IDENTIFICATION OF 2 AMINO-ACIDS CONTRIBUTING THE HIGH ENZYME-ACTIVITYIN THE ALKALINE PH RANGE OF AN ALKALINE ENDOGLUCANASE FROM A BACILLUSSP

An alkaline cellulase (beta-1,4-endoglucanase; NK1) from an alkalophil ic Bacillus sp. shows great similarity in amino acid sequence to a neu tral cellulase (BSC) from Bacillus subtilis, despite a considerable di fference in their pH activity profiles. Multiple amino acid exchanges by site-directed mutagenesis, using BSC as the reference, were perform ed on the residues in region 5 of NK1, which was previously shown to b e responsible for the high enzyme activity of this alkaline cellulase in a broad alkaline pH range. Two amino acid residues, Ser287 and Ala2 96, were identified as being responsible for the activity in the alkal ine range. The double mutation, Ser287 to Asn and Ala2% to Ser, of NK1 made its pH activity profile almost the same as that of BSC. On the o ther hand, the pH activity profile in the acidic range was not signifi cantly affected by various amino acid replacements including these two positions in region 5. This observation, together with the informatio n 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 ca talytic site.