Structural and enzymatic analysis of soybean β-amylase mutants with increased pH optimum

Comparison of the architecture around the active site of soybean beta-amylase and Bacillus cereus beta-amylase showed that the hydrogen bond networks (Glu(380)- (Lys(295)-Met(51)) and Glu(380)-Asn(340)-Glu(178)) in soybean beta-amylase around the base catalytic residue, Glu(380), seem to contribute to the lower pH optimum of soybean beta-amylase. To convert the pH optimum of soybean beta-amylase (pH 5.4) to that of the bacterial type enzyme (pH 6.7), three mutants of soybean beta-amylase, M51T, E178Y, and N340T, were constructed such that the hydrogen bond networks were removed by site-directed mutagenesis. The kinetic analysis showed that the pH optimum of all mutants shifted dramatically to a neutral pH (range, from 5.4 to 6.0 - 6.6). The K-m values of the mutants were almost the same as that of soybean beta-amylase except in the case of M51T, while the V-max values of all mutants were low compared with that of soybean beta-amylase. The crystal structure analysis of the wild type-maltose and mutant-maltose complexes showed that the direct hydrogen bond between Glu(380) and Asn(340) was completely disrupted in the mutants M51T, E178Y, and N340T. In the case of M51T, the hydrogen bond between Glu(380) and Lys(295) was also disrupted. These results indicated that the reduced pK(a) value of Glu(380) is stabilized by the hydrogen bond network and is responsible for the lower pH optimum of soybean beta-amylase compared with that of the bacterial beta-amylase.