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Stabilization of beta-galactosidase from {\it Kluyveromyces marxianus\/} by histidine
Abstract
The objective of this research was to examine and investigate the stabilization of $\beta$-galactosidase by histidine. Of the four $\beta$-galactosidases tested, histidine stabilized the enzyme from Kluyveromyces marxianus more than the enzyme from Streptococcus thermophilus, Escherichia coli or Aspergillus niger.^ The enzyme from K. marxianus was purified to electrophoretic homogeneity on a non-denaturing PAGE (pH 8.0) and its kinetic stability determined at 45$\sp\circ$C. All the twenty amino acids (1 mM) tested, except proline, stabilized the enzyme. Histidine was the most effective stabilizer. It enhanced the half-life of the enzyme 58-fold in the presence of 5% lactose. Increasing the lactose concentration up to 15% increased histidine stabilization. Glucose and maltose did not affect the histidine stabilization while galactose and sucrose enhanced it. Histidine also stabilized the enzyme in the absence of sugars but to a lesser extent.^ The $\alpha$-amino group and the N-1 nitrogen on the imidazole ring of histidine were essential for histidine stabilization while the carboxylic group played a role in the extent of stabilization. Histidine stabilization decreased with increasing ionic strength. The energy of activation for inactivation of this enzyme in the temperature range of 45 to 51$\sp\circ$C was unaffected by histidine.^ Binding of histidine to the enzyme was not observed by equilibrium dialysis and gel filtration experiments. Further, the melting temperature (51.4$\sp\circ$C) of the enzyme as detected by differential scanning calorimetry was not affected by histidine. Under isothermal conditions a delay in unfolding of the enzyme in the presence of histidine was observed by absorbance spectroscopy. The delay was temperature dependent and was not detected at 47.5$\sp\circ$C. Histidine is probably acting in the inital stages of unfolding on a partially unfolded molecule but the nature of its action is not completely understood.^ Lactose alone, delayed the unfolding and increased the melting temperature but did not effectively enhance the half-life of the enzyme. The K$\sb{\rm m}$ for lactose and the apparent binding constant for magnesium were unaffected by histidine. Histidine increased the half-life of this enzyme by 44% in milk at 45$\sp\circ$C but was not effective in milk ultrafiltrate. ^
Subject Area
Agriculture, Food Science and Technology|Chemistry, Biochemistry
Recommended Citation
Sanjog Shankar Surve,
"Stabilization of beta-galactosidase from {\it Kluyveromyces marxianus\/} by histidine"
(January 1, 1993).
Electronic Doctoral Dissertations for UMass Amherst.
Paper AAI9408351.
http://scholarworks.umass.edu/dissertations/AAI9408351