Diversity And Molecular Engineering Of Glycoside Hydrolyase Family 3β-glucosidases From Thermophilic Fungi

Biofuel is one of the few alternatives to fossil fuel. During the cellulose saccharification process, β-glucosidase plays a vital role in the efficiency enhancement. Evaluations of new alternatives for the current β-glucosidase preparations are very necessary for sustainable development. The current study systematically investigated the properties, functional diversity and key residues related to substrate binding and catalytic efficiency of glycoside hydrolase (GH) family 3 β-glucosidase from thermophilic fungi H. insolens Y1 and T. leycettanus. And part of the cloned β-glucosidases were modified to obtain better properties. The main results were as follow:(1) Functional diversity and substrate binding of family 3β-glucosidases from thermophilic cellulolytic fungus H. insolens Y1We analyzed the functional diversity of three distant family 3β-glucosidases from H. insolens strain Y1, which belonged to different evolutionary clades, by heterogeneous expression in Pichia pastor is strain GS115. The recombinant enzymes shared similar enzymatic properties including thermophilic and neutral optima (50-60℃ and pH 5.5-6.0) and high glucose tolerance, but differed in substrate specificities and kinetics.HiBgl3B was solely active towards aryl-glucosides while HiBgl3A and HiBgl3C showed broad substrate specificities including both disaccharides and aryl-glucosides. Of the three enzymes,HiBgl3C exhibited the highest specific activity (158.8 U/mg on pNPG and 56.4 U/mg on cellobiose) and catalytic efficiency and had the capacity to promote cellulose degradation. Substitutions of three key residues Ile48, Ile278 and Thr484 of HiBgl3B to the corresponding residues of HiBgl3A conferred the enzyme activity towards sophorose, and vice versa. This study reveals the relationship between sequence diversity and functional diversity of GH3 β-glucosidases as well as the key residues in recognizing +1 subsite of different substrates.(2) Engineering pH stability of thermophilic β-glucosidase Bgl3A from T. leycettanusThe newly identified β-glucosidase of GH3, Bg13A, from T. leycettanus JCM12802, was overexpressed in yeast strain Pichia pastoris GS115, yielding a crude enzyme activity of 6000 U/ml in a 3 L fermentation tank. The purified enzyme exhibited outstanding enzymatic properties, including favorable temperature and pH optima (75℃ and pH 4.5), good thermostability (maintaining stable at 60℃), and high catalytic performance (with specific activity and catalytic efficiency of 905 U/mg and 9096/s/mM on pNPG, respectively; specific activity and catalytic efficiency of 265 U/mg and 75.8/s/mM on cellobiose, respectively). However, the narrow stability of Bg13A at pH 4.0-5.0 would limit its industrial applications. Further site-directed mutagenesis indicated the role of excessive O-glycosylation in pH liability. By removing the potential O-glycosylation sites, two mutants showed improved pH stability over a broader pH range (3.0-10.0). Besides, with better stability under pH 5.0 and 50℃ compared with wild type Bg13A, saccharification efficiency of mutant M1 was improved substantially cooperating with cellulase Celluclast 1.5L. And mutant M1 reached approximately equivalent performance to commercial β-glucosidase Novozyme 188 with less mass of protein, suggesting its great prospect in biofuels production.(3)Improving the catalytic efficiency on cellobiose of thermophilic β-glucosidase Bg13A from T. leycettanusGH3 β-glucosidase Bg13A exhibited great potential application value due to its superior enzymatic properties. Experimental data indicated that Bgl3A had much lower catalytic efficiency toward cellobiose than pNPG, which could be attribute to the rather low affinity. In order to improve its catalytic efficiency by molecular modification, several key residues closely related to the enzyme catalysis were found out by analysis of protein structure and docking result. As results, Km values on cellobiose of mutants M36E and M36N, which were designed to deconstruct the rigid hydrophobic region near the catalytic pocket, were decreased to 5.0mM and 4.7mM, respectively, and catalytic efficiency were both improved to 2.3 fold of that of wild type enzyme. And affinities toward cellobiose of mutants F66Y and E168Q, which were designed to enhance the bonding capability of key residues for hydrogen bonding network Arg65 and Arg167, were improved to 2.4 fold and 2 fold of that of wild type, and catalytic efficiency were improved to 1.6 fold and 1.4 fold, respectively. The result of molecule dynamic (MD) simulation demonstrated that mutants M36E and M36N increased the flexibility of hydrophobic binding sites Phe258, and thus improved the catalytic efficiency by increasing substrate acquisition probability of the enzyme. The improvements of mutants M36E and M36N were mainly due to the strengthened affinities toward cellobiose cause by the stabilization of cation-π interaction between Arg65-Phe67 and Arg167-Tyr202. The calculation of binding energies also showed that the combination of substrate molecule and enzyme was more stable in mutants than wild type Bg13A.