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. 2023 Aug 16;12(16):3081.
doi: 10.3390/foods12163081.

Improving the Thermostability of Serine Protease PB92 from Bacillus alcalophilus via Site-Directed Mutagenesis Based on Semi-Rational Design

Huabiao Miao  1   2 , Xia Xiang  2 , Nanyu Han  1   2 , Qian Wu  1   2 , Zunxi Huang  1   2
Affiliations

Improving the Thermostability of Serine Protease PB92 from Bacillus alcalophilus via Site-Directed Mutagenesis Based on Semi-Rational Design

Huabiao Miao et al. Foods. .

Abstract

Proteases have been widely employed in many industrial processes. In this work, we aimed to improve the thermostability of the serine protease PB92 from Bacillus alcalophilus to meet the high-temperature requirements of biotechnological treatments. Eight mutation sites (N18, S97-S101, E110, and R143) were identified, and 21 mutants were constructed from B-factor comparison and multiple sequence alignment and expressed via Bacillus subtilis. Among them, fifteen mutants exhibited increased half-life (t1/2) values at 65 °C (1.13-31.61 times greater than that of the wild type). Based on the composite score of enzyme activity and thermostability, six complex mutants were implemented. The t1/2 values of these six complex mutants were 2.12-10.05 times greater than that of the wild type at 65 °C. In addition, structural analysis revealed that the increased thermal stability of complex mutants may be related to the formation of additional hydrophobic interactions due to increased hydrophobicity and the decreased flexibility of the structure. In brief, the thermal stability of the complex mutants N18L/R143L/S97A, N18L/R143L/S99L, and N18L/R143L/G100A was increased 4-fold, which reveals application potential in industry.

Keywords: B-factor; protease; site-directed mutagenesis; thermostability; weighted analysis.

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Conflict of interest statement

The authors have no financial or other conflicts of interest to declare.

Figures

Figure 1
Figure 1
B-factor analysis and multisequence alignment of protease PB92: (a) B-factor values of Cα atoms for protease PB92; (b) 146 thermolysin sequences from different species of Bacillus via multiple sequence alignment.
Figure 2
Figure 2
Temperature stability of the wild type and its mutants: (a) N18L, S97H, and S97A; (b) G98E, G98R, S99A, and S99L; (c) G100E, G100A, S101G, and S101V; (d) E110L, R143V, R143L, and R143G. WT represents an expression vector for the protease PB92. The enzymes were incubated for 30.0 min at 65 °C and demonstrated activity at pH 10.5 and 55 °C. The enzyme activity of protease without any heat treatment was established as the reference value of 100%.
Figure 3
Figure 3
Expression and properties of complex mutants. (a) SDS–PAGE analysis of complex mutants. Lane M: protein molecular weight marker. Lane 1: the strain with an empty expression vector. Lanes 2–7: different mutants. Lane 8: wild-type protease PB92. (b) Activity comparison. (c) Thermostability comparison at 65 °C. (d) Thermostability comparison at 70 °C. WT represents an expression vector for the protease PB92, indicated by a red column or black lines. Mutants are indicated by a black column or lines of different colors. The high activity of wild-type protease PB92 was 3548.05 ± 80.39 U/mL at pH 10.5 and 55 °C, which was defined as 100%. Relative activity was defined as the percentage of measured high enzyme activity.
Figure 4
Figure 4
Structural analysis of complex mutants: (a) hydrophobicity of amino acid residues; (b) average flexibility of amino acid residues; (c) hydrogen bond and hydrophobic interaction analysis of wild-type and mutant proteins. Hydrophobicity and average flexibility analysis of amino acid residues were determined using the online software ProtScale (http://www.expasy.org/tools/protscale.html (accessed on 12 February 2023)).
Figure 5
Figure 5
Surface electrostatic potential map of wild type and mutant G98R: (a) wild type; (b) mutant G98R. Positive, negative, and neutral values of electrostatic potentials are represented by shades of blue, red, and white, respectively.
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