Enhancing the thermostability of carboxypeptidase A by rational design of disulfide bonds

Abstract

Carboxypeptidase A(CPA) has a great potential application in the food and pharmaceutical industry due to its capability to hydrolyze ochratoxin A(OTA) and remove the bitterness of peptide. However, CPA is a mesophilic enzyme that cannot adequately exert its catalytic activity at elevated temperatures, which seriously restricts its industrial application. In this study, the rational design of disulfide bonds was introduced to improve the thermostability of CPA. The highly flexible regions of CPA were predicted through the HotSpot Wizard program and molecular dynamics (MD) simulations. Then, DbD and MODIP online servers were conducted to predict potential residue pairs for introducing disulfide bonds in CPA. After the conservativeness analysis of the PSSM matrix and the structural analysis of the MD simulation, two mutants with potentially enhanced thermostability were screened. Results showed that these mutants D93C/F96C and K153C/S251C compared to the wild-type(WT) exhibited increase by 10 and 10 °C in T _opt , 3.4 and 2.7 min in t _1/2 at 65 °C, in addition to rise of 8.5 and 11.4 °C in T ₅₀ ¹⁵ , respectively. Furthermore, the molecular mechanism responsible for thermostability was investigated from the perspective of advanced structure and molecular interactions. The enhanced thermostability of both mutants was not only associated with the more stable secondary structure and the introduction of disulfide bonds but also related to the changes in hydrogen bonds and the redistribution of surface charges in mutant regions. This study showed for the first time that the rational design of disulfide bonds is an effective strategy to enhance the thermostability of CPA, providing in this way a broader industrial application.

Keywords

Disulfide bonds / Thermostability / Carboxypeptidase A / Rational design

References

[1] Mohammadi M, Shareghi B, Saboury AA. Comparative studies on the interaction of spermidine with carboxypeptidase A using multispectroscopic and docking methods International Journal of Biological Macromolecules. 2020, 147, 821-31

[2] Mohammadi M, Shareghi B, Farhadian S, Saboury AA. The effect of sorbitol on the structure and activity of carboxypeptidase A: Insights from a spectroscopic and computational approach Journal of Molecular Liquids. 2021, 330, 115710

[3] Dai L, Niu D, Huang JW, Li X, Shen P, et al. Cryo-EM structure and rational engineering of a superefficient ochratoxin A-detoxifying amidohydrolase Journal of Hazardous Materials. 2023, 458, 131836

[4] Manzo RM, Ceruti RJ, Bonazza HL, Adriano WS, Sihufe GA, et al. Immobilization of Carboxypeptidase A into Modified Chitosan Matrixes by Covalent Attachment Applied Biochemistry and Biotechnology. 2018, 185 4 1029-43

[5] Nezhad NG, Rahman RNZRA, Normi YM, Oslan SN, Shariff FM, et al. Recent advances in simultaneous thermostability-activity improvement of industrial enzymes through structure modification International Journal of Biological Macromolecules. 2023, 232, 123440

[6] Xu Z, Cen YK, Zou SP, Xue YP, Zheng YG. Recent advances in the improvement of enzyme thermostability by structure modification Critical Reviews in Biotechnology. 2020, 40 1 83-98

[7] Nezhad NG, Rahman RNZRA, Normi YM, Oslan SN, Shariff FM, et al. Thermostability engineering of industrial enzymes through structure modification Applied Microbiology and Biotechnology. 2022, 106, 4845-66

[8] Song Z, Zhang Q, Wu W, Pu Z, Yu H. Rational design of enzyme activity and enantioselectivity Frontiers in Bioengineering and Biotechnology. 2023, 11, 1129249

[9] Xu S, Zheng P, Sun P, Chen P, Wu D. Biosynthesis of 3-hydroxyphloretin using rational design of 4-hydroxyphenylacetate 3-monooxygenase Journal of Agricultural and Food Chemistry. 2023, 71( 49): 19457-64

[10] Ban X, Wu J, Kaustubh B, Lahiri P, Dhoble AS, et al. Additional salt bridges improve the thermostability of 1,4-α-glucan branching enzyme Food Chemistry. 2020, 316, 126348

[11] Zhou X, Xu Z, Li Y, He J, Zhu H. Improvement of the Stability and Activity of an LPMO Through Rational Disulfide Bonds Design Frontiers in Bioengineering and Biotechnology. 2022, 9, 815990

[12] Dotsenko AS, Dotsenko GS, Rozhkova AM, Zorov IN, Sinitsyn AP. Rational design and structure insights for thermostability improvement of Penicillium verruculosum Cel7A cellobiohydrolase Biochimie. 2020, 176, 103-9

[13] Lu Z, Zhong Q, Li J, Zhou B, Xing Y, et al. Glycine Substitution of Residues with Unfavored Dihedral Angles Improves Protein Thermostability Catalysts. 2022, 12 8 898

[14] Pu M, Xu Z, Peng Y, Hou Y, Liu D, et al. Protein crystal quality oriented disulfide bond engineering Protein & Cell. 2018, 9 7 659-63

[15] Wu H, Chen Q, Zhang W, Mu W. Overview of strategies for developing high thermostability industrial enzymes: Discovery, mechanism, modification and challenges Critical Reviews in Food Science and Nutrition. 2023, 63 14 2057-73

[16] Zhang Z, Yang J, Xie P, Gao Y, Bai J, et al. Characterization of a thermostable phytase from Bacillus licheniformis WHU and further stabilization of the enzyme through disulfide bond engineering Enzyme and Microbial Technology. 2020, 142, 109679

[17] Yang P, Wang X, Ye J, Rao S, Zhou J, et al. Enhanced thermostability and catalytic activity of Streptomyces mobaraenesis transglutaminase by rationally engineering its flexible regions Journal of Agricultural and Food Chemistry. 2023, 71 16 6366-75

[18] Li L, Wu W, Deng Z, Zhang S, Guan W. Improved thermostability of lipase Lip2 from Yarrowia lipolytica through disulfide bond design for preparation of medium-long-medium structured lipids LWT. 2022, 166, 113786

[19] Ni ZF, Li N, Xu P, Guo ZW, Zong MH, et al. Enhancement of thermostability and catalytic properties of ammonia lyase through disulfide bond construction and backbone cyclization International Journal of Biological Macromolecules. 2022, 219, 804-11

[20] Niu C, Zhu L, Xu X, Li Q. Rational design of disulfide bonds increases thermostability of a mesophilic 1,3-1,4-β-glucanase from Bacillus terquilensis PLoS ONE. 2016, 11 4 0154036

[21] Ming Y, Zhang H, Zhao Z, Zhang Z, Wang H, et al. Enhancing the thermostability of carboxypeptidase A by a multiple computer-aided rational design based on amino acids preferences at β-turns International Journal of Biological Macromolecules. 2023, 245, 125447

[22] Bi J, Chen S, Zhao X, Nie Y, Xu Y. Computation-aided engineering of starch-debranching pullulanase from Bacillus thermoleovorans for enhanced thermostability Applied Microbiology and Biotechnology. 2020, 104 17 7551-62

[23] Peng M, Zhang Z, Xu X, Zhang H, Zhao Z, et al. Purification and characterization of the enzymes from Brevundimonas naejangsanensis that degrade ochratoxin A and B Food Chemistry. 2023, 419, 135926

[24] Sun Z, Liu Q, Qu G, Feng Y, Reetz M. Utility of B-Factors in Protein Science: Interpreting Rigidity, Flexibility, and Internal Motion and Engineering Thermostability Chemical Reviews. 2019, 119 3 1626-65

[25] Cao J, Fan F, Lyu C, Hu S, Zhao W, et al. Pocket modification of ω-amine transaminase AtATA for overcoming the trade-off between activity and stability toward 1-acetonaphthone Engineering. 2023, 30, 203-14

[26] Ribeiro AJM, Tyzack JD, Borkakoti N, Holliday GL, Thornton JM. A global analysis of function and conservation of catalytic residues in enzymes The Journal of Biological Chemistry. 2020, 295 2 314-24

[27] Zheng Y, Du Z, Li H, Zheng M, Hong T, et al. Enhancing thermostability of alkaline κ-carrageenase from Pseudoalteromonas tetraodonis by rational design of disulfide bonds Process Biochemistry. 2023, 134, 304-15

[28] Yang J, Zhang X, Sun Q, Li R, Wang X, et al. Modulation of the catalytic activity and thermostability of a thermostable GH7 endoglucanase by engineering the key loop B3 International Journal of Biological Macromolecules. 2023, 248, 125945

[29] Li C, Ban X, Zhang Y, Gu Z, Hong Y, et al. Rational Design of Disulfide Bonds for Enhancing the Thermostability of the 1,4-α-Glucan Branching Enzyme from Geobacillus thermoglucosidans STB02 Journal of Agricultural and Food Chemistry. 2020, 68 47 13791-97

[30] Yang M, Yang S, Deng Z, Zhang Y, Yuan Z, et al. Improving the thermostability of glycosyltransferase UGT76G1 by computer-aided target analysis for highly efficient biosynthesis of rebaudioside M Food Bioscience. 2023, 56, 103119

[31] Arabnejad H, Dal Lago M, Jekel PA, Floor RJ, Thunnissen AMWH, et al. A robust cosolvent-compatible halohydrin dehalogenase by computational library design Protein Engineering Design and Selection. 2016, 30 3 175-89

This research was funded by the National Key Research and Development Program of China (No. 2023YFD1301000); and the Shandong Province Natural Science Foundation (No. ZR202102260301).

RIGHTS & PERMISSIONS

2024 Editorial Office of Food Innovation and Advances