Single-site substitutions improve cold activity and increase thermostability of the dehairing alkaline protease (DHAP)

Abstract

To engineer dehairing alkaline protease (DHAP) variants to improve cold activity and increase thermostability so these variants are suitable for the leather processing industry. Based on previous studies with bacterial alkaline proteases, double-site mutations (W106K/V149I and W106K/M124L) were introduced into the DHAP from Bacillus pumilus. Compared with the wild-type DHAP hydrolytic activity, the double-site variant W106K/V149I showed an increase in specific hydrolytic activity at 15 °C by 2.3-fold toward casein in terms of hydrolytic rate and 2.7-fold toward the synthetic peptide AAPF-pN by means of k/K value. The thermostability of the variant (W106K/V149I) was improved with the half-life at 60 and 70 °C increased by 2.7- and 5.0-fold, respectively, when compared with the thermostability of the wild-type DHAP. Conclusively, an increase in the cold activity and thermostability of a bacterial alkaline protease was achieved by protein engineering.

Engineering Bacillus pumilus alkaline serine protease to increase its low-temperature proteolytic activity by directed evolution

“…Since the α-helix and β-sheet in a protein are usually regarded as more rigid than the loop region, a large sidechain group of threonine may dominate the space, potentially perturbing the local structure. A similar result was also observed for another variant (W106 K) on the same helix, which showed an increase in the catalytic turnover number, without loss of thermostability [ 24 ]. T243 is localized on the seventh α-helix, which is buried far behind the catalytic center.…”

“…For example, in silico analyses of the homologous counterparts between mesophilic and psychrophilic proteins have been performed, along with experimental studies including in vitro random mutations as well as site-directed mutagenesis [ 32 , 33 ]. Previously, several variants of DHAP have been engineered to improve the activity of the enzyme at cold temperatures by site-directed mutagenesis [ 23 , 24 ]. In this study, through in vitro molecular evolution and extensive screening, we obtained several new variants with improved catalytic activity towards both of the substrates, casein and AAPF- p N, especially at lower temperature (15 °C).…”

“…Finally, four combined variants were constructed from three sites: P9, K27, and T162, and catalytic activity with the macromolecular substrate casein was found to be further increased, especially at low temperature. For example, the variant (P9S/K27Q) showed an increase in caseinolytic activity of more than 5-fold compared with the wt, which was higher than that of the previous variant (W106 K/V149I) [ 24 ]. By contrast, the catalytic efficiency of variant P9S/K27Q was lower than that of variant W106 K/V149I for the hydrolysis of AAPF- p N. Therefore, the substrate specificity of these variants may be different.…”

“…The mutation library of DHAP was constructed following the protocol described previously [ 26 ]. Briefly, the error PCR was performed in a 100-μL mixture containing 5 U Taq DNA polymerase, 50 μM dNTPs, 150 μM dITP, 400 nM primers (DEAP-Hin.F/DEAP-Bam.R, Additional file 1 : Table S1), and 10 ng template DNA of pAPN8B, which harbored the full-length sequence encoding the alkaline serine protease DHAP as well as its promoter and terminal sequences [ 24 ]. The PCR conditions were set as: predenaturation for 2 min at 94 °C; then 30 cycles of denaturation for 30 s at 94 °C, annealing for 30 s at 52 °C, and extension for 1 min at 72 °C; followed by a final extension for 10 min at 72 °C.…”

“…Therefore, there is a need to improve the catalytic activity of this protease at lower temperatures, especially for industrial applications. Previously, we have engineered several DHAP variants by site-directed mutagenesis based on homologous sequence alignments and structural modeling and demonstrated improvements in their catalytic performance compared with the wt enzyme [ 23 , 24 ].…”