Abstract
Soybean ( Glycine max cv. Maple Arrow) was infected with Bradyrhizobium japonicum (strain 61-A-101) and grown under semi-sterile conditions. Trehalase was extracted from nodules under acidic conditions (pH 3.7) and purified 530-fold by chromatography on concanavalin A-agarose, anion exchange chromatography and gel filtration. Its native molecular weight was close to 54 kDa. Activity staining after electrophoresis yielded only one band. Isoelectric focussing under non-denaturing conditions indicated an acidic isoelectric point (ca. pH 5.2). The enzyme had a broad pH-optimum (pH 3.5-7) and a temperature optimum of 59 °C. The K m value for trehalose was 0.28 mM, close to the data given previously for crude nodule extracts. Trehalase was found to be relatively insensitive to chelators and divalent cations, indicating that it does not require a metal cofactor. Validamycin A, a fungitoxic antibioticum isolated from Streptomyces , competitively inhibited trehalase with a K i value of 2 nM.
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