CN114940984A - Eutectic solvent prepared based on organic acid hydrogen bond donor and method for improving catalytic activity of omega-transaminase - Google Patents
Eutectic solvent prepared based on organic acid hydrogen bond donor and method for improving catalytic activity of omega-transaminase Download PDFInfo
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Abstract
The invention discloses a eutectic solvent prepared based on an organic acid hydrogen bond donor and a method for improving catalytic activity of omega-transaminase, and particularly belongs to the technical field of catalysts. And S02, mixing the eutectic solvent and the buffer solution to obtain the DES-buffer solution. The eutectic solvent prepared by the method can promote the dissolution of the substrate to a large extent. In the process of the enzymatic reaction of the omega-transaminase, the problem of low catalytic efficiency caused by poor solubility of reaction substrates in the enzymatic reaction is solved, and the catalytic activity, thermal stability, storage time and other enzyme activity properties of the omega-transaminase are improved. The DES-buffer solvent disclosed by the invention is simple to prepare, mild in reaction conditions, non-toxic and degradable, conforms to the development of green chemistry, and has a wide industrial production prospect.
Description
Technical Field
The invention belongs to the technical field of catalysts, and particularly relates to a eutectic solvent prepared based on an organic acid hydrogen bond donor and a method for improving catalytic activity of omega-transaminase.
Background
Green chemistry and sustainable chemistry have been the hot and leading direction in the chemical research field, and research and development of green solvents and catalysts are more important core content. In some chemical reactions, complicated operation steps, harsh reaction conditions, expensive catalysts and severely polluted organic solvents have become major resistance restricting further development and application thereof. The enzyme catalysis reaction has the obvious characteristics of mild condition, environmental friendliness, high efficiency and specificity and the like, and is an ideal green catalyst. The biological enzyme catalyst has the characteristics of natural source, mild reaction conditions and less side reactions, so that the reaction process is more environment-friendly, and the reaction process and products are safer.
Chiral amines are an important pharmaceutical intermediate and have wide applications in the agricultural and chemical industries, and at present, almost half of active pharmaceutical ingredients contain at least one chiral amine unit, so that the chiral amines become very valuable structural blocks in the pharmaceutical industry. The industrial production of amines relies mainly on the metal-catalyzed hydrogenation of enamides, but this process is expensive, dangerous, and polluting, and the amines produced are mostly achiral. The synthesis of chiral amine by the biological enzyme method is gradually concerned with economy, environmental protection, mild reaction conditions, high preparation efficiency and the like, wherein the chiral amine is asymmetrically synthesized by catalyzing corresponding precursor ketone by using omega-aminotransferase, and becomes one of the most potential alternative means for the industrial production of the chiral amine by using the excellent stereoselectivity of the chiral amine.
The biocatalysts currently available for chiral amine synthesis mainly include amine dehydrogenase, artificial metalloenzyme, monoamine oxidase, imine reductase, ω -transaminase, and the like. Wherein, the omega-transaminase uses a former chiral ketone compound as a substrate, the biosynthesis of chiral amine is realized through the stereoselective transamination, and the product has high purity and relatively better industrial application prospect.
Omega-aminotransferases (transaminases) are a class of transferases that require reversible catalysis of amino acid to ketone transfers via pyridoxal phosphate (PLP), are ubiquitous in animals, plant tissues and microorganisms, and can reversibly transfer amino groups derived from amino donors to ketone amino acid acceptors.
In the case of transaminase-catalyzed reactions, this type of reaction does not give satisfactory results when carried out in conventional buffers, due to the constraints of certain conditions (low enzyme stability, severe hydrolysis, poor solubility, etc.).
Disclosure of Invention
The invention aims to provide a eutectic solvent prepared based on an organic acid hydrogen bond donor and a method for improving the catalytic activity of omega-transaminase. In the process of the enzymatic reaction of the omega-transaminase, the problem of low catalytic efficiency caused by poor solubility of reaction substrates in the enzymatic reaction is solved, and the catalytic activity, thermal stability, storage time and other enzyme activity properties of the omega-transaminase are improved. The DES-buffer solvent disclosed by the invention is simple to prepare, mild in reaction conditions, non-toxic and degradable, conforms to the development of green chemistry, and has a wide industrial production prospect.
In order to achieve the purpose, the invention provides the following technical scheme:
the eutectic solvent prepared on the basis of the organic acid hydrogen bond donor comprises a hydrogen bond donor reagent and a hydrogen bond acceptor reagent, wherein the hydrogen bond acceptor reagent is selected from at least one of choline chloride or betaine, and the hydrogen bond donor reagent is selected from at least one of formic acid, oxalic acid, citric acid and tartaric acid.
Preferably, the molar ratio of hydrogen bond donor agent to hydrogen bond acceptor agent is 1:1 or 1:2 or 2:1 or 2: 3. The invention also provides a method for improving the catalytic activity of the omega-transaminase, which comprises the following steps:
s01, mixing the hydrogen bond donor reagent and the hydrogen bond acceptor reagent according to a preset proportion, heating to 70-80 ℃, reacting for 3-8h to obtain a clear solution state, and obtaining the eutectic solvent.
And S02, mixing the eutectic solvent and the buffer solution to obtain the DES-buffer solution.
Preferably, in step S01, the heating temperature is 75 ℃.
Preferably, in step S01, the reaction time is 2-3 h.
Preferably, in step S02, the buffer solution is at least one selected from a mixture of choline chloride and formic acid at a molar ratio of 1:2, a mixture of choline chloride and acetic acid at a molar ratio of 1:2, a mixture of choline chloride and oxalic acid at a molar ratio of 1:2, and a mixture of choline chloride and glycine at a molar ratio of 1: 2.
Preferably, in step S02, the buffer solution is selected from a mixed solution of choline chloride and acetic acid at a molar ratio of 1:1, and the molar ratio of DES to the buffer solution is 1: 9.
Preferably, in step S02, the ratio of the eutectic solvent to the buffer solution is 1-10: 1-10.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention solves the problem of poor solubility of reaction substrates in enzymatic reaction, promotes the transfer of amino groups between amino acids and ketones, and optimizes the reaction environment by using the eutectic solvent.
2. Compared with the traditional buffer solvent, the preparation method firstly carries out the preparation of the DES-buffer solvent, and then the traditional buffer solvent is changed into the DES-buffer solvent in the process of the omega-transaminase, and the eutectic solvent has the advantages of low volatility, low flammability, low melting point, low vapor pressure, high solubility, strong stability, easy storage and synthesis, low raw material cost and the like.
3. The eutectic solvent provided by the invention is prepared by mixing choline chloride, formic acid, choline chloride, acetic acid, choline chloride and oxalic acid according to a certain proportion, heating and stirring at 80 ℃ for more than 3 hours until colorless transparent liquid is formed. After cooling to room temperature, the mixture was dried in a drying dish. The low cosolvent has the advantages of low price, no toxicity, degradability, easy preparation, easy separation of reaction products and the like, and is an important application conforming to green chemistry.
4. The choline chloride, the formic acid, the acetic acid and the oxalic acid are all low in price, and the eutectic solvent is non-toxic and degradable, so that the buffer solvent has great advantages compared with the traditional buffer solvent.
Drawings
FIG. 1 is a high performance liquid chromatogram of acetophenone.
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. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The invention provides a eutectic solvent prepared based on an organic acid hydrogen bond donor and a method for improving catalytic activity of omega-transaminase, solves the problem of poor solubility of reaction substrates, promotes transfer of amino groups between amino acid and ketone, and optimizes a reaction environment by using the eutectic solvent. The eutectic solvent is prepared by mixing choline chloride and acetic acid with purity of more than or equal to 98% according to the proportion of 1: 2.
The method for preparing the low cosolvent comprises the following steps of mixing choline chloride and acetic acid with the purity of more than or equal to 98% in a blue-neck flask according to the proportion of 1:2, and stirring the mixture by a magnetic stirrer at the temperature of 70-80 ℃ for 2 hours until colorless transparent liquid is formed. After cooling to room temperature, collected in a desiccator for drying. And mixing the dried eutectic solvent with a traditional buffer solvent in a ratio of 1:9 to obtain the DES-buffer solution.
The embodiment provides a eutectic solvent prepared based on an organic acid hydrogen bond donor and a method for improving catalytic activity of omega-transaminase, and the specific method comprises the following steps:
a certain amount of the strain was inoculated in LB liquid medium containing kanamycin and cultured overnight in a shaker at 37 ℃. Subsequently, the bacterial suspension was inoculated into 200mL of LB liquid medium containing kanamycin and cultured on a shaker for 2 to 4 hours. When OD concentration reaches the index, IPTG is added for induction for 18-20 h.
The cells after induction expression were stored in a centrifuge tube, and the cells were collected by a refrigerated centrifuge. The somatic cells were resuspended in imidazole, the cells were disrupted by a high-pressure homogenizer, and the supernatant was taken. The crude enzyme is filtered by a filter membrane, and then the recombinant protein is separated and purified by using an affinity chromatography column. And (3) concentrating the obtained enzyme solution to remove imidazole components, and replacing the traditional buffer solution with DES-buffer solution in the concentration process to obtain the omega-transaminase. Taking the diluted purified enzyme solution, adding 180 mu L of reaction substrate into an EP tube, mixing, taking phosphate buffer solution as a control, carrying out three groups of parallel experiments, and reacting for 3 min. The specific activity of the omega-transaminase and the catalytic efficiency of the omega-transaminase are calculated by detecting the peak area at 245nm by a high performance liquid chromatograph.
Example 2
The invention provides a eutectic solvent prepared based on an organic acid hydrogen bond donor and a method for improving catalytic activity of omega-transaminase, solves the problem of poor solubility of reaction substrates, promotes transfer of amino groups between amino acid and ketone, and optimizes a reaction environment by using the eutectic solvent. The eutectic solvent is prepared by mixing choline chloride and formic acid with purity of more than or equal to 98% according to the proportion of 1: 2.
The method for preparing the low cosolvent comprises the following steps of mixing choline chloride and formic acid with the purity of more than or equal to 98% in a blue-neck flask according to the ratio of 1:2, and stirring the mixture by a magnetic stirrer at the temperature of 70-80 ℃ for 2 hours until colorless transparent liquid is formed. After cooling to room temperature, collected in a desiccator for drying. And mixing the dried eutectic solvent with a traditional buffer solvent in a ratio of 1:9 to obtain the DES-buffer solution.
The embodiment provides a eutectic solvent prepared based on an organic acid hydrogen bond donor and a method for improving catalytic activity of omega-transaminase, and the specific method is as follows:
a certain amount of the strain was inoculated in LB liquid medium containing kanamycin and cultured overnight in a shaker at 37 ℃. Subsequently, the bacterial suspension was inoculated into 200mL of LB liquid medium containing kanamycin and cultured on a shaker for 2 to 4 hours. When OD concentration reaches the index, IPTG is added for induction for 18-20 h.
The cells after induction expression were stored in a centrifuge tube, and the cells were collected by a refrigerated centrifuge. The somatic cells were resuspended in imidazole, the cells were disrupted by a high-pressure homogenizer, and the supernatant was taken. The crude enzyme is filtered by a filter membrane, and then the recombinant protein is separated and purified by using an affinity chromatography column. And (3) concentrating the obtained enzyme solution to remove imidazole components, and replacing the traditional buffer solution with a DES-buffer solution in the concentration process to obtain the omega-transaminase. Taking the diluted purified enzyme solution, adding 180 mu L of reaction substrate into an EP tube, mixing, taking phosphate buffer solution as a control, carrying out three groups of parallel experiments, and reacting for 3 min. And detecting the peak area at 245nm by using a high performance liquid chromatograph, calculating the specific activity of the omega-transaminase, and calculating the catalytic efficiency of the omega-transaminase.
Example 3
The invention provides a eutectic solvent prepared based on an organic acid hydrogen bond donor and a method for improving catalytic activity of omega-transaminase, solves the problem of poor solubility of reaction substrates, promotes transfer of amino groups between amino acid and ketone, and optimizes a reaction environment by using the eutectic solvent. The eutectic solvent is prepared by mixing choline chloride and oxalic acid with the purity of more than or equal to 98% according to the proportion of 1: 2.
The method for preparing the low cosolvent comprises the steps of mixing choline chloride and oxalic acid with the purity of more than or equal to 98% in a blue-neck flask according to the proportion of 1:2, and stirring the mixture by using a magnetic stirrer at the temperature of 70-80 ℃ for 2 hours until colorless transparent liquid is formed. After cooling to room temperature, collected in a desiccator for drying. And mixing the dried eutectic solvent with a traditional buffer solvent in a ratio of 1:9 to obtain the DES-buffer solution.
The embodiment provides a eutectic solvent prepared based on an organic acid hydrogen bond donor and a method for improving catalytic activity of omega-transaminase, and the specific method comprises the following steps:
a certain amount of the strain was inoculated in LB liquid medium containing kanamycin and cultured overnight in a shaker at 37 ℃. Subsequently, the bacterial suspension was inoculated into 200mL of LB liquid medium containing kanamycin and cultured on a shaker for 2 to 4 hours. When OD concentration reaches the index, IPTG is added for induction for 18-20 h.
The cells after induction expression were stored in a centrifuge tube, and the cells were collected by a refrigerated centrifuge. The somatic cells were resuspended in imidazole, the cells were disrupted by a high-pressure homogenizer, and the supernatant was taken. The crude enzyme is filtered by a filter membrane, and then the recombinant protein is separated and purified by using an affinity chromatography column. And (3) concentrating the obtained enzyme solution to remove imidazole components, and replacing the traditional buffer solution with a DES-buffer solution in the concentration process to obtain the omega-transaminase. Taking the diluted purified enzyme solution, adding 180 mu L of reaction substrate into an EP tube, mixing, taking phosphate buffer solution as a control, carrying out three groups of parallel experiments, and reacting for 3 min. The specific activity of the omega-transaminase and the catalytic efficiency of the omega-transaminase are calculated by detecting the peak area at 245nm by a high performance liquid chromatograph.
Results of this example:
through detection, the method solves the problem of poor solubility of reaction substrates in the enzymatic reaction, promotes the transfer of amino groups between amino acids and ketones, and optimizes the reaction environment by using the eutectic solvent. And the catalytic activity, the thermal stability and the storage time of the omega-transaminase are greatly improved.
The application of the invention is that in the reaction catalyzed by omega-transaminase, because of the restriction of certain conditions (low enzyme stability, serious hydrolysis, poor solubility, etc.), the reaction of the type is carried out in the traditional buffer solution, and the satisfactory result cannot be obtained. In order to solve the problems of low catalytic efficiency, poor stability and the like and promote the transfer of amino groups between amino acid and ketone, the invention optimizes the reaction environment by using the eutectic solvent.
As shown in FIG. 1, the chromatogram is a high performance liquid chromatogram for detecting acetophenone at 245 nm. As shown in the figure, the peak appeared about 8.7min is the target peak of acetophenone, and the amount of acetophenone generated is judged according to the peak area.
The specific activity of the enzyme was calculated by the following formula:
wherein, Δ OD245 is a change in absorbance per minute at 245nm
V total volume of enzyme reaction System (200. mu.L)
d optical path (0.6cm)
ε is the molar absorptivity (12000L/(mol. cm))
VE volume of enzyme in reaction System (20. mu.L)
[E] Concentration of enzyme (mg/mL)
The relationship between the reaction rate [ V ] and the substrate concentration [ S ] is shown in the formula
Catalytic efficiency kcat/Km, where [ E ] is the molar concentration of the enzyme.
k cεt ＝V max /[E]。
Claims (8)
1. The eutectic solvent prepared on the basis of the organic acid hydrogen bond donor is characterized by comprising a hydrogen bond donor reagent and a hydrogen bond acceptor reagent, wherein the hydrogen bond acceptor reagent is selected from at least one of choline chloride or betaine, and the hydrogen bond donor reagent is selected from at least one of formic acid, oxalic acid, citric acid and tartaric acid.
2. The eutectic solvent prepared based on organic acid hydrogen bond donor, according to claim 1, wherein the molar ratio of the hydrogen bond donor agent to the hydrogen bond acceptor agent is 1:1 or 1:2 or 2:1 or 2: 3.
3. A method for improving catalytic activity of omega-transaminase, which is characterized by comprising the following steps:
s01, mixing the hydrogen bond donor reagent and the hydrogen bond acceptor reagent according to a preset proportion, heating to 70-80 ℃, reacting for 3-8h to obtain a clear solution state, and obtaining a eutectic solvent;
and S02, mixing the eutectic solvent and the buffer solution to obtain the DES-buffer solution.
4. The method of claim 3, wherein the heating temperature is 75 ℃ in step S01.
5. The method of claim 3, wherein in step S01, the reaction time is 2-3 h.
6. The method of claim 3, wherein in step S02, the buffer solution is at least one selected from the group consisting of a mixture of choline chloride and formic acid at a molar ratio of 1:2, a mixture of choline chloride and acetic acid at a molar ratio of 1:2, a mixture of choline chloride and oxalic acid at a molar ratio of 1:2, and a mixture of choline chloride and glycine at a molar ratio of 1: 2.
7. The method of claim 1, wherein in step S02, the buffer solution is selected from a mixture of choline chloride and acetic acid at a molar ratio of 1:1, and the molar ratio of DES to buffer solution is 1: 9.
8. The method of claim 1, wherein the ratio of the eutectic solvent to the buffer solution is (1-10) - (1-10) in step S02.
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