Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei beta-mannanase gene containing a cellulose binding domain.
beta-Mannanase (endo-1,4-beta-mannanase; mannan endo-1,4-beta-mannosidase; EC 3.2.1.78) catalyzes endo-wise hydrolysis of the backbone of mannan and heteromannans, including hemicellulose polysaccharides, which are among the major components of plant cell walls. The gene man1, which encodes beta-mannanase, of the filamentous fungus Trichoderma reesei was isolated from an expression library by using antiserum raised towards the earlier-purified beta-mannanase protein. The deduced beta-mannanase consists of 410 amino acids. On the basis of hydrophobic cluster analysis, the beta-mannanase was assigned to family 5 of glycosyl hydrolases (cellulase family A). The C terminus of the beta-mannanase has strong amino acid sequence similarity to the cellulose binding domains of fungal cellulases and is preceded by a serine-, threonine-, and proline-rich region. Consequently, the beta-mannanase is probably organized similarly to the T. reesei cellulases, having a catalytic core domain separated from the substrate-binding domain by an O-glycosylated linker. Active beta-mannanase was expressed and secreted by using the yeast Saccharomyces cerevisiae as the host. The results indicate that the man1 gene encodes the two beta-mannanases with different isoelectric points (pIs 4.6 and 5.4) purified earlier from T. reesei.
Stålbrand
H
H
VTT Biotechnology and Food Research, Espoo, Finland.
Saloheimo
A
A
Vehmaanperä
J
J
Henrissat
B
B
Penttilä
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D002480:Cellulase / Q000235:genetics; D002482:Cellulose / Q000378:metabolism*; D003001:Cloning, Molecular; D016000:Cluster Analysis; D015971:Gene Expression Regulation, Enzymologic; D005800:Genes, Fungal / Q000235:genetics*; D007526:Isoelectric Point; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D008361:Mannosidases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
D000596:Amino Acids; D007527:Isoenzymes; D002482:Cellulose; D008361:Mannosidases; D002480:Cellulase; C041867:endo-1,4-beta-D-mannanase