Functional analysis of a hybrid endoglucanase of bacterial origin having a cellulose binding domain from a fungal exoglucanase.
A cellulose binding domain (CBD) of an endo-beta-1,4-glucanase (Ben) from the bacterium Bacillus subtilis BSE616 was replaced with the CBD of exoglucanase I (TexI) from the fungus Trichoderma viride HK-75. The resultant hybrid enzyme Ben'-CBDTexI, comprising the catalytic domain (Ben') of Ben and the CBD (CBDTexI) of TexI, was highly expressed at 20% of the total protein in Escherichia coli. The molecular mass of the hybrid enzyme was estimated to be ca. 38 kDa by SDS-PAGE, which was in good agreement with that calculated from 305 amino acids of Ben and 42 amino acids of CBDTexI. The hybrid enzyme exhibited almost the same activity as that of the original Ben toward soluble substrates, such as cellooligosaccharides. The hybrid enzyme showed higher binding ability and hydrolysis activity toward microcrystalline cellulose (Avicel), even though the length of the CBD of TexI was four times smaller than that of Ben. The hybrid enzyme was more resistant to tryptic digestion than the original Ben. The efficient binding ability of the hybrid enzyme to Avicel permitted purification of the enzyme using an Avicel-affinity column to the extent of ca. 90% purity.
Kim
H
H
Department of Agricultural Chemistry, Sunchon National University, Korea.
Goto
M
M
Jeong
H J
HJ
Jung
K H
KH
Kwon
I
I
Furukawa
K
K
D000327:Adsorption; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D001709:Biotechnology; D020134:Catalytic Domain / Q000235:genetics; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D010450:Endopeptidases; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D043326:Glucan 1,3-beta-Glucosidase; D006868:Hydrolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014242:Trichoderma / Q000201:enzymology / Q000235:genetics; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D002482:Cellulose; D001617:beta-Glucosidase; D002480:Cellulase; D043326:Glucan 1,3-beta-Glucosidase; D010450:Endopeptidases