Computer-assisted semi-rational design enhanced the enzymatic activity and protein stability of Proteinase K in calcium-free conditions.

Wild-type Proteinase K binds to two Ca2+ ions, which play an important role in regulating enzymaticactivity and maintaining protein stability. Therefore, a predetermined concentration of Ca2+ must be added during the use of Proteinase K, which increases its commercial cost. Herein, we addressed this challenge using a computational strategy to engineer a Proteinase K mutant that does not require Ca2+ and exhibits high enzymatic activity and protein stability. In the absence of Ca2+, the best mutant, MT24 (S17W-S176N-D260F), displayed an activity approximately 9.2-fold higher than that of wild-type Proteinase K. It also exhibited excellent protein stability, retaining 56.2 % of its enzymatic activity after storage at 4 °C for 5 days. The residual enzymatic activity was 65-fold higher than that of the wild-type Proteinase K under the same storage conditions. Structural analysis and molecular dynamics simulations suggest that the introduction of new hydrogen bond and π-π stacking at the Ca2+ binding sites due to the mutation may be the reasons for the increased enzymatic activity and stability of MT24.

Hygroscopic growth and CCN activity of secondary organic aerosol produced from dark ozonolysis of γ-terpinene.

The hygroscopic growth factor (GF) and cloud condensation nuclei (CCN) activity of secondary organic aerosol (SOA) particles produced during dark ozonolysis of γ-terpinene under different reaction conditions were investigated. The SOA particles were produced in the presence or absence of cyclohexane, an OH scavenger; 1,3,5-trimethylbenzene, an anthropogenic volatile organic compound; and (NH4)2SO4 seed particles. A hygroscopicity tandem differential mobility analyzer was used to determine the GFs of the SOA particles at RHs ≤ 93%. For some experiments, a CCN counter was used for size-resolved measurement of CCN activation at supersaturation (S) in the range of 0.1 to 1%. The single hygroscopicity parameter κ was derived from both the GF and CCN measurements. Under subsaturated conditions, all the SOA (except those in the presence of the (NH4)2SO4 seeds) showed small GF values. These GFs demonstrated that SOA mass loading affected the GF. A decrease in the SOA mass loading led to increased GF and corresponding κGFvalues. However, in a supersaturation regime, the SOA mass loading and the size of the particles did not significantly alter the CCN activity of the SOA. Our CCN measurements showed higher κCCN values (κCCN = 0.20-0.24) than those observed in most monoterpene ozonolysis studies (κCCN = 0.1-0.14). This difference may have been due to the presence of the two endocyclic double bonds in the γ-terpinene structure, which may have affected the SOA chemical composition, in contrast to monoterpenes that contain an exocyclic double bond. Our comparisons of sub- and supersaturated conditions showed a larger range of κ values than other experiments. Average κCCN/κGF ratios of ~7 and 14 were obtained in the unseeded SOA experiments at low and high SOA mass loadings, respectively. The average κCCN of 0.23 indicated that the SOA produced during ozonolysis of γ-terpinene exhibited fairly high CCN activity.