Study On The Characteristics Of Endoproteases And Changes Of Main Nitrogenous Components During Brown Rice Germination

Using casein or gelatin as substrate, the characterization of endoproteases from germinated brown rice was investigated with an "in solution" or gel electrophoresis assay in this paper. Then, the physiological characters and the correlation between the changes of protease activity and nitrogen compounds during germination of brown rice were discussed. The main results were summarized as follows:Using casein as substrate, the proteases from geminated brown rice had two catalytic groups, an acidioid with optimal pH of 3.5 and an alkaloid of 8.0. They were hydrolytically most active 40℃and had a Km of 0.404 mg/mL and 3.889 mg/mL, respectively. The acidic protease activity was inhibited by Ba2+ and Pb2+, stimulated by Zn2+. The alkaline one was inhibited by Ca2+ and Pb2+, stimulated by Mg2+ and Zn2+. Inhibition assay revealed that pepstatin A and IAA inhibited most of the protease activity at pH 3.5, indicating the presence of aspartic protease and cysteine protease. Metalloproteases played a major role in alkaline condition (88.37% and 74.82% inhibition with EDTA and 1,10-Phe, respectively). L-Cys,β-ME and DTT can remarkably improve the acidic protease activity, while, the alkaline protease activity was not influenced by L-Cys andβ-ME and impaired by the presence of DTT.Using gel electrophoretic system with gelatin as substrate, the results showed that two protease activity bands were obvious at pH 3.5 and another two with higher molecular weights were observed at pH 8.0. The alkaline bands were detected earlier than the acidic ones. Inhibition assay revealed that the main acidic activity band was cysteine protease and the alkaline one belonged to the metalloproteas class. Reducing agents enhanced the acidic bands, but they had contrary effects on the alkaline ones. After the alkaline proteases were inhibited with EDTA, the activities were recovered in the presence of Zn2+ and Mn2+. The maximum recoveries with Zn2+ occurred at concentrations of 0.5-1.0 mmol/L and an excess of Zn2+(5.0～7.5 mmol/L) inhibited most of the activities.The length of seedlings, germination rate, respiration rate and consume in dry matter content increased gradually during brown rice gernination. The acidic protease activity increased at the start, and then decreased. The alkaline protease activity remarkably increased during 4 d germination, after which it kept constant. Stored proteins hydrolysis lead to the improvement on the content of peptides. The content of soluble protein and free amino acids increased at the start, and then decreased. Albumin and globulin hydrolyzed rapidly at the beginning 5 d of germination. SDS-PAGE showed that the high molecular weight proteins subunits of them were degraded completely. After 5 d, the content of glutelin began to decrease and the subunits of 57 KDa were remarkably reduced. Positive correlations were found between acidic protease activity and soluble protein, peptides, free amino acids during one week germination of brown rice. The correlations between alkaline protease activity and soluble protein, peptides were also positive, while negative correlations were found between globulin and the two kinds of protease groups.