Hydrolysis of beta-1,3/1,6-glucan by glycoside hydrolase family 16 endo-1,3(4)-beta-glucanase from the basidiomycete Phanerochaete chrysosporium.
When Phanerochaete chrysosporium was grown with laminarin (a beta-1,3/1,6-glucan) as the sole carbon source, a beta-1,3-glucanase with a molecular mass of 36 kDa was produced as a major extracellular protein. The cDNA encoding this enzyme was cloned, and the deduced amino acid sequence revealed that this enzyme belongs to glycoside hydrolase family 16; it was named Lam16A. Recombinant Lam16A, expressed in the methylotrophic yeast Pichia pastoris, randomly hydrolyzes linear beta-1,3-glucan, branched beta-1,3/1,6-glucan, and beta-1,3-1,4-glucan, suggesting that the enzyme is a typical endo-1,3(4)-beta-glucanase (EC 3.2.1.6) with broad substrate specificity for beta-1,3-glucans. When laminarin and lichenan were used as substrates, Lam16A produced 6-O-glucosyl-laminaritriose (beta-D-Glcp-(1->6)-beta-D-Glcp-(1->3)-beta-D-Glcp-(1->3)-D-Glc) and 4-O-glucosyl-laminaribiose (beta-D-Glcp-(1->4)-beta-D-Glcp-(1->3)-D-Glc), respectively, as one of the major products. These results suggested that the enzyme strictly recognizes beta-D-Glcp-(1->3)-D-Glcp at subsites -2 and -1, whereas it permits 6-O-glucosyl substitution at subsite +1 and a beta-1,4-glucosidic linkage at the catalytic site. Consequently, Lam16A generates non-branched oligosaccharide from branched beta-1,3/1,6-glucan and, thus, may contribute to the effective degradation of such molecules in combination with other extracellular beta-1,3-glucanases.
Kawai
Rie
R
Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
Igarashi
Kiyohiko
K
Yoshida
Makoto
M
Kitaoka
Motomitsu
M
Samejima
Masahiro
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002480:Cellulase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002855:Chromatography, Thin Layer / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D015394:Molecular Structure; D020075:Phanerochaete / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D047071:beta-Glucans / Q000737:chemistry / Q000378:metabolism*
D018076:DNA, Complementary; D005656:Fungal Proteins; D047071:beta-Glucans; C064197:beta-1,6-glucan; C033363:beta-1,3-glucan; D006026:Glycoside Hydrolases; D002480:Cellulase