CN119592636A - Catalytic system and method for catalyzing hydrolysis of halogenated organic matters by enzyme at oil-water interface - Google Patents
Catalytic system and method for catalyzing hydrolysis of halogenated organic matters by enzyme at oil-water interface Download PDFInfo
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Abstract
The invention discloses a catalytic system and a method for catalyzing hydrolysis of halogenated organic matters at an oil-water interface by enzyme. The catalytic system comprises an organic solvent, an aqueous solution and enzyme-polymer coupled enzyme, wherein the enzyme-polymer coupled enzyme is a conjugate formed by covalently combining halogenated alkane dehalogenase and amphiphilic block copolymer, and each molecule of enzyme in the enzyme-polymer coupled enzyme is connected with 2-3 molecules of amphiphilic block copolymer. The invention builds an oil-water two-phase reaction system, and concentrates enzyme at an oil-water interface through the amphiphilic block polymer and the conjugate synthesized by the enzyme, improves accessibility of a substrate in an organic solvent, avoids enzyme inactivation caused by the organic solvent, and improves hydrolysis activity of the enzyme to halogenated organic matters.
Description
Technical Field
The invention relates to a catalytic system and a method for catalyzing hydrolysis of halogenated organic matters by enzyme at an oil-water interface, belonging to the field of biocatalysis.
Background
Halogenated organics are often used widely in the chemical industry as raw materials, intermediates, solvents, etc., which also make many halogenated organics inevitably discharged into the environment, a common type of industrial pollutants. The halogenated organic pollutants have strong durability and are difficult to biodegrade, and the halogenated organic pollutants cause serious harm to the environment and human health.
At present, the treatment of halogenated organic pollutants mainly comprises an incineration method, a catalytic oxidation method, a catalytic reduction method and the like, and the application of the incineration method is wider. Incineration processes decompose halogenated organic contaminants at high temperatures, potentially producing dioxins/furans, which enhance organic toxicity. Although high temperature and high pressure incineration can inhibit the generation of dioxin/furan, the technology is costly and equipment corrosion is serious. Catalytic oxidation and chemical catalytic reduction generally require a noble metal catalyst and also require an oxidizing or reducing substance such as oxygen or hydrogen.
Dehalogenation is the primary means of reducing the toxicity of halogenated organic contaminants. The reaction is carried out at normal temperature and normal pressure by means of the hydrolytic dehalogenation catalyzed by the halogenated alkane dehalogenase, oxygen or hydrogen is not needed, and additional coenzyme/prosthetic group is not needed, so that the method is a simple and effective mode for reducing the toxicity of halogenated organic pollutants.
The enzyme-catalyzed hydrolytic dehalogenation process needs to be performed in an aqueous environment, however, most halogenated organics are poorly water-soluble materials. If catalyzed in the aqueous phase, the accessibility of the substrate is poor and the reaction rate is low, while catalyzed in the organic solvent results in substantial water removal of the enzyme and low activity of the enzyme. In addition, hydrogen halide generated by hydrolysis of halogenated organics is a polar molecule and is also difficult to dissolve in organic solvents, resulting in product inhibition. The enzyme-catalyzed hydrolysis dehalogenation reaction is most desirably carried out at the interface of the oil-water phases.
Common oil-water two-phase enzyme catalysis technology comprises catalysis by adopting free enzyme and immobilized enzyme. When free enzymes are used, an aqueous oil dispersion is typically prepared, and the enzymes are then catalyzed by dissolution in the aqueous phase. For example, the invention patent CN201610529540.8 discloses a technology for catalyzing transesterification reaction by using free lipase in an oil-water two-phase system, and the invention patent CN202311447751.3 discloses a technology for catalyzing debenzylation to synthesize a key intermediate of sitafloxacin in the oil-water two-phase system by using free laccase. The use of the immobilized enzyme can reduce the influence of the organic solvent on the enzyme to a certain extent, and simultaneously is helpful for the recovery and the reuse of the enzyme. As described in patent CN202211616988.5, a two-phase catalytic method of immobilizing enzymes in a packed column is reported. The technology loads enzyme on porous particles and fixes the porous particles on the filler, and the oil-water two-phase is operated according to the mode of a filler extraction tower to realize two-phase dispersion and make the two-phase dispersion contact with the enzyme. These techniques have difficulty in avoiding the decrease in enzyme activity caused by direct contact of the organic solvent with the enzyme, and in sufficiently contacting the enzyme in the aqueous phase with the substrate in the organic phase, resulting in lower enzyme activity.
In order to realize the catalysis of the oil-water interface more efficiently, a plurality of novel technologies for constructing an oil-water two-phase system and immobilized enzymes are also developed. For example, an immobilized enzyme Pickering emulsion reaction system, wherein the enzyme is immobilized on solid particles serving as a Pickering emulsion stabilizer, so that the enzyme is catalyzed at an oil-water interface. For example, document [ Acs Sustainable Chemistry & Engineering 2019,7 (8): 7619-7629] reports the preparation of Pickering emulsions by adsorption immobilization of lipases on mesoporous carbon spheres for use in esterification of phytosterin and alpha-linolenic acid. The literature and the invention patent CN202210057297.X report the technology of catalyzing reaction at oil-water phase interface by adopting amphiphilic macromolecule-enzyme-inorganic hybridization nanoflower as immobilized enzyme preparation. Although these methods can obtain higher enzyme activity and catalytic efficiency, the preparation technology of the immobilized enzyme needs to be very complex, the large-scale preparation is very difficult, and the stability of the immobilized carriers still faces the examination.
In summary, no additional emulsifier or auxiliary agent is used, and a relatively simple and reliable immobilized enzyme mode capable of being prepared in a large scale is adopted, so that the enzyme is enriched at the oil-water two-phase interface, and the technology for maintaining high enzyme activity and obtaining high enzyme catalytic efficiency is still to be further developed.
Disclosure of Invention
The invention aims to provide a catalytic system and a method for catalyzing hydrolysis of halogenated organic matters by enzyme at an oil-water interface, which can obviously improve the activity of the dehalogenation reaction of halogenated hydrocarbon by enzyme catalysis.
In order to construct an oil-water interface enzyme catalytic system, the invention firstly provides an enzyme-polymer coupled enzyme capable of being enriched at an oil-water interface, which is a conjugate of enzyme and amphiphilic block copolymer in covalent bonding.
Wherein the amphiphilic block copolymer is a polyoxyethylene-polyoxypropylene block copolymer, preferably a polyoxyethylene-polyoxypropylene block copolymer Pluronic F127;
The amphiphilic block copolymer may specifically be a polyoxyethylene-polyoxypropylene block copolymer.
The enzyme is halogenated alkane dehalogenase and is used for catalyzing halogenated alkane hydrolysis reaction of an oil-water interface;
Preferably, the haloalkane dehalogenase is sphingomonas paucimobilis hydrolysis dehalogenase LinB;
in the enzyme-polymer coupled enzyme, each molecule of the enzyme is connected with 2-3 molecules of the amphiphilic block copolymer.
The enzyme-polymer coupled enzyme may be prepared as follows:
And carrying out covalent connection on the obtained amphiphilic block copolymer subjected to terminal aldehyde modification and free amino groups on the surface of the enzyme in a solution.
Preferably, terminal aldehyde group modification is performed with dess-martin oxidizer;
The covalent connection comprises the steps of dissolving the enzyme in a phosphate buffer solution, adding the phosphate buffer solution into an organic solution containing the amphiphilic block copolymer modified by terminal aldehyde, stirring for reaction, and then adding a reducing agent (such as sodium cyanoborohydride) for continuous reaction.
On the basis of the enzyme-polymer coupled enzyme, the invention further provides a catalytic system for catalyzing hydrolysis of halogenated organic matters at an oil-water interface by utilizing the enzyme, which comprises an organic solvent, an aqueous solution and the enzyme-polymer coupled enzyme.
Preferably, the organic solvent is a high boiling point organic solvent which is partially miscible with water, has high solubility for the reactants and is selected from n-dodecane or paraffin oil, and further preferably n-dodecane.
The aqueous solution is pure water, glycine-NaOH buffer solution or phosphate buffer solution;
the volume ratio of the organic solvent to the aqueous solution is 1:20-1:5.
The catalytic system provided by the invention can catalyze the hydrolysis of halogenated organic matters, and can be carried out according to the following steps:
S1, dissolving the enzyme-polymer coupled enzyme in the aqueous solution of the catalytic system to obtain an aqueous phase;
s2, dissolving halogenated organic matters into the organic solvent of the catalytic system to serve as an oil phase;
s3, mixing the oil phase and the water phase to form an oil-water dispersion system, and carrying out enzyme catalysis on the hydrolysis of the halogenated organic matters at an oil-water interface.
Preferably, the halogenated organic matter is halogenated alkane, including chain halogenated hydrocarbon, cyclic halogenated hydrocarbon, halogenated ester, halogenated nitrile and halogenated amide containing benzene ring structure.
Preferably, the mass concentration of the enzyme-polymer coupled enzyme (based on the mass of the enzyme contained therein) and the mass concentration ratio of the halogenated organic compound are 1:200-1:1000, and the ratio affects the reaction rate.
The oil-water interface enzyme catalyzed reaction is characterized in that a substrate (reactant) of the enzyme catalyzed reaction is a water insoluble substance and is dissolved in an organic solvent for reaction, a product of the enzyme catalyzed reaction is an organic solvent insoluble substance and needs to be dissolved in an aqueous solution, and the catalyzed reaction occurs at an oil-water interface.
The preparation modes of the oil-water two-phase of the catalytic system are various, namely an emulsion formed by suspending an organic solvent (aqueous solution) in the aqueous solution (organic solvent) in the form of liquid drops, and an oil-water dispersion system formed by forming a flowing liquid film on the surface of a filler by the aqueous solution (organic solvent) and attaching the organic solvent to the surface of the liquid film of the aqueous solution.
The oil-water interface enzyme catalyzed reaction has the following advantages:
1) By constructing an oil-water two-phase reaction system and by coupling the amphiphilic block polymer and enzyme synthesis, the enzyme is enriched at the oil-water interface, thereby improving the accessibility of the substrate in the organic solvent, avoiding the enzyme inactivation caused by the organic solvent and improving the hydrolytic activity of the enzyme on halogenated organic matters.
2) By constructing an oil-water two-phase reaction system, hydrogen halide which is an enzyme catalytic reaction product is dissolved in an aqueous solution, so that the reduction of the enzyme catalytic activity caused by the retention of halogen ions in an enzyme activity pocket is avoided.
3) The conjugate synthesized by the amphiphilic block polymer and the enzyme is used as a surface active substance to stabilize an oil-water interface, and no additional surfactant is needed to be added to stabilize an oil-water dispersion system.
The hydrolysis reaction equation of halogenated hydrocarbon RX (R represents hydrocarbon chain and X represents halogen) catalyzed by halogenated hydrocarbon dehalogenase is RX+H 2 O=R-OH+HX, wherein the halogenated hydrocarbon RX is usually insoluble in water, and needs to be dissolved in an organic solvent for reaction, and water is needed for reaction, and the HX generated by the reaction is insoluble in the organic solvent and also needs to be dissolved in an aqueous solution. Therefore, the reaction needs to construct an oil-water two-phase dispersed reaction system. In general, in an oil-water two-phase dispersion system, the free enzyme is generally uniformly dispersed in the water phase, so that accessibility to the organic reaction substrate RX is poor, and the apparent catalytic activity of the enzyme is low. The enzyme is combined with the amphiphilic block copolymer, and the constructed enzyme-copolymer conjugate can be distributed at an oil-water interface (but is mainly positioned at one side of a water phase), so that the accessibility of the enzyme to a substrate RX in an organic phase is greatly improved. Meanwhile, the amphiphilic enzyme-copolymer conjugate has the function of a surfactant and can stabilize an oil-water interface.
In the invention, through the specific selection of the polymer and the organic solvent, the protein main body part of the coupled enzyme molecule is positioned in the water environment instead of the organic solvent at the oil-water two-phase interface, so that the structural flexibility and activity of the enzyme are ensured. For this purpose, for the amphiphilic copolymer constituting the enzyme-copolymer conjugate, the amphiphilic polymer Pluronic F127 (hydrophilic-lipophilic balance coefficient HLB of 22) with slightly higher hydrophilicity was selected in the present invention. Meanwhile, an organic solvent is preferred, and besides better dissolving the halogenated hydrocarbon substrate, the preferred organic solvent has the characteristics of 1) less influence on the enzyme activity, 2) proper viscosity and density and easiness in constructing a more stable oil-water dispersion system. 3) The copolymer linked to the enzyme has a suitable solubility so that the coupled enzyme is mainly enriched at the oil-water interface and mainly on the aqueous side. According to the invention, through molecular simulation of the distribution of polyoxyethylene-polyoxypropylene block copolymers of different types on an oil-water interface, the fact that Pluronic F127 with poor solubility in organic phase n-dodecane is adopted can ensure that the main body part of the coupling enzyme molecules is distributed in a water phase, so that the water environment necessary for enzyme is ensured. The laser confocal micrograph of panel a in FIG. 1 shows that the fluorescently labeled conjugated enzyme is distributed at the interface of the oil-water phases and predominantly on the aqueous phase side.
In addition, the product HX generated by the halogenated hydrocarbon dehalogenation reaction can stay in an active pocket of the enzyme to show obvious product inhibition, and the main body of the coupled enzyme molecule is placed in an aqueous solution to be beneficial to the separation of HX and reduce the inhibition of the product.
Drawings
FIG. 1 is a confocal microscope photograph of the enzyme in oil-water two-phase distribution, wherein a graph a shows rhodamine B label LinB-PluronicF127 distributed in n-dodecane-glycine buffer, and B graph B shows FITC label LinB-PluronicF127 distributed in toluene-glycine buffer.
FIG. 2 shows the catalytic activity of the coupled enzyme in an oil-water two-phase reaction system of n-dodecane-glycine buffer.
FIG. 3 shows the catalytic activity of the coupled enzyme in a toluene-glycine buffer oil-water two-phase reaction system.
FIG. 4 shows the hydrolysis rate of 1-bromobutane by coupled enzyme catalysis in an oil-water two-phase reaction system.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
In the examples described below, the haloalkane dehalogenase was Sphingomonas paucimobilis hydrolytic dehalogenase B (LinB, commercially available) and each of the other chemicals was commercially available.
Example 1 Synthesis of LinB-Pluronic F127 conjugate
First, the polymer Pluronic terminal aldehyde group modification 10g Pluronic F127 was dissolved in 500mL of methylene chloride, and 5 times the amount of the substance (1.7 g) of dess-Martin oxidant was added thereto, followed by stirring at room temperature for 24 hours. Removing most of the organic solvent by using a rotary evaporator to obtain white turbid liquid, adding cold diethyl ether to form precipitation, filtering, collecting and washing the precipitation with the cold diethyl ether for multiple times to remove excessive dess-Martin oxidant, thus obtaining the terminal aldehyde modified Pluronic F12, and measuring the aldehyde modification rate by using a Purpald method, wherein the aldehyde modification rate is about 30%.
The haloalkane dehalogenase LinB was dissolved in 10mmol/L phosphate buffer (ph=8.0) to give a solution with an enzyme content of 2 mg/L. And (3) dissolving the solid powder of the terminal aldehyde Pluronic F127 in the phosphate buffer solution, and ensuring that the molar ratio of the terminal aldehyde group of the finally added Pluronic F127 to the surface amino group of LinB is 1.5-2. Then stirring at room temperature for 2h, adding Pluronic F127, adding 10% of reducing agent sodium cyanoborohydride by mass, and stirring at room temperature for 12h. The product was isolated by a protein purification system to give LinB-Pluronic F127 conjugate.
The grafting rate of LinB-Pluronic F127 molecules was determined and calculated using the TNBS method, with an average of 2 Pluronic F127 molecules grafted per LinB enzyme molecule.
Example 2, construction of LinB-Pluronic F127 conjugate oil-Water reaction System and catalytic Properties
The distribution of LinB-Pluronic F127 at the oil-water interface was observed with a laser confocal microscope.
The rhodamine B labeled LinB-Pluronic F127 is dissolved in 0.1mol/L glycine buffer (50 mmol/L, pH=9.8) by using a fluorescent dye rhodamine B labeled LinB-Pluronic F127, and an enzyme-containing solution with the enzyme mass content of 0.05mg/mL is obtained. 200 mu L of the enzyme-containing solution is dripped into a 96-well plate with optical precision, and then 200 mu L of n-dodecane (without reactants) is continuously dripped into the 96-well plate to construct an oil-water two-phase system. And carrying out horizontal slice scanning on the sample in the pore plate along the oil-water interface direction under the z-stack mode of the laser confocal microscope, wherein the excitation wavelength is 595nm, and the detection excitation wavelength is 561nm. The obtained confocal laser photograph is shown in a graph in FIG. 1, wherein red fluorescent spots are rhodamine B marks LinB-PluronicF127, and the fluorescent spots are focused at an oil-water interface. By contrast, the same oil-water two-phase system was constructed with the same mass content of free LinB enzyme solution, and it was seen that the free LinB enzyme was evenly distributed in glycine buffer.
In a two-phase reaction system of n-dodecane-glycine buffer solutions with different oil-water ratios, the relative activity (relative to the enzyme activity of free enzyme in the water phase) of the coupled enzyme is measured by taking 1-bromobutane as a substrate. The reaction produced bromide was measured by mercury thiocyanate-ferric ammonium sulfate colorimetry to determine the reaction rate (the reaction rate was measured for 30min at the beginning of the reaction). The results are shown in FIG. 2. It can be seen that by constructing LinB-Pluronic F127 conjugate, the relative enzyme activity of LinB in an oil-water two-phase reaction system is remarkably improved. The kinetic constants K m and K cat of the enzyme-catalyzed reactions were determined and the results are shown in Table 1 (oil to water ratio 1:10). It can be seen that the K m of LinB-Pluronic F127 conjugate was significantly reduced compared to the free enzyme, indicating significantly improved accessibility to the substrate, while K cat was similar to K cat of the free enzyme in the aqueous phase, indicating significantly reduced enzyme activity.
TABLE 1 kinetic constants of enzyme-catalyzed reactions
| Enzyme/reaction system | Km(mmol/L) | kcat(s-1) |
| Free enzyme/glycine buffer | 0.59 | 6.26 |
| Free enzyme/n-dodecane-glycine buffer | 22.14 | 4.70 |
| Coupling enzyme/n-dodecane-glycine buffer | 3.21 | 7.39 |
By adopting the same method, paraffin oil is used as a solvent to construct an oil-water two-phase system (the oil-water ratio is 1:10), and the LinB-Pluronic F127 coupling enzyme is also enriched in the water phase. The enzyme activities were measured in the same manner as above, and the relative enzyme activities of the coupled enzyme and the free enzyme in the oil-water two-phase system (with the free enzyme activity in the pure water phase being 100%) were 114% and 72%, respectively. It can be seen that the catalytic activity is also significantly improved in paraffin oil-water systems.
Comparative example 1 influence on organic solvent
Toluene with high Pluronic F127 solubility is used as an organic solvent to construct an oil-water two-phase reaction system, and the composition of the water phase and the proportion of the oil-water two phases are the same as in example 2.
Using the fluorescent dye FTIC label LinB-Pluronic F127, the distribution of the coupling enzyme LinB-Pluronic F127 in both oil and water phases was observed by laser confocal microscopy as in example 2, as shown in FIG. 1b, and it was seen that the coupling enzyme LinB-Pluronic F127 was distributed in large amounts in the organic solvent and not enriched at the oil-water interface. The enzyme activity was measured as in example 2, and the results (FIG. 3) showed that the catalytic activity of the coupled enzyme in the oil-water two-phase system was almost the same as that of the free enzyme, which was significantly lower than that of example 2 using n-dodecane as the oil phase.
The above results show that the solubility of the organic solvent in the system for constructing the oil-water two-phase is an important factor affecting the distribution of the coupling enzyme in the reaction system, and the organic solvent with lower solubility to the selected copolymer must be selected according to the selected copolymer so as to ensure that the coupling enzyme is enriched on the oil-water interface and the main body of the coupling enzyme exists in the water phase in the constructed oil-water two-phase reaction system.
Comparative example 2 influence on Polymer
A polymer-enzyme conjugate was prepared as in example 1 using the more lipophilic polyoxyethylene-polyoxypropylene block copolymer Pluronic P123 (Pluronic P123 having a hydrophilic-lipophilic balance of HLB of 8 and Pluronic F127 having an HLB of 22) and the non-amphiphilic polyethylene glycol PEG10K (relative molecular mass 10000, close to Pluronic F127). An oil-water two-phase reaction system was constructed in the same manner as in example 2, wherein the ratio of the oil-water two phases was 1:10, and the enzyme activities and kinetic constants K m and K cat were measured in the same manner as in example 2, and the results are shown in Table 2. It can be seen that the polymer with strong lipophilicity is selected, and the relative enzyme activity in the oil-water two-phase catalytic system is obviously reduced due to the fact that the enzyme-polymer main body is dissolved in the oil phase, and the polymer-enzyme conjugate is constructed by adopting the non-amphiphilic polymer PEG10K, so that the polymer-enzyme conjugate cannot be enriched at an oil-water interface, has poor accessibility to a substrate, and the relative enzyme activity in the oil-water two-phase catalytic system is obviously reduced (even lower than that of free enzyme).
Table 2 comparison of enzyme activities for the preparation of enzyme-polymer conjugates using different polymers
| Polymer | HLB | Relative enzyme Activity (%) | Km(mmol/L) | kcat(s-1) |
| Pluronic P123 | 8 | 89 | 6.33 | 5.88 |
| Pluronic F127 | 22 | 121 | 3.21 | 7.39 |
| PEG10K | — | 47 | 18.32 | 3.47 |
EXAMPLE 3 construction of LinB-Pluronic F127 conjugate oil-Water reaction System and catalysis of 1-bromobutane hydrolysis
50Mmol/L glycine buffer (pH=9.75) was used as the aqueous phase and n-dodecane was used as the oil phase. The volume ratio of the oil phase to the water phase is 1:10.LinB-Pluronic F127 conjugate was dissolved in glycine buffer at a level of 0.03mg/L (based on LinB mass) and 1-bromobutane was dissolved in n-dodecane at a level of 15mg/mL. N-dodecane is dripped into glycine buffer solution and stirred slowly to form an oil-water dispersion system with oil drops dispersed in the glycine buffer solution. The hydrolysis rate of 1-bromobutane was measured, and the results are shown in FIG. 4.
In contrast, linB-Pluronic F127 conjugate was replaced with the same amount of free LinB, with the same conditions. It can be seen that by constructing an oil-water reaction system and simultaneously using LinB-Pluronic F127 conjugate to realize interfacial catalysis, the hydrolysis rates of 60min and 90min are improved by 21% and 17% compared with the hydrolysis rate separation catalyzed by free LinB enzyme in the water phase.
Example 4, linB-Pluronic F127 catalyzed hydrolysis of 4-chloro-1-butene by an enzyme-coupled oil-water reaction System
An oil-water two-phase reaction system and a construction method thereof are the same as in example 3.
The content of the substrate 4-chloro-1-butene in the organic phase was 10mg/mL, 15mg/mL and 25mg/mL, respectively, and the content of LinB-Pluronic F127 (based on LinB mass) in the aqueous phase was 0.03mg/L. In contrast, 4-chloro-1-butene hydrolysis was catalyzed in glycine buffer at the same level of free LinB, except for the same conditions. The hydrolysis rate was measured after 90 minutes of hydrolysis reaction, and the results are shown in Table 3. The conversion rate of the coupled enzyme oil-water two-phase reaction is higher than that of the free enzyme water phase reaction for different substrate concentrations, and the difference is more clear when the substrate concentration is lower.
TABLE 34 hydrolysis percentage of chloro-1-butene (%)
| Substrate content (mg/mL) | Coupled enzyme oil-water two-phase system | Free enzyme aqueous phase reaction system |
| 10 | 28 | 17 |
| 15 | 29 | 21 |
| 25 | 19 | 16 |
Example 5, influence of the oil-Water volume ratio in LinB-Pluronic F127 conjugate oil-Water reaction System
The oil-water reaction system was the same as in example 2, wherein the volume ratio of n-dodecane solvent to carbonate buffer was 1:20, 1:10, 3:20, and 1:5 in order.
The catalytic activity of LinB-Pluronic F127 was determined as in example 2 and the results are shown in Table 4. The catalytic activity is obviously reduced after the oil-water ratio is more than 15%.
TABLE 4 influence of the oil-water volume ratio on the catalytic Activity
| Volume ratio of oil to water | Specific activity | Km(mmol/L) | kcat(s-1) |
| 1:20 | 126% | 1.25 | 7.83 |
| 1:10 | 119% | 3.21 | 7.39 |
| 3:20 | 112% | 5.05 | 6.89 |
| 1:5 | 93% | 10.01 | 6.63 |
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