Purification and characterization of bifunctional alginate lyase from Alteromonas sp strain no. 272 and its action on saturated oligomeric substrates
Purification and characterization of bifunctional alginate lyase from Alteromonas sp strain no. 272 and its action on saturated oligomeric substrates
复制标题
DOI：
10.1271/bbb.65.133
复制
发表时间：
2001-01-01
影响因子：
1.6
通讯作者：
Muramatsu, T
中科院分区：
文献类型：
--
作者：
Iwamoto, Y;Araki, R;Muramatsu, T
从大村湾海泥中分离出一种产褐藻酸解酶的海洋细菌（菌株272），属异单胞菌。该酶经DEAE-Cellulofine、Sephadex G-100凝胶层析纯化，在SDS存在和不存在的情况下均为电泳均匀状态，经Sephadex G-100柱层析和SDS-聚丙烯酰胺凝胶电泳，酶的分子量分别为23和33.9 kDa，等电点为3.8。经圆二色性分析，该酶的主要二级结构为β -结构，在pH 7.5 ~ 8.0范围内活性最强，在pH 5 ~ 11范围内稳定。该酶在Tris-HCl缓冲液（pH 7.0）中比在磷酸盐缓冲液（pH 7.0）中对热处理更不稳定。没有金属离子对酶活性有显著影响。通过常规紫外法（235 nm）和底物的圆二向色光谱变化判断，该酶以内型方式作用于海藻酸钠，并对海藻酸盐的两种成分poly- α 1,4- l - gulurate和poly- β 1,4- d - mannurate起作用。然而，共存的聚α 1,4- l -谷露醛酸酯和聚α 1,4- d -甘露醛酸酯显然以竞争的方式与酶相互作用。虽然该酶以内聚方式解聚海藻酸盐，但它不作用于三聚体古卢醛酸盐和甘露醛酸盐，而作用于四聚体或更多。动力学分析表明，古鲁醛酸酯低聚物的k(cat)/ k -m均大于甘露醛酸酯低聚物的k(cat)/ k -m，并且该酶的亚位点结构很可能由内在反应速率常数（k -int）和内在底物结合常数（k -int）组成6个结合位点。
A marine bacterium (strain No. 272) isolated from sea mud in Omura Bay produced an alginate lyase and was classified as an Alteromonas species. The enzyme was purified from the culture medium of the bacterium by DEAE-Cellulofine, Sephadex G-100 gel chromatography to an electrophoretically homogeneous state in the presence and absence of SDS, The molecular mass of the enzyme was 23 and 33.9 kDa on Sephadex G-100 column chromatography and SDS-polyacrylamide gel electrophoresis, respectively, with an isoelectric point of 3.8. The predominant secondary structure of the enzyme was found to be most likely beta -structure by circular dichroism, The enzyme was most active at pH 7.5-8.0 and stable around pH 5-11. The enzyme was more labile in Tris-HCl buffer (pH 7.0) to heat treatment, than in phosphate buffer (pH 7.0). No of metal ions significantly affected the enzyme activity. The enzyme acted on sodium alginate in an endo-type manner and on two components of alginate, poly-alpha1,4-L-guluronate and polyp-beta1,4-D-mannuronate, as judged by routine ultraviolet assay (235 nm) and circular dichroic spectral changes of the substrates. However, the coexisting poly-alpha1,4-L-guluronate and poly-beta1,4-D-mannuronate apparently interacted with the enzyme in a competitive manner. Although the enzyme depolymerized alginate in an endo-type, it did not act on trimeric guluronate and mannuronate, but on the tetramers or more. The kinetic analyses showed that k(cat)/K-m for each oligomer was larger for the guluronate oligomers than for the mannuronate ones, and that the subsite structure of the enzyme most likely consisted of six binding sites from the intrinsic reaction rate constant (K-int) and intrinsic substrate binding constant (K-int).