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

TitleLignocellulose degradation by Phanerochaete chrysosporium: purification and characterization of the main alpha-galactosidase
Publication TypeJournal Article
Year of Publication1999
AuthorsBrumer, H, Sims, PFG, Sinnott, ML
Date PublishedAPR 1
Type of ArticleArticle

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 peptide chains, which gives the N-terminal sequence ADNGLAITPQMG(?W)NT( ?W)NHFG-(?W)DIS(?W)DTI. It is remarkably stable, with crude extracts losing no activity over 3 h at 80 degrees C, and the purified enzyme retaining its activity over several months at 4 degrees C. The kinetics of hydrolysis at 25 degrees C of various substrates by this retaining enzyme were measured, absolute parameters being obtained by active-site titration with 2',4',6'-trinitrophenyl 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-nitrophenyl-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 effect at optimal pH. There is no,alpha-H-2 effect on binding galactose {[}(alpha D)(K-i) = 0.994 +/- 0.013]. The enzyme hydrolyses p-nitrophenyl beta-L-arabinopyranoside similar to 510 times slower than the galactoside, but has no detectable activity on the alpha-D-glucopyranoside or alpha-D-mannopyranoside. Hydrolysis of a-galactosides with poor leaving groups is Michaelian, but that of substrates with good leaving groups exhibits pronounced apparent substrate inhibition, with K-is values similar to K-m values. We attribute this to the binding of the second substrate molecule to a beta-galacropyranosyl-enzyme intermediate, forming an E(.)beta Gal(.)alpha GalX complex which turns over slowly, if at all. 1-Fluoro-alpha-D-galactopyranosyl fluoride, unlike alpha-D-galactopyranosyl fluoride, is a Michaelian substrate, indicating that the effect of l-fluorine substitution is greater on the first than on the second step of the enzyme reaction.