Kim Dong-Eun, Kim Kyung-Hwa, Bae Yu-Jin, Lee Jung-Hee, Jang Yhon-Hwa, Nam Soo-Wan
Department of Biotechnology and Bioengineering, Dong-Eui University, Busan 614-714, Republic of Korea.
Protein Expr Purif. 2005 Jan;39(1):107-15. doi: 10.1016/j.pep.2004.09.007.
Arylsulfatase cloned from a marine aerobic Gram-negative bacterium, Pseudoalteromonas carrageenovora, was overexpressed in Escherichia coli with 10 microM IPTG induction. The expressed recombinant arylsulfatase was purified to homogeneity from the harvested cells through osmotic disruption and column chromatography methods, such as DEAE-cellulose anion exchange chromatography and Heparin-Sepharose affinity chromatography. The purified arylsulfatase was kinetically characterized using the synthetic substrate of phenolic ester, p-nitrophenyl sulfate (pNPS). One unit of arylsulfatase catalyzes the liberation of 1.0 micromol p-nitrophenol from pNPS per minute. The purified enzyme has a specific activity of 468 U/mg with a purification yield of 27% from the cell lysate, and exhibited an estimated molecular mass of 33 kDa in SDS-PAGE analysis. The precursor polypeptide of 36 kDa was processed by releasing a putative signal peptide, and the mature arylsulfatase of 33.1 kDa with a N-terminal sequence of S-E-T-K-N was trafficked to periplasmic space. The enzyme had optimum reaction conditions for activity at pH 7.0 and at a temperature range of 40-45 degrees C. The apparent K(M) and k(cat) of the enzyme for hydrolysis of pNPS at pH 7.0 and at 45 degrees C were determined to be 1.15 mM and 1000 s-1, respectively. Based on inhibitor studies along with optimal pH values and preferential periplasmic location of the enzyme, we suggest that the recombinant arylsulfatase from P. carrageenovora is probably similar to the Klebsiella sulfatase with serine residue in the active site.
从海洋需氧革兰氏阴性细菌角叉菜假交替单胞菌（Pseudoalteromonas carrageenovora）克隆的芳基硫酸酯酶，在大肠杆菌中经10微摩尔异丙基-β-D-硫代半乳糖苷（IPTG）诱导实现了过表达。通过渗透破碎和柱色谱法（如DEAE-纤维素阴离子交换色谱法和肝素-琼脂糖亲和色谱法），从收获的细胞中纯化出表达的重组芳基硫酸酯酶，使其达到均一性。使用酚酯类合成底物对硝基苯硫酸酯（pNPS）对纯化的芳基硫酸酯酶进行动力学表征。1个单位的芳基硫酸酯酶每分钟催化从pNPS释放1.0微摩尔对硝基苯酚。纯化后的酶比活性为468 U/mg，从细胞裂解物中的纯化产率为27%，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析中显示估计分子量为33 kDa。36 kDa的前体多肽通过释放一个假定的信号肽进行加工，成熟的33.1 kDa芳基硫酸酯酶（N端序列为S-E-T-K-N）被转运到周质空间。该酶在pH 7.0和40 - 45℃温度范围内具有最佳活性反应条件。在pH 7.0和45℃下，该酶水解pNPS的表观米氏常数（K(M)）和催化常数（k(cat)）分别测定为1.15 mM和1000 s-1。基于抑制剂研究以及酶的最佳pH值和优先周质定位，我们认为来自角叉菜假交替单胞菌的重组芳基硫酸酯酶可能与活性位点含有丝氨酸残基的克雷伯氏菌硫酸酯酶相似。
