Isolation and analysis of two cellulase cDNAs from Orpinomyces joyonii.
Two cellulase cDNAs, celB29 and celB2, were isolated from a cDNA library derived from mRNA extracted from the anaerobic fungus, Orpinomyces joyonii strain SG4. The nucleotide sequences of celB2 and celB29 and the primary structures of the proteins encoded by these cDNAs were determined. The larger celB29 cDNA was 1966bp long and encoded a 477 amino acid polypeptide with a molecular weight of 54kDa. Analysis of the 1451bp celB2 cDNA revealed an 1164bp open reading frame coding for a 44kDa protein consisting of 388 amino acids. Both deduced proteins had a high sequence similarity in central regions containing putative catalytic domains. Primary structure analysis revealed that CelB29 contained a Thr/Pro-rich sequence that separated the N-terminal catalytic domain from a C-terminal reiterated region of unknown function. Homology analysis showed that both enzymes belong to glycosyl hydrolase family 5 and were most closely related to endoglucanases from the anaerobic fungi Neocallimastic patriciarum, Neocallimastix frontalis and Orpinomyces sp. The classification of CelB29 and CelB2 as endoglucanases was supported by enzyme assays. The cloned enzymes had high activities towards barley beta-glucan, lichenan and carboxymethylcellulose (CMC), but not Avicel, laminarin, pachyman, xylan and pullulan. In addition, CelB29 and CelB2 showed activity against p-nitrophenyl-beta-D-cellobioside (pNP-G(2)) to p-nitrophenyl-beta-D-cellopentaoside (pNP-G(5)) but not p-nitrophenyl-beta-D-glucopyranoside (pNP-G(1)) with preferential activity against p-nitrophenyl-beta-D-cellotrioside (pNP-G(3)). Based on these results, we proposed that CelB29 and CelB2 are endoglucanases with broad substrate specificities for short- and long-chain beta-1,4-glucans.
Qiu
X
X
Lethbridge Research Station, Agriculture and Agri-Food Canada, Lethbridge, Canada. xqui@pbi.nrc.ca
Selinger
B
B
Yanke
L
L
Cheng
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D020085:Neocallimastigales / Q000201:enzymology / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D018076:DNA, Complementary; D007527:Isoenzymes; D002480:Cellulase