基于蛋白智能模型提升溶菌酶RPL187的热稳定性

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

摘要/Abstract

摘要：

目的溶菌酶可作为抑菌剂而广泛应用于食品、生物、医药等领域。但溶菌酶作为一种生物活性物质，其稳定性受温度影响较大，难以满足不同行业的需求。因此采用一种结合人工智能模型设计并筛选蛋白质突变体的改造策略，提高溶菌酶的热稳定性，扩大溶菌酶的实际应用范围。方法研究材料为瘤胃原虫基因组来源的溶菌酶RPL187，通过大肠杆菌异源表达蛋白质进行后续实验，利用国标法检测溶菌酶RPL187在不同温度（37、45、50、55 ℃）处理不同时间（0、1、2、4、8 h）下剩余酶比活的变化情况；基于人工智能模型生成并筛选RPL187的多点突变体，同样利用国标法检测溶菌酶突变体在不同温度处理不同时间下剩余酶比活的变化情况，并通过测定野生型与突变体T_m值、自由能、氢键数量、二级结构含量等变化进而研究热稳定性提高的机制。结果 RPL187在37 ℃和pH 6.5条件下的酶比活为（142 000±2 000）U/mg，比蛋清溶菌酶高5倍；RPL187在37 ℃和45 ℃条件下较稳定，但随着温度升高和热处理时间延长，酶比活出现明显下降的情况，在55 ℃条件下孵育1 h，酶比活下降88%左右；为了提高RPL187的热稳定性，基于人工智能模型共筛选11个RPL187多点突变体；有7个突变体成功在大肠杆菌中可溶性表达，其中RPL187-592和RPL187-209具有抑制藤黄微球菌的活性；进一步热稳定性的检测结果显示，RPL187-592和RPL187-209在50 ℃下经热处理8 h后剩余酶比活较野生型分别高4.43倍和2.29倍，T_m值较野生型分别提高2.06 ℃和2.41 ℃，并且自由能较野生型分别降低1.57 kcal/mol和0.43 kcal/mol，表现出较野生型更稳定的构象和更高的热稳定性。与野生型相比，突变体RPL187-592的分子内氢键增加了3个，且氨基酸由亲水向疏水的转变（K2V和K137V）均有助于提高蛋白质的热稳定性；而在RPL187-209中，可能是由于氨基酸由柔性向刚性（K78P和K108P）以及由亲水向疏水（K137A）的转变而增加热稳定性。结论本改造策略可以有效提高蛋白质热稳定性，对于扩大溶菌酶应用范围的实际需求有着重要意义，同时也为相关研究提供可参考的依据。

关键词: 溶菌酶, 藤黄微球菌, 酶比活, 热稳定性改造, 人工智能模型

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

王辉, 范灵熙, 孙纪录, 王苑, 伍宁丰, 田健, 关菲菲. 基于蛋白智能模型提升溶菌酶RPL187的热稳定性[J]. 生物技术通报, 2025, 41(7): 336-346.

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.

图/表 8

图1 RPL187表达与活性测定A： SDS-PAGE蛋白凝胶电泳图，粗酶液为诱导后破碎产生的杂蛋白，纯酶液为经纯化获得的纯蛋白；B：粗酶液牛津杯抑菌圈实验图，NC为阴性对照，PC为阳性对照，CK为空白对照；C：国标法检测溶菌RPL187酶比活图，NC为阴性对照，PC为阳性对照。****P<0.000 1

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

图2 国标法测定溶菌酶RPL187热稳定性A：37 ℃下不同热处理时间的溶菌酶RPL187和蛋清溶菌酶HEWL酶比活变化图；B：45 ℃下不同热处理时间的溶菌酶RPL187和蛋清溶菌酶HEWL酶比活变化图；C：50 ℃下不同热处理时间的溶菌酶RPL187和蛋清溶菌酶HEWL酶比活变化图；D：55 ℃下不同热处理时间的溶菌酶RPL187和蛋清溶菌酶HEWL酶比活变化图。ns： P≥0.05； *P<0.05； **P<0.01； ***P<0.001； ****P<0.000 1。下同

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

图3 RPL187突变体的表达与活力检测A： RPL187及突变体粗酶液SDS-PAGE蛋白凝胶电泳图；B：粗酶液牛津杯抑菌圈实验图

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

图4 国标法测定RPL187及突变体的热稳定性A：50 ℃不同热处理时间的蛋清溶菌酶、RPL187及突变体RPL187-209和RPL187-592的酶比活变化图；B：55 ℃不同热处理时间的蛋清溶菌酶、RPL187及突变体RPL187-209和RPL187-592的酶比活变化图

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 ℃

图5 热稳定性提高的机制分析图A：Pymol可视化展示突变体RPL187-592相对于野生型增加的氢键图；B：利用Pymol计算突变体RPL187-209与野生型RPL187的RMSD （root mean square deviation）值；C：利用Pymol计算突变体RPL187-592与野生型RPL187的RMSD值；基于Pymol绘制的突变体（灰色）与野生型（绿色）的蛋白结构叠合图，直观展示突变引起的构象变化；RMSD（均方根偏差）用于衡量两个蛋白质结构之间的相似性，值越小表示结构差异越小；D：利用圆二色谱仪（CD）检测野生型RPL187及突变体RPL187-209、RPL187-592的二级结构含量变化图

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)

表1 多模型结合筛选RPL187突变体评价分值表

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

表2 RPL187稳定性改造突变体的突变位点变化表

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	-

表3 RPL187与突变体的Tm值与自由能变化

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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