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The aim of this study was to clarify the roles of endogenous proteases and higher molecular weight thiols in the release of bound β-amylase, which occurs during barley germination. In resting barley grains (Hordeum vulgare L. cv. Torrent) five major β-amylase monomers were found in the thiol reduced, salt soluble extract. Only the two smallest monomers were present in large amounts in the bound form of the enzyme that had been released with 2-mercaptoethanol. In vitro, bound β-amylase was solubilised by “releasing factors” extracted from the endosperm of decorticated grains germinated for three days. The releasing factors were in the higher molecular weight fraction (>5 kDa) and their formation was induced in degermed grains by gibberellic acid. When an endosperm extract, containing only higher molecular material (>5 kDa) prepared from three day germinated grain, was incubated with a preparation of bound β-amylase, about 75% of the release of the enzyme could be prevented by a mixture of proteolytic inhibitors. The dominant class of proteases in malt were the sulphydryl proteases. These were not fully active when extracted, but could be activated approximately six fold by the addition of 2-mercaptoethanol. Heating the extract to a limited extent destroyed all the proteolytic activity, but 10% of the bound β-amylase could still be released by the extract. |
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