SITE-DIRECTED MUTAGENESIS OF THE PUTATIVE ACTIVE-SITE OF ENDOGLUCANASE-K FROM BACILLUS SP KSM-330

The roles of one Glu and four Asp residues of endoglucanase K from Bac illus sp. KSM-330, which are conserved in all the endo-beta-glucanases in the family D, were analyzed by site-directed mutagenesis. The gene for endoglucanase K was mutated to replace Asp-154, Asp-191, Asp-193 or Asp-300 by Asn, or to replace Glu-130 by Gin in the encoded enzyme. Mutant and wild-type genes were separately expressed in Bacillus subt ilis and the resultant enzymes were purified from the culture broth. A ll mutant enzymes exhibited the same mobility on SDS-polyacrylamide ge l electrophoresis as the wild-type enzyme and gave similar circular di chroism spectra to that of the wild-type enzyme. Substitution of Glu-1 30, Asp-191, Asp-193 or Asp-300 significantly decreased the specific a ctivity of the enzyme toward CM-cellulose. Kinetic analysis of the abi lities of these mutant enzymes to liberate p-nitrophenol from p-nitrop henylcellotrioside revealed that all the mutant enzymes had very much lower k(cat) values than that of the wild-type enzyme, while the K-m v alues of these mutant enzymes were almost the same as that of the wild -type enzyme. Of these Glu and Asp residues, Glu-130 and Asp-191 seem to be most likely to be catalytic residues because substitutions of th ese residues resulted in the lowest k(cat) values of the mutant enzyme s.