Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul, South Korea.
Appl Microbiol Biotechnol. 2010 Apr;86(3):879-90. doi: 10.1007/s00253-009-2327-7. Epub 2009 Nov 12.
Recombinant heparinase III (rHepIII) from Bacteroides stercoris HJ-15 was cloned, expressed, and characterized. The full-length heparinase III gene from B. stercoris HJ-15 was identified by Southern blotting, and the sequence was deposited in GenBank. The heparinase III gene, which is 2,001-bp long, was cloned and overexpressed in Escherichia coli; highly active rHepIII was easily purified using only one step of immobilized Ni(2+) affinity column chromatography. Enzymatic properties and substrate specificities of rHepIII were assessed, and its kinetic constants were calculated. rHepIII was most active in 50 mM sodium phosphate buffer with 350 mM NaCl (pH 6.6) at 45 degrees C. Through amino acid modification studies and site-directed mutagenesis assay, cysteines and histidines were identified as crucial residues for enzymatic activity. Moreover, this enzyme digested not only heparan sulfate but also heparin and hyaluronic acid, and their degradation products were verified by strong anion exchange/high-performance liquid chromatography. These characteristics, including active residues and substrate specificities were interesting compared with those of existing heparinase III from other species. We anticipate that the convenience of purification and the characteristics of this enzyme will make it a powerful tool for studies of glycosaminoglycans and their lyases.
从类杆菌属 HJ-15 中克隆、表达和表征了重组肝素酶 III（rHepIII）。通过 Southern 印迹鉴定了来自 B. stercoris HJ-15 的全长肝素酶 III 基因，并将其序列提交到 GenBank。肝素酶 III 基因长 2001bp，在大肠杆菌中克隆并过表达；使用固定化 Ni(2+)亲和柱层析一步即可轻松纯化高活性 rHepIII。评估了 rHepIII 的酶学性质和底物特异性，并计算了其动力学常数。rHepIII 在 45°C 下，在 50mM 磷酸钠缓冲液中（pH 6.6），在 350mM NaCl 条件下最活跃。通过氨基酸修饰研究和定点突变分析，鉴定出半胱氨酸和组氨酸是酶活性的关键残基。此外，该酶不仅消化肝素聚糖硫酸盐，还消化肝素和透明质酸，其降解产物通过强阴离子交换/高效液相色谱得到验证。与来自其他物种的现有肝素酶 III 相比，这些特征，包括活性残基和底物特异性都很有趣。我们预计，这种酶的纯化便利性和特性将使其成为研究糖胺聚糖及其裂解酶的有力工具。
