Substrate-dependent change in the pH-activity profile of alkaline endo-1,4-beta-glucanase from an alkaline Bacillus sp.
A neutral cellulase (BSC) from Bacillus subtilis and an alkaline cellulase (NK1) from alkalophilic Bacillus sp. N-4 show significant amino acid sequence homology. Despite the high homology, the pH-activity profiles of the two enzymes for carboxymethyl cellulose (CMC) hydrolysis are quite different; BSC shows a sharp optimum pH at 6, whereas NK1 shows its full activity in a broad range, from pH 6 to 10.5. For elucidation of the reasons for the difference in their pH-activity profiles, their activities were examined at various pHs using a series of cellooligosaccharides and their derivatives, cellotetraose (G4), cellopentaose (G5), cellohexaose (G6), cellopentaitol (G5OH), and cellohexaitol (G6OH), as substrates. The optimum pH of BSC was around 6 for all the cellooligosaccharides examined. On the other hand, the optimum pH of NK1 varied depending on the substrate, i.e., a sharp optimum at pH 6 with G4 and G5OH, and a broad optimum of pH 6 to 10.5 with G5, G6, and G6OH. Comparison of the kinetic parameters of the two cellulases at pH 7 and 9 using G6OH as a substrate revealed that NK1 showed similar values at both pHs, while BSC showed a greatly increased Km value for this substrate at pH 9. In addition, NK1 showed a greatly increased Km value for G5OH hydrolysis at pH 9. Both enzymes cleaved these substrates at the same position, which suggests the same productive binding mode of these substrates with both enzymes. All these observations suggest that the reduced enzyme activity of BSC in the alkaline pH range can be attributed to a decrease in the affinity of a subsite for the third glucose moiety from the scissile site of these substrates.
Department of Biotechnology, University of Tokyo.
J
J
Hitomi
J S
JS
Park
M
M
Nishiyama
S
S
Horinouchi
T
T
Beppu
D000468:Alkalies / Q000737:chemistry*; D001412:Bacillus subtilis / Q000201:enzymology*; D002480:Cellulase / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009844:Oligosaccharides / Q000378:metabolism; D010084:Oxidation-Reduction; D013379:Substrate Specificity
D000468:Alkalies; D009844:Oligosaccharides; D002480:Cellulase