Efficacy of sugar alcohols and sugars in protein stabilization during freezing, freeze-drying, and air-drying

Wendell Q. Sun; Yongqi Luo

doi:10.1515/fzm-2025-0007

Efficacy of sugar alcohols and sugars in protein stabilization during freezing, freeze-drying, and air-drying

doi: 10.1515/fzm-2025-0007

Wendell Q. Sun^,,
Yongqi Luo

Institute of Biothermal Science and Technology, School of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai 200093, China

Funds:

a research grant from the National University of Singapore to WQS RP-3960366

a collaborative research grant from Sichuan Zhongke Organ Co. Ltd (Chengdu, China)

More Information

Corresponding author: Wendell Q. Sun, E-mail: wendell.q.sun@gmail.com

摘要

Abstract: Objectives Cold-acclimated organisms accumulate low molecular weight organic solutes such as sugar alcohols and soluble sugars. This study aimed to compare the efficacy of five sugar alcohols and 14 soluble sugars in stabilizing proteins under freezing, freeze-drying, and air-drying stresses. Materials and methods Glucose-6-Phosphate Dehydrogenase (G6PD) was used as the model protein. G6PD solutions with or without sugar alcohols and or sugars were subjected to freezing, freeze-drying, and air-drying stresses. The recovery of G6PD activity was measured to evaluate the protective efficacy of these compounds. Results Without stabilizers, freezing G6PD at -20℃ or -80℃ reduced enzyme activity by around 24%, while freeze-drying or air-drying reduced activity by 90%-95%. Among the five sugar alcohols tested, pinitol, quebrachitol and sorbitol stabilized G6PD, whereas mannitol and myo-inositol destabilized it. Among 14 soluble sugars, trehalose and raffinose showed slightly lower enzyme recovery after repeated freeze-thaw cycles at -20℃. Most soluble sugars (except arabinose and xylose) protected G6PD during freeze-drying, with di-, tri-, and oligosaccharides generally outperforming monosaccharides. During air-drying, lactose was ineffective, while arabinose, galactose, and xylose were detrimental. Conclusion The study highlights the diverse mechanisms of sugar alcohols and sugars in protein stabilization under stress, offering insights for formulating stable protein- and cell-based drugs.
- desiccation tolerance /
- freezing tolerance /
- protein stabilization /
- sugar alcohols /
- sugars

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Figure 1. Chemical structures of five sugar alcohols that used in the protein stabilization study

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Figure 2. Recovered activity of glucose-6-phosphate dehydrogenase (in 50 mmol/L Tris-HCl buffer, pH 7.5) after five freeze/thaw cycles at -20℃ or one freeze/thaw cycle at -80℃ in the presence of sugar alcohols and sugars

The recovery of enzyme activity was 76% in the absence of a protectant (the dashed line). Data are means SE of at least six replicate.

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Figure 3. Recovered activity of glucose-6-phosphate dehydrogenase (in 50 mmol/L Tris-HCl buffer, pH 7.5) after five freeze/thaw cycles at -20℃ or one freeze/thaw cycle at -80℃ in the presence of sugar alcohols and sugars

In the absence of a protectant, the recovery of enzyme activity was 4% and 10% after freeze-drying and air-drying respectively. Data are means SE of at least six replicates.

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Table 1. The adaptive accumulation of sugar alcohols in stress-tolerant organisms in response to freezing (< 0℃), chilling (> 0℃), salinity and desiccation

Sugar alcohol	Stress Factor
	Freezing	Chilling	Salinity	Desiccation
Mannitol	ns	+	+ + +	+ + +
Sorbitol	+ + +	+ + +	+ + +	+ + +
Myo-Inositol	+	+ +	+ +	+ +
Ononitol	ns	ns	+ + +	+ + +
Quebrachitol	ns	ns	ns	ns
Pinitol	+ +	+ +	+ + +	+ + +
+: The adaptive accumulation of such a solute was reported in some organisms; + +: The adaptive accumulation of such a solute is observed in a number of cases; + + +: The adaptive accumulation of such a solute has been widely observed; ns: No data was reported in the subject.

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