Construction and characterization of the chimeric enzymes between the Bacillus subtilis cellulase and an alkalophilic Bacillus cellulase.
The amino acid sequences of cellulase from Bacillus subtilis (BSC) and that from an alkalophilic Bacillus sp. N-4 (NK1) show significant homology in most parts except for the C-terminal portions. Despite the high homology, the pH activity profiles of the two enzymes are quite different; BSC has its optimum pH at 6-6.5, whereas NK1 is active over a broad pH range from 6 to 10.5. In order to identify the structural features which determine such pH activity profiles, chimeric cellulases between BSC and NK1 were constructed using four restriction sites commonly present within the homologous coding sequences, and were produced in Escherichia coli. The chimeric cellulases showed various chromatographic behaviors, reflecting the origins of their C-terminal regions. The pH activity profiles of the chimeric enzymes in the alkaline range could be classified into either the BSC or NK1 type mainly depending on the origins of the fifth C-terminal regions. In the acidic range, the profile was determined only by the origin of the fourth enzyme region from the N terminus. Comparison of the kinetic parameters between pH 5 and 6 using p-nitrophenyl cellobioside as a substrate indicated that the fourth region is responsible for the pH-dependent change of the kcat value. Only a limited number of amino acids in the fourth region may affect on deprotonation of catalytic residues of the cellulases and modulate the catalytic activity in the acidic pH values.
Nakamura
A
A
Department of Biotechnology, Faculty of Agriculture, University of Tokyo, Japan.
Fukumori
F
F
Horinouchi
S
S
Masaki
H
H
Kudo
T
T
Uozumi
T
T
Horikoshi
K
K
Beppu
T
T
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D013045:Species Specificity; D013329:Structure-Activity Relationship
D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D002480:Cellulase