Abstract
BackgroundMesophilic 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.ResultsSeven 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.ConclusionsDirected 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.
Highlights
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
Cold-adapted proteases with higher catalytic efficiency at low temperature have been purified from psychrophilic bacteria, these enzymes are commonly thermosensitive [7, 8]
Construction and evaluation of a mutation library Using a protocol involving dITP for error-prone PCR and DNA shuffling [26], a mutation library for the alkaline protease dehairing alkaline protease (DHAP) was constructed that contained more than 100,000 independent clones
Summary
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. These proteases have an optimal catalytic temperature of approximately 50 °C, and their activity decreases significantly at low temperature. The alkaline serine proteases from various bacteria are commercially important enzymes that are widely used in a variety of industries such as laundry, leather, and silk processing [1, 2]. Reported improvements in the cold activity of bacterial serine proteases have attracted great attention because such enzymes are commercially important in various biotechnological processes [5, 6]. The P3C9 mutant of the mesophilic serine-like protease (SSII) from Bacillus sphaericus was constructed through molecular evolution and exhibited a 9.6-fold increase in catalytic efficiency (kcat/Km) compared with the wild-type (wt) enzyme [12]
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