For normal growth adaptation to life under low-temperature conditions, cold-adapted microorganisms have evolved a set of gene expression sequences and special molecular mechanisms specific to low-temperature environments, in which the high expression of low-temperature active enzymes plays a key role during the process of cold adaptation. In recent decades, research on cold-adapted proteases has initially focused on exploring enzymatic characteristics, including the optimal temperature, pH, and Effects of metal ions (e.g., Hg²⁺, Cd²⁺, Co²⁺, Mn²⁺, Ca²⁺, K⁺, Na⁺, Fe²⁺, etc.) and compounds (such as SDS, PMSF, EDTA, LAS, etc.) on enzyme activity. Their optimal catalytic temperature is 10–60 °C, whilst their optimal pH is usually in the neutral to alkaline pH range of 7–10 [1]. To date, thermostable alkaline proteases primarily originate from Bacillus subtilis and Bacillus licheniformis, whilst proteases from Geomyces panorum, and yeast Candida humicola are rare acid thermostable proteases with an optimal pH of 3.0 [1,56,60]. Presently, the reported molecular weight of cold-adapted proteases is small, typically in the range of 25–60 kDa; only a few cold-adapted proteases have large molecular weights [39,50,51,57]. Organic reagents such as EDTA, PMSF, and DFP often inhibit the activity of cold-adapted protease [41,42,43], as do metal ions Hg²⁺, Cu²⁺ and Co²⁺ [40,66]. Metal ions, such as Mg²⁺, Mn²⁺, and Ca²⁺ can improve the activity of cold-adapted proteases to varying degrees [46,58].

The structure of cold-adapted proteases can be analysed from their molecular (amino acid composition and sequence, terminal composition, disulphide bond position, etc.) and three-dimensional structure. Nuclear magnetic resonance spectroscopy, site-directed mutagenesis, X-ray crystal diffraction, and freeze electron microscopy are effective methods that can be used to understand the crystal structure. Additionally, the high structural homology of enzymes at different temperatures provides an opportunity to study the structural characteristics of their adaptation to different temperatures. Through comparative analysis of the structural characteristics of cold-adapted, mesophilic, and thermophilic proteases (Figure 1), as well as previous studies, we can understand how proteases become adapted to low temperatures. These groups comprise protein surfaces that determine the important interactions between proteins and water, which is expected to significantly impact protein function in adapting to high and low temperatures. In many thermophilic proteins, a larger proportion of non-polar surfaces is believed to help increase stability [67,68]. The proportion of serine residues in cold-adapted enzyme molecules of cold-adapted microorganisms is high, while the proportion of proline and acid residues is low. Owing to the high number of polar amino acid residues, these types of enzymes have a more hydrophilic interaction with solvents [49,69]. Therefore, the active sites of these enzymes are usually more accessible to compensate for substrate diffusion at lower temperatures. The difference between cold-adapted Vibrio proteases and more stable proteases is their strong anionic properties due to the numerous uncompensated negatively-charged residues on their surface. In some studies, the difference in surface charge distribution or the increase in the non-polar surface area has been considered to enable adaptation to low temperatures [70,71,72]. On the other hand, the weakness of non-covalent interactions (such as salt bridges, hydrophobic interactions, aromatic interactions, main chains, side chains, and side chain hydrogen bonds) in some special positions can increase the core volume of enzyme molecules and make the whole molecule looser [46], improving the conformational flexibility of proteases [37]. However, the relationship between the number of non-covalent bonds and the temperature adaptation of enzymes cannot be determined at present. For example, the number of salt bridges in cold-adapted protease 1SH7, thermophilic protease 1IC6, and thermophilic protease 1THM was compared [73]. The number of salt bridges in 1SH7 and 1IC6 was the same and was only two less than that in thermophilic protease 1THM. Although there have been many reported instances of salt bridges and hydrogen bonds introduced in the modification of thermal stability enhancement of many enzymes, an important aspect of their contribution to protein stability lies in their location and distribution [73]. However, this increase in flexibility is often accompanied by decreased stability. Cold-adapted enzymes generally show low thermal stability, short half-life, and high sensitivity to low pH [74]. In contrast, thermostable enzymes have a more rigid and compact conformation, which is more likely to protect them from instability at higher temperatures [75].

From a kinetic point of view, a very common feature of all low-temperature active enzymes is the reduction of activation enthalpy, accompanied by negative entropy, which reduces the exponential decline of the chemical reaction rate resulting from the reduction in temperature. It has been reported that the surface flexibility of low-temperature active enzymes is the source of cold adaptability [76]. For example, Isaksen discussed the role of protein surface mobility in determining the enthalpy centre equilibrium and subsequently proved that the surface of the enzyme is rigid through a single remote mutation of the key residue of protein water surface interaction. Therefore, cold-denatured trypsin can be transformed into a variant with medium-temperature characteristics without changing the amino acid sequence [77]. It has been suggested that the rigidity of the surface of low-temperature active enzymes may produce a higher activation enthalpy of the catalytic reaction with fewer negative entropy components.