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The Use of Disaccharides in Inhibiting Enzymatic Activity Loss and Secondary Structure Changes in Freeze-Dried β-Galactosidase during Storage

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ABSTRACT

Purpose

The purpose of this study is to show how disaccharides differ in their ability to protect lyophilized β-galactosidase from enzymatic activity loss and secondary structure changes during storage.

Methods

β-galactosidase was lyophilized with trehalose, sucrose, cellobiose or melibiose at 2:1, 20:1 and 40:1 excipient/protein weight ratios, and stored up to 90 days at 45°C. Protein enzymatic activity was studied using o-nitrophenyl-β-d-galactopyranoside cleavage test, and its secondary structure in lyophilizates analyzed using Fourier transform infrared spectroscopy. The crystallization tendencies, glass transition temperatures and water contents of lyophilizates were evaluated using x-ray powder diffractometry, differential scanning calorimetry and thermogravimetry, respectively.

Results

The enzymatic activity of β-galactosidase decreased more slowly in lyophilizates containing trehalose or melibiose at 2:1 excipient/protein weight ratio when compared to those containing sucrose or cellobiose. Similar behavior was observed when analyzing the protein’s secondary structure in lyophilizates. In 20:1 and 40:1 excipient/protein weight ratio lyophilizates the decrease of enzymatic activity was less dependent on the excipient, but activity was always amongst the highest in melibiose lyophilizates.

Conclusions

Melibiose was shown to be effective in protecting lyophilized β-galactosidase during storage. The protein secondary structure was shown to change at comparable rate in lyophilizates as its enzymatic activity after rehydration.

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ACKNOWLEDGMENTS

The authors wish to acknowledge Vetcare Oy for providing material funding and Amano Enzymes Inc. for donating a sample of the model protein to be used in the study.

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Authors and Affiliations

  1. Faculty of Pharmacy, Industrial Pharmacy, University of Helsinki, Helsinki, Finland

    Ville Petteri Heljo & Anne M. Juppo

  2. Department of Food Technology, Faculty of Agriculture and Forestry, University of Helsinki, Helsinki, Finland

    Kirsi Jouppila

  3. Department of Chemistry, Laboratory of Inorganic Chemistry, University of Helsinki, Helsinki, Finland

    Timo Hatanpää

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  1. Ville Petteri Heljo
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  2. Kirsi Jouppila
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Correspondence to Ville Petteri Heljo.

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Heljo, V.P., Jouppila, K., Hatanpää, T. et al. The Use of Disaccharides in Inhibiting Enzymatic Activity Loss and Secondary Structure Changes in Freeze-Dried β-Galactosidase during Storage. Pharm Res 28, 540–552 (2011). https://doi.org/10.1007/s11095-010-0300-x

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  • DOI: https://doi.org/10.1007/s11095-010-0300-x

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