Endoglucanase I from the edible straw mushroom, Volvariella volvacea. Purification, characterization, cloning and expression.
We isolated an endoglucanase, EG1, from culture fluid of Volvariella volvacea grown on crystalline cellulose by ion-exchange and gel filtration chromatography, and preparative PAGE. EG1 has a molecular mass of 42 kDa as determined by SDS/PAGE and an isoelectric point of 7.7. Enzyme-catalysed hydrolysis of carboxymethyl-cellulose (CM-cellulose) is maximal at pH 7.5 and 55 degrees C. EG1 also hydrolysed phosphoric acid-swollen cellulose and filter paper (at rates of 29% and 6%, respectively, compared with CM-cellulose), but did not hydrolyse crystalline cellulose, cotton, oat spelt xylan, and birchwood xylan. Degenerate primers based on the N-terminal sequences of purified EGI and a protease-generated fragment were used to generate cDNA fragments encoding a portion of the EG1 gene (eg1), and RACE was used to obtain full-length cDNA clones. The cDNA of eg1 contained an ORF of 1167 bp encoding 389 amino acids. The amino-acid sequence from Ala24 to Thr40 corresponded to the N-terminal sequence of the purified protein. The first 23 amino acids are presumed to be a signal peptide. V. volvacea EG1 has been assigned to glycoside hydrolase family 5 according to the classification of glycohydrolases based on amino-acid sequence similarities. Transcripts of eg1 were detected in total RNA from mycelium grown on cellulose but not from mycelium grown on glucose. Cellobiose also induced eg1 expression in 1- to 4-day-old cultures but the signal intensity was lower than that obtained with cellulose. Catabolite repression was observed 24 h after addition of 1% (w/v) glucose, alpha-lactose, beta-lactose, xylose, mannose, sorbose or fructose to medium containing 1% (w/v) crystalline cellulose. Eg1 was expressed at a high level in the yeast, Pichia pastoris, and the catalytic activity of the recombinant EG1 was confirmed.
Ding
S J
SJ
Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China.
Ge
W
W
Buswell
J A
JA
D000363:Agaricales / Q000201:enzymology*; D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D011994:Recombinant Proteins; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase