CN115181734A - Novel glucose oxidase with high thermal stability based on saturation mutation and composite evaluation design - Google Patents
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Abstract
The invention discloses a novel glucose oxidase with high thermal stability, which is designed based on saturation mutation and composite evaluation, and comprises the following steps of 1: structure acquisition, namely acquiring a three-dimensional structure of glucose oxidase from a PDB database; step 2: selecting mutation sites, calculating RMSF of each amino acid residue in a glucose oxidase structural model by using kinetic simulation, and determining the residue sites with relative flexibility by calculation; and 3, step 3: calculating the unfolding free energy (delta G) change of each mutant amino acid of the glucose oxidase by using a plurality of online server predictions in combination with mutation analysis of a plurality of thermal stability prediction algorithms; and 4, step 4: constructing and expressing the glucose oxidase mutant screened in the step 3, rapidly detecting through DSF to obtain the melting temperature of the mutant, and evaluating the thermal stability of the mutant; and 5: measuring and evaluating the activity of the glucose oxidase mutant screened in the step 3; step 6: mutant thermostability test.
Description
Technical Field
The invention relates to the technical field of glucose oxidase, in particular to novel glucose oxidase with high thermal stability, which is designed based on saturation mutation and composite evaluation.
Background
Glucose Oxidase (GOD), in the presence of oxygen, catalyzes the conversion of beta-D-Glucose to gluconic acid and hydrogen peroxide as a byproduct. Glucose oxidase is often present in microorganisms, plants and mammals. The glucose oxidase is mainly used in the fields of medicines, foods, veterinary feeds and the like to complete the tasks of removing, sterilizing and deoxidizing glucose. The low activity and yield of the glucose oxidase and the challenge of the enzyme activity determination technology are important factors for restricting the industrialization of the glucose oxidase. It is therefore of great importance to find ways to increase the stability of glucose oxidase and to reduce its industrial costs.
Like most enzymes, glucose oxidase is selective in that it can oxidize substrates and only oxidizes beta glucose and not alpha glucose, and the mass fraction of glucose oxidase is typically in the range of 130kD to 175 kD. Both subunits of glucose oxidase have binding sites for flavin adenosine trinucleotide (FAD). The redox reaction of FAD follows the mode of action of ping-pong kinetics, and subunits can only be separated under conditions of enzyme denaturation, in which case FAD is destroyed. FAD is an integral part of the independent, reversible oxidation reaction. The cofactor flavin adenosine trinucleotide in GOD is then converted to FADH2, and the reduced GOD-FADH2 is then oxidized to GOD-FAD by reaction with the oxygen molecules in the oxidation reaction, which produces hydrogen peroxide. GOD can oxidize glucose to gluconate in a reduction half-reaction step, which can be converted to gluconic acid under certain conditions.
The key factor contributing to the importance of microorganisms as a source of glucose oxidase is their rapid propagation and numerous sources, e.g., penicillium, aspergillus niger being the most important source strains. The penicillium can express GOD outside cells, and is simple to purify; the extracted enzyme preparation has high yield and wide application. And in appropriate cases, the productivity of the enzyme is excellent, facilitating mass production. However, since fermentation of aspergillus niger is mainly used for the production of gluconic acid or gluconate, glucose oxidase only occurs as a by-product. And the enzyme activity of the produced glucose oxidase is low. By taking pichia as a host, high-purity GOD can be produced, and the difficulty of further purification is reduced, so that the purified GOD can be widely applied to the industries of food, beverage, feed, analysis and detection and the like.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a novel glucose oxidase with high thermal stability, which is designed based on saturation mutation and composite evaluation, and aims to solve the problem of poor thermal stability of the glucose oxidase on the market, and provide a glucose oxidase mutant V404F with high thermal stability. Based on a 3D model of glucose oxidase, a thermal stability prediction algorithm strategy is adopted, 3 delta G calculation tools (FoldX, dDFIRE and I-Mutant 3.0) are combined to screen potential forward mutation sites, a site-specific mutation technology is adopted to carry out molecular modification and explore a thermal stability mechanism of a positive Mutant, and purified Mutant glucose oxidase is utilized to determine enzymatic parameters and enzymatic reaction kinetic parameters.
In order to realize the purpose, the invention is realized by the following technical scheme:
a novel glucose oxidase with high thermal stability, which is designed based on saturation mutation and composite evaluation, comprises the following steps:
step 1: obtaining a structure, namely obtaining the three-dimensional structure of the glucose oxidase from a PDB database, further performing dynamic simulation on the three-dimensional structure of the target glucose oxidase by using dynamic simulation software, optimizing an enzyme protein structure, eliminating unreasonable factors in the structure and obtaining the most stable three-dimensional structure;
and 2, step: selecting mutation sites, calculating RMSF of each amino acid residue in a glucose oxidase structural model by using kinetic simulation, and determining the relative flexible residue sites by calculation;
and step 3: calculating the unfolding free energy (delta G) change of each mutant amino acid of the glucose oxidase by using a plurality of online server predictions in combination with mutation analysis of a plurality of thermal stability prediction algorithms;
and 4, step 4: constructing and expressing the glucose oxidase mutant screened in the step 3, rapidly detecting through DSF to obtain the melting temperature of the mutant, and evaluating the thermal stability of the mutant;
and 5: measuring and evaluating the activity of the glucose oxidase mutant screened in the step 3;
step 6: and testing the thermal stability of the mutant, and measuring the half-life period of the purified glucose oxidase mutant under the high-temperature condition by taking glucose as a substrate to measure the change of the thermal stability. Finally, the optimal glucose oxidase mutant is obtained by integrating enzyme activity and thermal stability screening.
Wherein: the dynamic simulation in the step 1 can be realized by software such as Amber and Gromacs, generally needs to be performed in an explicit solvent environment, processes including energy minimization, constant-pressure temperature rise, pre-balancing and the like need to be performed before formal simulation, and the simulation time is generally longer than 20ns.
Wherein: the flexible domain in the protein structure in step 2 is determined by analysis of the three-dimensional structure of the protein and the RMSF throughout the kinetic simulation.
Wherein: and 3, carrying out saturation mutation on the flexible region by the glucose oxidase mutant in the step 3 through software such as Amber, discovery studio and the like.
Wherein: the folding free energy change in the step 3 can be calculated by various software or online servers, including FoldX, dDFIRE, I-Mutant 3.0. In order to ensure the reliability of the calculation result, the calculation result of various software is generally required to be analyzed and determined.
Wherein: in the step 4, the expression and purification of the glucose oxidase mutant strain adopt a general protease experimental technology, the thermodynamic stability test needs to be carried out under the medium-high temperature condition, and the temperature range is generally 25-95 ℃.
Wherein: the specific activity and enzymatic kinetic parameters of the glucose oxidase in the step 5 can be detected by a dianisidine spectrophotometry and a protein concentration determinator. As a method for measuring the enzyme activity, a3,5-dinitrosalicylic acid colorimetric method (red DNS method) or the like can be used.
The invention provides a novel glucose oxidase with high thermal stability, which is designed based on saturation mutation and composite evaluation, and has the following beneficial effects:
1. the invention provides a method for quickly and reliably designing the glucolase with high thermal stability by combining a calculation simulation theory method, various algorithm calculation tools, protein mutation design and simulation, dynamics simulation and the like.
2. The design method provided by the invention is a general enzyme design method, and can be used for dealing with the thermal stability improvement task of the glucolase in different application scenes.
3. The invention finally designs the most valuable and studied high thermal stability glucolase mutant V404F, which simultaneously maintains high thermal stability and higher level of enzyme activity.
Drawings
FIG. 1 is a three-dimensional structure model of glucose oxidase (3 QVP);
FIG. 2 is a glucose oxidase amino acid sequence;
FIG. 3 shows the free energy changes of 10 stable mutants calculated from FoldX, I-Mutant 3.0 and Ddfire;
FIG. 4 is a comparison of Tm for wild-type glucose oxidase and 10 selected mutants;
FIG. 5 shows half-lives t1/2 of wild-type glucose oxidase and 7 mutants;
FIG. 6 shows the specific activities of wild-type glucose oxidase and 7 mutants.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
(1) The three-dimensional structure of the target glucose oxidase (3 QVP) was obtained by searching the protein database (PDB database), and the corresponding amino acid sequence is shown in fig. 1 and fig. 2, respectively.
(2) The target glucose oxidase (3 QVP) was simulated by kinetic simulation software (Amber, gromacs, NAMD), and RMSF (root mean square fluctuation) was calculated for each amino acid residue in the three-dimensional structure of 3 QVP. The simulation time is 30ns, the step length is set to be 2fs, an explicit solvent model is used in the simulation process, and proper K + ions are added to keep the system electrically neutral. The atomic coordinate information is stored at a frequency of 10 ps/frame. Flexibility plays an important role in allosteric regulation such as molecular recognition, protein docking and the like, so analysis and calculation results determine relatively flexible residue positions (more potential conformations), including K13, N66, A156, E194, R230, P311, A342, D360, V404, D492, Y509 and the like, and these positions are selected as key mutation regions to carry out local virtual saturation mutation through FoldX.
(3) The Δ Δ G (change in unfolding free energy) of each mutated amino acid of glucose oxidase in the last step was calculated using various online servers (FoldX, dDFIRE, I-Mutant 3.0), and the amino acid residue with the largest change in unfolding free energy was selected as the candidate mutation site. First, the folding free energy change of the virtual saturated mutant of glucose oxidase was evaluated by FoldX, and the results of FoldX output returning Δ Δ G as a negative value indicates a stable mutation, and 30 most stable mutants were selected, and the results are shown in table 1.
TABLE 1 Δ Δ G of stable mutants calculated from FoldX
Then, the selected 30 mutants were evaluated by using I-Mutant, and the returned value of the predicted result of I-Mutant is positive and represents stable mutation, and 20 stable mutants were selected, and the results are shown in Table 2.
TABLE 2 Δ Δ G for the stable mutants calculated from FoldX and I-Mutant
Finally, the 20 screened mutants are evaluated through Ddfire, the output result returns that delta Delta G is a negative value to represent stable mutation, and 10 potential stable mutants (A156K, A156C, A156W, E194I, E194L, A342I, V404F, V404W, V404Y and Y509A) are screened out. The folding free energy changes calculated by FoldX, I-Mutant 3.0 and dDFIRE for these 10 mutants are shown in FIG. 3.
(4) And (3) constructing, expressing and purifying the 10 glucose oxidase mutants screened in the last step. The melting temperature of the mutant is obtained by rapid detection of DSF (differential scanning fluorescence) to evaluate the thermal stability of the mutant. Assay was performed using a dye SYPRO Orange, 5. Mu.L SYPRO Orange mixed with 20. Mu.L purified protein sample at a concentration of 50. Mu.g/mL, centrifuged at 4 ℃ for 6min. The real-time fluorescent quantitative PCR rate is 1 ℃/min, the temperature range is 25 ℃ to 95 ℃, the fluorescent intensity is recorded every 1 ℃ to 3 ℃, three times of parallel tests are carried out, microCal Origin is used for drawing a change curve of the fluorescent intensity along with the temperature, the mutant V404F with the optimal thermal stability is obtained by fitting a Boltzmann equation, the Tm value is improved by 6.5 ℃, and the Tm values of 10 mutants are shown in figure 4.
(5) And (3) carrying out water bath on the purified mutant protein (with the concentration of 50 mu g/mL) at 80 ℃, setting the carrying time to be 0, 30, 60, 90, 120, 150, 180 and 240min, cooling the final residual enzyme activity in ice bath for 5min, and measuring the enzyme activity by adopting a red DNS method. Mixing glucose as substrate with enzyme solution, reacting at 60 deg.C for 30min, and immediately cooling in ice bath for 5min to terminate catalytic reaction. The control experiment was supplemented with glucose solution after the reaction. After cooling, 700. Mu.L of DNS reagent is added into the reaction solution, boiled in water bath for 5min, cooled in ice bath, and then measured for corresponding light absorption value. Protein half-life t 1/2 The measurement formula of (2) is as follows:
t 1/2 ＝ln2/k d
k d = residual enzyme activity/Water bath time
The activity of the three mutants, a156C, a342I and Y509A, was found to be almost lost in the test, and therefore were excluded in subsequent half-life assays. The assay results are shown in fig. 5, where the half-life of mutant V404F is significantly increased compared to wild-type glucose oxidase.
(6) Glucose solutions with different concentrations prepared by a buffer solution are used as substrates, and the specific activity of the mutant glucose oxidase is determined based on a dianisidine spectrophotometry and a protein concentration determinator. The enzyme activity was measured according to the DNS method in step 5, and the test results are shown in FIG. 6. The specific activity of the glucose oxidase mutant V404F is improved to a certain extent compared with that of a wild type while the highest thermal stability is achieved, and the mutant is proved to realize high enzyme activity and high thermal stability.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (7)
1. A novel glucose oxidase with high thermal stability based on saturation mutation and composite evaluation design is characterized in that: the method comprises the following steps:
step 1: obtaining a structure, namely obtaining the three-dimensional structure of the glucose oxidase from a PDB database, further performing dynamic simulation on the three-dimensional structure of the target glucose oxidase by using dynamic simulation software, optimizing an enzyme protein structure, eliminating unreasonable factors in the structure and obtaining the most stable three-dimensional structure;
and 2, step: selecting mutation sites, calculating RMSF of each amino acid residue in a glucose oxidase structural model by using kinetic simulation, and determining the residue sites with relative flexibility by calculation;
and step 3: calculating the unfolding free energy (delta G) change of each mutant amino acid of the glucose oxidase by using a plurality of online server predictions in combination with mutation analysis of a plurality of thermal stability prediction algorithms;
and 4, step 4: constructing and expressing the glucose oxidase mutant screened in the step 3, rapidly detecting through DSF to obtain the melting temperature of the mutant, and evaluating the thermal stability of the mutant;
and 5: measuring and evaluating the activity of the glucose oxidase mutant screened in the step 3;
step 6: and testing the thermal stability of the mutant, and measuring the half-life period of the purified glucose oxidase mutant under the high-temperature condition by taking glucose as a substrate to measure the change of the thermal stability. Finally, the optimal glucose oxidase mutant is obtained by integrating enzyme activity and thermal stability screening.
2. The novel glucose oxidase with high thermal stability designed based on saturation mutation and composite evaluation as claimed in claim 1, wherein: the dynamic simulation in the step 1 can be realized by software such as Amber and Gromacs, generally needs to be performed in an explicit solvent environment, processes including energy minimization, constant-pressure temperature rise, pre-balancing and the like need to be performed before formal simulation, and the simulation time is generally longer than 20ns.
3. The novel glucose oxidase with high thermal stability designed based on saturation mutation and composite evaluation according to claim 1, wherein: the flexible domain in the protein structure in step 2 is determined by analysis of the three-dimensional structure of the protein and the RMSF throughout the kinetic simulation.
4. The novel glucose oxidase with high thermal stability designed based on saturation mutation and composite evaluation according to claim 1, wherein: and 3, carrying out saturation mutation on the flexible region by the glucose oxidase mutant in the step 3 through software such as Amber, discovery studio and the like.
5. The novel glucose oxidase with high thermal stability designed based on saturation mutation and composite evaluation as claimed in claim 1, wherein: the folding free energy change in the step 3 can be calculated by various software or online servers, including FoldX, dDFIRE, I-Mutant 3.0. In order to ensure the reliability of the calculation result, the calculation results of various kinds of software are generally required to be analyzed and determined.
6. The novel glucose oxidase with high thermal stability designed based on saturation mutation and composite evaluation according to claim 1, wherein: in the step 4, the expression and purification of the glucose oxidase mutant strain adopt a general protease experimental technology, the thermodynamic stability test needs to be carried out under the medium-high temperature condition, and the temperature range is generally 25-95 ℃.
7. The novel glucose oxidase with high thermal stability designed based on saturation mutation and composite evaluation according to claim 1, wherein: the specific activity and enzymatic kinetic parameters of the glucose oxidase in the step 5 can be detected by a dianisidine spectrophotometry and a protein concentration determinator. The enzyme activity can be measured by a method such as 3, 5-dinitrosalicylic acid colorimetry (red DNS method).
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