CN114330025B

CN114330025B - A method for improving enzyme thermal stability and catalytic activity by cavity engineering technology

Info

Publication number: CN114330025B
Application number: CN202210059055.4A
Authority: CN
Inventors: 夏小乐; 张泽华; 龙梦飞; 高玲; 王颖妤; 郑楠
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-01-18
Filing date: 2022-01-18
Publication date: 2025-01-24
Anticipated expiration: 2042-01-18
Also published as: CN114330025A

Abstract

The invention discloses a method for improving enzyme thermal stability and catalytic activity by a cavity engineering technology, belonging to the fields of bioinformatics, computational chemistry, bioengineering, biophysics, protein engineering and the like. According to the method, firstly, molecular dynamics are used for simulating (MD) protein movement, representative conformations are selected through cluster analysis, then, simulation tracks are analyzed through AQUA-DUCT, key cavities in the protein are selected, amino acid residues nearby the key cavities are selected, amino acid residues with a certain range of binding active sites are removed, then, multi-scale free energy calculation software is combined, saturated mutation is carried out on the residue sites by taking energy as an index, and potential positive mutants are obtained through screening. The positive mutation rate obtained by the method of the invention reaches 90% at most, the thermal stability and catalytic activity of the enzyme are enhanced, and the method has very high application potential.

Description

Method for improving enzyme thermal stability and catalytic activity by cavity engineering technology

Technical Field

The invention belongs to the fields of bioinformatics, computational chemistry, bioengineering, biophysics, protein engineering and the like, and particularly relates to a method for improving enzyme thermal stability and catalytic activity by using a cavity-mediated engineering technology.

Background

Enzymes (enzymes) are generally proteins or RNAs produced by living cells that are highly specific and catalytic, and are susceptible to numerous external environmental factors such as temperature, pH, etc. Depending on the type of reaction catalyzed, these can be classified into six classes, oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. Because the enzyme has good catalytic function, the enzyme is widely applied to the industrial fields of food, medicine, oil production and processing, cosmetics and the like.

However, in actual industrial production, enzymes often undergo structural transformation under various external disturbances such as temperature, pressure, and solution due to the relatively severe and complicated industrial conditions, thereby affecting their stability and catalytic activity. Therefore, the enhancement of enzyme stability and activity is a necessary way in industrial production. However, in the process of enzyme evolution, a compromise between thermal stability and catalytic activity is often accompanied, and finally, only one of the two improvements is achieved. For example, mutation of the 189 th aspartic acid residue in the lid region of NTU 03 lipase resulted in an increase in the enzyme activity, but the thermostability was reduced (Shih T W,Pan T M.Substitution of Asp189 residue alters the activity and thermostability of Geobacillus sp.NTU 03lipase[J].Biotechnology Letters,2011,33(9):1841-1846.). but there were few reports on improvement of both the enzyme stability and the catalytic activity. For example, wong et al have modified D-amino acid oxidase derived from Trigonopsis variabilis by homology modeling and molecular docking to improve the thermostability and activity of the mutant Phe54Tyr (Kamal M Z,Ahmad S,Molugu T R,et al.In vitro evolved non-aggregating and thermostable lipase:structural and thermodynamic investigation[J].J Mol Biol,2011,413(3):726-741.)., and have not systematically studied the mechanism problems and thus have a great blindness, so that development of a general method for improving enzyme properties has been desired.

For natural proteins in which the amino acids are in a close arrangement, their unique atomic stacking has an important impact on the function, stability, activity, selectivity, etc. of the protein, researchers have called "cavity". They can be generally classified into 3 kinds according to functions, inter-domain cavities, intra-domain cavities, inter-subunit cavities. Among these, intra-domain cavities have been widely recognized as the contributors to the greatest thermal stability and catalytic activity of natural proteins. Its size and spatial arrangement have a great influence on the stability and catalytic activity of the protein. It is therefore important how to screen the proteins for critical cavities that have an important role in stability and catalytic activity. Regarding the use of cavities to increase enzyme stability and catalytic activity, it is currently the main practice to create cavities or reduce the volume of cavities by amino acid residue mutations, and there is no systematic screening of cavities.

Molecular dynamics (Molecular dynamics, MD) simulation refers to a computational method based on Newton's classical mechanical development to simulate the motion trajectories of all atoms in a protein system, which increases our knowledge of conformational changes in protein regulatory elements such as gates and loops. It enhances our understanding of the role of solvents in protein folding and stability, in modeling enzyme activity and selectivity, or in drug design. At present, the application method of molecular dynamics simulation is mainly to simulate and analyze the motion trail of systems such as proteins, small molecules and the like under the condition of constant temperature and constant pressure.

AQUA-DUCT is an open source Python software aimed at analyzing biological macromolecules from the point of view of intramolecular voids using small ligands as molecular probes, taking into account not only the time evolution of tunnel and hole geometry over time, but also the physicochemical properties of specific amino acids arranged inside the macromolecules. The method is mainly used for analyzing the structures (such as cavities and tunnels) of the whole protein and the functions of the tunnels and the cavities in the internal structure.

Rosetta is a comprehensive set of software for modeling macromolecular structures as a flexible, versatile application that includes tools for structural prediction, design and reconstruction of proteins and nucleic acids. The use of cartesian space coordinates to search for the side chain conformation with the lowest energy, and combining the experimental thermodynamic data of small molecules with the statistical function of high resolution structural data of macromolecules for saturation mutation (H.Park,P.Bradley,P.Greisen,Y.Liu,V.K.Mulligan,D.E.Kim,D.Baker,F.Dimaio,Simultaneous Optimization of Biomolecular Energy Functions on Features from Small Molecules and Macromolecules.Journal of Chemical Theory&Computation,6201(2016).). is currently commonly used in the development of vaccines, new materials, targeted protein binders and enzyme designs.

The FoldX Suit has been able to perform advanced protein design functions, which are empirically guided, and saturation mutagenesis (J.Schymkowitz,J.Borg,F.Stricher,R.Nys,F.Rousseau,L.Serrano,The FoldX web server:an online force field.Nucleic Acids Res 33,W382-388(2005)). is currently used for protein design and stability prediction without changing the amino acid structure around the mutation site.

Disclosure of Invention

The invention aims to provide a method for improving the thermal stability and catalytic activity of enzyme by using a cavity engineering technology, and aims to solve the problems of low thermal stability and low catalytic activity of enzyme in industrial production. The method has strong operability, achieves high positive rate with less workload, has certain universality and has high industrial application potential.

In order to achieve the above object, the present invention provides an evolution method for improving enzyme thermal stability and catalytic activity by using cavity engineering technology, which comprises the steps of firstly, performing molecular dynamics simulation on an enzyme protein structure to be evolved, analyzing an internal cavity/tunnel change process by using AQUD-DUCT program, calculating and screening an internal key cavity by combining McVol program, counting amino acid residues contributing more than 30% to the internal key cavity, and removing active sitesResidue sites in the range avoid great influence on activity, and residual amino acid sites are predicted through FoldX5.0 and Rosetta to screen potential mutants with high stability and catalytic activity. Specifically, the method of the invention comprises the following steps:

(1) Performing molecular dynamics simulation on enzyme proteins, wherein each protein is subjected to 5 parallels at random initial speed, and each parallels for 30ns;

(2) Screening internal key cavities of enzyme proteins, specifically, carrying out cluster analysis on molecular dynamics simulation tracks under all parallel conditions by taking a Root Mean Square Deviation (RMSD) matrix as a root, selecting a representative conformation (namely an average conformation of protein conformations), improving the accuracy of prediction, calculating the internal cavities of all the representative conformations by using a McVol program, and counting and screening out the cavities with occurrence frequency higher than 80 percent, wherein the cavities have an important effect on the stability and the functionality of the proteins as the internal key cavities;

(3) Virtually screening potential mutants of enzyme proteins, specifically, firstly sorting according to temperature factors B-factor, counting the top 80 amino acid residues of the B-factor in the representative conformation selected in the step (2), then combining the internal key cavities selected in the step (2), counting the amino acid residues with the occurrence frequency higher than 30% near each internal key cavity under different parallels, and simultaneously removing active sites Residue sites in the range avoid the influence of mutation on the catalytic activity as much as possible, virtual saturation mutation is carried out by utilizing Rosetta and FoldX5.0, a mutant library is constructed, and DeltaDeltaG <0 is screened out to be a potential mutant;

(4) In-vitro directed evolution experiments and enzymatic property characterization, specifically, selecting potential mutants screened by the method, mutating target site amino acid by utilizing a site-directed mutation experimental technology, gradually performing experiments such as transformation, sequencing verification, induced expression, separation and purification, enzymatic property measurement, T _m value measurement and the like, and finally, identifying the mutants with remarkably improved robustness.

Further, the molecular dynamics simulation performed in the step (1) of the invention comprises the specific steps of performing molecular dynamics simulation by using Gromacs (a molecular dynamics program package for researching a biological molecular system), selecting an AMBER99 force field during simulation, creating a cube box while placing protein in the center of the box (the shortest distance between the protein and the edge of the box is 1.0 nm), adding a solvent by using a water model (the TIP4P is adopted by the water model), subsequently neutralizing charges to enable the whole system to reach an equilibrium state, performing energy minimization on the whole system by using a steepest descent method to ensure that the structure is normal, the interatomic distance is proper and the geometric configuration is reasonable, then performing 400ps of position-limiting pre-balances (NVT and NPT) under the period boundary condition, heating the system temperature to 313K by using Berendsen temperature coupling, finally performing finished product simulation, integrating the whole simulation process by using a Leap-frog algorithm, calculating the long-distance electrostatic potential energy by using a Particle-MESH EWALD (PME) method, and performing five times at random initial speed, and performing simulation for 30ns.

Further, the cluster analysis in the step (2) is performed by using a Gromacs self-contained tool gmx _cluster. And selecting gromos algorithm for cluster analysis, testing, adjusting the minimum value of cutoff and root mean square deviation (rmsd), ensuring the clustering result to be 3-5 types, and selecting the most representative conformation (average conformation under each cluster).

Further, in step (2) of the present invention, the probe radius is set to be the same as that of the internal cavity of all representative conformations calculated by McVol procedure The minimum volume of the cavity is not less than(Smaller thanIs discarded), the volume of water molecules is set to

Further, the temperature factor B-factor in step (2) of the present invention is calculated by the B-fitter program.

Further, the virtual saturation mutation flows of Rosetta and FoldX in the step (3) of the present invention are as follows:

FoldX (http:// foldxite. Crg. Eu /) predicting that correcting and optimizing the enzyme by Repairpdb and Optimize modules before performing virtual saturation mutation, setting the operation times to 3 times to obtain an average result, and using other settings with default values;

The method comprises the steps of predicting Rosetta (https:// www.rosettacommons.org /), carrying out FastRelax on a protein structure before Rosetta calculation of DeltaG, so that the protein structure has certain relaxation, ensuring that atoms in a crystal structure are as close to original positions as possible, limiting a main chain and a branched chain of the protein structure when relaxing to avoid overlarge deflection, setting the relaxation times as 40, and then selecting one protein structure (a wild type and a mutant with optimal side chain arrangement mode) with the lowest energy score from 40 relaxation structures for next energy calculation, and selecting mutants with free energy difference DeltaG < 0;

Intersection of FoldX predicted results and Rosetta predicted results was taken as potential mutants for in vitro directed evolution experiments.

The invention relates to an in-vitro directed evolution experiment and enzymatic property characterization in step (4), which specifically comprises the following steps:

① Designing a primer sequence for mutating a target site, verifying correctness by PCR mutating a base sequence of the target site, connecting the sequence with correct mutation with an expression vector and transferring the sequence into escherichia coli or yeast for induced expression, and performing centrifugal collection to obtain supernatant after ultrasonic disruption if the sequence is intracellular protein, namely obtaining crude protein enzyme liquid if the sequence is extracellular protein;

② Purifying the crude enzyme solution by using a nickel ion affinity chromatographic column or an ion exchange chromatographic column and using a AKTA AVANT protein purifier;

③ Desalting the pure enzyme solution, namely performing ultrafiltration by using an ultrafiltration tube of 10kDa, ultrafiltering each sample for at least 3 times, diluting the ultrafiltered enzyme solution to a protein concentration of 0.01-0.1mg ml ^-1, and measuring the melting temperature (T _m) of the sample by using a circular dichroscope, wherein the temperature gradient is set to be 30-90 ℃.

The invention also provides an enzyme mutant with improved activity and thermostability obtained by the method, which comprises the following steps:

(1) Lipase mutant:

T17V/S23A/T145I is based on SEQ ID NO. 2, the substitution of threonine at position 17 for valine, the substitution of serine at position 23 for alanine and the substitution of threonine at position 145 for isoleucine,

T17V/S23A/T194L is based on SEQ ID NO.2, the threonine at position 17 is replaced by valine, the serine at position 23 is replaced by alanine and the threonine at position 194 is replaced by leucine, or,

T17V/S23A is based on SEQ ID NO. 2, the substitution of threonine at position 17 for valine and serine at position 23 for alanine, or,

T17V/T194L is based on SEQ ID NO. 2, the substitution of threonine at position 17 for valine and the substitution of threonine at position 194 for leucine, or,

S23A/T145I is based on SEQ ID NO. 2, wherein serine at position 23 is replaced by alanine and threonine at position 145 is replaced by isoleucine.

(2) Laccase mutants:

T102M is based on SEQ ID NO. 4, the threonine at position 102 is replaced by methionine, or,

S258A, wherein serine at 258 is replaced by alanine based on SEQ ID NO. 4.

(3) Glutamine transaminase mutant:

G263M, which is based on SEQ ID NO. 6, replaces glycine at position 263 with methionine, or,

N284F is formed by replacing 284 th asparagine with phenylalanine based on SEQ ID NO. 6, or,

T308F is based on SEQ ID NO. 6, wherein threonine at position 308 is replaced by phenylalanine.

Advantageous effects

In vitro experiments prove that the mutants screened by the method can improve the thermal stability and the catalytic activity at the same time, and the positive mutant rate is high. The method has good application prospect.

According to the invention, the protein simulation track is subjected to cluster analysis, and the representative conformation is selected, so that more systematic and comprehensive analysis of protein conformation fluctuation can be realized, and the accuracy of virtual prediction is improved.

The invention screens the internal key cavities of the protein representative conformation, takes the internal key cavities as probes, takes the amino acid residue sites forming the internal key cavities as potential mutation sites, screens the potential positive mutants, can realize more accurate positioning of amino acids with great influence on the protein structure and function, and further improves the positive rate of the screening result.

Since FoldX is entirely empirical, free energy calculations are performed using empirical formulas after ensuring that residues other than the mutation site do not shift. However, rosetta is a statistical function that uses cartesian space coordinates to find the lowest energy side chain conformation, combining empirical thermodynamic data with macromolecular structure data to make saturation mutations, predicted by interpreting the backbone changes of the mutant. The invention takes the intersection of the two prediction results, and further improves the accuracy of the prediction results.

Drawings

The screening flow diagram of the whole method of fig. 1.

FIG. 2 is a graph showing the results of screening amino acid sites near the cavity, the amino acids being represented by a stick model.

FIG. 3 shows the final screening result of mutants, in which the network represents cavities, the mutation sites are represented by black tags, the active centers are represented by red tags, and the broken lines represent the spatial distance of the cavities from the mutation sites.

FIG. 4 shows the 3D structure of laccase and the calculation of the internal cavity.

FIG. 5 shows the calculated results of the 3D structure of glutamine transaminase and the internal cavity.

Detailed Description

Example 1 Rhizomucor miehei lipase (Rhizomucor MIEHEI LIPASE RML)

1. Screening of Critical internal Cavity and mutation sites

This example shows the crystal structure of wild-type RML (PDB ID:3TGL,Resolution) was used as an initial model, and molecular dynamics simulation was performed using Gromacs (2019.03 th edition). The entire system was brought to equilibrium using the TIP4P water model, followed by the addition of 9 Na ⁺ to neutralize the charge. The system is subjected to energy minimization by adopting a 50000-step steepest descent method to ensure normal structure, proper interatomic distance and reasonable geometric configuration, then 400ps of position-limiting pre-equilibrium (NVT and NPT) is carried out under the periodic boundary condition, the temperature of the system is heated to 313K by adopting Berendsen temperature coupling, and the pressure coupling is carried out by using PARRINELL-Rahman. After the system is balanced, removing the limit to simulate the finished product, integrating the whole simulation process by adopting a Leap-frog algorithm, and calculating the remote electrostatic potential energy by using a PME method. Constraint algorithm selection Lincs is defined, and the precision is set to 1,4. The cut-off mode of the adjacent search is Verlet, the search mode is grid search (grid), simulation is executed five times at different initial speeds for ensuring the repeatability and fairness of the result, and the duration is 30ns.

Cluster analysis was performed to select representative conformations by gmx _cluster program. Then, the simulated trajectory is analyzed using the AQUA-DUCT procedure and the cavity is calculated using McVol (limiting the minimum volume of the cavity to not less thanWithin a range of (2) 6 internal key cavities were screened. 80 amino acid residues (aa) were selected by B-factor sequencing followed by cavity-mediated selection of 38 aa sites with a contribution rate to the cavity of greater than 30% (fig. 1). Removing active tripletsAa of the range was thus screened out 19 aa. Finally, using the complementarity of FoldX and Rosetta algorithms (i.e., taking the intersection of the two calculations), we obtained 6 aa potential mutation sites. As a result, 8 potential mutants were designed as shown in Table 1, S20V/T145L, Y F/S20V, S A/T145I, T V/T194L, T V/S23A, T145I/T194I, T V/S23A/T145I, T V/S23A/T194L.

2. Construction of mutants and characterization of enzymatic Properties

Lipase (RML) from Rhizomucor miehei sources was selected as a subject (Genbank: KP164599.1, the nucleotide and amino acid sequences of its coding region were shown in the sequence Listing). The RML adopts an escherichia coli expression system to carry out protein expression, and the selected plasmid pET-28a is E.coli JM109 as a host. Firstly, constructing a recombinant plasmid pET-28a-wt, and taking the recombinant plasmid pET-28a-wt as a template for the subsequent construction of a T1 lipase mutant. BamH I and EcoR I restriction sites are added to the 5 'and 3' ends of the RML lipase gene, respectively, and the designed gene sequence is sent to the Jinweizhi biotechnology Co. The synthesized full-length RML lipase gene and pET-28a vector were digested with BamH I and EcoR I, respectively, in a reaction system of 50. Mu.L for RML lipase gene (or pET-28a plasmid) 20. Mu.L, 10 XQ Buffer 5. Mu.L, bamH I and EcoR I each 2. Mu.L, and dd H2O 21. Mu.L. The enzyme cutting condition is 37 ℃ and 2h. And after enzyme digestion, performing nucleic acid gel electrophoresis verification on the enzyme digestion product, cutting gel according to the size of a target strip, and performing gel recovery treatment on the double enzyme digestion product of the RML gene and the pET-28a plasmid by using a DNA gel recovery kit. The RML gene and the vector pET-28a were ligated with T4 DNA ligase in a reaction system of 10. Mu.L of the target fragment 6. Mu.L, pET-28a plasmid 2. Mu.L, 10x T4 DNA Ligase Buffer 1. Mu.L, T4 DNA LIGASE. Mu.L, and placed in a 16℃metal bath overnight for 10-12h. After ligation, the ligation product of the target gene and the vector was purified using a PCR product purification kit, and E.coli JM109 was transferred to 200r/min for 7h, followed by plating the transformation solution with LB plate containing kanamycin sulfate (50. Mu.g/mL) for 12-16h. Single colony is picked up and shake-cultured in LB culture medium containing kanamycin for 6-8h, bacterial liquid PCR verification and sequencing verification are carried out, and correct recombinants are verified to carry out subsequent experiments.

Construction of mutant recombinant plasmids. Primers were designed and sent to the Jinwei Biotechnology Co., ltd for synthesis, and the results of the primer design are shown in Table 1. After the primer synthesis, the recombinant plasmid pET-28a-RML is used as a template for full plasmid PCR amplification to construct mutant recombinant plasmid, wherein the PCR reaction system is 50 mu L ddH ₂ O18 mu L,2 xMax Buffer 25 mu L, dNTP Mix (10 mM) 1 mu L, pET-28a-wt template 1 mu L, and upstream and downstream primers (10 mM) 2 mu L respectively, phanta Max Super-FIDELITY DNA Ploymerase mu L. PCR reaction conditions were 95℃30s, 95℃15s,68℃15s,72℃5min,30 cycles, 72℃5min,4℃storage. After the reaction is finished, the PCR product is digested by using Dpn I enzyme, the digested product is transferred into a plasmid amplification strain E.coli JM109, finally, the recombinant with correct sequencing is subjected to amplification culture, plasmids are extracted, and the plasmid is transferred into a protein expression strain E.coli BL21 (DE 3) and is stored at the temperature of minus 20 ℃.

TABLE 1 mutant primer sequences

The 8 RML mutants were cloned, expressed and purified, and the crude enzyme solution was purified using a nickel ion affinity column (1 ml/5ml His Trap FF) and AKTA AVANT protein purifier, the purification steps were (1) flushing the system lines, since the system lines were all kept in 20% ethanol, the lines and pumps were preferentially flushed with ultrapure water. (2) The column was equilibrated with ultrapure water (10 column volumes), and then with a final concentration of imidazole of 20mM in the binding buffer (10 column volumes). (3) And (3) loading the crude enzyme liquid at a flow rate of 1mL min ^-1 (or 5mL min ^-1) by adopting an automatic sample feeding mode of a sample feeding pump. (4) Eluting by washing 10 column volumes with buffer solution with imidazole final concentration of 20mM to remove part of impurity protein, washing 30 column volumes with eluent with imidazole final concentration of 500mM, collecting eluting product under target peak, and labeling. (5) And (3) regenerating the column, namely, due to the loss of nickel ions in the purification process, after the purification is finished for a plurality of times, regenerating the nickel column by using a pre-prepared regeneration solution, so that the next use is convenient. (6) And (3) performing SDS-PAGE verification on the purified and collected enzyme solution, and performing enzymatic property measurement after the verification is successful.

The enzyme activity is detected by using p-nitrophenyl palmitate (p-npp) as a substrate, and the enzyme activity detection system is 3mL, wherein the detection steps are that 1.8mL of 50 mol.L ^-1 Tris-HCl buffer solution with the pH of 8.0 is mixed with 100 mu L of substrate, and the mixture is reacted for 10min at 45 ℃. 100 mu L of purified enzyme solution (diluted to a proper concentration) is added into a sample tube, 100 mu L of inactivated enzyme solution is added into a control tube, and the mixture is immediately and evenly mixed for accurate reaction at 45 ℃ for 10min. Immediately after the completion of the reaction, 500. Mu.L of 10% trichloroacetic acid was added to terminate the reaction. Then, 500. Mu.L of a 10% Na ₂CO₃ solution was added thereto for color development, and the absorbance was measured at 405 nm.

Lipase enzyme activity calculation formula:

A-sample enzyme activity (U.mL ^-1)

A ₁ -absorbance OD value of sample

A ₀ -blank absorbance OD value

Slope of k-para-nitrophenol standard curve

Intercept of C ₀ -para-nitrophenol Standard Curve

N-dilution factor

V ₁ -volume of reaction solution (mL)

V ₂ -volume of enzyme solution (mL)

T-reaction time (min)

Definition of Lipase Activity Unit the amount of enzyme required to hydrolyze a substrate to release 1. Mu. Mol of free fatty acid per minute under fixed temperature and pH conditions is defined as one enzyme activity unit (U). In the present invention, 1U refers to the amount of enzyme required for hydrolyzing p-nitrophenyl palmitate to produce 1. Mu. Mol of free p-nitrobenzene per minute by the wild-type and mutant T1 lipases at 45℃and pH 8.0.

Table 2 shows that the specific activities of the mutants S20V/T145L, S A/T145I, T V/T194L, T V/S23A, T I/T194I, T V/S23A/T145I, T V/S23A/T194L were increased by 2.86 fold, 5.56 fold, 4.51 fold, 3.73 fold, 4.95 fold, 5.5 fold and 9.95 fold, respectively, relative to the wild type. Wherein the T _m value of S23A/T145I, T V/T194L, T V/S23A, T145I/T194I, T V/S23A/T145I, T V/S23A/T194L is increased by 8.89 ℃, 8.9 ℃, 10.47 ℃, 0.7 ℃, 11.98 ℃ and 11.01 ℃ respectively. The comprehensive positive rate is up to 75%. Meanwhile, the kinetic parameter results show that the catalytic efficiency of S23A/T145I, T V/T194L, T V/S23A, T I/T194I, T V/S23A/T145I, T V/S23A/T194L is respectively improved by 99%, 92%, 113%, 140%, 81% and 386%.

Table 2 specific enzyme activity, melting temperature, half-life and kinetic parameters of mutants

EXAMPLE 2 Bacillus Subtilis Laccase (BSL)

In order to prove the universality of the method, laccase which is strong in industrial application is selected according to enzyme classification, belongs to the oxidation-reduction enzyme main class (RML belongs to hydrolase), and is very widely applied to foods. For example, improving the texture, appearance, taste of the bread after baking, removing some of the adverse phenolic compounds in the processing of beer, white spirit, apple juice, grape juice, maintaining their flavor, reducing the rate of their discoloration and deterioration.

1 Screening of Critical internal Cavity and mutation sites

This example shows the crystal structure of the wild-type laccase (PDB ID:1W8R,Resolution) was used as an initial model, and molecular dynamics simulation was performed using Gromacs (2019.03 th edition). And (3) adopting a TIP4P water model, and adding Na ⁺/Cl^-1 to neutralize charges so as to enable the whole system to reach an equilibrium state. The system was energy minimized by 50000 steps of steepest descent to ensure proper structure, proper interatomic distance and reasonable geometry, then 400ps of position-limiting pre-equilibrium (NVT, NPT) was performed, the system temperature was heated to 313K by Berendsen temperature coupling, and the pressure was adjusted by PARRINELL-Rahman. After the system is balanced, the limit is removed for the final product simulation. The whole simulation process adopts the Leap-frog algorithm to integrate, and the PME method is utilized to calculate the remote electrostatic potential energy. Constraint algorithm selection Lincs is defined, and the precision is set to 1,4. The cut-off mode of the adjacent search is Verlet, the search mode is grid search (grid), simulation is executed five times at different initial speeds for ensuring the repeatability and fairness of the result, and the duration is 30ns.

Cluster analysis selects a representative conformation of the different parallels. Protein trajectories were then analyzed by AQUC-DUCT and cavity calculations were performed using McVol (the minimum volume of the cavity was limited to no less thanTo a range of 8) internal key cavities (fig. 4). Amino acid residues with cavity contribution rate of more than 30% are screened out by combining key cavities according to the sequence from large to small of B-factor values. The 25 amino acid residues were further screened by restriction activity triplets. Finally, 6 potential mutants were identified, T102V, T102M, S258A, T340L, T102F, G271M, using the complementarity of FoldX and Rosetta algorithm.

Construction of 2 mutant

Laccase (BSL) from Bacillus subtili source was selected as a study (Genbank: CP 053102.1). The BSL adopts an escherichia coli expression system to carry out protein expression, and the selected plasmids pET-28a are E.coli JM109 and E.coli BL21 (DE 3) as hosts. Firstly, constructing a recombinant plasmid pET-28a-wt, and taking the recombinant plasmid pET-28a-wt as a template for constructing laccase mutants subsequently. BamH I restriction enzyme cutting sites and EcoR I restriction enzyme cutting sites are respectively added at the 5 'end and the 3' end of laccase gene, and the designed gene sequence is sent to Jinweizhi biotechnology limited company for synthesis. The synthesized laccase full-length gene and pET-28a vector were digested with BamH I and EcoR I, respectively, in a reaction system of 50. Mu.L, 20. Mu.L of laccase gene (or pET-28a plasmid), 5. Mu.L of 10 XQ Buffer, 2. Mu.L of BamH I and EcoR I, respectively, and 21. Mu.L of dd H ₂ O. The enzyme cutting condition is 37 ℃ and 2h. After the enzyme digestion is finished, performing nucleic acid gel electrophoresis verification on the enzyme digestion product, cutting gel according to the size of a target strip, and performing gel recovery treatment on the double enzyme digestion products of the BSL gene and the pET-28a plasmid by using a DNA gel recovery kit. The BSL gene and the vector pET-28a were ligated with T4 DNA ligase in a reaction system of 10. Mu.L of the target fragment 6. Mu.L, pET-28a plasmid 2. Mu.L, 10x T4 DNA Ligase Buffer 1. Mu.L, T4 DNA LIGASE. Mu.L, and placed in a 16℃metal bath overnight for 10-12h. After ligation, the ligation product of the target gene and the vector was purified using a PCR product purification kit, and E.coli JM109 was transferred to 200r/min for 7h, followed by plating the transformation solution with LB plate containing kanamycin sulfate (50. Mu.g/mL) for 12-16h. Single colony is picked up and shake-cultured in LB culture medium containing kanamycin for 6-8h, bacterial liquid PCR verification and sequencing verification are carried out, and correct recombinants are verified to carry out subsequent experiments. Construction of mutant recombinant plasmids. Primers were designed and sent to the Jinwei Biotechnology Co., ltd for synthesis, and the results of the primer design are shown in Table 3. After the primer synthesis, the recombinant plasmid pET-28a-wt is used as a template for full plasmid PCR amplification to construct mutant recombinant plasmid, wherein the PCR reaction system is 50 mu L ddH ₂ O18 mu L, 2 xMax Buffer 25 mu L, dNTP Mix (10 mM) 1 mu L, pET-28a-wt template 1 mu L, and upstream and downstream primers (10 mM) 2 mu L respectively, phanta Max Super-FIDELITY DNA Ploymerase mu L. PCR reaction conditions were 95℃30s, 95℃15s,68℃15s,72℃5min,30 cycles, 72℃5min,4℃storage. After the reaction is finished, the PCR product is digested by using Dpn I enzyme, the digested product is transferred into a plasmid amplification strain E.coli JM109, finally, the recombinant with correct sequencing is subjected to amplification culture, plasmids are extracted, and the plasmid is transferred into a protein expression strain E.coli BL21 (DE 3) and is stored at the temperature of minus 20 ℃.

TABLE 3 mutant primer sequences

3 Protein induced expression, purification and enzymatic Property determination

The protein induction expression condition is 20 ℃,200 r.min ^-1,2mM CuSO₄, and the final concentration of IPTG is 0.5mM, and the induction is carried out for 12-16h. After the induction is finished, the supernatant is collected by centrifugation and is the crude enzyme solution. The carrier pET-28a adopted in the experiment is provided with a His tag, so that the nickel ion affinity chromatography principle is adopted to purify the crude enzyme liquid, the crude enzyme liquid is centrifuged for 10min at 4 ℃ and 12000r min ^-1 before purification, and then a water filter with the concentration of 0.22 mu M is used for filtering and purifying the crude enzyme liquid, so that impurities are further removed. The 6 BSL mutants were cloned, expressed and purified, and the crude enzyme solution was purified using a nickel ion affinity column (1 ml/5ml His Trap FF) and AKTA AVANT protein purifier, the purification steps were (1) flushing the system lines, since the system lines were all kept in 20% ethanol, the lines and pumps were preferentially flushed with ultrapure water. (2) The column was equilibrated with ultrapure water (10 column volumes), and then with a final concentration of imidazole of 20mM in the binding buffer (10 column volumes). (3) And (3) loading the crude enzyme liquid at a flow rate of 1mL min ^-1 (or 5mL min ^-1) by adopting an automatic sample feeding mode of a sample feeding pump. (4) Eluting by washing 10 column volumes with buffer solution with imidazole final concentration of 20mM to remove part of impurity protein, washing 30 column volumes with eluent with imidazole final concentration of 500mM, collecting eluting product under target peak, and labeling. (5) And (3) regenerating the column, namely, due to the loss of nickel ions in the purification process, after the purification is finished for a plurality of times, regenerating the nickel column by using a pre-prepared regeneration solution, so that the next use is convenient. (6) And (3) performing SDS-PAGE verification on the purified and collected enzyme solution, and performing enzymatic property measurement after the verification is successful.

And detecting absorbance after the laccase reacts with the substrate by using a spectrophotometer, and calculating laccase activity. The enzyme activity is defined as the amount of enzyme required to oxidize 1. Mu. Mol of substrate per minute, which is 1 enzyme activity unit (U). When 1.5ml of the total reaction system and 2-Azino bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) are used as substrates, the final concentration of the system is 1mM ABTS, the purified enzyme solution and the phosphate buffer solution are contained, OD value is measured at 420nm after water bath at 30 ℃ for 3min, and all reactions are repeated for 3 times.

Table 4 shows that the specific activities of the mutants T102V, T102M, S258A, T340L, T102F and G271M are respectively improved by 37%,170%,113%,97%,10% and 73% relative to the wild type. Meanwhile, the kinetic constants of all mutants are improved, which indicates that the catalytic efficiency of laccase is improved. The half lives of the mutants T102V, T102M, S258A and G271M are respectively improved by 11min,58min,16min and 14min at 70 ℃, which shows that the heat stability is obviously improved.

TABLE 4 specific enzyme activities, T _1/2 and kinetic parameters of wild type and mutant

EXAMPLE 3 microbial-derived glutamine transaminase (microbial transglutaminase, MTG)

Similarly, in order to prove the universality of the method, according to enzyme classification, glutamine transaminase which is industrially applied to stronger microorganism sources is selected, belongs to the class of transferase, is known as a 21 st century super adhesive, and is widely applied to foods. For example, it is most widely used in meat products, and it is possible to bind ground meat into pieces, improve the texture of food, and improve the mouthfeel, flavor texture and nutrition of meat products.

1 Screening of Critical internal Cavity and mutation sites

The present example shows the crystal structure of the wild-type glutamine transaminase (PDB ID:3IU0,Resolution) was used as an initial model, and molecular dynamics simulation was performed using Gromacs (2019.03 th edition). And (3) adopting a TIP4P water model, and adding Na ⁺/Cl^-1 to neutralize charges so as to enable the whole system to reach an equilibrium state. The system was energy minimized by 50000 steps of steepest descent to ensure proper structure, proper interatomic distance and reasonable geometry, then 400ps of position-limiting pre-equilibrium (NVT, NPT) was performed, the system temperature was heated to 313K by Berendsen temperature coupling, and the pressure was adjusted by PARRINELL-Rahman. After the system is balanced, the limit is removed for the final product simulation. The whole simulation process adopts the Leap-frog algorithm to integrate, and the PME method is utilized to calculate the remote electrostatic potential energy. Constraint algorithm selection Lincs is defined, and the precision is set to 1,4. The cut-off mode of the adjacent search is Verlet, the search mode is grid search (grid), simulation is executed five times at different initial speeds for ensuring the repeatability and fairness of the result, and the duration is 30ns.

And selecting different parallel simulation structures, and selecting a representative conformation through cluster analysis. Protein trajectories were analyzed using AQUC-DUCT and cavity calculations were performed using McVol (the minimum volume of the cavity was limited to no less thanTo a range of 7) internal key cavities (fig. 5). The amino acid residues with the cavity contribution rate of more than 30% are then screened out according to the sequence from the large value to the small value of the B-factor. The 36 amino acid residues were further screened by restriction activity triplets. Finally, using the complementarity of FoldX and Rosetta algorithms, 7 potential mutants were identified, G264F, G264L, G264M, N285F, T309L, T367M.

Construction of 2 mutant

A glutamine transaminase (STG) from Streptomyces mobaraensis sources was selected as a subject (Genbank: Y18315.1). The STG adopts an escherichia coli expression system to carry out protein expression, and the selected plasmids pET-28c are E.coli JM109 and E.coli BL21 (DE 3) as hosts. Firstly, constructing a recombinant plasmid pET-28c-wt, and taking the recombinant plasmid pET-28c-wt as a template for constructing laccase mutants subsequently. Xba I and EcoR I restriction enzyme sites are added at the 5 'end and the 3' end of laccase gene respectively, and the designed gene sequence is sent to the Jinwei Zhi Biotech Co. The synthesized STG full-length gene and pET-28c vector were digested with Xba I and EcoR I, respectively, in a reaction system of 50. Mu.L of STG gene (or pET-28c plasmid) 20. Mu.L, 10 XQ Buffer 5. Mu.L, 2. Mu.L of each of Xba I and EcoR I, and dd H ₂ O21. Mu.L. The enzyme cutting condition is 37 ℃ and 2h. After the enzyme digestion is finished, performing nucleic acid gel electrophoresis verification on the enzyme digestion product, cutting gel according to the size of a target strip, and performing gel recovery treatment on the double enzyme digestion product of the STG gene and the pET-28c plasmid by using a DNA gel recovery kit. The STG gene and the vector pET-28C were ligated with T4 DNA ligase in a reaction system of 10. Mu.L of the target fragment 6. Mu.L, pET-28C plasmid 2. Mu.L, 10x T4 DNA Ligase Buffer 1. Mu.L, T4 DNA LIGASE. Mu.L, and placed in a 16℃metal bath overnight for 10-12h. After ligation, the ligation product of the target gene and the vector was purified using a PCR product purification kit, and E.coli JM109 was transferred to 200r/min for 7h, followed by plating the transformation solution with LB plate containing kanamycin sulfate (50. Mu.g/mL) for 12-16h. Single colony is picked up and shake-cultured in LB culture medium containing kanamycin for 6-8h, bacterial liquid PCR verification and sequencing verification are carried out, and correct recombinants are verified to carry out subsequent experiments. Construction of mutant recombinant plasmids. Primers were designed and sent to the Jinwei Biotechnology Co., ltd for synthesis, and the results of the primer design are shown in Table 4. After the primer synthesis, the recombinant plasmid pET-28c-wt is used as a template for full plasmid PCR amplification to construct mutant recombinant plasmid, wherein the PCR reaction system is 50 mu L ddH ₂ O18 mu L, 2 xMax Buffer 25 mu L, dNTP Mix (10 mM) 1 mu L, pET-28c-wt template 1 mu L, and upstream and downstream primers (10 mM) 2 mu L respectively, phanta Max Super-FIDELITY DNA Ploymerase mu L. PCR reaction conditions were 95℃30s, 95℃15s,68℃15s,72℃5min,30 cycles, 72℃5min,4℃storage. After the reaction is finished, the PCR product is digested by using Dpn I enzyme, the digested product is transferred into a plasmid amplification strain E.coli JM109, finally, the recombinant with correct sequencing is subjected to amplification culture, plasmids are extracted, and the plasmid is transferred into a protein expression strain E.coli BL21 (DE 3) and is stored at the temperature of minus 20 ℃.

TABLE 5 mutant primer sequences

3 Protein induced expression, purification and enzymatic Property determination

The protein induction expression condition is 20 ℃,200 r.min ^-1, and the final concentration of IPTG is 0.5mM, and the induction is carried out for 12-16h. After the induction is finished, the supernatant is collected by centrifugation and is the crude enzyme solution. The carrier pET-28C adopted in the experiment is provided with a His tag, so that the nickel ion affinity chromatography principle is adopted to purify the crude enzyme liquid, the crude enzyme liquid is centrifuged for 10min at 4 ℃ and 12000r min ^-1 before purification, and then a water filter with the concentration of 0.22 mu M is used for filtering and purifying, so that impurities are further removed. The 6 BSL mutants were cloned, expressed and purified, and the crude enzyme solution was purified using a nickel ion affinity column (1 ml/5ml His Trap FF) and AKTA AVANT protein purifier, the purification steps were (1) flushing the system lines, since the system lines were all kept in 20% ethanol, the lines and pumps were preferentially flushed with ultrapure water. (2) The column was equilibrated with ultrapure water (10 column volumes), and then with a final concentration of imidazole of 20mM in the binding buffer (10 column volumes). (3) And (3) loading the crude enzyme liquid at a flow rate of 1mL min ^-1 (or 5mL min ^-1) by adopting an automatic sample feeding mode of a sample feeding pump. (4) Eluting by washing 10 column volumes with buffer solution with imidazole final concentration of 20mM to remove part of impurity protein, washing 30 column volumes with eluent with imidazole final concentration of 500mM, collecting eluting product under target peak, and labeling. (5) And (3) regenerating the column, namely, due to the loss of nickel ions in the purification process, after the purification is finished for a plurality of times, regenerating the nickel column by using a pre-prepared regeneration solution, so that the next use is convenient. (6) And (3) performing SDS-PAGE verification on the purified and collected enzyme solution, and performing enzymatic property measurement after the verification is successful.

The absorbance (600 nm) after the reaction of STG with the substrate was measured by a spectrophotometer, and the STG activity was calculated. The enzyme activity unit (U) is defined as the amount of enzyme catalyzing the production of 1. Mu. Mol L-glutamic acid-. Gamma. -monohydroxamate per minute of substrate at 37 ℃. Specific enzyme activity (U/mg) is defined as the unit of enzyme activity per mg of protein. The enzyme activity of the sample was determined by mixing 200. Mu.L of the properly diluted enzyme solution with 500. Mu.L of a substrate reagent, rapidly adding 200. Mu.L of 10% trichloroacetic acid after 10min in a 37 ℃ water bath, centrifuging the reaction solution at 4000r/min for 5min in a 37 ℃ water bath, taking the supernatant, measuring the absorbance at 525nm, calculating the enzyme activity, and repeating all the reactions for 3 times.

Table 6 shows that the specific activities of the mutants G263L, G263M, N284F, T308F and T366M are respectively improved by 14%,125%,78%,64% and 44% compared with the wild type. Meanwhile, the catalytic constants are respectively improved by 7.84%,2.57%,64.71%,49.02% and 29.41%, which shows that the catalytic efficiency of the glutamine transaminase is improved. The improvement of T _1/2 of G263L, G263M, N284F, T308F and T366M by 22%,214%,8%,72% and 48% respectively shows that the thermal stability of the mutants is obviously improved.

TABLE 6 specific enzyme activities and kinetic parameters of wild type and mutant

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> University of Jiangnan

<120> A method for improving enzyme thermal stability and catalytic Activity by Cavity engineering technique

<130> BAA211766A

<160> 48

<170> PatentIn version 3.3

<210> 1

<211> 795

<212> DNA

<213> Rice Black root hair

<400> 1

ggtatccgtg ctgctacttc ccaggaaata aatgaactta cctactacac cactctttct 60

gcaaattctt actgtaggac agtcattcca ggtgccactt gggactgtat tcattgcgat 120

gctactgagg atttgaaaat catcaagact tggtctactt tgatctatga tacaaacgcc 180

atggttgcca gaggagattc agagaaaaca atttacattg tattccgagg atcttcctct 240

attagaaact ggattgccga cttgacattt gtacctgttt cttatccacc tgtttccgga 300

acgaaggtac acaagggttt cctggattct tacggtgagg tccagaacga attagtcgct 360

actgtcctgg accagtttaa acagtaccca agttacaaag tggccgtgac tggtcatagt 420

ttgggtgggg caactgctct tttatgtgct ctggatctat atcaaagaga agaaggccta 480

tcttcttcaa atttattcct ttacacccaa ggacaaccaa gagtaggtga tcccgctttc 540

gctaactatg ttgtttccac aggtattccc tacagaagaa ctgtgaatga aagggatatt 600

gttccccacc taccaccagc agcctttggt ttccttcatg caggagagga gtattggatt 660

accgacaatt ctcccgaaac tgtgcaagtt tgtacctcag atttggaaac gtctgattgt 720

tctaactcaa tagtcccttt tacttcagtg ttagaccact tgagttattt tggaataaat 780

accgggttgt gcacg 795

<210> 2

<211> 265

<212> PRT

<213> Rice Black root hair

<400> 2

Gly Ile Arg Ala Ala Thr Ser Gln Glu Ile Asn Glu Leu Thr Tyr Tyr

1 5 10 15

Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val Ile Pro Gly Ala

20 25 30

Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp Leu Lys Ile Ile

35 40 45

Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala Met Val Ala Arg

50 55 60

Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg Gly Ser Ser Ser

65 70 75 80

Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro Val Ser Tyr Pro

85 90 95

Pro Val Ser Gly Thr Lys Val His Lys Gly Phe Leu Asp Ser Tyr Gly

100 105 110

Glu Val Gln Asn Glu Leu Val Ala Thr Val Leu Asp Gln Phe Lys Gln

115 120 125

Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser Leu Gly Gly Ala

130 135 140

Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg Glu Glu Gly Leu

145 150 155 160

Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln Pro Arg Val Gly

165 170 175

Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly Ile Pro Tyr Arg

180 185 190

Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu Pro Pro Ala Ala

195 200 205

Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile Thr Asp Asn Ser

210 215 220

Pro Glu Thr Val Gln Val Cys Thr Ser Asp Leu Glu Thr Ser Asp Cys

225 230 235 240

Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp His Leu Ser Tyr

245 250 255

Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210> 3

<211> 1539

<212> DNA

<213> Bacillus subtilis

<400> 3

atgacacttg aaaaatttgt ggatgctctc ccaatcccag atacactaaa gccagtacag 60

caatcaaaag aaaaaacata ctacgaagtc accatggagg aatgcactca tcagctccat 120

cgcgatctcc ctccaacccg cctgtggggc tacaacggct tatttccggg accgaccatt 180

gaggttaaaa gaaatgaaaa cgtatatgta aaatggatga ataaccttcc ttccacgcat 240

ttccttccga ttgatcacac cattcatcac agtgacagcc agcatgaaga gcccgaggta 300

aagactgttg ttcatttaca cggcggcgtc acgccagatg atagtgacgg gtatccggag 360

gcttggtttt ccaaagactt tgaacaaaca ggaccttatt tcaaaagaga ggtttatcat 420

tatccaaacc agcagcgcgg ggctatattg tggtatcacg atcacgccat ggcgctcacc 480

aggctaaatg tctatgccgg acttgtcggt gcatatatca ttcatgaccc aaaggaaaaa 540

cgcttaaaac tgccttcaga cgaatacgat gtgccgcttc ttatcacaga ccgcacgatc 600

aatgaggatg gttctttgtt ttatccgagc gcaccggaaa acccttctcc gtcactgcct 660

aatccttcaa tcgttccggc tttttgcgga gaaaccatac tcgtcaacgg gaaggtatgg 720

ccatacttgg aagtcgagcc aaggaaatac cgattccgtg tcatcaacgc ctccaataca 780

agaacctata acctgtcact cgataatggc ggagatttta ttcagattgg ttcagatgga 840

gggctcctgc cgcgatctgt taaactgaat tctttcagcc ttgcgcctgc tgaacgttac 900

gatatcatca ttgacttcac agcatatgaa ggagaatcga tcattttggc aaacagcgcg 960

ggctgcggcg gtgacgtcaa tcctgaaaca gatgcgaata tcatgcaatt cagagtcaca 1020

aaaccattgg cacaaaaaga cgaaagcaga aagccgaagt acctcgcctc atacccttcg 1080

gtacagcatg aaagaataca aaacatcaga acgttaaaac tggcaggcac ccaggacgaa 1140

tacggcagac ccgtccttct gcttaataac aaacgctggc acgatcccgt cacagaaaca 1200

ccaaaagtcg gcacaactga aatatggtcc attatcaacc cgacacgcgg aacacatccg 1260

atccacctgc atctagtctc cttccgtgta ttagaccggc ggccgtttga tatcgcccgt 1320

tatcaagaaa gcggggaatt gtcctatacc ggtccggctg tcccgccgcc gccaagtgaa 1380

aagggctgga aagacaccat tcaagcgcat gcaggtgaag tcctgagaat cgcggcgaca 1440

ttcggtccgt acagcggacg atacgtatgg cattgccata ttctagagca tgaagactat 1500

gacatgatga gaccgatgga tataactgat ccccataaa 1539

<210> 4

<211> 510

<212> PRT

<213> Bacillus subtilis

<400> 4

Met Thr Leu Glu Lys Phe Val Asp Ala Leu Pro Ile Pro Asp Thr Leu

1 5 10 15

Lys Pro Val Gln Gln Ser Lys Glu Lys Thr Tyr Tyr Glu Val Thr Met

20 25 30

Glu Glu Cys Thr His Gln Leu His Arg Asp Leu Pro Pro Thr Arg Leu

35 40 45

Trp Gly Tyr Asn Gly Leu Phe Pro Gly Pro Thr Ile Glu Val Lys Arg

50 55 60

Asn Glu Asn Val Tyr Val Lys Trp Met Asn Asn Leu Pro Ser Thr His

65 70 75 80

Phe Leu Pro Ile Asp His Thr Ile His His Ser Asp Ser Gln His Glu

85 90 95

Glu Pro Glu Val Lys Thr Val Val His Leu His Gly Gly Val Thr Pro

100 105 110

Asp Asp Ser Asp Gly Tyr Pro Glu Ala Trp Phe Ser Lys Asp Phe Glu

115 120 125

Gln Thr Gly Pro Tyr Phe Lys Arg Glu Val Tyr His Tyr Pro Asn Gln

130 135 140

Gln Arg Gly Ala Ile Leu Trp Tyr His Asp His Ala Met Ala Leu Thr

145 150 155 160

Arg Leu Asn Val Tyr Ala Gly Leu Val Gly Ala Tyr Ile Ile His Asp

165 170 175

Pro Lys Glu Lys Arg Leu Lys Leu Pro Ser Asp Glu Tyr Asp Val Pro

180 185 190

Leu Leu Ile Thr Asp Arg Thr Ile Asn Glu Asp Gly Ser Leu Phe Tyr

195 200 205

Pro Ser Ala Pro Glu Asn Pro Ser Pro Ser Leu Pro Asn Pro Ser Ile

210 215 220

Val Pro Ala Phe Cys Gly Glu Thr Ile Leu Val Asn Gly Lys Val Trp

225 230 235 240

Pro Tyr Leu Glu Val Glu Pro Arg Lys Tyr Arg Phe Arg Val Ile Asn

245 250 255

Ala Ser Asn Thr Arg Thr Tyr Asn Leu Ser Leu Asp Asn Gly Gly Asp

260 265 270

Phe Ile Gln Ile Gly Ser Asp Gly Gly Leu Leu Pro Arg Ser Val Lys

275 280 285

Leu Asn Ser Phe Ser Leu Ala Pro Ala Glu Arg Tyr Asp Ile Ile Ile

290 295 300

Asp Phe Thr Ala Tyr Glu Gly Glu Ser Ile Ile Leu Ala Asn Ser Ala

305 310 315 320

Gly Cys Gly Gly Asp Val Asn Pro Glu Thr Asp Ala Asn Ile Met Gln

325 330 335

Phe Arg Val Thr Lys Pro Leu Ala Gln Lys Asp Glu Ser Arg Lys Pro

340 345 350

Lys Tyr Leu Ala Ser Tyr Pro Ser Val Gln His Glu Arg Ile Gln Asn

355 360 365

Ile Arg Thr Leu Lys Leu Ala Gly Thr Gln Asp Glu Tyr Gly Arg Pro

370 375 380

Val Leu Leu Leu Asn Asn Lys Arg Trp His Asp Pro Val Thr Glu Thr

385 390 395 400

Pro Lys Val Gly Thr Thr Glu Ile Trp Ser Ile Ile Asn Pro Thr Arg

405 410 415

Gly Thr His Pro Ile His Leu His Leu Val Ser Phe Arg Val Leu Asp

420 425 430

Arg Arg Pro Phe Asp Ile Ala Arg Tyr Gln Glu Ser Gly Glu Leu Ser

435 440 445

Tyr Thr Gly Pro Ala Val Pro Pro Pro Pro Ser Glu Lys Gly Trp Lys

450 455 460

Asp Thr Ile Gln Ala His Ala Gly Glu Val Leu Arg Ile Ala Ala Thr

465 470 475 480

Phe Gly Pro Tyr Ser Gly Arg Tyr Val Trp His Cys His Ile Leu Glu

485 490 495

His Glu Asp Tyr Asp Met Met Arg Pro Met Asp Ile Thr Asp

500 505 510

<210> 5

<211> 1131

<212> DNA

<213> Streptomyces mobaraensis

<400> 5

gacaatggcg cgggggaaga gacgaagtcc tacgccgaaa cctaccgcct cacggcggat 60

gacgtcgcga acatcaacgc gctcaacgaa agcgctccgg ccgcttcgag cgccggcccg 120

tcgttccggg cccccgactc cgacgacagg gtcacccctc ccgccgagcc gctcgacagg 180

atgcccgacc cgtaccgtcc ctcgtacggc agggccgaga cggtcgtcaa caactacata 240

cgcaagtggc agcaggtcta cagccaccgc gacggcagga agcagcagat gaccgaggag 300

cagcgggagt ggctgtccta cggctgcgtc ggtgtcacct gggtcaattc gggtcagtac 360

ccgacgaaca gactggcctt cgcgtccttc gacgaggaca ggttcaagaa cgagctgaag 420

aacggcaggc cccggtccgg cgagacgcgg gcggagttcg agggccgcgt cgcgaaggag 480

agcttcgacg aggagaaggg cttccagcgg gcgcgtgagg tggcgtccgt catgaacagg 540

gccctggaga acgcccacga cgagagcgct tacctcgaca acctcaagaa ggaactggcg 600

aacggcaacg acgccctgcg caacgaggac gcccgttccc cgttctactc ggcgctgcgg 660

aacacgccgt ccttcaagga gcggaacgga ggcaatcacg acccgtccag gatgaaggcc 720

gtcatctact cgaagcactt ctggagcggc caggaccggt cgagttcggc cgacaagagg 780

aagtacggcg acccggacgc cttccgcccc gccccgggca ccggcctggt cgacatgtcg 840

agggacagga acattccgcg cagccccacc agccccggtg agggattcgt caatttcgac 900

tacggctggt tcggcgccca gacggaagcg gacgccgaca agaccgtctg gacccacgga 960

aatcactatc acgcgcccaa tggcagcctg ggtgccatgc atgtctacga gagcaagttc 1020

cgcaactggt ccgagggtta ctcggacttc gaccgcggag cctatgtgat caccttcatc 1080

cccaagagct ggaacaccgc ccccgacaag gtaaagcagg gctggccgtg a 1131

<210> 6

<211> 376

<212> PRT

<213> Streptomyces mobaraensis

<400> 6

Asp Asn Gly Ala Gly Glu Glu Thr Lys Ser Tyr Ala Glu Thr Tyr Arg

1 5 10 15

Leu Thr Ala Asp Asp Val Ala Asn Ile Asn Ala Leu Asn Glu Ser Ala

20 25 30

Pro Ala Ala Ser Ser Ala Gly Pro Ser Phe Arg Ala Pro Asp Ser Asp

35 40 45

Asp Arg Val Thr Pro Pro Ala Glu Pro Leu Asp Arg Met Pro Asp Pro

50 55 60

Tyr Arg Pro Ser Tyr Gly Arg Ala Glu Thr Val Val Asn Asn Tyr Ile

65 70 75 80

Arg Lys Trp Gln Gln Val Tyr Ser His Arg Asp Gly Arg Lys Gln Gln

85 90 95

Met Thr Glu Glu Gln Arg Glu Trp Leu Ser Tyr Gly Cys Val Gly Val

100 105 110

Thr Trp Val Asn Ser Gly Gln Tyr Pro Thr Asn Arg Leu Ala Phe Ala

115 120 125

Ser Phe Asp Glu Asp Arg Phe Lys Asn Glu Leu Lys Asn Gly Arg Pro

130 135 140

Arg Ser Gly Glu Thr Arg Ala Glu Phe Glu Gly Arg Val Ala Lys Glu

145 150 155 160

Ser Phe Asp Glu Glu Lys Gly Phe Gln Arg Ala Arg Glu Val Ala Ser

165 170 175

Val Met Asn Arg Ala Leu Glu Asn Ala His Asp Glu Ser Ala Tyr Leu

180 185 190

Asp Asn Leu Lys Lys Glu Leu Ala Asn Gly Asn Asp Ala Leu Arg Asn

195 200 205

Glu Asp Ala Arg Ser Pro Phe Tyr Ser Ala Leu Arg Asn Thr Pro Ser

210 215 220

Phe Lys Glu Arg Asn Gly Gly Asn His Asp Pro Ser Arg Met Lys Ala

225 230 235 240

Val Ile Tyr Ser Lys His Phe Trp Ser Gly Gln Asp Arg Ser Ser Ser

245 250 255

Ala Asp Lys Arg Lys Tyr Gly Asp Pro Asp Ala Phe Arg Pro Ala Pro

260 265 270

Gly Thr Gly Leu Val Asp Met Ser Arg Asp Arg Asn Ile Pro Arg Ser

275 280 285

Pro Thr Ser Pro Gly Glu Gly Phe Val Asn Phe Asp Tyr Gly Trp Phe

290 295 300

Gly Ala Gln Thr Glu Ala Asp Ala Asp Lys Thr Val Trp Thr His Gly

305 310 315 320

Asn His Tyr His Ala Pro Asn Gly Ser Leu Gly Ala Met His Val Tyr

325 330 335

Glu Ser Lys Phe Arg Asn Trp Ser Glu Gly Tyr Ser Asp Phe Asp Arg

340 345 350

Gly Ala Tyr Val Ile Thr Phe Ile Pro Lys Ser Trp Asn Thr Ala Pro

355 360 365

Asp Lys Val Lys Gln Gly Trp Pro

370 375

<210> 7

<211> 30

<212> DNA

<213> Artificial sequence

<400> 7

gaactgacct acttcaccct gtctgcgaac 30

<210> 8

<211> 30

<212> DNA

<213> Artificial sequence

<400> 8

cagggtgaag taggtcagtt cgttgatttc 30

<210> 9

<211> 33

<212> DNA

<213> Artificial sequence

<400> 9

ctgacctact atgtgaccct gtctgcgaac agc 33

<210> 10

<211> 33

<212> DNA

<213> Artificial sequence

<400> 10

agacagggtc acatagtagg tcagttcgtt gat 33

<210> 11

<211> 33

<212> DNA

<213> Artificial sequence

<400> 11

ctgggtggtg cgatcgctct gctgtgcgcg ctg 33

<210> 12

<211> 33

<212> DNA

<213> Artificial sequence

<400> 12

cagcagagcg atcgcaccac ccagagagtg gcc 33

<210> 13

<211> 30

<212> DNA

<213> Artificial sequence

<400> 13

accaccctgg ttgcgaacag ctactgccgt 30

<210> 14

<211> 30

<212> DNA

<213> Artificial sequence

<400> 14

gctgttcgca accagggtgg tatagtaggt 30

<210> 15

<211> 33

<212> DNA

<213> Artificial sequence

<400> 15

tctgcgaacg catactgccg taccgtgatt ccg 33

<210> 16

<211> 33

<212> DNA

<213> Artificial sequence

<400> 16

acggcagtat gcgttcgcag acagggtggt ata 33

<210> 17

<211> 30

<212> DNA

<213> Artificial sequence

<400> 17

ggtggtgcgc tcgctctgct gtgcgcgctg 30

<210> 18

<211> 30

<212> DNA

<213> Artificial sequence

<400> 18

cagcagagcg agcgcaccac ccagagagtg 30

<210> 19

<211> 30

<212> DNA

<213> Artificial sequence

<400> 19

taccgtcgta tcgttaacga acgtgatatc 30

<210> 20

<211> 30

<212> DNA

<213> Artificial sequence

<400> 20

ttcgttaacg atacgacggt acgggatacc 30

<210> 21

<211> 30

<212> DNA

<213> Artificial sequence

<400> 21

taccgtcgtc tcgttaacga acgtgatatc 30

<210> 22

<211> 30

<212> DNA

<213> Artificial sequence

<400> 22

ttcgttaacg agacgacggt acgggatacc 30

<210> 23

<211> 34

<212> DNA

<213> Artificial sequence

<400> 23

cgaggtaaag gttgttgttc atttacacgg cggc 34

<210> 24

<211> 32

<212> DNA

<213> Artificial sequence

<400> 24

caacaacctt tacctcgggc tcttcatgct gg 32

<210> 25

<211> 34

<212> DNA

<213> Artificial sequence

<400> 25

cgaggtaaag atggttgttc atttacacgg cggc 34

<210> 26

<211> 32

<212> DNA

<213> Artificial sequence

<400> 26

caaccatctt tacctcgggc tcttcatgct gg 32

<210> 27

<211> 38

<212> DNA

<213> Artificial sequence

<400> 27

caacgccgcc aatacaagaa cctataacct gtcactcg 38

<210> 28

<211> 32

<212> DNA

<213> Artificial sequence

<400> 28

ttgtattggc ggcgttgatg acacggaatc gg 32

<210> 29

<211> 32

<212> DNA

<213> Artificial sequence

<400> 29

gtcttaaaac cattggcaca aaaagacgaa ag 32

<210> 30

<211> 38

<212> DNA

<213> Artificial sequence

<400> 30

gccaatggtt ttaagactct gaattgcatg atattcgc 38

<210> 31

<211> 32

<212> DNA

<213> Artificial sequence

<400> 31

acaaacttta cctcgggctc ttcatgctgg ct 32

<210> 32

<211> 30

<212> DNA

<213> Artificial sequence

<400> 32

cagcagagcg agcgcaccac ccagagagtg 30

<210> 33

<211> 41

<212> DNA

<213> Artificial sequence

<400> 33

ctcgataatg gcatggattt tattcagatt ggttcagatg g 41

<210> 34

<211> 32

<212> DNA

<213> Artificial sequence

<400> 34

tccatgccat tatcgagtga caggttatag gt 32

<210> 35

<211> 32

<212> DNA

<213> Artificial sequence

<400> 35

aagtacttcg acccggacgc cttccgcccc gc 32

<210> 36

<211> 32

<212> DNA

<213> Artificial sequence

<400> 36

tccgggtcga agtacttcct cttgtcggcc ga 32

<210> 37

<211> 32

<212> DNA

<213> Artificial sequence

<400> 37

agaggaagta cctcgacccg gacgccttcc gc 32

<210> 38

<211> 32

<212> DNA

<213> Artificial sequence

<400> 38

gtcgaggtac ttcctcttgt cggccgaact cg 32

<210> 39

<211> 32

<212> DNA

<213> Artificial sequence

<400> 39

aagtacatgg acccggacgc cttccgcccc gc 32

<210> 40

<211> 32

<212> DNA

<213> Artificial sequence

<400> 40

tccgggtcca tgtacttcct cttgtcggcc ga 32

<210> 41

<211> 32

<212> DNA

<213> Artificial sequence

<400> 41

agggacaggt tcattccgcg cagccccacc ag 32

<210> 42

<211> 32

<212> DNA

<213> Artificial sequence

<400> 42

ggaatgaacc tgtccctcga catgtcgacc ag 32

<210> 43

<211> 32

<212> DNA

<213> Artificial sequence

<400> 43

gcccagttcg aagcggacgc cgacaagacc gt 32

<210> 44

<211> 32

<212> DNA

<213> Artificial sequence

<400> 44

tccgcttcga actgggcgcc gaaccagccg ta 32

<210> 45

<211> 32

<212> DNA

<213> Artificial sequence

<400> 45

gcccagttag aagcggacgc cgacaagacc gt 32

<210> 46

<211> 32

<212> DNA

<213> Artificial sequence

<400> 46

tccgcttcta actgggcgcc gaaccagccg ta 32

<210> 47

<211> 34

<212> DNA

<213> Artificial sequence

<400> 47

aagagctgga acatggcccc cgacaaggta aagc 34

<210> 48

<211> 32

<212> DNA

<213> Artificial sequence

<400> 48

gccatgttcc agctcttggg gatgaaggtg at 32

Claims

1. A method for simultaneously evolving enzyme thermal stability and catalytic activity, characterized in that it comprises the following steps:

(1) Molecular dynamics simulation of the evolving enzyme protein structure,

(2) Analyze the internal cavities of the enzyme protein structure and screen out the key internal cavities, which means that all parallel molecular dynamics simulation trajectories are clustered based on the root mean square deviation matrix and representative conformations are selected. Then, the internal cavities of all representative conformations are calculated using the McVol program, and the cavities with a frequency of occurrence higher than 80% are counted and screened as the key internal cavities. The cluster analysis uses the Gromacs built-in tool gmx_cluster, selects the gromos algorithm for cluster analysis, and performs tests to adjust the cutoff and the minimum value of the root mean square deviation to ensure that the clustering results are 3-5 categories, and select representative conformations.

(3) Select amino acid residues with a frequency of more than 30% near each internal key cavity, and exclude residues within 5Å of the active site to minimize the effect of mutation on catalytic activity. Then use Rosetta and FoldX5.0 to perform virtual saturation mutagenesis, construct a mutant library, and screen out potential mutants with ΔΔG < 0;

Before using FoldX to perform virtual saturation mutation on the enzyme, the enzyme was first corrected and optimized using the Repairpdb and Optimize modules. The number of runs was set to 3 to obtain the average result, and other settings used the default values;

Before performing Rosetta calculation of ΔΔG, FastRelax was first performed on the protein structure to relax the protein structure to a certain extent, ensuring that the atoms in the crystal structure were as close to their original positions as possible. During relaxation, the backbone and side chains of the protein structure were restricted to avoid excessive deflection, and the number of relaxations was set to 40. Then, the protein structure with the lowest energy score was selected from the 40 relaxed structures for the next energy calculation, and the mutant with a free energy difference of ΔΔG < 0 was selected.

(4) Using site-directed mutagenesis technology, the potential mutation site is mutated into the target amino acid, and the mutated sequence is sequenced for verification. It is then expressed using genetically engineered bacteria and the enzyme properties are tested after separation and purification to screen out mutants with improved thermal stability and catalytic activity.

2. A method for simultaneously evolving enzyme thermal stability and catalytic activity according to claim 1, characterized in that in step (1), the enzyme protein is subjected to molecular dynamics simulation, and each protein is subjected to 5 parallel simulations at a random initial speed, and each parallel simulation lasts 30 ns.

3. A method for simultaneously evolving enzyme thermal stability and catalytic activity according to claim 1, characterized in that step (3) first sorts according to the temperature factor B-factor, counts the top 80 amino acid residues ranked by B-factor in the representative conformation selected in step (2), and then combines the internal key cavity screened out in step (2) to count the amino acid residues with a frequency of occurrence higher than 30% near each internal key cavity under different parallels, and excludes residue sites within 5 Å of the active site to avoid the effect of mutation on catalytic activity, and then uses Rosetta and FoldX5.0 to perform virtual saturation mutation, construct a mutant library, and screen out potential mutants with ΔΔG<0.

4. A method for simultaneously evolving enzyme thermal stability and catalytic activity according to claim 2, characterized in that the molecular dynamics simulation performed in step (1) comprises the following specific steps: using Gromacs to perform molecular dynamics simulation, selecting the AMBER99 force field during the simulation, creating a cubic box while placing the protein at the center of the box, adding solvent using a water model, and then neutralizing the charge to allow the entire system to reach equilibrium; using the steepest descent method to minimize the energy of the entire system to ensure that the structure is normal, the interatomic distance is appropriate, and the geometric configuration is reasonable; then, performing a position-restricted pre-equilibrium for 400 ps under periodic boundary conditions, and heating the system temperature to 313 K using Berendsen temperature coupling; finally, performing a finished product simulation, the entire simulation process is integrated using the Leap-frog algorithm, and the long-range electrostatic potential energy is calculated using the Particle-Mesh Ewald method, and the simulation is performed five times with a random initial velocity, and the simulation time is 30 ns.

5. A method for simultaneously evolving enzyme thermal stability and catalytic activity according to claim 1, characterized in that, when the McVol program is used to calculate the internal cavities of all representative conformations in step (2), the probe radius is set to 1.1 Å, the minimum cavity volume is not less than 7 Å ³ , and the volume of water molecules is set to 18 Å ³ .

6. The lipase mutant obtained by the method of claim 1, characterized in that:

T17V/S23A/T145I, based on SEQ ID NO: 2, the threonine at position 17 is replaced by valine, the serine at position 23 is replaced by alanine, and the threonine at position 145 is replaced by isoleucine; or,

T17V/S23A/T194L, based on SEQ ID NO: 2, the threonine at position 17 is replaced by valine, the serine at position 23 is replaced by alanine, and the threonine at position 194 is replaced by leucine; or,

T17V/S23A, based on SEQ ID NO: 2, the threonine at position 17 is replaced by valine and the serine at position 23 is replaced by alanine; or

T17V/T194L, based on SEQ ID NO: 2, the threonine at position 17 is replaced by valine and the threonine at position 194 is replaced by leucine; or

S23A/T145I is based on SEQ ID NO: 2, wherein the serine at position 23 is replaced by alanine and the threonine at position 145 is replaced by isoleucine.

7. The laccase mutant obtained by the method of claim 1, characterized in that:

T102M, based on SEQ ID NO: 4, the threonine at position 102 is replaced by methionine; or

S258A: Based on SEQ ID NO: 4, the serine at position 258 is replaced by alanine.

8. A glutamine aminotransferase mutant obtained by the method of claim 1, characterized in that:

G263M, based on SEQ ID NO: 6, the glycine at position 263 is replaced by methionine; or

N284F, based on SEQ ID NO: 6, the asparagine at position 284 is replaced by phenylalanine; or

T308F is based on SEQ ID NO: 6, with the threonine at position 308 replaced by phenylalanine.