Abstract
Objective
To evaluate an analog library of betaine-type cellular metabolites, which are naturally found in polar fish for survival in subzero temperatures, for preventing denaturation of enzymes during freezing.
Results
Comparison of the cryoprotective ability of reported cryoprotectants, such as dimethylsulfoxide, glycerol, ectoine, hydroxyectoine, and trehalose, with betaine-type analogs using α-glucosidase revealed that analogs introducing C3–C6 alkyl chains into an ammonium cation retained 20 % higher activity than the control cryoprotectants at the same concentration. In particular, the analog possessing triplicate n-butyl chains showed a profound effect. It allowed retention of enzyme activity to 95 % even after 100 freeze–thaw cycles, while addition of the control cryoprotectants decreased the activity to 10–20 %. The cryoprotective ability of betaine-type analogs can be applied not only to α-glucosidase but also other enzymes such as β-glucosidase, alkaline phosphatase, lactose dehydrogenase, sulfatase, and horseradish peroxidase.
Conclusion
Synthetic betaine-type metabolite analogs possess practicable cryoprotective ability for various enzymes, and are considerably superior to previously reported cryoprotectants.
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Supporting information
Supplementary Fig. 1—Plot of remaining activity of α-GLU in the enzyme stock solution as a function of freeze–thaw cycles. Cryopreservation conditions: [α-GLU] = 25 µg/ml, [phosphate buffer (pH 7.0)] = 100 mM, freeze–thaw cycles (fast freezing) = 0–50 times. Error bars represent the standard deviation of five measurements.
Supplementary Fig. 2—Plots of remaining activities of α-GLU in the stock solution as a function of concentration of analogs 7–14 after 12 freeze–thaw cycles. Cryopreservation conditions: [α-GLU] = 25 µg/ml, [phosphate buffer (pH 7.0)] = 100 mM, [analogs] = 0–1000 mM, freeze–thaw cycles (fast freezing) = 12 times. Error bars represent the standard deviation of five measurements.
Supplementary Fig. 3—Plots of remaining activities of LDH, ALP, HRP, β-GLU, and SUL in the enzyme stock solutions as a function of freeze–thaw cycles (fast freezing) (0–50 times). Cryopreservation conditions: α-GLU: [α-GLU] = 25 µg/ml, [phosphate buffer (pH 7.0)] = 100 mM, [analog 4] = 300 mM, β-GLU: [β-GLU] = 50 µg/ml, [phosphate buffer (pH7.0)] = 100 mM, [analog 4] = 300 mM, ALP: [ALP] = 50 µg/ml, [Tris buffer (pH 7.5)] = 100 mM, [analog 4] = 300 mM, SUL: [SUL] = 625 µg/ml, [phosphate buffer (pH 7.0)] = 100 mM, [analog 4] = 300 mM, LDH: [LDH] = 50 µg/ml, [Tris buffer (pH 7.5)] = 80 mM, [analog 4] = 300 mM, HRP: [HRP] = 1.0 µg/ml, [citric acid buffer (pH 4.2)] = 10 mM, [analog 4] = 300 mM. Error bars represent the standard deviation of five measurements.
Supplementary Fig. 4—Comparison of circular dichroisim (CD) spectra of α-GLU in the (a) absence and (b) presence of analog 4 before (solid line) and after 12 freeze–thaw cycles (broken line). Conditions: [α-GLU] = 0.5 mg/ml, [phosphate buffer (pH 7.0)] = 100 mM, [analog 4] = 0 or 300 mM at 30°C, freeze–thaw cycles (fast freezing) = 0 (solid line) or 12 times (broken line).
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Nakagawa, Y., Sota, M. & Koumoto, K. Cryoprotective ability of betaine-type metabolite analogs during freezing denaturation of enzymes. Biotechnol Lett 37, 1607–1613 (2015). https://doi.org/10.1007/s10529-015-1841-1
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DOI: https://doi.org/10.1007/s10529-015-1841-1