Lignocellulose degradation by Phanerochaete chrysosporium: purification and characterization of the main alpha-galactosidase

The main cr-galactosidase was purified to homogreneity, in 30% yield, from a solid culture of Phanerochaete chrysosporium on 1 part wheat bran/2 parts thermomechanical softwood pulp. It is a glycosylated tetramer of 50 kDa pe ptide chains, which gives the N-terminal sequence ADNGLAITPQMG(?W)NT( ?W)NH FG-(?W)DIS(?W)DTI. It is remarkably stable, with crude extracts losing no a ctivity over 3 h at 80 degrees C, and the purified enzyme retaining its act ivity over several months at 4 degrees C. The kinetics of hydrolysis at 25 degrees C of various substrates by this retaining enzyme were measured, abs olute parameters being obtained by active-site titration with 2',4',6'-trin itrophenyl 2-deoxy-2,2-difluoro-alpha-D-galactopyranoside. The variation of k(cat)/K-m for 1-naphthyl-alpha-D-galactopyranoside with pH is bell-shaped , with pK(1) = 1.9(1) and pK(2) = 5.5(4). The (alpha D)(V/K) value for p-ni trophenyl-alpha-D-glucopyranoside is 1.031 +/- 0.007 at the optimal pH of 3 .75 and 1.114 +/- 0.006 at pH 7.00, indicating masking of the intrinsic eff ect at optimal pH. There is no,alpha-H-2 effect on binding galactose [(alph a D)(K-i) = 0.994 +/- 0.013]. The enzyme hydrolyses p-nitrophenyl beta-L-ar abinopyranoside similar to 510 times slower than the galactoside, but has n o detectable activity on the alpha-D-glucopyranoside or alpha-D-mannopyrano side. Hydrolysis of a-galactosides with poor leaving groups is Michaelian, but that of substrates with good leaving groups exhibits pronounced apparen t substrate inhibition, with K-is values similar to K-m values. We attribut e this to the binding of the second substrate molecule to a beta-galacropyr anosyl-enzyme intermediate, forming an E(.)beta Gal(.)alpha GalX complex wh ich turns over slowly, if at all. 1-Fluoro-alpha-D-galactopyranosyl fluorid e, unlike alpha-D-galactopyranosyl fluoride, is a Michaelian substrate, ind icating that the effect of l-fluorine substitution is greater on the first than on the second step of the enzyme reaction.