Method for excavating protease catalytic performance far-end regulation and control of key amino acid by utilizing molecular dynamics simulation and combining with protein network structure

Abstract

According to the method, molecular dynamics simulation is combined with a protein network structure to excavate protease catalytic performance far-end regulation and control key amino acids, firstly, a far-end regulation and control path possibly existing in papain is excavated through temperature disturbance iterative conformation dynamics in combination with shortest path analysis; determining far-end regulation amino acid residues of the key path based on the shortest path residue contribution rate, the intermolecular interaction and the residue solvation area accessibility; and finally, expressing the papain mutant by using a heterologous expression technology, obtaining a pure papain mutant product through separation, purification and in-vitro activation, and verifying the influence of key amino acid residues on the catalytic specificity of papain by using a liquid chromatography-mass spectrometry technology. A potential regulation path and key amino acid residues of the catalytic performance of the papain are obtained. According to the invention, the aims of improving the hydrolysis specificity of the papain and further performing targeted degradation on main allergen epitopes of alpha s1-CN can be fulfilled, and a theoretical support is provided for strengthening targeted reduction of CN antigenicity by the papain.

Description

Technical Field

[0001] The present application relates to the field of protein and enzyme engineering technology, and in particular to a method for mining key amino acids for remote regulation of protease catalytic performance by combining molecular dynamics simulation with protein network structure. Background Art

[0002] At present, proteolytic enzymes play an important role in the food industry, such as tender meat, beverage production, dairy processing to improve food nutritional value, solubility and improve metabolic processes. It is estimated that the protease market will reach 3.12 billion US dollars in 2024, of which the lactase market is estimated to be 730 million US dollars ( DS, Trends Food Sci. Tech., 2022).

[0003] Usually, proteases have significant specificity for homologous substrates. The enzyme and substrate (8 amino acid residues, where the N-terminal direction of the cleavage site is P4-P1, and the C-terminal direction of the cleavage site is named P1'-P4') use the pre-existing conformation to bind through the induced fit effect (Fernández-Lucas, J. Trends Food Sci Technol, 2017); but some enzymes also show weak catalytic ability for non-homologous substrates. These unstable and low catalytic functions are secondary functions of the enzyme, also known as enzyme catalytic promiscuity, which has a continuous range of substrate preferences ranging from narrow (specific) to broad (universal). The existence of enzyme promiscuity greatly weakens its ability to hydrolyze specificity.

[0004] Milk protein is considered to be an important source of antihypertensive peptides. However, in traditional food processing, such as the preparation of bioactive peptides, functional peptides are usually prepared at the cost of high energy consumption, time and high hydrolysis degree. For example, Espejo-Carpio FJ, Int Dairy J, 2013) used subtilisin, trypsin and their combined enzymatic hydrolysis technology to hydrolyze milk protein at 50°C for 3 hours. The ACE inhibition rate of the hydrolyzate was 33% to 64%. After component separation and interception, the Ic50 of the smallest molecular weight component of the composite enzymatic hydrolysis was the best at 5.12ug / ml, but its hydrolysis degree was as high as about 32%. The smallest molecular weight component with a high hydrolysis degree is bound to contain bitter peptides, thus producing a bad taste. Based on this, domestic and foreign scholars began to study the technology of directional preparation of functional peptides, such as Lin K, J Agri Food Chem, 2018) based on the fact that casein is rich in aromatic amino acid residues, and the thermophilic protease-alkaline protease step-by-step enzymatic hydrolysis technology (50℃→70℃, 2h) was established by computer simulation of enzyme cutting. The ACE inhibitory activity Ic50 of the component less than 3kDa was 2.63ug / ml. Although the processing time was reduced to a certain extent, its hydrolysis degree was still very high (28.9%). In addition, Mazorra-Manzano, TC, (Food Chem. 2013) reported that whey protein was basically completely degraded in the crude extract of melon enzyme from the crude enzymes isolated from citrus flowers, silver leaf bricks and muskmelon. Among them, the hydrolyzate less than 10kDa could inhibit the angiotensin-converting enzyme by 90%, but the technology took up to 24h, indicating that the activity of the enzyme needs to be improved.

[0005] Usually when existing commercial proteases cannot meet production needs, scholars will use key proteins to re-screen proteases from nature through various means, which is tantamount to looking for a needle in a haystack, so the workload is huge and the cost is expensive. Even if a protease with specific hydrolysis ability is screened out by luck, problems such as low host gene expression level, enzyme instability, and inappropriate pH and temperature will cause low enzyme activity and low yield. Summary of the invention

[0006] The present application provides a method for mining key amino acids for remote regulation of protease catalytic performance by combining molecular dynamics simulation with protein network structure, so as to solve the problem of low success rate of enzyme directed evolution in the prior art, achieve the purpose of improving the hydrolysis specificity of papain and then targeting the degradation of the main allergen epitope of αs1-CN, and provide theoretical support for strengthening papain's targeted reduction of CN antigenicity.

[0007] The present application provides a method for mining the key amino acids for remote regulation of protease catalytic performance by combining molecular dynamics simulation with protein network structure, comprising the following steps:

[0008] A. First, we explored the possible distal regulatory pathways of papain by combining iterative conformational dynamics with temperature perturbation and shortest path analysis;

[0009] B. Using Escherichia coli BL21 as the engineering strain and plasmid pET28a as the papain gene expression vector, the papain mutant was expressed by heterologous expression technology. The pure papain mutant was obtained by separation, purification and in vitro activation, and casein hydrolysate was prepared based on it. The effect of key amino acid residues on the catalytic specificity of papain was verified by liquid chromatography-mass spectrometry.

[0010] Preferably, the four potential distal regulatory pathways are:

[0011] Pathway 1: The 96+95 site affects the catalytic triad (C25) via the Loop2→α3→α2 pathway;

[0012] Pathway 2: Sites 83+82+81 affect the catalytic triad (C25) via the Loop2→Loop1→α3→α2 pathway;

[0013] Pathway 3: Loop1→α2 at site 44+45 affects the catalytic triad (C25);

[0014] Pathway 4: The 101+102 site affects the catalytic triad (N175) through the Loop2→reverse hinge→β-barrel structure pathway.

[0015] Preferably, the key distal regulatory amino acid residues are: N44, Y82, R83 and R96.

[0016] Preferably, the distal SPM is mainly located in Loop1 and Loop2. Three high-frequency SPM sites appear in Loop1, namely N44, L45 and E50, among which E50 is judged to be an internal amino acid residue according to the SASA value, which is not conducive to modification; N44 has a relatively large hydrogen bond interaction force inside papain, and the catalytic center of papain is regulated through the hydrogen bond network structure. N44 can be used as a candidate amino acid residue for distal regulation.

[0017] Preferably, there are the most SPM sites in Loop2, and SPM is mainly concentrated in three sections, namely 80-83, 95-96 and 102-108. In the 80-83 section, according to the SASA value of 80-81, it can be determined that it is an internal amino acid residue and the possibility of being modified is extremely low; R83 shows a strong interaction with the papain network structure through salt bonds and hydrogen bonds, and the interaction force between Y82 and the papain network structure is relatively weak; in the 95-96 section, R96 shows a strong interaction with the papain network structure through salt bonds and hydrogen bonds, and the interaction force between C95 and the papain network structure is relatively weak; thus, Y82, R83 and R96 are candidate amino acid residues for distal regulation.

[0018] Preferably, in the 102-108 segment, the frequency of SPM occurrence is relatively low, wherein according to the SASA value, 102, 105 and 107 are internal amino acid residues, 103 interacts weakly with the overall network structure of papain, 106 and 108 show strong interactions with the papain network structure, and the 101-102 site affects the catalytic triad (N175) through the Loop2→reverse hinge→β-barrel structure path.

[0019] Preferably, the selectivity of the tyrosine 83+arginine 82 combined mutation located in loop 2 for tyrosine residues (log probability binomial probability value) increased from 1.4 to 6.6, an increase of 4.7 times.

[0020] Beneficial effects: The present invention can achieve the purpose of improving the hydrolysis specificity of papain and thus targeted degradation of the main allergen epitope of αs1-CN, providing theoretical support for strengthening the targeted reduction of CN antigenicity of papain; the present invention firstly mines the main remote regulatory pathways and key regulatory amino acid residues of papain based on the combination of molecular dynamics simulation-semi-rational design-heterologous expression combined with liquid chromatography-mass spectrometry technology, and compared with the existing traditional enzyme directed evolution technology, it reduces the workload and cost and improves the success rate of enzyme directed evolution.

[0021] The above description is only an overview of the technical solution of the embodiment of the present application. In order to more clearly understand the technical means of the embodiment of the present application, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the embodiment of the present application more obvious and easy to understand, the specific implementation methods of the present application are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present application, a brief introduction will be given below to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 Schematic diagram of the secondary and tertiary structures of papain;

[0024] Figure 2 The effect of temperature disturbance on the network structure of papain. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0026] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase "embodiments" in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0027] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0028] See also Figure 1-Figure 2 The present application discloses a method for mining the key amino acids for remote regulation of protease catalytic performance by combining molecular dynamics simulation with protein network structure, comprising the following steps:

[0029] A. First, we explored the possible distal regulatory pathways of papain by combining iterative conformational dynamics with temperature perturbation and shortest path analysis;

[0030] B. Using Escherichia coli BL21 as the engineering strain and plasmid pET28a as the papain gene expression vector, the papain mutant was expressed by heterologous expression technology. The pure papain mutant was obtained by separation, purification and in vitro activation, and casein hydrolysate was prepared based on it. The effect of key amino acid residues on the catalytic specificity of papain was verified by liquid chromatography-mass spectrometry.

[0031] Among them, the four potential distal regulatory pathways are:

[0032] Pathway 1: The 96+95 site affects the catalytic triad (C25) via the Loop2→α3→α2 pathway;

[0033] Pathway 2: Sites 83+82+81 affect the catalytic triad (C25) via the Loop2→Loop1→α3→α2 pathway;

[0034] Pathway 3: Loop1→α2 at site 44+45 affects the catalytic triad (C25);

[0035] Pathway 4: The 101+102 site affects the catalytic triad (N175) through the Loop2→reverse hinge→β-barrel structure pathway.

[0036] The key distal regulatory amino acid residues are: N44, Y82, R83 and R96.

[0037] Depend on Figure 2 It can be seen that in the figure, A: 300K, B: 315K, C: 340K, D: 374K; Overall, the structure of papain has a certain ability to withstand high temperatures, but high temperatures will cause the unstable interaction network structure inside papain to break. Under room temperature conditions, papain has four distal regulatory pathways located in Loop1 and Loop2, namely, Pathway 1: 96+95 sites affect the catalytic triad (C25) through the Loop2→α3→α2 path; Pathway 2: 83+82+81 sites affect the catalytic triad (C25) through the Loop2→Loop1→α3→α2 path; Pathway 3: 44+45 sites Loop1→α2 path affect the catalytic triad (C25); Pathway 4: 101+102 sites affect the catalytic triad (N175) through the Loop2→reverse hinge→β-barrel structure path. As the temperature rises, the overall regulation path of the catalytic center of the 96+95 site does not change; the 83+82+81 site establishes a Loop2→Loop1→β-barrel structure path at 315K to affect the catalytic triad (N175), but at 340 and 370K, its path returns to the same as that at room temperature. The regulation path of the 44+45 site becomes less obvious at 315K; at 370K, the regulation path becomes Loop1→α2→α1→β-barrel structure→catalytic triad (N175). Similarly, the regulation path of 101+102 disappears at 340K.

[0038] In general, among the four distal regulatory pathways, the 96+95 site located in Loop2 is the most stable (path 1) and has the shortest path, followed by the 83+82+81 site (path 2); the 44+45 site located in Loop1 is relatively unstable (path 3); and the 101+102 site located in Loop2 has the longest regulatory path (path 4), involving the L-subdomain crossing to the R-subdomain, which is the most complex. It is speculated that pathways 1 and 2 may produce better distal regulatory effects.

[0039] The effect of temperature on the intramolecular interaction forces (intramolecular hydrogen bonds and salt bonds) of papain Loop 1-3 was further analyzed to screen the key amino acid residues in SPM. As shown in Table 1, the distal SPM is mainly located in Loop 1 and Loop 2. Three high-frequency SPM sites appear in Loop 1, namely N44, L45 and E50. Among them, E50 is judged to be an internal amino acid residue based on the SASA value, which is not conducive to modification; compared with L45, N44 has a relatively large hydrogen bond interaction force inside papain, and regulates the catalytic center of papain through the hydrogen bond network structure. N44 can be used as a candidate amino acid residue for distal regulation.

[0040] Table 1 Intramolecular interactions and SASA of SPM residues in papain Loop 1-3

[0041]

[0042] Loop2 has the most SPM sites, probably because Loop2 is the largest and most flexible. SPM is mainly concentrated in three segments, namely 80-83, 95-96 and 102-108. In the 80-83 segment, according to the SASA value of 80-81, it can be judged that it is an internal amino acid residue (SASA < 0.25), and the possibility of being modified is extremely low. R83 shows strong interaction with the papain network structure through salt bonds and hydrogen bonds, and the interaction force between Y82 and the papain network structure is relatively weak. In the 95-96 segment, R96 also shows strong interaction with the papain network structure through salt bonds and hydrogen bonds, while the interaction force between C95 and the papain network structure is relatively weak (weaker than Y82). Therefore, Y82, R83 and R96 can be used as candidate amino acid residues for distal regulation. In the 102-108 segment, the frequency of SPM occurrence is relatively low (75%). According to the SASA values, 102, 105 and 107 are internal amino acid residues, while 103 has a weaker interaction with the overall network structure of papain, and 106 and 108 show a stronger interaction with the papain network structure. The 101-102 site can affect the catalytic triad (N175) through the Loop2→reverse hinge→β-barrel structure path.

[0043] In loop 3, 148 has a weak overall interaction with Papain, while D158 is close to the catalytic triad R159, so neither is suitable as a distal regulatory amino acid residue. Given that the distal regulatory pathway of 101-102 is long and complex, N44, Y82, R83 and R96 were comprehensively selected as the key distal AARs for regulating the catalytic specificity of Papain.

[0044] Mutants were prepared by heterologous expression to further verify the effectiveness of the distal regulatory amino acid residues. To reduce the workload of the experiment, the four candidate amino acid residues were first subjected to virtual mutation to determine the mutants for heterologous expression. The protein stability enhancement was used as the evaluation index. Among them, N44D and R96E had the best effect on papain stability enhancement after virtual mutation, while Y82P and R83P mutants had the worst effect on papain stability. Therefore, these four were selected as heterologous expression objects. At the same time, considering that Y82-R83 are adjacent key amino acid residues, the composite mutant Y82P-R83P was also determined, a total of 5 mutants. After separation and purification, 6 papain precursor proteins (basically without catalytic activity) were obtained, and the autocatalytic maturation of papain precursors was promoted by sodium acetate solution containing a reducing agent at pH 4-6.

[0045] The enzyme was added to a 4% casein solution at a ratio of 1:2000 and hydrolyzed at pH 7 and 42°C for 60 min. After the reaction, the enzyme was inactivated at 95°C for 5 min and centrifuged (4000 r / min) at low temperature (4°C-30 min). The supernatant was ultrafiltered (10 kDa) and desalted for peptide analysis.

[0046] From the binomial probability logarithm ratio (preference) of the mutants for the casein hydrolysis amino acid residue cleavage sites in Table 2 below, it can be seen that compared with the wild-type papain, the number of hydrolysis combinations of the mutant P1|P1' of papain has decreased significantly, and the smallest number of combinations of Mut3 and Mut4 is (3×4)12, which is much lower than the hydrolysis combination number of the wild type (8×6), which shows that its specificity has improved to a certain extent. Among them, the more noteworthy is that the selectivity of the mutants for Tyr has changed significantly, especially Mut3, whose selectivity for tyrosine residues (logarithmic probability binomial probability value) increased from 1.4 to 6.6, and the selectivity for Tyr increased by 4.6 times; in addition, the selectivity of Mut2 and Mut5 for Tyr also increased by 2.8 times and 2.6 times; while the increase of Mut1 and Mut4 is relatively small, but overall it shows that the selected distal amino acid residues (N44, Y82, R83 and R96) have the ability to regulate the hydrolysis specificity of papain.

[0047] Table 2 Logarithmic probability of wild-type and mutant Papain cleavage sites at P1 and P1'

[0048]

[0049] In summary, the present invention can achieve the purpose of improving the hydrolysis specificity of papain and thus targeting the degradation of the main allergen epitope of αs1-CN, and provide theoretical support for strengthening the targeted reduction of CN antigenicity of papain; the present invention firstly mines the main remote regulatory pathways and key regulatory amino acid residues of papain based on the combination of molecular dynamics simulation-semi-rational design-heterologous expression combined with liquid chromatography-mass spectrometry technology, and compared with the existing traditional enzyme directed evolution technology, it reduces the workload and cost and improves the success rate of enzyme directed evolution.

[0050] As described above, the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for mining the key amino acids for remote regulation of protease catalytic performance by combining molecular dynamics simulation with protein network structure, characterized in that: The following steps are involved: A. First, the possible distal regulatory pathways of papain were explored through iterative conformational dynamics perturbed by temperature (300K, 315K, 340K, 374K) combined with shortest path analysis; B. Using Escherichia coli BL21 as the engineering strain and plasmid pET28a as the papain gene expression vector, the papain mutant was expressed by heterologous expression technology. The pure papain mutant was obtained by separation, purification and in vitro activation, and casein hydrolysate was prepared based on it. The effect of key amino acid residues on the catalytic specificity of papain was verified by liquid chromatography-mass spectrometry.

2. According to claim 1, a method for mining the key amino acids for remote regulation of protease catalytic performance by combining molecular dynamics simulation with protein network structure, characterized in that: The four potential distal regulatory pathways are: Pathway 1: The 96+95 site affects the catalytic triad (C25) via the Loop2→α3→α2 pathway; Pathway 2: Sites 83+82+81 affect the catalytic triad (C25) via the Loop2→Loop1→α3→α2 pathway; Pathway 3: Loop1→α2 at site 44+45 affects the catalytic triad (C25); Pathway 4: The 101+102 site affects the catalytic triad (N175) through the Loop2→reverse hinge→β-barrel structure pathway.

3. According to claim 2, a method for mining the key amino acids for remote regulation of protease catalytic performance by combining molecular dynamics simulation with protein network structure, characterized in that: The key distal regulatory amino acid residues are: N44, Y82, R83 and R96.

4. According to claim 2, a method for mining the key amino acids for remote regulation of protease catalytic performance by combining molecular dynamics simulation with protein network structure, characterized in that: The distal SPM is mainly located in Loop1 and Loop2. Three high-frequency SPM sites appear in Loop1, namely N44, L45 and E50. Among them, E50 is judged to be an internal amino acid residue based on the SASA value, which is not conducive to modification; N44 has a relatively large hydrogen bond interaction force inside papain, and regulates the catalytic center of papain through the hydrogen bond network structure. N44 can be used as a candidate amino acid residue for distal regulation.

5. According to claim 2, a method for mining the key amino acids for remote regulation of protease catalytic performance by combining molecular dynamics simulation with protein network structure, characterized in that: There are the most SPM sites in Loop2, and SPM is mainly concentrated in three segments, namely 80-83, 95-96 and 102-108. In the 80-83 segment, according to the SASA value of 80-81, it can be determined that it is an internal amino acid residue and the possibility of being modified is extremely low; R83 shows a strong interaction with the papain network structure through salt bonds and hydrogen bonds, and the interaction force between Y82 and the papain network structure is relatively weak; in the 95-96 segment, R96 shows a strong interaction with the papain network structure through salt bonds and hydrogen bonds, and the interaction force between C95 and the papain network structure is relatively weak; therefore, Y82, R83 and R96 are candidate amino acid residues for distal regulation.

6. The method of mining the key amino acids for remote regulation of protease catalytic performance by combining molecular dynamics simulation with protein network structure according to claim 2, characterized in that: In the 102-108 segment, the frequency of SPM was relatively low. According to the SASA values, 102, 105 and 107 were internal amino acid residues, 103 had a weak interaction with the overall network structure of papain, 106 and 108 showed a strong interaction with the papain network structure, and the 101-102 site affected the catalytic triad (N175) through the Loop2→reverse hinge→β-barrel structure path.

7. The method of mining the key amino acids for remote regulation of protease catalytic performance by combining molecular dynamics simulation with protein network structure according to claim 2, characterized in that: The selectivity of the tyrosine 83+arginine 82 combined mutation for tyrosine residues in step B (logarithmic probability binomial probability value) increased from 1.4 to 6.6, an increase of 4.7 times.

Images