Applications of cold-adapted proteases in the food industry

Structure-Guided Engineering of a Protease to Improve Its Activity under Cold Conditions

“…To study the effect of temperature on the dynamics of the enzyme, MD simulations of the WT-AAPF complex and the MT6-AAPF complex were performed and explored by means of root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), radius of gyration ( R g ), and solvent accessible surface area (SASA) values. During the whole process of MD simulation, the RMSD values of MT6-AAPF were greater than that of WT-AAPF, suggesting the improved flexibility of the MT6 structure at 10 °C (Figure A), which was similar to the psychrophilic serine hydroxymethyltransferase from Psychromonas ingrahamii, presenting higher RMSD values than that of the mesophilic counterpart from Escherichia coli . In addition, MT6-AAPF had higher values of SASA and R g than WT-AAPF throughout the MD simulation, implying a larger size of the structure and a less compact packing of MT6-AAPF in comparison to the WT-AAPF structure, thus enhancing the MT6’s structural flexibility (Figure B,C).…”

“…To study the effect of temperature on the dynamics of the enzyme, MD simulations of the WT-AAPF whole process of MD simulation, the RMSD values of MT6-AAPF were greater than that of WT-AAPF, suggesting the improved flexibility of the MT6 structure at 10 °C (Figure 2A), which was similar to the psychrophilic serine hydroxymethyltransferase from Psychromonas ingrahamii, presenting higher RMSD values than that of the mesophilic counterpart from Escherichia coli. 10 In addition, MT6-AAPF had higher values of SASA and R g than WT-AAPF throughout the MD simulation, implying a larger size of the structure and a less compact packing of MT6-AAPF in comparison to the WT-AAPF structure, thus enhancing the MT6's structural flexibility (Figure 2B,C). All these data verified that MT6 derived from the multimutation of six residues had significant beneficial effects on the overall structural flexibility in comparison to WT.…”

“…Typically, the proteases derived from Bacillus species generally display higher catalytic efficiency between 40 and 60 °C, but their activities drop sharply at low temperatures . Compared with the mesophilic (40–60 °C) and thermophilic (>60 °C) counterparts, the cold-adapted proteases demonstrate optimum activities between 0 and 40 °C and can keep a constant catalytic efficiency under such low-temperature conditions, which is beneficial to usage in processing fresh foods that require the protein digestion at low temperatures, for example, cheese-making, beer, and beverage clarification . In general, it is suggested that one of the determinant factors for cold adaptation is the increased flexibility of cold-adapted proteases by destabilizing their structures, either globally or locally, by decreasing the stabilizing interactions of different classes. , This flexibility is also associated with the observed reduced structural thermostability of cold-active proteases. , …”