Identification and characterization of cellulose-binding domains in xylanase A of Clostridium stercorarium.
The xynA gene encoding a major xylanase of Clostridium stercorarium F-9 was sequenced. The structural gene consists of an open reading frame of 1533 bp encoding a protein of 511 amino acids with an M(r) of 56,519. XynA consists of a catalytic domain belonging to family G at the NH2-terminus and two direct repeats of about 90 amino acids with a short spacing at the COOH-terminus. The repeated sequences, CBDI and CBDII, were not homologous with amino acid sequences of the CBDs classified into families I to V. Nevertheless, XynA showed an affinity for insoluble cellulose such as Avicel. Binding of XynA to Avicel was strongly dependent on the concentration of the incubation buffer and was inhibited by Triton X-100. XynA bound to Avicel (2.4 nmol/g-cellulose) and acid-swollen cellulose (180 nmol/g-cellulose), suggesting that this enzyme has higher affinity for amorphous cellulose than for crystalline cellulose. Functions of CBDI and CBDII were investigated by constructing the mutant enzymes and evaluating the cellulose-binding ability of each of them. XynA4 lacking CBDI and XynA5 lacking CBDII bound to Avicel to a lesser extent than the parental enzyme XynA; but XynA6, devoid of both CBDs, did not bind at all, indicating that CBDI and CBDII each functioned independently as CBD in XynA and their binding capacity was additive. Although the Ruminococcus albus endoglucanase EgIV that was joined to CBDs of XynA acquired cellulose-binding ability, the substrate specificity of EgIV was not altered in the presence or absence of CBDs.
Sakka
K
K
Laboratory of Applied Microbiology, Faculty of Bioresources, Mie University, Tsu, Japan. sakka@bio.mieu.ac.jp
Takada
G
G
Karita
S
S
Ohmiya
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002482:Cellulose / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D043364:Endo-1,4-beta Xylanases; D004795:Enzyme Stability; D004926:Escherichia coli; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014995:Xylosidases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*
D011994:Recombinant Proteins; D002482:Cellulose; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases