Enzymic And Structural Analyses Of A 1,4-β-endoglucanase From Ganoderma Lucidum Expressed By Pichia Pastoris

Endoglucanase is one of the key enzyme which hydrolyze cellulose, The endoglucanase GlCel5 A from Ganoderma lucidum is a cellulase which is classified to glycoside hydrolases subfamily GH5, With the purpose to research the catalytic and functional mechanism of GlCel5 A furtherly, we determined the crystal structure of GlCel5 A with structural biotechnology, and this research is of great significance for industry application.The sequences of catalytic domain without signal peptide of the candidates were synthesized adapting P. pastoris codon usage and cloned into pPICZαA vector. The recombinant plasmids were linearized and transformed to P. pastoris X33 strain by electroporation, The proteins were purified by FPLC using DEAE column and Phenyl Sepharose FF. The enzyme activity was determined by dinitrosalicylic acid(DNS) method, The enzyme is capable of hydrolyzing CMC(104±3 U mg-1) and β-glucan(516±13 U mg-1). GlCel5 A exhibits optimal CMC-hydrolyzing activity at 60℃ and pH3-4, The thermostability of GlCel5 A at 80 and 90℃ was investigated. The enzyme remains 50% activity at 80 and 90℃ for at least 15 and 10 min. More than 20% and 17% residual activity was observed after heated at 80 and 90℃ for 1 h.Initial crystallization screening was performed using Hampton Research Crystal Screens with the sitting-drop vapor-diffusion method at room temperature,The initial crystals were obtained in solution containing 0.2 M magnesium chloride hexahydrate, 0.1 M bis-tris pH 5.5, 25%(w/v) PEG3350. The condition was further optimized to 0.25 M MgCl2, 23%(w/v) PEG3350,5 mM CaCl2, 0.1 M bis-tris pH 5.5. The cellobiose-bound crystal was obtained by soaking the native crystal with mother liquor containing 10 mM cellobiose for 3 h. The X-ray diffraction datasets were collected at beam line BL15A1 of the National Synchrotron Radiation Research Center(NSRRC, Hsinchu,Taiwan). The diffraction images were processed using the program HKL2000. The crystal structure of GlCel5 A was solved by molecular replace method with Phaser program,using a hypothetical model which was generated by Phyre2 web portal using Trichoderma reesei endoglucanase structure as a template(PDB ID, 3QR3;sequence identity,65%). Subsequent structure refinement was carried out by using Coot and Refmac5 programs. The complex structure GlCel5A-CBI was determined by MR method with Phaser using the refined apo-structure as a searching model. Structure refinement and water/ligand incorporation were carried out as described for the apo-structure.the PDB ID of GlCel5 A is 5D8 W and the PDB ID of GlCel5A-CBI is 5D8 Z.GlCel5A folds into a typical(β/α)8 TIM-barrel structure. GlCel5 A forms four pairs of disulfide bonds with three anchoring outer helices and one located in the long β1-α1 loop. A shallow open cleft across the protein surface is found to accommodate substrate in the complex structure. The groove is exposed to the solvent and lined with several aromatic and hydrophilic residues. Two glutamates, E164 and E276, that were identified as the catalytic residues according to sequence alignment are located on the C-terminus of the central barrel. Based on these results, GlCel5 A is proposed to operate a hydrolysis reaction through a retaining mechanism.