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Stabilizing Effect of Four Types of Disaccharide on the Enzymatic Activity of Freeze-dried Lactate Dehydrogenase: Step by Step Evaluation from Freezing to Storage

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Abstract

Purpose

In order to understand the stabilizing effects of disaccharides on freeze-dried proteins, the enzymatic activity of lactate dehydrogenase (LDH) formulations containing four types of disaccharide (trehalose, sucrose, maltose, and lactose) at two relative humidity (RH) levels (about 0 and 32.8%) was investigated after three processes: freeze-thawing, freeze-drying, and storage at three temperatures (20, 40, and 60°C) above and/or below the glass transition temperature (T g).

Materials and Methods

The enzymatic activity was determined from the absorbance at 340 nm, and T g of the samples was investigated by differential scanning calorimetry.

Results

At each RH condition, T g values of sucrose formulations were lower than those of other formulations. Although effects of the disaccharides on the process stability of LDH were comparable, storage stability was dependent on the type of disaccharide. All the formulations were destabilized significantly during storage at temperature above T g. During storage at temperature below T g, the LDH activity decreased with increases in the storage temperature and moisture. Maltose and lactose formulations showed significant destabilization with the change of color to browning.

Conclusions

Taking the storage stability of freeze-dried proteins under the various conditions (temperature and RH) into consideration, trehalose is better suited as the stabilizer than other disaccharides.

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Abbreviations

DSC:

differential scanning calorimetry

LDH:

lactate dehydrogenase

PFK:

phosphofructokinase

RH:

relative humidity (%)

T g :

glass transition temperature (°C)

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Acknowledgements

The authors gratefully acknowledge the financial support from a Grant-in-Aid for JSPS Fellows provided by The Ministry of Education, Culture, Sports, Science and Technology. The authors would also like to thank Drs. Rikuo Takai and Tomoaki Hagiwara of the Tokyo University of Marine Science and Technology for their valuable discussions, Drs. Munehiko Tanaka and Shoichiro Ishizaki of the Tokyo University of Marine Science and Technology for the use of UV-spectrometer, and Dr. Hiroto Chaen of Hayashibara Biochemical Lab. Inc., Japan, for providing the trehalose dihydrate reagent.

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Authors and Affiliations

  1. Tokyo University of Technology Institution, School of Bionics, 1404-1 Katakura, Hachioji, Tokyo, 192-0982, Japan

    Kiyoshi Kawai

  2. Department of Food Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo, 108-8477, Japan

    Toru Suzuki

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  1. Kiyoshi Kawai
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Correspondence to Kiyoshi Kawai.

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Kawai, K., Suzuki, T. Stabilizing Effect of Four Types of Disaccharide on the Enzymatic Activity of Freeze-dried Lactate Dehydrogenase: Step by Step Evaluation from Freezing to Storage. Pharm Res 24, 1883–1890 (2007). https://doi.org/10.1007/s11095-007-9312-6

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  • DOI: https://doi.org/10.1007/s11095-007-9312-6

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