃ and 7.0,respectively.The enzyme was stable in the range of 20-50℃ and pH 5.0-10.0,and displayed a broad substruate spectrum.The Km is 0.31 mg/mL,and Vmax is 135.14 μmol/min/mg.Thin Layer chromatography(TLC)analysis showed that the action mode of SaHyaL is an exo-cleavage manner.In the process of enzymatic hydrolysis of HA monitored by HPLC,the maxium conversion rate achieved 97.4%at 6 h with unsaturated hyaluronic acid disaccharide as the only product,which was further confirmed by Electrospray Ionization Mass Spectrometry(ESI-MS).Our results suggested that SaHyaL has the great potential in enzymatic conversion of HA to unsaturated hyaluronic acid disaccharide in industry.