【作者】 马素；

【导师】 于文功；

【作者基本信息】 中国海洋大学 ， 生药学， 2013， 博士

【摘要】 卡拉胶酶是一类能够专一性降解卡拉胶分子中β-1,4糖苷键的糖水解酶，根据其降解底物的不同主要分为κ-、ι-和λ-卡拉胶酶。根据序列和结构的特征，ι-卡拉胶酶被归属为糖水解酶82家族（GH82），目前已经发现了九种ι-卡拉胶酶，但只有四种进行了部分的性质研究。关于该家族酶的结构与功能研究目前还处于起步阶段，只有和催化相关的部分氨基酸残基有所报道。因此新ι-卡拉胶酶的发现和深入研究不仅可以丰富GH82家族ι-卡拉胶酶资源，而且对GH82家族酶的结构与功能阐释有重要意义。来自Cellulophaga sp. QY3的ι-卡拉胶酶CgiA的研究工作是在本实验室前期工作的基础上继续进行的。首先研究了CgiA降解不同类型底物的降解方式。CgiA降解非胶态底物时表现为随机内切酶的特点，而降解胶态底物时采用持续性降解的方式，在GH82家族的ι-卡拉胶酶中属于首次报道。其次，利用同源模建的方法获得了CgiA的三维结构。结合荧光辅助碳水化合物电泳分析表明，CgiA降解ι-卡拉胶的终产物为新ι-卡拉四糖和新ι-卡拉二糖，最小降解底物为新ι-卡拉六糖。该酶的催化腔大小为八（-4~+4）六糖在催化腔中占据-4~+2位点。利用序列标识图（sequence logo）的方式展示出GH82家族催化腔中与底物相距6以内的氨基酸残基。从GH82家族催化腔的保守分析可以明显看出催化腔-1和+1位点处的残基保守性高于其他位点。根据氨基酸残基的保守性及残基在结构中的空间位置，我们选择了六个位点(G228，Y229，R254，R314，K335和K480)进行突变实验。通过GH82家族多个底物结合位点的突变及性质比较，验证了功能残基的保守性与其重要性是正相关的。根据突变前后酶活力变化，米氏常数变化及高级结构分析，证明了催化腔-1，+1位点处G228, Y229，R254和R314是该酶与底物结合相关的残基。其中G228, Y229和R254在GH82家族中完全保守，因此推测它们为该家族最重要的与底物结合相关的残基。GH82家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。更多还原

【Abstract】 Carrageenases are kinds of glycoside hydrolases which could cleave the internalβ (1→4) linkages of carrageenans. Based on the substrate specificity, the enzymes areclassified into three groups, namely, κ-, ι-, and λ-carrageenases. ι-Carrageenases havebeen classified into family GH82according to the sequence and structure. Up to now,nine gene sequences of family GH82ι-carrageenases have been found, but only fourof them have been characterized. The relationship between structures and functions ofGH82is 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 richof GH82but also for the interpretation of the relationship between structures and theirfunctions.The study on ι-carrageenase CgiA from Cellulophaga sp. QY3was continued onthe basis of the preliminary work of our lab. At first, we studied the degradationmethods of CgiA on substrates in different conformations. The degradation of solubleι-carrageenans was by random-type cleaving. In contrast, the degradation of gelsubstrates was by processive-type. It was the first time to report that GH82ι-carrageenase showed different types of degradations which depended on the formsof substrates.Secondly, the three-dimensional structure of CgiA was obtained byhomology modeling. Combined the structure and the electrophoresis analysis, wefound that CgiA cleaved ι-carrageenan yielding neo-ι-carrabiose andneo-ι-carratetraose as the main end products, and neo-ι-carrahexaose was theminimum substrate. The number of subsites for CgiA was eight (-4~+4) and thehexasaccharide bound to subsites4to+2.The sequence logo of GH82showed the amino acid residues in catalyticcavity which closed to the substrate within6. The amino acid residues at subsite-1and+1were more conserved than those at other subsites. Based on the conservation and position in three-dimensional structure, we chose six residues(G228, Y229, R254, R314, K335and K480) for site-directed mutagenesis.Based on the site-directed mutagenesis of the substrate-binding residues infamily GH82, we could infer that the conservations of residues were positivecorrelation to the the importance of them. Site-directed mutagenesis followed bykinetic and structure analyses identified four residues at subsite-1and+1of CgiA,G228, Y229, R254and R314, which played important roles for substrate binding.G228, Y229and R254were strictly conserved in family GH82ι-carrageenases,therefore we inferred they were the important substrate binding residues for GH82.The most important binding residues (Y229and R254in CgiA) were interactspecifically with the sulfate groups of the sugar substrate located at subsite-1and+1and this may shed light on the mechanism of the substrate specificity for family GH82toward ι-carrageenan. It was the first time to identify the substrate binding residuesand explain the mechanism of the substrate specificity for GH82.The gene for a new ι-carrageenase, CgiB, from Cellulophaga sp. QY3wascloned by degenerate PCR and Sitefinding PCR. It comprised an open readingframe of1386bp, which encodes a protein consisting of461amino acid residues.According to the sequence analysis, CgiB was categorized as a new member ofGH family82and shared the highest identity of32%in amino acids withι-carrageenase CgiA2from Zobellia galactanovorans except an uncharacterizedι-carrageenase.The recombinant CgiB showed maximum specific activity at45°C and pH6.5.The enzyme retained70%of the original activity after incubation at the temperaturesbelow40°C for1h. It was stable between pH6.0-9.0. Cations Na+and K+increasedthe activity of CgiB significantly. CuCl2, SDS and EDTA reduced the enzyme activitysignificantly, while most divalent metal cations, such as Mg2+, Fe2+and Mn2+,increased the enzyme activity notably.According to the size-exclusion chromatography analysis on the degradationproducts, CgiB was an endo-type ι-carrageenase that hydrolyzes β-1,4-linkages ofι-carrageenan, yielding tetrasaccharide as the main product (more than80%of the total product). By the secondary mass spectrometry analysis, we identified the mainproduct was neo-ι-carratetraose showing CgiB cleaved the internal β (1→4) linkagesof ι-carrageenan. The characterization of CgiB was different from other reportedι-carrageenases, which showing great benefit not only for the rich of ι-carrageenasesbut also for the production and purification of carrageenan oligosaccharides.更多还原