海洋细菌cellulophaga sp.qy3ι——卡拉胶酶的研究-marine bacterium cellulose ophaga sp.qy3 ι - study on carrageenan enzyme.docx

海洋细菌海洋细菌 Cellulophaga sp. QY3 ι-卡拉胶酶的研究IIII族最重要的两个底物结合残基（Y229 和 R254）是与底物的硫酸根直接作用的，推测这是该家族酶专一性降解 ι-卡拉胶的关键。这是首次对 GH82 家族ι-卡拉胶酶的底物结合残基的研究，进一步阐释了该家族的底物专一性机制。 接下来，利用简并 PCR 和 Sitefinding PCR 成功从 Cellulophaga sp. QY3中克隆得到了一种新的 ι-卡拉胶酶 CgiB 的编码序列。该基因含有一段长度为 1383bp 的 ORF 编码框，编码得到 461aa 的蛋白序列。CgiB 归属于 GH82 家族，除了一个未进行表达和研究的序列，与其相似度最高的蛋白序列是来 自 Zobellia galactanovora DsiJT 的 CgiA2，相似度仅为 32%。对重组酶 CgiB 的性质研究表明：该酶的最适反应条件为 45℃、pH6.5 磷 酸盐缓冲液；在低于 40℃条件下存放 1h 该酶还能保留 70%的活性；在 pH6.0-9.6 的缓冲体系中存放较稳定；Na+和 K+对酶活力有促进作用，CuCl2, SDS 和 EDTA 都非常显著的抑制了酶活性，而一些二价金属离子，如 Mg2+, Fe2+及 Mn2+对酶活 力有明显的促进。利用凝胶色谱分 析酶解产物随 时间的变化 可知该酶是一种典 型的内 切 酶，降解主产物四糖在终产物中约占 80%。利用二级质谱技术鉴定出酶解主 产物为新 ι-卡拉四糖，表明该酶通过断裂卡拉胶分子中的 β-1,4 糖苷键实现 对 ι-卡拉胶的降解。CgiB 具有与已报道的 ι-卡拉胶酶不同的性质特点，不仅 丰富了 ι-卡拉胶酶的资源，而且对卡拉寡糖的制备和纯化具有重要意义。关键词：嗜纤维菌属；卡拉胶酶；酶学性质；底物结合；底物识别IIIIIIStudy on the ι-carrageenases of marine bacteriumCellulophaga sp. QY3AbstractCarrageenases are kinds of glycoside hydrolases which could cleave the internal β (1→4) linkages of carrageenans. Based on the substrate specificity, the enzymes are classified into three groups, namely, κ-, ι-, and λ-carrageenases. ι-Carrageenases have been classified into family GH82 according to the sequence and structure. Up to now, nine gene sequences of family GH82 ι-carrageenases have been found, but only four of them have been characterized. The relationship between structures and functions of GH82 is still in its infancy. Only the catalytic residues have been identified. Therefore, the discovery and study of the new ι-carrageenases are important not only for the rich of GH82 but also for the interpretation of the relationship between structures and their functions.The study on ι-carrageenase CgiA from Cellulophaga sp. QY3 was continued on the basis of the preliminary work of our lab. At first, we studied the degradation methods of CgiA on substrates in different conformations. The degradation of soluble ι-carrageenans was by random-type cleaving. In contrast, the degradation of gel substrates was by proces