| WF146 protease is a thermostable subtilase from thermophilic Bacillus sp. WF146. This enzyme shares high degree of amino acid sequence identity (above 60%) with two mesphilic subtilases (SSII and sphericase) and two psychrophilic subtilases (S41 and S39). The high identity between thermophilic WF146 protease and its psychrophilic and mesophilic homologs provides us an opportunity to compare molecular mechanisms of subtilases adapted to different temperatures, and to improve our understanding of structure-stability-activity relationship in enzymes. Previously, we obtained several cold-adapted variants of WF146 protease using directed evolution method. The varint R29 exhibited a～3-fold increase in caseinolytic activity at 25℃, but its thermostability decreased significantly.In this study, the mutation T7I in R29 was reverted back to Thr, resulting in a one-fold increase in half-life at 80℃, and no decrease in low-temperature activity was observed in the generated variant RT29. After homolog alignment and structure analysis, mutation S246N was subsequently introduced into RT29, generating a variant RTN29, which displayed a six-fold increase in caseinolytic activity at 25℃compared with wild-type enzyme. It is worth mentioning that RT29 and RTN29 remain the properties of thermophilic enzymes. For example, no loss of activity was observed in these two variants after being incubated at 60℃for 2 h.Cold-adapted variants generated by directed evolution and site-directed mutagenesis generally follows the general principle of trade-off between activity and stability. Nevertheless, variant ET45 with a～1.8-fold increase in caseinolytic activity at 25℃is actually more stable than WAM, implying there is no intrinsic correlation between stability and low-temperature activity. Moreover, none of mutations identified in cold-adapted variants match the corresponding sites in naturally psychrophilic and other artificially cold-adapted subtilases, implying there are multiple routes to cold adaptation. This phenomenon could also be explained in terms of the difference between laboratory evolution and natural evolution.The major difference between the cold-adapted variants of WF146 protease and those of subtilisin SSII, as well as BPN’ resides in their catalytic behaviors. The variant RTN29 showed about 6-fold increase in caseinolytic activity in the temperature range of 15-25℃. However, the hydrolytic activity of this variant against suc-AAPF-pNA were much lower than that of wild-type enzyme, resulting from a decrease in kcat and an increase in Km. All mutations identified in the cold-adapted variants occurred within or near the substrate-binding region, showing this region played a significant role in hydrolysis of enzymes toward macrosubstrate. Improvement of low-temperature activity toward macromolecular substrates is of particular interest in the use of thermostable subtilases for industrial applications, such as detergent additives and food processing. Our study may provide valuable information needed to develop enzymes coupling high stability and high low-temperature activity, which are highly desired for industrial use.To investigate and compare the molecular mechanisms of temperature adaptation in homologous proteases from the same bacterium, we have cloned WFT2 protease gene from Bacillus sp. WF146. WFT2 protease was expressed in E. coli as inclusion bodies, which could slowly convert into active protease under certain conditions. WFT2 protease displayed an optimal temperature of 50℃and a half-life of 14 min at 70℃, and suffered autolysis easily. These results indicate WFT2 protease is less stable than WF146 protease, although they are homologous proteases produced by the same host. These results enable us to futher probe the molecular mechanism underlying the temperature adaptation of two proteases from the same bacterium. |
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