Lipase / polymer mixed lyophilized formulations for gas phase catalysis and uses thereof

Abstract

The application discloses a kind of lipase / polymer mixed freeze-dried preparation for gas phase catalysis and application thereof.The lipase / polymer mixed freeze-dried preparation is prepared from lipase and hydrophilic-hydrophobic-hydrophilic triblock polymer, wherein the melting point of the hydrophilic-hydrophobic-hydrophilic triblock polymer is 40-60 DEG C;The solution of the lipase and the hydrophilic-hydrophobic-hydrophilic triblock polymer is respectively prepared using phosphate buffer solution;The obtained solution is mixed, then pre-cooled and placed in freeze dryer for freeze-drying to obtain the freeze-dried preparation of the application.The mixed freeze-dried preparation of the application has high activity of gas phase catalysis, simple preparation method and good thermal stability.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of lipase / polymer mixed freeze-dried preparation for gas phase catalysis and its application. BACKGROUND

[0002] There are a large number of gas phase catalytic reactions in industrial scenarios. On the one hand, many raw materials are in the gas phase under normal conditions, and the reaction is directly carried out in the gas phase, avoiding the energy consumption and higher requirements for equipment caused by pressurization and cooling; on the other hand, the activation energy of the gas phase reaction is lower, and the diffusion coefficient is higher, so a higher reaction rate can be obtained.

[0003] However, the necessary water on the surface of the solid-state enzyme in the gas phase is lost, so the catalytic activity of the enzyme is often very low or even loses activity, which greatly limits the application of enzyme catalytic reaction in the gas phase. By adding water vapor to the reaction gas to maintain the basic water on the enzyme surface, the gas phase enzyme activity can be improved, but the introduction of water vapor has an adverse effect on the kinetics and selectivity of some reactions, and the increase in water vapor content also causes the stability of the enzyme to decrease.

[0004] Another possible technical path is to improve the gas-phase catalytic activity of enzymes in the form of immobilized enzymes. The current immobilization methods for improving the catalytic activity of enzymes in the gas phase mainly replace the interaction between water and enzymes through hydrogen bonds between the carrier and enzyme molecules. For example, the document Engineered surface-immobilized enzyme that retains high levels of catalytic activity in air (Journal of the American Chemical Society, 2017, 139(8): 2872-2875) reports that the immobilization of alkyl halide dehalogenase by poly (sorbitol methacrylate) makes its activity in air humidity increase by 40 times; the document Graphene oxide enabled long-term enzymatic transesterification in an anhydrous gas flux (Nature Communications, 2019, 10: 2684) immobilizes lipase by graphene oxide, which makes its activity in anhydrous conditions increase by 10 times. The document Production of Natural Esters at the Pre-Industrial Scale by Solid / Gas Biocatalysis (Biocatalysis and Biotransformation 19, 2001) reports that the immobilized lipase novozym435 is used in the gas-phase catalytic esterification of alcohol and acid in a fixed bed, the reaction temperature is 80-100℃, the conversion rate of acid reaches more than 95%, and the ester synthesis rate can reach 1 kg / (kg catalyst·h -1 )。

[0005] The above immobilized enzymes improve the catalytic activity of enzymes in the gas phase to some extent, but the preparation process is complex, and there is a risk of enzyme inactivation during the preparation process. Enzyme preparations with simple preparation process and high activity in the gas phase and their preparation technology still need to be developed. SUMMARY

[0006] The purpose of the present application is to provide a lipase / polymer mixed freeze-dried preparation for gas-phase catalytic esterification and transesterification reactions.

[0007] The lipase / polymer mixed freeze-dried preparation provided by the present application is made of lipase and hydrophilic-hydrophobic-hydrophilic triblock polymer, and the melting point of the polyhydrophilic-hydrophobic-hydrophilic triblock copolymer is 40-60℃.

[0008] The preparation method of the mixed freeze-dried preparation comprises the following steps:

[0009] S1, a phosphate buffer solution is used to prepare solutions of the lipase and the hydrophilic-hydrophobic-hydrophilic triblock polymer respectively;

[0010] S2, the solutions obtained in step S1 are mixed, then pre-cooled and placed in a freeze dryer for freeze-drying.

[0011] Preferably, the hydrophilic-hydrophobic-hydrophilic triblock polymer can be a polyoxyethylene-polyoxypropylene block copolymer; specifically, the polyoxyethylene-polyoxypropylene block copolymer can be any one or any combination of Pluronic F127, Pluronic F123, Pluronic F108 and Pluronic P68.

[0012] The lipase is a lipase of microbial origin, such as Candida antarctica lipase B, Candida rugosa lipase, etc.

[0013] Preferably, the mass ratio of the hydrophilic-hydrophobic-hydrophilic triblock polymer to the lipase is 2:1 to 6:1.

[0014] The mass fraction of water in the lipase / polymer mixed freeze-dried preparation is not more than 5%.

[0015] According to the above preparation method of the freeze-dried preparation, the content of the lipase in the solution prepared in step S1 is 1 to 4 mg / mL, and the content of the hydrophilic-hydrophobic-hydrophilic triblock polymer is 4 to 20 mg / mL.

[0016] The pH of the phosphate buffer solution can be 7.0, and the concentration can be 10 mol / m 3 ;

[0017] According to the above preparation method of the freeze-dried preparation, in step S2, the solution is mixed and stirred at 10 to 30℃, and the stirring time is not less than 1h.

[0018] The pre-cooling temperature is -40 to -20℃.

[0019] The lipase / polymer mixed freeze-dried preparation of the present application can be used for gas phase catalytic esterification and transesterification, and has the following advantages:

[0020] 1. High activity of gas phase catalysis. The catalytic activity of free lipase in the gas phase is very low, while the catalytic activity of the lipase / polymer mixed freeze-dried preparation prepared by the present application can be increased by tens of times compared with that of free lipase. For example, in Example 2, the F68 / CALB mixed freeze-dried preparation at 25℃ is 71 times the catalytic activity of free CALB at the same temperature.

[0021] 2. The preparation method is simple. The lipase / polymer mixed freeze-dried preparation of the present application can be prepared only by mixing and freeze-drying in a solution.

[0022] 3. Good thermal stability: compared with free enzymes, the interaction of the polymer carrier with the enzyme and the reduction of water increase the thermal stability of the enzyme.

[0023] The principle by which the lipase / polymer mixed freeze-dried preparation of the present application can achieve the above-mentioned effects is as follows:

[0024] The flexible conformation of enzyme molecules is the molecular basis for the enzyme to show high enzyme activity. It is generally believed that the flexible conformation necessary for enzyme catalytic activity is maintained by water molecules directly or indirectly through hydrogen bonds and other non-covalent interactions. In an aqueous solution, enzyme molecule folding follows the principle of minimum energy, which makes the molecules exposed on the surface of the enzyme have more hydrophilic polar groups and charged groups. Water molecules are adsorbed on the surface of the protein by forming hydrogen bonds with these hydrophilic groups, shielding the interaction between the polar groups on the surface of the protein molecules, so that the protein molecules have a certain flexible conformation in water, thereby showing high enzyme activity. The minimum amount of water necessary for the enzyme to maintain the flexible conformation necessary for catalytic activity is called essential water. When the essential water is lost, the enzyme conformation shows high rigidity and loses catalytic activity.

[0025] In a gaseous environment, when the partial pressure of water vapor in the air is less than the equilibrium partial pressure of the essential water combined with the enzyme, the essential water on the surface of the enzyme evaporates and is lost, and the catalytic activity of the enzyme is greatly reduced. Therefore, some methods add water vapor to improve the activity of enzymes in the gas phase. However, the present application, without losing the essence, utilizes the flexibility of polymers in a near-melting state to maintain and enhance the conformation dynamics and flexibility of enzymes through the interaction of polymers and enzymes, so that the enzyme can have high catalytic activity even in the case of loss of essential water on the surface in a gaseous environment.

[0026] Therefore, the present application selects hydrophilic-hydrophobic-hydrophilic triblock polymers, preferably Pluronic F123, F127, F68 and F108, etc. The hydrophilic-hydrophobic-hydrophilic triblock polymers not only have hydrophilic groups that interact with enzymes, but also generally have a lower melting point. The melting point of the preferred polymer of the present application is in the range of 40-60℃, which is close to the suitable temperature range for enzyme catalysis.

[0027] In the preparation process, the lipase CALB solution is mixed and stirred with the polymer solution, and the particle size of the obtained solution particles is significantly increased compared with that of the free CALB solution at the same concentration (Example 1, Figure 1), indicating that the interaction between CALB and polymer makes it possible to be encapsulated in the micelles of polymer. The lower melting point of the freeze-dried powder of CALB and polymer than that of polymer also indicates that CALB and polymer form a mixture with certain solid dispersion properties. The water content of CALB and polymer / CALB mixed freeze-dried powder can be obtained by thermogravimetric curve, and it is found that the water content in the freeze-dried powder is lower, which indicates that the polymer may replace part of the bound water of CALB through hydrogen bond interaction.

[0028] In Example 1 of the specific embodiment of the present application, the conformational dynamics in the ms time scale is detected by low-field nuclear magnetic resonance, and it is found that the catalytic activity of the enzyme is positively correlated with the transverse relaxation time, indicating that the conformational dynamics in this time scale has an important influence on the gas phase catalytic reaction; it is further found that the flexibility of the polymer is also positively correlated with the catalytic activity of the polymer / CALB, which indicates that the interaction between the flexible polymer and the enzyme strengthens the conformational dynamics of the enzyme, so that the catalytic activity is restored. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Fig. 1 is the particle size distribution of colloids in CALB solution and CALB / polymer mixed solution, wherein 1 represents CALB; 2 represents CALB / F123; 3 represents CALB / F68; 4 represents CALB / F127; 5 represents CALB / F108; and 6 represents CALB / PEG10000.

[0030] Figure 2 Fig. 5 is the low-field nuclear magnetic spectrum of CALB / Pluronic F123 and Pluronic F123.

[0031] Figure 3 Fig. 6 is a flow chart of the gas phase catalytic reaction. DETAILED DESCRIPTION

[0032] The lipase in the following examples is lipase B of Candida antarctica (CALB, produced by recombinant Aspergillus oryzae, CAS 9001-62-1, which can be purchased through commercial channels), and other chemicals can be purchased through commercial channels.

[0033] Example 1, preparation and characterization of lipase / polymer mixed freeze-dried preparation

[0034] A CALB solution of 2 mg / mL was prepared with a phosphate buffer solution (pH=7.0, concentration of 10 mol / m 3 ) ; and Pluronic F123, F127, F68 and F108 solutions of 8 mg / mL were prepared respectively.

[0035] Take 1 mL of CALB solution and 1 mL of Pluronic F123 solution, mix in a 20 °C water bath and stir with a magnetic stirrer at 50 r / min for 1 h. The particle size distribution of colloidal particles in the CALB solution and the mixed solution is measured by a Malvin laser particle size analyzer (Malvin Zetasizer ZS-90), respectively. The particle size distribution of colloidal particles in the CALB solution and the CALB / polymer mixed solution is shown in Figure 1 The above mixture is placed in a refrigerator and pre-cooled at -20 °C for 4 h, and then vacuum freeze-dried in a freeze dryer for 48 h to obtain a CALB / polymer mixture powder. The freeze-dried mixed powders of CALB and F123, F68 and F108 are prepared by the same method.

[0036] As a comparison, a polyethylene glycol PEG10000 (non-hydrophilic-hydrophobic block polymer, melting point > 60 °C) is used to prepare a CALB / polymer mixed freeze-dried preparation by the above method.

[0037] The water content of the enzyme powder is measured by measuring the weight loss curve of the enzyme powder on a thermal gravimetric analyzer (Mettler Toledo) at a rate of 10 °C / min from 25 °C to 200 °C. The differential scanning calorimetry curve of CALB, polymer, CALB / polymer mixture powder is measured on a thermal analyzer (TA / Q5000) to determine the melting point. The sample is scanned twice at a rate of 10 °C / min from 20 °C to 70 °C. The water content and melting point of the freeze-dried powder are shown in Table 1.

[0038] Low-field nuclear magnetic resonance (LF-NMR) analysis is performed on a variable-temperature nuclear magnetic resonance analyzer (Suzhou Nu Mai) with a field strength of 0.5 T, a 90° radio frequency pulse (p90) of 2.32 μs, and a 180° radio frequency pulse (p180) of 5.12 μs. The temperature is set in the range of 25-55 °C at intervals of 10 °C. The enzyme and polymer powders are placed in clean glass tubes and pre-stabilized at the target temperature for 1 h before being placed in the corresponding temperature setting nuclear magnetic spectrometer for measurement. The MSE-CPMG sequence is used to detect the conformational dynamics of proteins and polymers. In the MSE decay, τMSE=4p90+7τΦ, where τΦ=2 μs; the CPMG decay is through τCPMG=0.1 ms interval 500 echoes. All decay signals are transformed by Laplace transform, and the SIRT algorithm is used on the Nu Mai analysis software to fit the multi-exponential decay at 10000 iterations. The inversion method of all enzyme and polymer samples is consistent. The low-field nuclear magnetic resonance spectra of CALB / Pluronic F123 and Pluronic F123 are shown in Figures Figure 2 (as an example, other examples are not listed one by one). The transverse relaxation time is calculated by weighted average method according to the signal intensity. The transverse relaxation times of lipase and lipase / polymer freeze-dried preparations under different stabilization are shown in Table 2.

[0039] Table 1 Formulation, water content and melting point of lipase / polymer mixed lyophilized preparations

[0040]

[0041] Table 2 Transverse relaxation time (ms) of lipase and lipase / polymer lyophilized preparations

[0042]

[0043] From Figure 1 It can be seen that, except for the mixed solution of CALB / PEG10000, the particle size of the colloidal particles in the other CALB / polymer mixed solutions is significantly increased compared with the particle size in the free CALB solution at the same concentration, indicating that the interaction between CALB and the polymer makes it possible to be coated in the micelles of the polymer.

[0044] As can be seen from Table 1, the melting point of the lipase / polymer lyophilized preparation is reduced compared with the melting point of the polymer, indicating that the interaction between the enzyme and the polymer molecules forms a mixture with certain solid dispersion properties; compared with the CALB / Pluronic lyophilized powder, the melting point of the CALB / PEG10000 mixed lyophilized powder is reduced less than the melting point of the polymer.

[0045] As can be seen from Table 2, the transverse relaxation time of the lipase / polymer lyophilized preparation is significantly increased compared with the free lipase, indicating that the interaction between the polymer and the enzyme strengthens the conformational dynamics of the enzyme, thereby restoring the catalytic activity. The transverse relaxation time of the CALB / PEG10000 mixed lyophilized powder is increased to a lesser extent than the free lipase.

[0046] Example 2,

[0047] The lipase CALB / polymer mixed lyophilized preparation was used to catalyze the transesterification reaction of methyl propionate [CH3CH2COOCH3] and n-propanol [CH3(CH2)2OH] to synthesize propyl propionate [CH3CH2COO(CH2)2CH3]:

[0048] CH3CH2COOCH3+ CH3(CH2)2OH→ CH3CH2COO(CH2)2CH3+ CH3OH

[0049] Reaction scheme (as Figure 3) and conditions as follows: dry nitrogen gas 100 (temperature 30°C, pressure 101 kPa, absolute pressure) as carrier gas, nitrogen gas was respectively introduced into the storage tank of n-propanol (1) and methyl propionate (2, temperature 40°C) to obtain n-propanol and methyl propionate saturated nitrogen gas (101 and 102). The flow rate of n-propanol containing nitrogen gas was 0.3 L / min, and the partial pressure of n-propanol was 8 kPa; the flow rate of methyl propionate containing nitrogen gas was 0.2 L / min, and the partial pressure of methyl propionate was 23 kPa. The reactor 3 was a tubular reactor with a volume of 2 L, and a jacket 4 was provided outside to adjust and control the temperature of the reaction (the reaction temperature was 25°C, 35°C and 45°C, respectively). The lipase CALB / polymer mixed freeze-dried preparation prepared according to the embodiment (40 mg of CALB) was uniformly loaded on glass wool, and the glass wool was loaded into the tubular reactor. The gas 103 after the reaction in the reactor was absorbed in 5 with low-temperature ethanol (-4°C). The product propyl propionate was quantified by gas chromatography (using methyl butyrate as an internal standard).

[0050] The catalytic activity of the enzyme preparation was characterized by turnover frequency (TOF, min -1 ) under the same reaction conditions without loading the enzyme preparation in the reactor. The TOF of the four lipase CALB / polymer mixed freeze-dried preparations prepared according to Example 1 (compared with free enzyme) is shown in Table 3. It can be seen that the catalytic activity of the CALB / polymer mixed freeze-dried preparation prepared by the present application is significantly improved compared with free enzyme, and the maximum TOF reaches 582 min -1 (CALB / F68, 35°C), which is 83 times the catalytic activity of free enzyme at the same temperature.

[0051] Table 3 Turnover frequency (TOF, min -1 )

[0052]

[0053] Comparative Example 1,

[0054] The catalytic activity of the lipase CALB / PEG10000 mixed freeze-dried preparation prepared in Example 1, the catalytic reaction and reaction conditions were the same as in Example 2, and the results are shown in Table 4. Comparing the data in Tables 3 and 4, it can be seen that the catalytic activity of the lipase / polymer freeze-dried powder prepared by PEG10000 is improved to a certain extent compared with free CALB, but it is significantly lower than that of CALB / Pluronic freeze-dried powder, and the catalytic activity is low.

[0055] Comparative Example 2,

[0056] The solid powder of CALB and the solid powder of Pluronic F68 were directly mixed in a mass ratio of 1:3, and the TOF at different temperatures was determined according to the method and conditions in Example 2. The results are shown in Table 4. It can be seen that the direct physical mixing of the two solid powders cannot achieve the effect of the present application.

[0057] Table 4 Conversion frequency (TOF, min) of the mixture of CALB and polymer solid powders directly mixed -1 )

[0058]

[0059] Example 3, Effect of the ratio of enzyme and polymer in the mixed freeze-dried preparation of lipase CALB / polymer on catalytic activity

[0060] The enzyme preparation was prepared according to the method of Example 1, the polymer was Pluronic F68, and the catalytic reaction and the evaluation method and conditions of catalytic activity were the same as those of Example 2, and the reaction temperature was 35°C. The catalytic activity of the mixed freeze-dried preparation with different ratios of enzyme and polymer is shown in Table 5. It can be seen that the ratio of enzyme and polymer has a significant effect on catalytic activity. When the ratio of polymer is too high or too low, the catalytic activity decreases to different degrees. Therefore, the mass ratio of enzyme to polymer is preferably 1:3-8, and more preferably 1:3-4.

[0061] Table 5 Conversion frequency (TOF, min) of the mixed freeze-dried preparation of CALB / polymer -1 )

[0062]

[0063] Example 4, Synthesis of butyl acetate by ester exchange reaction of ethyl acetate and n-butanol using the mixed freeze-dried preparation of lipase CALB / polymer as catalyst

[0064] The catalytic reaction process and the evaluation method and conditions of catalytic activity were the same as those of Example 2, and the reaction temperature was 45°C. Among them, the nitrogen flow containing ethyl acetate was 50 mL / min, and the partial pressure of ethyl acetate was 30.6 kPa; the nitrogen flow containing n-butanol was 200 mL / min, and the partial pressure of n-butanol was 4.5 kPa; the amount of catalyst (CALB) loaded in the reactor was 80 mg, 100 mg and 120 mg, respectively. The mixed preparation of lipase CalB / Pluronic F68 was prepared according to the formulation and method in Example 1. The TOF of the mixed freeze-dried preparation of lipase CalB / polymer at different catalyst loading amounts (compared with the same amount of free enzyme) is shown in Table 6.

[0065] Table 6 Conversion frequency (TOF, min) of the mixed freeze-dried preparation of CALB / polymer -1 )

[0066]

[0067] As can be seen from Table 6, the lipase CALB / polymer mixed lyophilizates also showed high catalytic activity in the transesterification of ethyl acetate with n-butanol.

Claims

1. A lipase / polymer mixed lyophilized preparation for gas phase catalytic transesterification reaction, characterized in that: 1) prepared from a lipase and a hydrophilic-hydrophobic-hydrophilic triblock polymer, wherein the melting point of the hydrophilic-hydrophobic-hydrophilic triblock polymer is 40-60℃; the hydrophilic-hydrophobic-hydrophilic triblock polymer is a polyoxyethylene-polyoxypropylene block copolymer; the polyoxyethylene-polyoxypropylene block copolymer is selected from any one of Pluronic F127, Pluronic F123, Pluronic F108 and Pluronic F68; the mass ratio of the hydrophilic-hydrophobic-hydrophilic triblock polymer to the lipase is 2:1-6:1; the mass fraction of water in the mixed lyophilized preparation is not more than 5%; 2) prepared according to the following steps: S1, a phosphate buffer solution is used to prepare solutions of the lipase and the hydrophilic-hydrophobic-hydrophilic triblock polymer, respectively; S2, the solutions obtained in step S1 are mixed, then pre-cooled and placed in a freeze dryer for lyophilization to obtain the mixed lyophilized preparation; in step S2, the solutions are mixed and stirred at 10-30℃, and the stirring time is not less than 1h; the pre-cooling temperature is-40- -20℃.

2. The mixed lyophilized formulation according to claim 1, characterized in that: the lipase is a lipase of microbial origin.

3. The mixed lyophilized formulation according to claim 2, characterized in that: the lipase is Candida antarctica lipase B or Candida rugosa lipase.

4. The mixed lyophilized formulation according to any one of claims 1-3, characterized in that: in the solutions prepared in step S1, the content of the lipase is 1-4 mg / mL, and the content of the hydrophilic-hydrophobic-hydrophilic triblock polymer is 4-20 mg / mL.

5. Use of the mixed lyophilized preparation of any one of claims 1-4 in gas phase catalytic transesterification reaction.

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