Enhanced thermal stability and hydrolytic ability of Bacillus subtilis aminopeptidase by removing the thermal sensitive domain in the non-catalytic region

Abstract

Besides the catalytic ability, many enzymes contain conserved domains to perform some other physiological functions. However, sometimes these conserved domains were unnecessary or even detrimental to the catalytic process for industrial application of the enzymes. In this study, based on homology modeling and molecular dynamics simulations, we found that Bacillus subtilis aminopeptidase contained a thermal sensitive domain (protease-associated domain) in the non-catalytic region, and predicted that deletion of this flexible domain can enhance the structure stability. This prediction was then verified by the deletion of protease-associated domain from the wild-type enzyme. The thermal stability analysis showed that deletion of this domain improved the T50 (the temperature required to reduce initial activity by 50% in 30 min) of the enzyme from 71 °C to 77 °C. The melting temperature (Tm) of the enzyme also increased, which was measured by thermal denaturation experiments using circular dichroism spectroscopy. Further studies indicated that this deletion did not affect the activity and specificity of the enzyme toward aminoacyl-p-nitroanilines, but improved its hydrolytic ability toward a 12-aa-long peptide (LKRLKRFLKRLK) and soybean protein. These findings suggested the possibility of a simple technique for enzyme modification and the artificial enzyme obtained here was more suitable for the protein hydrolysis in food industry than the wild-type enzyme.

Figure 1. Sequences alignment of BSAP, SGAP, and SSAP.

BSAP, Bacillus substilis aminopeptidase; SGAP, Streptomyces griseus aminopeptidase; SSAP, Streptomyces septatus TH-2 aminopeptidase. All sequences are full-length. The α helices and β sheets of BSAP were indicated by α 1–11 and β 1–16. The box indicates the PA domain (α 4–5 and β 2–9). The filled triangles indicate the five residues in metal-binding sites, and the open tetragons indicate the two catalytic residues. The alignment was performed with COBALT and the figure was produced with ESPript.

Figure 2. Structure analysis of BSAP.

A: The predicted structure of BSAP. The structure was obtained by homology modeling, using AP from Aneurinibacillus sp. strain AM-1 (PDB ID. 2EK8) as the template. The PA domain (raspberry), catalytic domain (cyan), zinc atoms (red), N-terminus (purple), and C-terminus (green) were labelled, respectively. B: Schematic domain structures of full-length BSAP and BSAP-ΔPA.

Figure 3. RMSF values of BSAP and BSAP-ΔPA at various temperatures.

A: RMSF values of BSAP. B: RMSF values of BSAP-ΔPA. The residues in the structure of BSAP-ΔPA were renumbered from 1 to 330 after deleting the PA domain. The different temperatures were indicated by blue (300 K), red (320 K), and olive (340 K), respectively.

Figure 4. Effect of temperature on the stability of BSAP and BSAP-ΔPA.

A: Thermal stability of the enzymes was determined by monitoring residual enzymatic activity after incubating for 30 min at various temperatures. Data points correspond to the mean values of three independent experiments. B: Temperature-induced unfolding measured using CD spectroscopy of BSAP and BSAP-ΔPA.

Figure 5. HPLC chromatogram and mass spectrum of the hydrolysate from Peptide A.

Peptide A (4 mmol) were hydrolyzed by BSAP and BSAP-ΔPA at 37°C for 30 min. The reaction without the enzyme was performed as the control. The insets show the mass spectrum of Peak 1 and Peak 2.

Figure 6. The peptide molecular mass distribution estimates for the hydrolysate of soybean protein.

The reaction without AP was used as the control. The values present the means of three independent experiments.