Identification of two amino acids contributing the high enzyme activity in the alkaline pH range of an alkaline endoglucanase from a Bacillus sp.
An alkaline cellulase (beta-1,4-endoglucanase; NK1) from an alkalophilic Bacillus sp. shows great similarity in amino acid sequence to a neutral cellulase (BSC) from Bacillus subtilis, despite a considerable difference in their pH activity profiles. Multiple amino acid exchanges by site-directed mutagenesis, using BSC as the reference, were performed on the residues in region 5 of NK1, which was previously shown to be responsible for the high enzyme activity of this alkaline cellulase in a broad alkaline pH range. Two amino acid residues, Ser287 and Ala296, were identified as being responsible for the activity in the alkaline range. The double mutation, Ser287 to Asn and Ala296 to Ser, of NK1 made its pH activity profile almost the same as that of BSC. On the other hand, the pH activity profile in the acidic range was not significantly affected by various amino acid replacements including these two positions in region 5. This observation, together with the information available on other endoglucanases, suggests that the above two amino acid substitutions caused a profound effect through rearrangement of the hydrogen bond network forming the substrate-binding site or the catalytic site.
Park
J S
JS
Department of Agricultural Chemistry, University of Tokyo, Japan.
Hitomi
J
J
Horinouchi
S
S
Beppu
T
T
D000468:Alkalies; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D005798:Genes, Bacterial / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007231:Infant, Newborn; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D000468:Alkalies; D000596:Amino Acids; D011993:Recombinant Fusion Proteins; D002480:Cellulase