Theoretical and experimental studies on the conformational changes of organic solvent-stable protease from Bacillus sphaericus DS11 in methanol/water mixtures

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Abstract

If natural proteases are used in organic synthesis, they are often inactivated or give a low rate of reaction in non-aqueous or aqueous-organic media. Therefore, to reveal the molecular mechanism governing the stability of proteases in organic solvents and increase protease stability in those systems is of intriguing interest. In the present study, the activity and conformational changes of an organic solvent-stable protease (OSP) from Bacillus sphaericus DS11 in different concentrations of methanol were investigated by measuring fluorescence, UV–Vis spectra, circular dichroism (CD), and conducting molecular dynamics (MD) simulations. The OSP expanded with increasing methanol concentration. The methanol molecules were able to enter into the OSP, leading to microenvironmental changes around the aromatic amino acids. More hydrophobic groups were exposed to the solvents at high methanol concentrations, and the original hydrophobic interaction in the protein decreased, thus resulting in the secondary and tertiary structure change in the OSP. Our results provide helpful insight into the molecular mechanism of the OSP tolerance to organic solvent and indicate directions for future work to design and engineer proteases that are stable at high organic solvent concentrations.

Introduction

Proteases are industrial enzymes that are widely used in peptide synthesis, protein processing, detergent addition, food processing, pharmaceuticals, tanning, sewage treatment, and other fields [[1], [2], [3]]. In recent years, there has been greater interest focused on the application of protease in organic synthesis because there are many advantages associated with the application of proteases for the synthesis of short peptides [[4], [5], [6]]. There are wide applications in the food and pharmaceutical industries for short peptides synthesized by proteases in non-aqueous or aqueous-organic media, but they cannot be produced in sufficient quantities because they are often inactivated or give low reaction rates during organic synthesis in non-aqueous or aqueous-organic media [7,8]. Therefore, several labor-intensive and expensive strategies have been formulated to overcome these limitations, such as enzyme immobilization and solvent engineering [[9], [10], [11]]. Rational protein design technology has developed into a powerful alternative to overcome limitations in solvent stability [12,13]. However, the molecular mechanism of deactivation of proteases by organic solvent has not been well disclosed.

Ogino et al. found that the organic solvent-stable protease PST-01 had two more disulfide bonds than thermolysin, which mainly affected protease stability in organic solvents [14]. The amino acids located on the surface of the enzyme molecule were related to the organic solvent stability of the enzyme [15]. Circular dichroism studies found that the amount of helix and folding significantly affects the stability of protease PST-01 in organic solvents [16]. Zhuang obtained 5 mutants that improved the organic solvent stability through directed evolution of the organic solvent-stable protease PT121. The results of molecular dynamics simulation showed that the protein N-terminus and the region between 200 and 225 pairs played an important role in the organic solvent stability of protease PT121. Further analysis of PT121 and 5 mutants and thermolysin revealed that the thermolysin partial loop region (122–225) had greater flexibility, resulting in a loose structure of the enzyme molecules in organic solvents [17]. Gupta et al. discovered that organic solvent-stable protease PseA exhibited a higher hydrophobic surface area [18].

Molecular dynamics is a set of molecular simulation methods that can provide dynamic properties of macromolecules in single molecule or even atomic layer structures. Molecular dynamics simulations have evolved into a mature technique that can be effectively used to investigate the catalysis and stability mechanisms of enzymes [19].

In our previous study, the protease of Bacillus sphaericus DS11 retained approximately 98% of its initial activity at 24 h after treatment at 37 °C with 25% (v/v) toluene (log P = 2.5) and shaking at 150 r/min [20]. In this work, we have investigated the activity and conformational changes of the protease OSP from Bacillus sphaericus DS11 at different concentrations of methanol by measuring fluorescence and UV–Vis spectra, and by molecular dynamics simulations. Since aromatic amino acids are very sensitive to solvents, the molecular mechanism and microenvironmental changes of OSP tolerance to organic solvents was explored through molecular dynamics simulations combined with assays.

The gene of protease (OSP) from Bacillus sphaericus DS11 was cloned and heterogeneously expressed in Bacillus subtilis W800. In this work, the molecular mechanisms of deactivation of the OSP by methanol were studied with molecular dynamics simulation, together with examination of circular dichroism, fluorescence spectra, and UV–Vis spectra to determine the changes in the three-dimensional structure and secondary structure in different concentrations of methanol.

Section snippets

Materials

Chromatography-grade methanol was purchased from Millipore Sigma (St. Louis, MO, USA). All of the other chemicals used, such as KH2PO4, K2HPO4, Na2CO3, and Tris, were of analytically pure grade and were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Molecular dynamics simulation

Molecular dynamics simulation of the OSP was performed with the method described by Park et al. [21]. In this study, we used GROMACS 5.1.4 for molecular dynamics simulation, as well as the TIP3P water molecule model and the

Overall conformation

In different concentrations of methanol, there was no large conformational change in the tertiary structure (domain) of the protein. However, there was a large difference in the local secondary structure, which suggested that the surface methanol concentration can cause local protein structure fluctuations (Fig. 1). The tertiary structure of OSP does not significantly change in organic solvents because it is solvent-resistant. However, with the addition of organic solvents, the secondary

Conclusion

This study used circular dichroism, fluorescence spectra, UV–Vis spectra, and molecular dynamics simulations to research the conformational changes of OSP in different concentrations of methanol. Combined with the results of molecular dynamics simulation and experimental results, it is known that with the addition of methanol molecules, the secondary structure of the OSP was destroyed, and the proportion was significantly reduced. Since the methanol molecules entered the inside of the enzyme

Acknowledgement

This work was supported by the National Natural Science Foundation of China (grant no. 31772016), Jiangsu Province Marine Science and Technology Innovation Project (HY2018-10), the Priority Academic Program Development of Jiangsu Higher Education Institutions, Six Talent Peaks Project in Jiangsu Province (2016-SWYY-195), and Project “333” of Jiangsu Province.

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