Construction And Characterization Of Chimeras Derived From Thermophilic, Mesophilic And Psychrophilic Subtilases

Members of subtilases (subtilisin-like serine proteases) are widely distributed in archaea, bacteria, fungi, animals and plants. They not only play important roles in various physiological processes, such as protein turnover, activation, and metabolism, but also hold a great potential for application in the detergent and leather industries. Psychrophilic enzymes are characterized as cold-active but heat-laible, and thermophilic enzymes generally exhibit enhanced conformational rigidity, which contributes to the increased thermostability and decreased low-temperature activity of these enzymes. Systematic investigations on subtilases from microorganisms adapted to different temperatures will not only contribute to the understanding of temperature adaptation mechanisms of microorganisms, but also provide valuable theoretical basis for engineering novel biocatalysts.Thermophilic protease WF146 (from thermophilic Bacillus sp. WF146), mesophilic subtilisin Sphericase (Sph, from B. sphaericus) and psychrophilic subtilisin S41 (from Antarctica psychrotrophic Bacillus TA41) belong to subtilases. Although these three enzymes adapt to different temperatures, they share high similarities in amino acid composition, sequence (-70% identity), and molecular structure, thus making them ideal for the investigation of the temperature adaptation mechanisms and evolution strategies of enzymes. In this study, we performed systematic investigation of WF146 protease by substituting variable sites or regions (VRs) in the alignment of the trio of enzymes with those of S41 and Sph in an effort to gain insights into temperature adaptation and evolutionary strategies of subtilases.First, the genes encoding the WF146 protease, S41, and Sph, were subjected to random recombination by StEP PCR to construct a chimera library of the three enzymes. By using Escherichia coli BL21(DE3) as the host, the chimera library was screened on milk plate for chimeras with increased activity and stability according to the size of clear halo around the colonies. Sequencing of the target chimera genes revealed that some of the amino acid residues from psychrophilic enzymes could improve low-temperature activity but others led to a decrease in low-temperature activity of thermophilic enzymes, implying that structural elements of psychrophilic enzymes may not always endow thermophilic enzymes with "cold-adapted" features. On the other hand, some amino acid residues from thermophilic enzymes could improve high-temperature activity of mesophilic enzymes, while some others could improve low-temperature activity of them. These results demonstrate that psychrophilic enzymes possess stability-enhancing structural elements, and thermophilic enzymes possess low-temperature activity-enhancing structural elements.Subsequently, each of the VRs of thermophilic protease WF146 were substituted with the corresponding VRs of psychrophilic S41 and mesophilic Sph to construct chimera libraries WA (contains VRs from S41) and WB (contains VRs from Sph). All chimeras were produced in E. coli BL21(DE3) and were purified. The activities and thermostabilities of chimeras were analyzed and compared with wild-type WF146 protease (WT). Several S41-and Sph-derived structural elements that have significant effects on enzyme thermostability and activity were identified. In terms of activity-stability relationship, there is a "trade-off" between activity and stability in some chimeras. However, some other chimeras showed a trend of increase or decrease in both activity and thermostability, implying that there is no intrinsic correlation between stability and activity.Then, structural elements from S41 and Sph that significantly improved the thermostability and/or activity of WF146 protease variants were subjected to successive rounds of recombination in WF146 protease and selection, and a chimera (named PBL5X) containing 7 VRs from S41 was obtained. PBL5X displayed much longer half-lives at 80? (265 min) and 85? (76 min) than wild-type WF146 protease (WT; 60 and 6 min, respectively). The substitutions also led to an increase in the apparent Tm of the enzyme by 5.5?, as determined by differential scanning calorimetry analysis. Compared to WT, PBL5X exhibited higher caseinolytic activity (25-95?) and higher values of Km and kcat (25-80?). The results demonstrate that thermophilic subtilase could be further stabilized by incorporating structural elements from its psychrophilic or mesophilic counterparts, and the stabilization was accompanied by an increase in catalytic activity.Finally, models of the three-dimensional structures of PBL5X and WT were constructed based on the crystal structures of S41 and Sph. Structural analysis of PBL5X demonstrates that the accumulative effects of secondary structure stabilization, additional hydrogen bonds, and increased local hydrophobicity contributed to the enhancement in global structure stability. Notably, PBL5X "mimicked" the low-temperature adaptation strategy of psychrophilic S41, i.e., the improvement in activity was achieved by rigidifying catalytic triad residues and increasing the mobility of the substrate-binding region. These findings not only contribute to a better understanding of temperature adaptation mechanisms of enzymes in terms of structure-stability-activity relationship, but also may provide a rational basis for developing highly stable and active subtilases, which are highly desired in industrial application.