Enhanced extracellular recombinant keratinase activity in Bacillus subtilis SCK6 through signal peptide optimization and site-directed mutagenesis

Abstract

Keratinase has a great commercial value owing to its applications in the enzymatic dehairing of goatskins. In this study, we adopted a combined strategy to enhance the extracellular recombinant keratinase activity in Bacillus subtilis SCK6. First, nine signal peptides were screened to enhance the expression of extracellular keratinase. The recombinant strain with SP_LipA exhibited the highest extracellular keratinase activity of 739.03 U per mL, which was two-fold higher activity of the wild type. Second, based on the multiple sequence alignment with the bacterial alkaline proteases, the mutant (M123L/V149I/A242N) was introduced into the keratinase. Comparing with the wild type of keratinase, the mutant M123L/V149I/A242N showed an increase in the extracellular keratinase activity, which was about 1.2-fold higher activity of the wild type. Finally, the keratinase expression vector with SP_LipA and mutant M123L/V149I/A242N was constructed, and the extracellular keratinase activity reported at 830.91 U per mL was a 2.2-fold activity of the wild type. Then, the mutant keratinase was purified and characterized. The mutant exhibited properties similar to those of the wild type at an optimal temperature of 60 °C and pH 10.0. Conclusively, the extracellular expression of keratinase was enhanced via a combined strategy, and the mutant keratinase demonstrated properties similar to that of the wild type of keratinase.

Fig. 1. SDS-PAGE analysis of extracellular keratinase of the recombinant strains with different signal peptide. 1: wild-type; 2: SP_{Npr E}; 3: SP_Vpr; 4: SP_{Ywe A}; 5: SP_{Lip A}; 6: SP_{Wap A}; M: marker.

Fig. 2. The extracellular keratinase activities of the recombinant strains with different signal peptide. 1: wild-type; 2: SP_{Npr E}; 3: SP_Vpr; 4: SP_{Ywe A}; 5: SP_{Lip A}; 6: SP_{Wap A}, M: marker.

Fig. 3. Protein sequence alignment of keratinase and other proteases by ESPript 3.0. “α” represents α helix; “β” represents β sheet; the substitution sites (M123, V149 and A242) are indicated by “★”.

Fig. 4. The extracellular keratinase activities of the recombinant strains by site-directed mutagenesis. (1) Wild type; (2) M123L; (3) V149I; (4) A242N; (5) M123L/V149I; (6) M123L/A242N; (7) V149I/A242N; (8) M123L/V149I/A242N.

Fig. 5. SDS-PAGE analysis of the extracellular keratinase of the recombinant strains by site-directed mutagenesis. (1) Wild type; (2) M123L; (3) V149I; (4) A242N; (5) M123L/V149I; (6) M123L/A242N; (7) V149I/A242N; (8) M123L/V149I/A242N.

Fig. 6. The extracellular keratinase activities and SDS-PAGE analysis of extracellular keratinase of the recombinant strains. (a) (1) Wild type, (2) SP_LipA, (3) SP_LipA with mutant (M123L/V149I/A242N); (b) M: marker; (1) wild type, (2) SP_{Lip A}, (3) SP_{Lip A} with mutant (M123L/V149I/A242N).

Fig. 7. Effect of pH and temperature on the activity of keratinase and mutant. (a) Effect of pH on activity; (b) effect of temperature on activity.