Development of limit dextrinase in germinated barley (Hordeum vulgare L.): Evidence of proteolytic activation

Barley (Hordeum vulgare L.) that had been malted for 5 d developed only a small amount of bound (inactive) limit dextrinase, and very little free (active) enzyme was detected. Continuation of malting for up to 10 d only slightly increased the amount of both bound and free forms. Grain grown under conditions of ample moisture (wet grown) for 5 d produced a much higher amount of bound enzyme but a similarly low amount of free enzyme compared to malting conditions. After 10 d of growth there was a decrease in the amount of bound enzyme and a large increase in the amount of free enzyme, such that almost all of the enzyme was present in the free form. A more detailed study of limit dextrinase development in wet-grown grains revealed that a bound form was rapidly produced soon after germination. Five to 6 d after germination the amount of bound enzyme decreased rapidly and a very low amount was found in grains 9 d after germination. Meanwhile, a free form appeared slightly later and its initial rate of development was slow. At about 5 d after germination, precisely when the bound enzyme began to decrease, the free form increased rapidly, so that by 9 d after germination nearly all the enzyme was in the free form. The release of bound limit dextrinase in vitro occurred by proteolytic modification through the action of cysteine proteinases that were kept active or activated by the presence of reduced thiols in the extraction medium. The presence of cysteine proteinases was confirmed by inhibition studies using the inhibitors iodoacetamide, N-ethylmaleimide, antipain, and leupeptin. In addition, most of the bound form of limit dextrinase was soluble in 0.2 M sodium acetate buffer (pH 5.0) following extraction at 30°C for 16 h and centrifugation at 3000g.