Mesophilic alkaline serine proteases from various bacteria have been commercially applied in a range of industries owing to their high catalytic efficiency and wide substrate specificity. However, these proteases have an optimal catalytic temperature of approximately 50 °C, and their activity decreases significantly at low temperature. Therefore, to enhance their cold activity, it is necessary to improve the catalytic performance of these proteases at low temperature. The alkaline serine protease (DHAP) from Bacillus pumilus BA06 is a typical mesophilic enzyme, which has demonstrated great potential in various industrial applications. Here we attempted to improve the cold activity of DHAP via directed evolution. Seven variants (P9S, A1G/K27Q, A38V, A116T, T162I, S182R, and T243S) of DHAP from B. pumilus were obtained via directed evolution. The results showed that all of the variants had increased proteolytic activity at 15 °C towards both the casein and synthetic peptide substrates. With the exception of variant T243S, the thermostability of these variants did not decrease in comparison with the wild-type enzyme. Kinetic analysis indicated that the increase in catalytic efficiency was largely attributed to the increase in turnover number (kcat). Furthermore, the combined variants generated by site-directed mutagenesis showed a further increase in specific caseinolytic activity and the kcat value for hydrolysis of the synthetic peptide. The combined variants of P9S/K27Q and P9S/T162I exhibited an approximate 5-fold increase in caseinolytic activity at 15 °C and almost no loss of thermostability. Finally, the possible mechanism responsible for the change in catalytic properties for these variants was interpreted based on structural modeling. Directed evolution and site-directed mutagenesis were combined to engineer variants of the DHAP from B. pumilus. All of the variants exhibited an increase in hydrolytic efficiency at low temperature towards both of the substrates, casein and synthetic peptide, without any loss of thermostability compared with the wild-type. These data suggest that engineering low-temperature activity for a bacterial protease is not always associated with the loss of thermostability. Furthermore, our findings demonstrate that enhanced cold activity and thermostability could be integrated into a single variant.

Rice bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the devastating diseases of rice. It is well established that the wild rice Oryza meyeriana is immune to BB. In this study, the transcriptomic analysis was carried out by RNA sequencing of O. meyeriana leaves, inoculated with Xoo to understand the transcriptional responses and interaction between the host and pathogen. Totally, 57,313 unitranscripts were de novo assembled from 58.7 Gb clean reads and 14,143 unitranscripts were identified after Xoo inoculation. The significant metabolic pathways related to the disease resistance enriched by KEGG, were revealed to plant-pathogen interaction, phytohormone signaling, ubiquitin mediated proteolysis, and phenylpropanoid biosynthesis. Further, many disease resistance genes were also identified to be differentially expressed in response to Xoo infection. Conclusively, the present study indicated that the induced innate immunity comprise the basal defence frontier of O. meyeriana against Xoo infection. And then, the resistance genes are activated. Simultaneously, the other signaling transduction pathways like phytohormones and ubiquitin mediated proteolysis may contribute to the disease defence through modulation of the disease-related genes or pathways. This could be an useful information for further investigating the molecular mechanism associated with disease resistance in O. meyeriana.