Automated Gas Chromatographic Monitoring of Ethylene Evolution out of Rice Seedlings Infected with Blast Fungus

To find amino acid residues which are required for glucoamylase activity, mutant glucoamylase genes were constructed by in vitro mutations of GLU1 DNA encoding Saccharomycopsis fibuligera glucoamylase and introduced into Saccharomyces cerevisiae, and the resulting mutant proteins were assayed for enzymatic activities. Eighteen mutant proteins were obtained by random insertions of a BamHX linker DNA. Six out of 7 proteins with mutations in conserved regions among divergent glucoamylases showed no activities, while 8 out of 11 proteins with mutations in unconserved regions had similar specific activities to a wild-type value, suggesting that the conserved regions are important to the activity. A series of amino-terminal deletion mutants were also constructed. A mutant protein with a deletion of only two amino acid residues from the amino terminus had a significant reduction in the activity, suggesting an essential role for the amino-terminal peptide. Ten mutant proteins with single amino acid replacements were produced by site-directed mutagenesis. Analyses for thermal stability and temperature dependency of these mutant proteins revealed that Ala81, Asp89, Trp94, Arg96, Asp97, and Trp166 are required for wild-type levels of activities, and that at least Ala81 and Asp89 are not essential to catalytic activities, but act in thermal stability.