【作者】 周鹏；

【导师】 江正强；

【作者基本信息】 中国农业大学 ， 食品生物技术， 2014， 博士

【摘要】 嗜热真菌是一小类真菌,它们的最高生长温度可高达55-60℃。嗜热真菌也是重要的耐热酶的来源。由于来源于嗜热真菌的酶具有最适温度高、稳定性好的优点,因此被广泛的研究。本论文研究了米黑根毛霉(R. miehei)β-1,3-葡聚糖酶和β-甘露糖苷酶的克隆、表达、酶学性质、晶体结构和催化机理。为了进一步开发米黑根毛霉资源,对米黑根毛霉的基因组与转录组进行了研究。本论文的主要结果如下：(1)从米黑根毛霉中克隆表达了一种新的β-1,3-葡聚糖酶((RmLam81A)。该酶可以水解β-1,3-葡聚糖,如昆布多糖、可德兰多糖和酵母葡聚糖。解析了它的两种蛋白结构(Form Ⅰ-native和Form Ⅱ-Se)以及一种酶-昆布五糖复合物(RmLam81A/D475A-G5)的结构,分辨率分别为2.3、2.0和2.7A。RmLam81A由三个结构域构成,包括一个β-sandwich结构域,一个(α/α)6结构域和两者之间的一个小的结构域,与已知的β-1,3-葡聚糖酶结构明显不同。根据复合物的结构推测,Asp475和Glu557分别作为广义酸和广义碱起到催化的作用。酶-底物复合物还揭示,每个(RmLam81A结合三条昆布寡糖糖链,糖链的排列与天然葡聚糖的三股螺旋结构一致,据此推测该酶可以直接结合三螺旋p-葡聚糖。(2)从米黑根毛霉中克隆表达了一种GH5家族β-甘露糖苷酶((RmMan5B)。RmMan5B对甘露寡糖具有高的催化能力,高于人工底物pNPM,另外,它还表现出强的转糖苷能力,可以把甘露糖残基转移到果糖或甘露寡糖上。为了深入研究该酶的底物特异性和转糖苷活性,解析了RmMan5B以及它的一种突变蛋白(E202A)与甘露二糖、甘露三糖和甘露果糖复合物的结构,分辩分别为1.3、2.6、2.0和2.4A。和以往的报道一致,RmMan5B呈一种典型的(β/α)8-桶结构。与GH5家族β-甘露聚糖酶相比,一些更长的loops修饰催化位点,使RmMan5B成为外切酶。其中loop354-392,构成-1位结合位点一侧的空间位阻,参与β-甘露糖苷酶的催化口袋的构成。通过比较可以还确定Trp119、Asn260和Glu380,是与β-甘露糖苷酶外切活性相关的重要氨基酸。另外,酶-甘露果糖复合物结构的解析,可以解释RmMan5B可以利用果糖作为转糖昔受体的机理。(3)研究了米黑根毛霉CAU432的基因组和转录组。组装完成的基因组共有27.6-million-base (Mb),预测含有10,345个蛋白质编码基因。全基因组和编码基因中的G+C含量分别只有43.8%和47.4%,小于50%,虽然该菌是一种嗜热真菌。进化树显示米黑根毛霉与布拉克须霉进化关系最近,而不是毛霉或根霉。米黑根毛霉基因组中具有大量的蛋白酶编码基因。转录组表明,不少淀粉酶、葡聚糖酶、蛋白酶和脂肪酶基因具有高的转录水平。米黑根毛霉基因组的研究为嗜热真菌的嗜热机制的进一步研究、嗜热酶资源的开发利用及工业化生产提供依据。更多还原

【Abstract】 Thermophilic fungi are a small assemblage in mycota that have a maximum temperature of growth extending up to55to60℃. They are also potential sources of enzymes with scientific and commercial interests. In the present dissertation, a (3-1,3-glucanase and a β-mannosidase were cloned from thermophilic fungi Rhizomucor miehei and expressed heterologously in E. coli. Purification of the recombinant enzymes and their structural and biochemical characterization was performed. To facilitate future investigations, we sequenced the genome of R. miehei CAU432. The main results are as follows:(1) A novel GH family81P-1,3-glucanase gene (RmLam81A) from Rhzmucor miehei was expressed in E. coli. The enzyme can hydrolyze laminarin, curdlan and yeast β-D-glucan. Purified RmLam8lA (Form I-native), its Selenomethionine-derivative (Form Il-Se) and an inactive mutant D475A in complex with laminaripentaose (RmLam81A/D475A-G5) were crystallized and determined at2.3,2.0and2.7A resolution, respectively. The overall structure of GH family81β-1,3-glucanase contains each monomer of the protein is arranged in a β-sandwich domain, a (α/α)6domain and an additional domain between them. Comparison with structures of β-1,3-glucanases from other GH families revealed differences in three-dimensional structure. Asp475and Glu557are proposed to serve as the proton donor and nucleophile, respectively, in a single-displacement mechanism. The structure of RmLam81A with laminaripentaose also showed binding details with three laminari-oligosaccharides, proving that the enzyme can recognize triplex β-glucan. The structure of first crystal structure of a GH family81member will be helpful to study the GH family81proteins and endo-β-1,3-glucanases.(2) A first fungal GH family5β-mannosidase(RmMan5B) from R. miehei was functionally and structurally characterized. RmMan5B exhibited much higher activity against mannan oligosaccharides compared with p-nitrophenyl β-D-mannopyranoside (pNPM) and had a transglycosylation action which transferred mannose residue to sugars such as fructose. To investigate its substrate specificity and transglycosylation activities, the crystal structures of RmMan5B and an inactive mutant E202A in complex with mannobiose, mannotriose and mannosyl-fructose have been determined at a resolution of1.3,2.6,2.0and2.4A, respectively. The enzyme adopts the (β/α)8barrel architecture common to the members of GH family5, but shows several differences in the loops around the active site. The extended loop between strand β8and helix a8(residues354-392) forms a "double" steric barrier to "block" the substrate binding cleft at the end of the-1subsite. Comparied with β-mannanases, Trp119, Asn260and Glu380which are involved in the hydrogen bonds contact with-1mannose might be essential for exo-catalytic activity. Moreover, the structure in complex with mannosyl-fructose has provided an evidence for the interactions between the β-mannosidase and the D-fructofuranose, and explains why fructose is an effective transglycosylation acceptor.(3) The assembled genome size of R. miehei CAU432is27.6-million-base (Mb) with10,345predicted protein-coding genes. Even being thermophilic, the G+C contents of fungal whole genome (43.8%) and coding genes (47.4%) are less than50%. Phylogenetically, R. miehei is more closerly related to Phycomyces blakesleeanus than to Mucor circinelloides and Rhizopus oryzae. The genome of R. miehei harbors a large number of genes encoding secreted proteases, which is consistent with the characteristics of R. miehei being a rich producer of proteases. The transcriptome profile of R. miehei showed that the genes responsible for degrading starch, glucan, protein and lipid were highly expressed. The genome information of R. miehei will facilitate future studies to better understand the mechanisms of fungal thermophilic adaptation and the exploring of the potential of R. miehei in industrial-scale production of thermostable enzymes.更多还原