Insecticidal Activity and Processing in Larval Gut Juices of Genetically Engineered 130-kDa Proteins of Bacillus thuringiensis subsp. aizawai

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The 130-kDa insecticidal protein (IP) of Bacillus thuringiensis subsp. aizawai is proteolytically processed in the gut juice of susceptible insect larvae to yield an insecticidally active 60-kDa fragment. Twenty-seven mutant IP genes with the replacement of codons for Arg and Lys with codons for Gln in the active fragment and its adjacent regions of the 130-kDa IP were constructed by site-directed mutagenesis and expressed in Escherichia coli cells. The produced mutant IPs at Arg87, Arg131, Arg198, Arg311, Arg368, Arg402, Arg458, Arg502, Arg512, Arg524, Arg526, Arg528, and Arg601 had reduced insecticidal activity against Spodoptera litura larvae. The mutant at Arg601 was sensitive to proteolytic digestion in the gut juice of S.litura larvae. Although the mutants at Arg619, Lys622, and Lys637 had nearly the same activity as that of the wild type, the mutant with the triple replacement at Arg619, Lys622, and Lys637 was 2.5 times more active against S.litura larvae than the wild type. This triple mutant showed a slightly different processing profile in the gut juice than that of the wild type.