Stabilization of beta-galactosidase from Kluyveromyces marxianus by histidine

1993 1993

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Abstract (summary)

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.

Indexing (details)

Food science;
0359: Food science
0487: Biochemistry
Identifier / keyword
Pure sciences, Biological sciences, galactosidase
Stabilization of beta-galactosidase from Kluyveromyces marxianus by histidine
Surve, Sanjog Shankar
Number of pages
Publication year
Degree date
School code
DAI-B 54/10, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
Mahoney, Raymond R.
University of Massachusetts Amherst
University location
United States -- Massachusetts
Source type
Dissertations & Theses
Document type
Dissertation/thesis number
ProQuest document ID
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
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