Source: Medical Research Scholar
Author: Xiaoyan, who loves conducting experiments
As a biological catalyst, the catalytic activity of enzymes depends on the integrity of their three-dimensional structure. The influence of temperature on enzymes is dual: high temperature accelerates the denaturation and inactivation of enzymes, while low temperature inhibits metabolic activities. Although extremely low temperatures (such as -80℃) are often used for enzyme preservation, they are not absolutely safe. The characteristics of the enzymes and the preservation requirements should be comprehensively considered.
1.1 Thermodynamic novelty of protein structure
Protein molecules maintain their conformation through non-covalent bonds such as hydrogen bonds and hydrophobic interactions. Although low temperatures can reduce the thermal motion of molecules, the formation of ice crystals at -80℃ will generate mechanical stress, destroying the hydration layer on the surface of enzyme molecules and causing distortion of the active center. Studies have shown that the volume change of ice crystals during repeated freeze-thaw cycles can cause a loss of enzyme activity of up to 30% to 50%. In the study of soil enzyme activities in different forest types of Changbai Mountain, it was found that the freeze-thaw process significantly affected soil enzyme activities. The activities of oxidase and reductase showed a trend of first increasing and then decreasing, which further demonstrated the complex influence of temperature changes on enzyme activities.
1.2 Stability differences in the time dimension
For short-term storage (within several weeks), the effect difference between -20℃ and -80℃ is relatively small. Long-term storage (several months to several years) at -80℃ can significantly delay chemical degradation and microbial contamination. It should be noted that the stability of freeze-dried preparations at -20℃ is usually better than that of liquid enzymes, as the dehydrated state can reduce ice crystal damage. For instance, in the production of pharmaceuticals, the freeze-drying process involves freezing liquid drugs and evaporating the moisture in a vacuum environment to transform them into solid powder. This avoids chemical reactions and microbial contamination at room temperature, thereby enhancing the stability of the drugs and extending their shelf life.
Not all enzymes need to be stored at ultra-low temperatures. Reverse transcriptase, due to the presence of easily oxidized thiol groups, is prone to oxidative inactivation at higher temperatures. The quaternary structure of restriction endonucleases makes them sensitive to conformational changes at room temperature, and subunit dissociation needs to be inhibited at -80℃. In contrast, hydrolases such as amylase and protease, due to their simple structure, can maintain stability at -20℃ and do not overly rely on ultra-low temperatures.
Ice crystal formation is inevitable during the freezing process, but temperature directly affects the size of ice crystals. Research shows that the size of ice crystals at -20℃ is 3 to 5 times that at -80℃, and large ice crystals will cut the three-dimensional structure of enzyme molecules during growth. The influence of freeze-thaw cycles on soil enzyme activities further verified the destructive nature of temperature fluctuations: the activity of oxidoreductase showed a trend of first increasing and then decreasing. To alleviate ice crystal damage, 10% to 50% glycerol is usually added as a protective agent to lower the freezing point. However, it should be noted that high concentrations of glycerol may interfere with the microenvironment of the active center.
The purchase cost of a -80℃ refrigerator is 3 to 5 times that of a -20℃ one, and its energy consumption increases significantly. Frequent access can cause a temperature fluctuation of ±5℃ inside the box, which may counteract the ultra-low temperature protection effect. It is recommended to adopt a small-volume portioning strategy (single use ≤10μL), reduce the number of accesses to minimize the impact of temperature fluctuations, and at the same time achieve a balance between cost and activity by combining freeze-drying processes or glycerol protectants.
Short-term preservation (<1 monthRefrigeration at 4℃ is a suitable choice. Adding a protective agent can further enhance stability.
BSA（0.1-1 mg/mL）
Trehalose (5-10%
Mid-term preservation (1-6 months)Freezing at -20℃ is a common method. It should be aliquoted into small portions to avoid repeated freezing and thawing.
Long-term storage (>6 monthsFreezing or freeze-drying at -80℃ is the ideal method.
Freeze-drying treatment
Resolubility optimization
Tris-HCl（pH7.5-8.0）与HEPES
EDTA（1-5 mM）
DTT（0.1-1 mM）
Vitrification freezing (liquid nitrogen rapid freezing)
Spray drying method
4.1 Taq DNA Polymerase
Taq DNA polymerase is a key enzyme in PCR technology and is widely used in molecular biology research. Commercial preparations usually add 50% glycerin and BSA as protective agents and can be stably stored for more than one year at -20℃. However, experiments have shown that when stored at -80℃, the mechanical stress generated by glycerol crystallization can disrupt the spatial conformation of the enzyme, leading to a decrease in activity. For instance, in the experiment comparing the activity of Taq DNA polymerase at different temperatures, the enzyme with 50% glycerol and BSA added had lower amplification efficiency and product quality after being stored at -80℃ than the enzyme stored at -20℃, indicating that -80℃ had a negative impact on its activity.
HRP is widely used in clinical testing and immunoassay kits, and its stability is crucial to the quality of the kits. The activity loss of freeze-dried preparations after being stored at -20℃ for 3 years is less than 10%, demonstrating good stability. However, liquid HRP will show significant aggregation after being stored at -80℃ for 6 months, resulting in the masking of active centers and a decline in activity. In the immunoassay experiment, when liquid HRP was stored at -80℃ for 6 months, the detection signal was significantly weakened and the accuracy of the results was affected, indicating that -80℃ is not suitable for the long-term storage of liquid HRP.
When storing alkaline phosphatase derived from Escherichia coli at -80℃, 10% DMSO should be added as a protective agent; otherwise, its oligomer structure is prone to dissociation, leading to loss of activity. The alkaline phosphatase derived from the intestinal mucosa of calves contains natural antifreeze proteins and can be stably stored at -20℃. For instance, in the research on the preservation conditions of alkaline phosphatases from different sources, the enzyme derived from Escherichia coli with 10% DMSO added had stable activity at -80℃, while the enzyme without DMSO added had significantly decreased activity. The enzymes derived from the intestinal mucosa of calves have stable activity at -20℃, reflecting the differentiated requirements of enzymes from different sources for preservation conditions and protective agents.
Establish a classification management system based on the source, structural characteristics and temperature sensitivity of enzymes. For temperature-sensitive enzymes (such as reverse transcriptases and restriction endonucleases), a dedicated temperature-sensitive grade file should be established, recording storage conditions, storage periods and revalidation cycles. Enzymes with a high temperature sensitivity level usually have a shorter shelf life, and the revalidation cycle also needs to be shortened. For instance, the shelf life of certain special restriction endonucleases may only be six months, and their activity needs to be revalidated every three months.
Install an intelligent temperature and humidity recorder in the enzyme preservation refrigerator to monitor the temperature and humidity in real time. Set the temperature alarm threshold (±2℃). When the temperature fluctuation exceeds the range, immediately trigger the sound and light alarm to remind the laboratory personnel to take measures. Regularly calibrate the temperature and humidity recorder to ensure the accuracy of the data, and upload the data to the cloud storage for subsequent analysis and traceability. For instance, large-scale research institutions use Internet of Things (iot) technology to centrally upload the data of refrigerators to cloud servers. Researchers can view the real-time operation status of the refrigerators through mobile phone apps.
When taking enzymes, the exposure time at room temperature should be minimized as much as possible. Thaw in an ice water bath (0-4℃), and avoid thawing directly at room temperature. For enzymes with smaller dosages, they can be aliquoted into small single-use portions (such as 10μL) to reduce the number of repeated freeze-thaw cycles. When operating, it is necessary to ensure the accuracy of the pipette to avoid sample addition errors affecting the experimental results. For instance, in PCR experiments, Taq DNA polymerase is aliquot into 10μL portions, and only one portion needs to be taken out each time it is used, reducing waste and loss of activity.
Regularly conduct activity tests on the preserved enzymes and establish quality control standards for enzyme activity. A certain number of enzyme samples are randomly selected for activity testing every quarter. For batches with activity deviations exceeding 15%, further analysis and evaluation are required. If it is confirmed that the activity has decreased, the enzyme of this batch should be eliminated in time to avoid affecting the experimental results. At the same time, strict quality inspections should be conducted on newly purchased enzymes to ensure that their activities meet the experimental requirements.
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