Crystallization and preliminary X-ray crystallographic analysis of recombinant β-mannosidase from Aspergillus niger

Demo G.^1,2, Friedlová B.³, Weignerová L.³, Wimmerová M.^1,2,4

¹National Center for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5/A4, 62500 Brno, Czech Republic

²Central European Institute of Technology-CEITEC, Masaryk University, Kamenice 5/A4, 62500 Brno, Czech Republic

³Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague, Czech Republic ⁴Department of Biochemistry, Masaryk University, Kamenice 5/A5, 62500 Brno, Czech Republic

guliver@mail.muni.cz

β-Mannosidase (β-D-mannoside mannohydrolase, EC 3.2.1.25) is an important glycoside hydrolase (GH) specific for the hydrolysis of terminal b-linked mannosides in various sugar chains. This enzyme plays an essential role in the complete hydrolysis of b-mannans to mannose and therefore b-mannosidases are very efficient in various industrial processes, such as hydrolysis of galactomannans used for the improved removal of drilled material in oil and gas drilling or coffee extraction, and as a bleach-boosting agent in the pulp and paper industries [1]. β-Mannosidases are also used in the synthesis of oligosaccharides or alkyl β-mannosides for medical and other purposes [2].

Here, we report the crystallization and preliminary X-ray crystallographic analysis of recombinant β-mannosidase overexpressed in Pichia pastoris. The initial screening showed two conditions for obtaining the crystals: (i) 0.2 M magnesium chloride, 25%(w/v) polyethylene glycol 3350, 0.1 M bis-tris pH 6.5 and (ii) 0.2 M calcium chloride, 25%(w/v) polyethylene glycol 4000, 0.1 M Tris pH 8.5. The best crystals were produced by further optimization using the hanging-drop vapour diffusion method. The main precipitants PEG 3350 and PEG 4000 were changed in small concentration steps. The trials showed the best possible concentration was 20%(w/v) for PEG 3350 and PEG 4000. The long needles were observed to be more compact with a small increase in the salt concentration (MgCl2 or CaCl2); the effective range of the concentration is 0.2–0.4 M. The crystals of the β-mannosidase were typically needles with dimensions 0.25x0.01x0.08 mm. Diffraction data were collected at BESSY II Berlin (14.1 and 14.2). The data were processed by XDSAPP [4]. The crystals belonged to space group P1. The β-mannosidase in the native data set diffracted to 2.41 Å resolution and had unit-cell parameters a = 62.37, b = 69.73, c = 69.90 Å, α = 108.20, β = 101.51, γ = 103.20 º. The calculated Matthews coefficient (VM) of 2.56 ųDa^-1 with a solvent content of 52.06% indicates the presence of one molecule in the asymmetric unit. The molecular-replacement method was performed with the structure of the b-mannosidase from Bacteroides thetaiotaomicron (PDB entry 2JE8) [4] as a model, but a suitable solution was not found. The β-mannosidase in the SAD data set diffracted to 2.44 Å resolution and had unit-cell parameters a = 61.82, b = 65.23, c = 68.72 Å , α = 108.63, β = 101.06, γ = 103.06 º. The calculated Matthews coefficient (VM) of 2.34 ųDa^-1 with a solvent content of 47.42% indicates the presence of one molecule in the asymmetric unit. Experimental phasing, model fitting and refinement are in progress [5].

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