CRYSTALLIZATION AND PRELIMINARY X-RAY ANALYSIS OF b-GLUCOSIDASE FROM Bacillus circulans sp. alkalophilus

Nina Hakulinen¹, Timo Korpela², Sari Paavilainen² and Juha Rouvinen¹

¹Department of Chemistry, University of Joensuu, P.O. Box 111, 80101 Joensuu, Finland
² Department of Biochemistry, University of Turku, P.O. Box , Turku, Finland

b-glucosidases are heterogenous group of enzymes, which are found in different organisms and have various biological functions. b-glucosidase from Bacillus circulans sp. alkalophilus (BG) is involved in the biodegradation of cellulose. It catalyzes the hydrolysis of b-glucosidic bond of cellobiose. The molecular mass of the monomeric enzyme is 51,3 kDa. BG belongs to the family 1 of glycosyl hydrolases based on the Henrissat classification. So far, only few three-dimensional structures of members of this family has been reported: a cyanogenic b-glucosidase from Trifolium repens, a 6-phospho-b-galactosidase from Lactococcus lactis, a myrosinase from Sinapis alba and recently, a b-glucosidase from Bacillus polymyxa.

BG has been crystallized by the hanging drop vapour diffusion method at room temperature. Crystals appeared to the drop after two weeks using PEG 8000 and sodium formate as precipitants and sodium acetate as the buffer at pH 6.5. In the presence of some detergents, like octaethylene glycol monododecyl ether crystals grow larger and attained their size of 0.4 x 0.3 x 0.3 mm. The Crystal was flash-frozen and diffraction data set at 100 K was measured. The crystal diffracted 2.7 A resolution and belonged to triclinic space group P1 with unit cell parameters a = 116.4 A, b = 122.3 A, c = 163.2 A, a = 89.3^o, b = 74.3^o and g = 68.0^o. The final data set consisted of 186 165 unique reflections with Rmerge 7.6% and the completeness was 84%. The Matthews coefficient suggest 16 protomers per asymmetric unit (Vm = 2.51 A³Da^-1) with a solvent content of 51%.