A single mutation within a Ca2+ binding loop increases proteolytic activity, thermal stability, and surfactant stability

Bioscience, Biotechnology, and Biochemistry

Liu

et al. 2016

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.

Section: Discussionmentioning

confidence: 89%

Section: Temperature-dependent Caseinolytic Activitymentioning

confidence: 99%

Section: Temperature-dependent Caseinolytic Activitymentioning

confidence: 99%

See 1 more Smart Citation

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

Bioscience, Biotechnology, and Biochemistry

Liu

et al. 2016

“…The residue Ser78 is located in this calcium‐binding loop. We speculated that mutation S78T affected the interaction with calcium and the packing of this loop at a low pH, consequently increasing the protein stability (Okuda et al, ). According to the results of the MD simulation, residue Tyr217 was away from the active site when it was mutated to lysine, and the mutation Y217K was more stable under acidic conditions.…”

Section: Discussionmentioning

confidence: 99%

Improvement of the acid resistance, catalytic efficiency, and thermostability of nattokinase by multisite‐directed mutagenesis

Liu

Biotech & Bioengineering

Han

et al. 2019

Nattokinase (NK) is a serine protease of the subtilisin family; as a potent fibrinolytic enzyme, it is potentially useful for thrombosis therapy. For NK to be applied as an oral medicine for the treatment of cardiovascular diseases, it must overcome the extremely acidic environments of the gastrointestinal tract despite its limited acidic stability. In this study, three strategies were adopted to improve the acid resistance of NK: (a) Surface charge engineering, (b) sequence alignment, and (c) mutation based on the literature. Eleven variants were constructed and four single-point mutations were screened out for their distinctive catalytic properties: Q59E increased the specific activity; S78T improved the acid stability; Y217K enhanced the acid and thermal stabilities; and N218D improved the thermostability. Based on these observations, multipoint variants were constructed and characterized, and one variant with better acid stability, catalytic efficiency, and thermostability was obtained. Molecular dynamics simulation was carried out to clarify the molecular mechanism of the increased stability of S78T and Y217K mutants under acidic conditions. This study explored effective strategies to engineer acid resistance of NK; moreover, the NK variants with better catalytic properties found in this study have potential applications for the medical industry.

“…However, the trade-off between catalytic activity and thermostability does not always occur. For example, substitution of Y195 with a cysteine residue in the alkaline serine protease KP-43 led to an increase in both proteolytic activity and thermostability [ 42 ]. The variant T162S achieved a large increase in catalytic activity by more than 2.5-fold with both substrates at 15 °C without loss of thermostability as measured by the t 2/1 value (Fig.…”

Section: Discussionmentioning

confidence: 99%

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

Hong

2018

BMC Biotechnol

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.Electronic supplementary materialThe online version of this article (10.1186/s12896-018-0451-0) contains supplementary material, which is available to authorized users.