具广泛结合能力的新家族碳水化合物结合组件的鉴定

Most carbohydrate-active enzymes are modular proteins that comprise two or more discrete modules, such as catalytic modules and non-catalytic carbohydrate binding modules (CBMs). The main function of CBMs is to recognize and bind polysaccharides, and to increase the hydrolytic activities of the enzymes against insoluble substrates by increasing the enzyme concentration on the surface of the substrate. CBMs were classified into 64 families according to amino acid similarities. Our previous work identified that the 134 amino acid sequence of the C- terminus of endoglucanase C5614-1 derived from the uncultured microorganism of content of buffalo rumen was novel CBM. The CBMC5614-1 bound to a broad range of substrates including Avicel, acid swollen cellulose, xylan, mannan, insoluble starch, barley glucan, 2-hydroxyethylcellulose, methylcellulose and carboxymethyl cellulose. CBMC5614-1 play important roles in hydrolyzing insoluble polysaccharides and maintaining enzyme stablility in pH and temperature. The aim of this project is to identify a novel family of CBMs with broad binding specificity and its application. Firstly，we plan to identify the detailed binding properties of CBMC5614-1 as the candidate member of novel family of CBMs and illuminate the mechanism of CBMC5614-1 potentiating enzyme activity against insoluble polysaccharides by enzymatic activity comparison between wild type endoglucanase rC5614-1 and CBM deletion enzyme rGHF5. Secondly, we plan to identify the homologs of the CBMC5614-1 are also CBMs, and carry out site-directed mutational analysis to determine the key aromatic amino acid residues necessary for carbohydrate binding in CBM binding site. Conservation of these residues in other similar sequences may then suggest those homologs are of the same CBM family. Finally, the aim of this project is to explore potential application of the novel family of CBMs in approving the properties of cellulase by constructing fused endoglucanase Umcel9B-CBM and comparing enzymatic property between wild type and fused enzyme. This project will increase the diversity of CBM families. Research on novel famiy of CBM will provide new mechanism in CBM binding to polysaccharides, and will give us the basis for modifying the properties of carbohydrate-active enzymes and utilizing the carbohydrates in the future.

碳水化合物结合组件（carbohydrate-binding module, CBM）的主要功能是识别和结合多糖，增强碳水化合物活性酶对不可溶底物的水解活性。我们前期工作鉴定了一个来源于水牛瘤胃未培养微生物内切葡聚糖酶C5614-1的长度为134个氨基酸的新的CBM，CBMC5614-1对广泛的多糖具有结合能力,且该CBM是酶保持对pH和温度稳定性及对不可溶底物降解活性起重要作用。本项目研究目的第一是详细研究代表成员CBMC5614-1的结合特性和作用于不可溶多糖的机制，第二是确定CBMC5614-1和它的同源多肽形成一个新家族CBM及及该家族配体结合位点中与多糖结合有关的关键氨基酸；第三是明确该新家族CBM应用于分子改性的潜力。该项目的开展将丰富CBM家族的多样性，对新家族CBM的详尽研究将提供新的CBM与多糖的结合机制，研究结果为将来改造碳水化合物水解酶和利用碳水化合物奠定基础。

纤维素酶通常由由催化组件（catalytic module, CM）和碳水化合物结合组件（carbohydrate-binding module, CBM）组成。CBM主要负责与底物的特异性结合，在碳水化合物活性酶水解不可溶底物的过程中起重要作用。我们从来源于水牛瘤胃未培养微生物的内切葡聚糖酶C5614-1中鉴定了一个新的CBM，命名为CBMC5614-1，该CBM的特性主要是能结合广泛的多糖。在NCBI数据库中搜索到CBMC5614-1的6个同源多肽，多序列比较发现它们有10个保守的芳香族氨基酸。首先我们检测了CBMC5614-1的同源多肽对多糖的结合，发现它们都具有与CBMC5614-1类似的广泛的多糖结合谱。然后我们将CBMC5614-110个保守芳香族氨基酸都定点突变为丙氨酸，发现W380、W423、Y414是结合位点的关键氨基酸位点。上述结果表明这些多肽形成了一个新的CBM家族（CBM72）。新家族CBM的确立为研究CBM和配体的结合提供了新素材。.在细菌中，糖基水解酶(glycoside hydrolase，GH)家族9的纤维素酶被认为是除了GH48纤维素酶之外的主要纤维素降解酶。从堆肥的未培养微生物中获得内切纤维素酶Umcel9A属于GH9的纤维素酶，它不含有CBM。通过对CAZy数据库中来源于细菌的GH 9的纤维素酶的结构域组成进行分析，我们选取CBM1、CBM2、CBM3、CBM4、CBM10和CBM72家族中的代表CBM与Umcel9A进行融合。融合酶和Umcel9A对纤维寡糖的水解模式也是一样的，都是偏好于以二糖为单位进行切割。6个CBM的融合显著提高了Umcel9A对不可溶纤维素的结合。相比与野生酶Umcel9A，CBM的融合酶对可溶底物如CMC和pNPC的比活力基本一致或稍有改变，但是融合酶对不可溶纤维素都表现出比活力显著的增加，其中CBM4和Umcel9A的融合效果最好，而CBM3和Umcel9A的融合酶对不可溶底物的活性与野生酶没有显著差别。Umcel9A-CBM72对不可溶底物磷酸膨胀的Avicel、碱处理的甘蔗渣、滤纸粉末和Avicel的水解活性与野生酶相比分别提高了2.9、2.0、2.3和2.9倍，说明该项目鉴定的CBM72家族也可以用于纤维素酶的遗传改造，这些融合酶都有一定的应用价值，为以后的结构域之间的分子重组提供了借鉴意义。