Effects of pH on Activity, Thermostability, and Aggregation of Glucose Isomerase from S.murinus A.K. Andersen S.B.Petersen* H.Frisner *Corresponding author Keywords: Glucose isomerase, melting temperature, aggregation
Glucose isomerase (GI) from S.murinus is a homotetrameric enzyme, which catalyses the reversible isomerisation of glucose into fructose. For industrial application of GI, high temperatures combined with acidic pH are desired. The aim of the present study was to investigate effects of pH on activity, thermostability, and aggregation of GI provided by Novo Nordisk A/S. Protein concentration in the activity measurements varied from 0.01µM to 0.03µM GI, 2mM MgSO4 was added. Thermostability and aggregation was investigated at 1.5µM GI, with 2mM MgSO4. At isomerisation temperatures at 45°C and 60°C, activity was investigated in the pH range 5.0 to 9.0. From being inactive at pH 5.0, activity increased at both investigated temperatures up to pH 8.0 at 45°C and pH 8.5 - 9.0 at 60°C. Thermostability was investigated in the same pH range by circular dichroism; temperature was scanned from 5°C to 100°C with a scan rate at 1.5°C/min, response was measured at 223nm. After differentiation of thermal scan curves, Tm appeared as peak tops. Tm increased from 72.4°C at pH 5.0 to 76.6°C at pH 6.0; then a pronounced increase took place between pH 6.0 and pH 7.0. At pH 7.0, Tm had reached a level at 90°C-91°C, which remained constant up to pH 9.0. Aggregation was investigated in the temperature range from 5°C to 80°C by dynamic light scattering (DLS). The GI has an isolelectric point of 4.7, and it was found that temperature induced heavy aggregation at pH 5.0 above 30°C. At pH 7.0, tendencies of aggregation were observed above 60°C. No aggregation was observed at pH 9.0 in the temperature range investigated. Thus, the larger surface charge, the minor aggregation. When pH was increased from 5.0 to 9.0, activity and thermostability increased, whereas aggregation decreased. From literature and the present experiments, explanation of low activity at acidic pH values might be a complex issue. |
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