Purification, characterization and sequencing of the major beta-1,3-glucanase from the midgut of Tenebrio molitor larvae.
The major beta-1,3-glucanase from Tenebrio molitor (TLam) was purified to homogeneity (yield, 6%; enrichment, 113 fold; specific activity, 4.4 U/mg). TLam has a molecular weight of 50 kDa and a pH optimum of 6. It is an endoglucanase that hydrolyzes beta-1,3-glucans as laminarin and yeast beta-1,3-1,6-glucan, but is inactive toward other polysaccharides (as unbranched beta-1,3-glucans or mixed beta-1,3-1,4-glucan from cereals) or disaccharides. The enzyme is not inhibited by high substrate concentrations and has low processivity (0.6). TLam has two ionizable groups involved in catalysis, and His, Tyr and Arg residues plus a divalent ion at the active site. A Cys residue important for TLam activity is exposed after laminarin binding. The cDNA coding for this enzyme was cloned and sequenced. It belongs to glycoside hydrolase family 16, and is related to other insect glucanases and glucan-binding proteins. Sequence analysis and homology modeling allowed the identification of some residues (E174, E179, H204, Y304, R127 and R181) at the active site of the enzyme, which may be important for TLam activity. TLam efficiently lyses fungal cells, suggesting a role in making available walls and cell contents to digestion and in protecting the midgut from pathogen infections.
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, C.P 26077, 05513-970, São Paulo, Brazil; Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil.
Fernando A
FA
Genta
Ivan
I
Bragatto
Walter R
WR
Terra
Clélia
C
Ferreira
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D044602:Cellulases / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D016384:Consensus Sequence; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D005260:Female; D041981:Gastrointestinal Tract / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007814:Larva / Q000201:enzymology; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D011487:Protein Conformation; D016415:Sequence Alignment; D013712:Tenebrio / Q000145:classification / Q000201:enzymology* / Q000235:genetics
D018076:DNA, Complementary; D044602:Cellulases; D004693:Glucan Endo-1,3-beta-D-Glucosidase