Analysis of the barley grain protease spectrum
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Malting is the controlled germination of barley grains, under specific conditions and up to the point of grain modification, and is the keystone of the brewing and distilling industries. Protease enzyme activity is an important constituent of this process as not only does this catalyse the degradation of the grains protein stores, but it also has involvement in other aspects of the germination process such as the activation of β – amylase. However, despite their importance, little is known of the identities and specific roles of the protease classes in the malting and germination process. With this in mind a biochemical and proteomic approach was taken in order to attempt to identify protease enzymes from malted and germinating barley grains and also to identify the roles of specific protease classes in barley grain malting and germination. FPLC based protein fractionation, SDS – PAGE analysis and MALDI – ToF mass spectroscopy were used in an attempt to purify metallo and serine class proteases from extracts of four day micro malted malt and germinating barley grains. A combination of class specific protease inhibitors, germination studies, enzyme assays of both barley grain proteases and specific starch degrading enzymes, and western blotting were employed to investigate the roles of the different protease classes in both overall grain physiology during germination and specifically, in the regulation of enzymes involved in starch breakdown. These investigations show that the serine and aspartate class proteases have a role in the positive regulation of the amounts of α – amylase present in barley grains during germination in a process that may involve gibberellic acid signalling. Furthermore, it was also shown that the serine class proteases have a role in the negative regulation of β – amylase activity during germination, thus revealing the complexity of the regulatory roles of barley grain proteases during grain germination. In addition, germination studies carried out in the presence of both the divalent cation chelator 1, 10 phenanthroline and different divalent cations revealed the potential importance of transition metal ions such as zinc, iron and manganese in the onset of grain germination.