DE NOVO SYNTHESIS OF α-AMYLASE BY BACILLUS STEAROTHERMOPHILUS

Abstract

Welker , N. E. (Western Reserve University, Cleveland, Ohio and University of Illinois, Urbana), and L. Leon Campbell . De novo synthesis of α-amylase by Bacillus stearothermophilus . J. Bacteriol. 86: 1202–1210. 1963.—The pH optimum for the synthesis of α-amylase by washed-cell suspensions was 6.7. α-Amylase synthesis began soon after the addition of the inducer (maltose, methyl-β- d -maltoside, or phenyl-α- d -glucoside, at 10 −3 m ), proceeded at a linear rate for 60 min, and then leveled off. Cell suspensions without inducer produced small amounts of α-amylase. The addition of glucose (2 × 10 −3 m ), sucrose (10 −3 m ), or glycerol (4 × 10 −3 m ) to washed-cell suspensions failed to stimulate the production of α-amylase. Nitrogen starvation of washed cells for 60 min with fructose as a carbon source or by induction with pure maltose showed that the ability to produce α-amylase was lost. Examination of the amino acid pool at this time showed a general depletion of amino acids and the complete disappearance of tyrosine, phenyl-alanine, proline, and valine. Replenishment of the amino acid pool with casein hydrolysate (0.5%) restored the ability of the cells to produce α-amylase. Chloramphenicol and 8-azaguanine were shown to inhibit α-amylase synthesis. Inhibition was observed immediately upon the addition of chloramphenicol to cell suspensions preinduced for varying periods of time. Actinomycin D and mitomycin C also inhibited α-amylase synthesis when added to induced washed-cell suspensions. The amino acid analogues, norvaline, norleucine, and ethionine, inhibited α-amylase formation by 72, 53, and 38%, respectively. p -Fluorophenylalanine inhibited the synthesis of active α-amylase by 92% and the incorporation of proline-C 14 into α-amylase and cellular proteins by 95 and 74%, respectively.