Crystallization and preliminary X-ray crystallographic studies of an exo-β-D-glucosaminidase from Trichoderma reesei

Chitosan is degraded to glucosamine (GlcN) by chitosanase and exo-beta-D-glucosaminidase (GlcNase). GlcNase from Trichoderma reesei (Gls93) is a 93 kDa extracellular protein composed of 892 amino acids. The enzyme liberates GlcN from the nonreducing end of the chitosan chain in an exo-type manner and belongs to glycoside hydrolase family 2. For crystallographic investigations, Gls93 was overexpressed in Pichia pastoris cells. The recombinant Gls93 had two molecular forms of similar to 105 kDa (Gls93-F1) and similar to 100 kDa (Gls93-F2), with the difference between them being caused by N-glycosylation. Both forms were crystallized by the hanging-drop vapour-diffusion method. Crystals of Gls93-F1 belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 98.27, b = 98.42, c = 108.28 angstrom, and diffracted to 1.8 angstrom resolution. Crystals of Gls93-F2 belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.84, b = 81.62, c = 183.14 angstrom, and diffracted to 2.4 angstrom resolution. Both crystal forms were suitable for X-ray structure analysis at high resolution.