# Cold-Adapted Fungi: Goldmine of Biomolecules Applicable in Industry
## 4 Biotechnological Application of Psychrophilic Fungi in Food and Agriculture
### Cold-Active Enzymes  
In 2021, the global enzyme market was valued at approximately $12.5 billion, with the food industry generating approximately $3.7 billion in revenue. It is estimated that this value will reach $5.8 billion in 2032, representing an approximate expansion of 6.8% for the total enzyme market. The expansion of technology in research and development, the dissemination of metagenomics, and the potential for genetically modified enzymes have collectively contributed to the growth of the enzyme market [4,50]. Furthermore, enzymatic reactions are safer, faster, and generate less waste, thus meeting the 12 principles of green chemistry [45]. In this regard, psychrophilic enzymes are of particular significance, exhibiting distinctive characteristics that make them applicable in a diverse array of industrial contexts (Figure 2) [45,50]. The cold-adapted enzymes are notable for their high turnover rates and intrinsic catalytic efficiency at low temperatures. Their flexible structures result in lower activation enthalpy and negative entropy of activation when compared to enzymes from mesophilic and thermophilic organisms. The increased flexibility of these enzymes is considered a key molecular mechanism in their evolution, involving several features: (i) reduced core hydrophobicity, increased surface hydrophobicity, a lower arginine to lysine ratio, weaker interactions between domains and subunits, and the presence of more and longer loops; (ii) a dominance of alpha-helices in their secondary structure; (iii) a reduction in secondary structure content, fewer and weaker metal-binding sites, and a decrease in the number of disulfide bridges and electrostatic interactions; and (iv) reduced oligomerization and increased non-conformational entropy in the unfolded state [51]. The incorporation of unique cold-active enzymes into processes offers a variety of advantages, including reduced emissions of environmentally harmful substances; the use of lower concentrations of psychrozymes compared to mesophilic or thermophilic counter  
Figure 1: Physiological adaptation of psychrophilic and psychrotrophic fungi. parts for given reaction conditions; running processes at tap water or ambient temperature, thus avoiding the costly heating step and significantly reducing costs; reduction in microbial contamination; minimization of side reactions and generation of by-products; maintenance of product functionality, nutritional value, and taste at low process temperatures; and efficient and selective post-process inactivation through a minimal increase in temperature, obviating the necessity for chemical agents. These unique features make cold-active enzymes indispensable for the food and feed industry. It is essential to avoid spoilage, flavor changes, and any loss of nutritional value in thermolabile substrates and products. Cold-active enzymes could be used in food applications such as meat tenderization, baking, brewing, flavoring, cheese production, and animal feed processing (Figure 3). Figure 3: Cold-active enzymes and their biotechnological application in the food and feed industry. Figure 2: Basic features of enzymes from psychrophilic microorganisms. Some of the cold-active enzymes produced by fungi are now used on a commercial scale in the food industry, such as Novoshape(r) (_Aspergillus oryzae_, pectin methyl esterase), Pectinase 62L (_Aspergillus sp._, polygalacturonase and pectin lyase), and Lallzyme(r) (_Aspergillus niger_, a mixture of polygalacturonase, pectin esterase, and pectin lyase). However, even though most of these enzymes can react at low temperatures, they are not derived from psychrophilic producers [50; 52]. It is important to acknowledge that the number of commercially available psychrozymes remains relatively limited. It is estimated that the global market for cold-active enzymes represents approximately 30-40% of the total enzyme market [50]. However, market analysis indicates a consistent and growing demand for novel and distinctive psychrozymes.