Structure And Functions Of β-1,3-glucanase And β-mannosidase From Rhizomucor Miehei And Its Genome Sequence Analysis

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.