Enhancing the Thermostability of Lysozyme RPL187 Based on Protein Intelligence Models

doi:10.13560/j.cnki.biotech.bull.1985.2025-0008

Abstract

Abstract:

Objective Lysozyme may be widely used as bacteriostatic agent in food, biomedicine and other fields. However, the stability of lysozyme as a biologically active substance is greatly affected by temperature, which makes it difficult to meet the needs of different industries. Therefore, a modification strategy incorporating the use of artificial intelligence models to design and screen protein mutants was used to improve the thermostability of lysozyme and expand the practical applications of lysozyme. Method The research material is lysozyme RPL187 from rumen protozoa genome. The variation of residual enzyme activities of lysozyme RPL187 at different temperatures (37, 45, 50, and 55 ℃) for different treating times (0, 1, 2, 4, and 8 h) were detected by the national standard method through the heterologous expression of the protein in Escherichia coli. The multipoint mutant of RPL187 was generated and screened based on the AI model, and the variations of residual enzyme activities of lysozyme mutant were detected at different temperatures and different times by the national standard method. The mechanism of the improved thermostability was investigated by determining the changes in T_m value, free energy, number of hydrogen bonds, and content of secondary structure between the wild type and the mutant. Result The specific activity of the RPL187 was (142 000±2 000) U/mg at 37 ℃ and pH 6.5, which was 5 times that of egg white lysozyme. RPL187 was more stable at 37 ℃ and 45 ℃, but with the increase of temperature and the prolongation of the heat treatment time, there was a significant decrease in the specific activity of the enzyme. After incubation for 1 h at 55 ℃, the specific activity of the enzyme decreased by about 88%. In order to improve the thermostability of RPL187, a total of 11 RPL187 multipoint mutants were screened based on the artificial intelligence model; seven mutants were successfully expressed solubilistically in E. coli, among which, RPL187-592 and RPL187-209 had the inhibitory activity against Micrococcus garciniae. Further results of the thermostability assay showed that RPL187-592 and RPL187-209 were stable at 50 ℃. The residual enzyme specific activities after heat treatment at 50 ℃ for 8 h were 4.43 times and 2.29 times that of the wild type, the T_m values were 2.06 ℃ and 2.41 ℃ higher than that of the wild type, and the free energies were 1.57 kcal/mol and 0.43 kcal/mol lower than that of the wild type, which demonstrated a more stable conformation and a higher thermostability than those of the wild type. Compared with the wild type, mutant RPL187-592 showed an increase of three intramolecular hydrogen bonds and the shift of amino acids from hydrophilic to hydrophobic (K2V and K137V) both contributed to the increase in thermostability of the protein; whereas, in RPL187-209, the increase in thermostability may be due to the shift of amino acids from flexible to rigid (K78P and K108P) and from hydrophilic to hydrophobic (K137A). Conclusion This modification strategy may effectively improve the thermostability of proteins, which is of great significance for the practical needs of expanding the application range of lysozyme, and also provides a referable basis for related research.

Key words: lysozyme, Micrococcus garciniae, specific activity, thermostability modification, artificial intelligence model

WANG Hui, FAN Ling-xi, SUN Ji-lu, WANG Yuan, WU Ning-feng, TIAN Jian, GUAN Fei-fei. Enhancing the Thermostability of Lysozyme RPL187 Based on Protein Intelligence Models[J]. Biotechnology Bulletin, 2025, 41(7): 336-346.

Figures/Tables 8

Fig. 1 Expression and activity assay of RPL187A: SDS-PAGE protein gel electrophoresis, crude enzyme solution was the heterogeneous protein produced by fragmentation after induction, and pure enzyme solution was obtained by purification. B: Oxford cup circle of inhibition experiment graph of crude enzyme solution, NC is the negative control, PC is the positive control, and CK is the blank control. C: Specific activity graph of lysin RPL187 enzyme detected by the national standard method, NC is the negative control, and PC is the positive control. ****P<0.000 1

Fig. 2 Thermostability of lysozyme RPL187 by national standard methodA: Specific activity of lysozyme RPL187 and egg white lysozyme HEWL at different durations of heat treatment at 37 ℃. B: Specific activity of lysozyme RPL187 and egg white lysozyme HEWL at different durations of heat treatment at 45 ℃. C: Specific activity of lysozyme RPL187 and egg white lysozyme HEWL at different durations of heat treatment at 50 ℃. D: Specific activity of lysozyme RPL187 and egg white lysozyme at different durations of heat treatment at 55 ℃. ns: P≥0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.000 1. The same below

Fig. 3 Expression and viability assay of the RPL187 mutantsA: SDS-PAGE protein gel electrophoresis of crude enzyme solution of RPL187 and mutants. B: Oxford cup circle of inhibition experiment graph of crude enzyme solution

Fig. 4 Thermostability of RPL187 and mutants by national standard methodA: Plots of changes in specific activity of egg white lysozyme, RPL187 and mutants RPL187-209 and RPL187-592 at different durations of heat treatment at 50 ℃. B: Plots of changes in specific activity of egg white lysozyme, RPL187 and mutants RPL187-209 and RPL187-592 at different durations of heat treatment at 55 ℃

Fig. 5 Analysis diagram of the mechanism of improving thermostabilityA: Pymol visualisation showing the increased hydrogen bonding map of mutant RPL187-592 related to the wild type. B: Calculating RMSD (root mean square deviation) values of mutant RPL187-209 versus wild type RPL187 using Pymol. C: Calculating RMSD values of mutant RPL187-592 versus wild type RPL187 using Pymol. Based on the Pymol (grey) and wild-type (green) protein structure superimposed diagrams to visually demonstrate the conformational changes caused by the mutation; RMSD was used to measure the similarity between the structures of the two proteins, with smaller values indicating smaller structural differences. D: Detection of the secondary structure content changes of the wild-type RPL187 and the mutants RPL187-209 and RPL187-592 by circular dichroism (CD)

Table 1 Multi-model combined screening of RPL187 mutant evaluation score sheet

名称 Name	模型性能评估值 Evaluated value of model performance	T_m值预测模型 T_m value predicting model	最适生长温度预测模型 Predicting model for optimal growth temperature	大肠杆菌表达量预测模型 Predicting model for E. coli expression	枯草芽孢杆菌表达量预测模型 Predicting model for B. subtilis expression	酿酒酵母表达量预测模型 Predicting model for S. cerevisiae expression	总和 Sum
RPL187-592	0.83	-0.69	2.42	2.08	1.32	1.60	6.72
RPL187-319	0.80	1.26	1.28	1.18	0.60	1.79	6.10
RPL187-496	0.80	0.49	0.89	1.98	0.34	2.18	5.88
RPL187-622	0.80	0.13	-0.25	2.31	0.81	2.63	5.63
RPL187-168	0.84	2.15	1.96	-0.65	1.70	-1.34	3.82
RPL187-857	0.82	0.95	1.78	-0.87	1.92	-0.38	3.40
RPL187-649	0.84	1.69	1.20	-0.65	1.29	-1.02	2.50
RPL187-954	0.83	1.09	0.43	-0.54	0.11	0.27	1.37
RPL187-209	0.80	-0.96	-0.34	1.71	0.64	-0.01	1.05
RPL187-963	0.83	0.22	0.33	0.18	0.04	-0.27	0.51
RPL187-700	0.82	-0.93	0.86	-0.60	0.82	-0.12	0.03
RPL187	-1.00	-1.37	-1.25	-1.12	-1.58	-1.35	-6.67

Table 2 Table of mutation site changes in RPL187 stability-modified mutants

名称 Name	突变数 Number of mutations	突变氨基酸 Mutated amino acids
RPL187-592	6	K2V__C26A__A43S__S115D__K137A__K150R
RPL187-319	8	N3R__Q11A__C26A__Y54T__S65A__I148V__V152I__F173I
RPL187-496	4	C26A__Y54T__K78P__C82R
RPL187-622	5	M10L__C26A__C82R__Y89L__M111Q
RPL187-168	6	V5I__R39A__L51S__F84Y__R109N__E146T
RPL187-857	7	V5I__K12A__R53Q__Q56R__S65A__K137A__E146T
RPL187-649	5	V5I__M10L__M13L__S118A__K132S
RPL187-954	7	Q11A__R39A__A50G__L51S__S65A__F84Y__K137A
RPL187-209	4	K78P__Q99T__K108P__K137A
RPL187-963	3	M10L__R39A__K132S
RPL187-700	8	K78P__A87R__Y89L__M111Q__S118A__K132S__E146T__F173I
RPL187	0	-

Table 3 Changes in Tm values and free energy changes in RPL187 and mutants

名称

Name

蛋白质熔化温度

T_m（℃）

自由能变化

Free energy change（kcal/mol）

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