Variability of the molecular size of extracellular amylase produced by intact cells and protoplasts of Bacillus caldolyticus

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Abstract
One of the typical properties of the extracellular amylase produced by Bacillus caldolyticus is the tendency to disintegrate into subunits with a molecular weight (MW) of less than 10 000, when the enzyme is subjected to ultrafiltration. Disintegration is due to a loss of Ca-ions, leading to nonactive subunits. Activity can be fully restored by addition of Ca-ions. Reactivation occurs also spontaneously if the low MW fraction is stored in glassware. Comparative ultrafiltration experiments with the subunit fraction with or without a supply of Ca revealed that in oresence of this divalen cation the subunit reaggregated to the active enzyme. The different distribution patterns obtained in absence or presence of Ca showed that reactivation is directly linked to the formation of a high MW form of the protein. Substitution of Ca by other divalent cations also led to reaggregation. These aggregates are, however, inactive. The enzyme was found to be formed intracellularly in its low MW form. Experiments with protoplasts revealed that these are capable to produce and release the amylase. When the production of the enzyme by protoplasts declined, full restoration could be achieved by a recovery treatment. Normally, the enzyme released by the protoplasts consisted of equal portions of the high, medium, and low MW form of the amylase. If the cells were, however, depleted as a result of continued incubations, the extruded enzyme consisted increasingly of the low MW form, which could finally represent more than 80%. This trend could be completely reversed by the supply of carbon and nitrogen sources during the recovery treatment, whereafter the enzyme consisted again of the initially observed equal amounts of the three MW forms. Vesicles prepared from the protoplasts were also found to release amylase, but on a lower level, and only for a very limited time, with no possibility to regain activity by a recovery treatment. Subunit formation was also observed during column chromatography, which could be counteracted by a sufficient supply with Ca-ions.