Cloning of the Trichoderma reesei cDNA encoding a glucuronan lyase belonging to a novel polysaccharide lyase family.
The filamentous fungus Trichoderma reesei produces glucuronan lyase (TrGL) when it is grown on beta-(1-->4)-polyglucuronate (cellouronate) as a sole carbon source. The cDNA encoding TrGL was cloned, and the recombinant enzyme was heterologously expressed in Pichia pastoris. The cDNA of TrGL includes a 777-bp open reading frame encoding a 20-amino-acid signal peptide and the 238-amino-acid mature protein. The amino acid sequence showed no similarity to the amino acid sequences of previously described functional proteins, indicating that the enzyme should be classified in a novel polysaccharide lyase (PL) family. Recombinant TrGL catalyzed depolymerization of cellouronate endolytically by beta-elimination and was highly specific for cellouronate. The enzyme was most active at pH 6.5 and 50 degrees C, and its activity and thermostability increased in the presence of Ca2+, suggesting that its calcium dependence is similar to that of other PLs, such as pectate lyases.
Konno
Naotake
N
Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, Japan.
Igarashi
Kiyohiko
K
Habu
Naoto
N
Samejima
Masahiro
M
Isogai
Akira
A
D002118:Calcium / Q000494:pharmacology; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D020536:Enzyme Activators / Q000494:pharmacology; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010843:Pichia / Q000235:genetics; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism; D021382:Protein Sorting Signals; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
D004271:DNA, Fungal; D020536:Enzyme Activators; D005656:Fungal Proteins; D011134:Polysaccharides; D021382:Protein Sorting Signals; D011133:Polysaccharide-Lyases; C109209:glucuronan lyase; D002118:Calcium