Purification of Glycolate Oxidase Enzymes from Peasm (Pisum sativum l.) and Sorghum (Sorghum sudanense J.) Using Ion-Exchange Chromatography and their Physicochemical Characteristics
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Abstract
The glycolate oxidase enzyme, a core photorespiratory enzyme in green plants, oxidizes glycolate to glyoxylate, playing an adaptive role in roots during hypoxia to normoxia transition. The present study aimed to purify glycolate oxidase enzymes from peas (Pisum sativumL.) and sorghum (Sorghum sudanense J.), and investigate their physicochemical and regulatory properties. The activity of one of the glycolate oxidase isoforms (LCO-like glycolate oxidase) was induced in pea and sorghum leaves. The enzymes were purified to an electrophoretically homogeneous condition. The purified enzymes' specific activity, molecular mass, and physicochemical characteristics were determined. The results showed that the LCO-like glycolate oxidase enzyme from pea leaves was 314.37E/mg proteins, the degree of purification was 207 times, and the yield was 11.5%. The sorghum leaf enzyme was 274.22E/mg proteins with the degree of purification and yield, estimated at 143 times and 13.8%, respectively. The molecular mass of the native enzymes was 182 and 168kDa for peas and sorghum, respectively. It was revealed that the enzyme consisted of four subunits with a molecular mass of 45 and 42kDa for glycolate oxidase from pea and sorghum leaves, respectively, indicating a homo-tetramer. The sorghum leaf enzyme exhibited a higher affinity for lactate substrate with an optimal temperature of 55°С and pH of 7.2. Meanwhile, the optimal temperature and pH of pea leaf enzyme were 50°C and 7.5, respectively. The availability of homogeneous specimens provides opportunities to investigate the role of LCO-like glycolate oxidase in the adaptive reactions of cellular plant metabolism.
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