Matsubara Y, Kawada R, Iwasaki K, Oda T, Muramatsu T
Kagawa Prefectural Fermentation and Food Experimental Station, Uchinomi, Japan.
J Protein Chem. 1998 Jan;17(1):29-36. doi: 10.1023/a:1022534429792.
Extracellular alginate lyase was purified from the culture supernatant of Corynebacterium sp. isolated from the sewage of a sea tangle processing factory in order to elucidate the structure-function relationship of alginate lyase. The electrophoretically homogeneous enzyme was shown to have a molecular mass of 27 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and by gel filtration, with an isoelectric point of 7.3. The molecular mass from amino acid analysis was 28.644 kDa. The optimal pH and temperature for the enzyme reaction were around 7.0 and 55 degrees C, respectively. Metal compounds such as MnCl2 and NiCl2 increased the enzyme activity. The enzyme was identified as the endolytic poly(alpha-L-guluronate)lyase, which was active on poly(alpha-L-1,4-guluronate) and caused a rapid decrease in the viscosity of alginate solution. Measurement of the far-UV circular dichroic spectrum of the enzyme molecule gave a spectrum with a deep trough at 215 nm accompanied by a shallow one at around 237 nm, and with a high peak at 197 nm and a much lower one at 230 nm. This spectrum was most likely to be that of the beta-form of the enzyme molecule and resembled poly(beta-D-mannuronate)lyase from Turbo cornutus (wreath shell) and poly(alpha-L-guluronate)lyase from Vibrio sp. (marine bacterium). The near-UV circular dichroic spectrum was characteristic for aromatic amino acid residues. In the presence of 6 M urea, these spectra changed drastically in the near-UV and a little in the far-UV with the disappearance of the enzyme activity. Removal of the denaturant in the enzyme solution by dialysis restored both the activity and inherent circular dichroic spectra. The beta-sheets observed in alginate lyases as the major ordered structure seem to be a common conformation for the lyases.
为阐明海藻酸盐裂解酶的结构-功能关系，从一家海带加工厂污水中分离得到的棒状杆菌属菌株的培养上清液中纯化出细胞外海藻酸盐裂解酶。通过十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳（PAGE）和凝胶过滤法显示，该电泳纯酶的分子量为27 kDa，等电点为7.3。氨基酸分析得出的分子量为28.644 kDa。酶反应的最适pH和温度分别约为7.0和55℃。MnCl2和NiCl2等金属化合物可提高酶活性。该酶被鉴定为内切聚（α-L-古洛糖醛酸）裂解酶，对聚（α-L-1,4-古洛糖醛酸）有活性，并能使海藻酸盐溶液的粘度迅速降低。对酶分子的远紫外圆二色光谱进行测定，得到的光谱在215 nm处有一个深谷，在约237 nm处有一个浅谷，在197 nm处有一个高峰，在230 nm处有一个低得多的峰。该光谱很可能是酶分子β-形式的光谱，类似于来自Turbo cornutus（花环螺）的聚（β-D-甘露糖醛酸）裂解酶和来自弧菌属（海洋细菌）的聚（α-L-古洛糖醛酸）裂解酶。近紫外圆二色光谱是芳香族氨基酸残基的特征光谱。在6 M尿素存在下，这些光谱在近紫外区发生剧烈变化，在远紫外区略有变化，同时酶活性消失。通过透析去除酶溶液中的变性剂可恢复活性和固有圆二色光谱。海藻酸盐裂解酶中观察到的β-折叠作为主要的有序结构似乎是裂解酶的常见构象。
