CN116904543B - Application of dehydrogenase in synthesis of R-configuration vitronectin and synthesis method - Google Patents

Application of dehydrogenase in synthesis of R-configuration vitronectin and synthesis method Download PDF

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CN116904543B
CN116904543B CN202311146033.2A CN202311146033A CN116904543B CN 116904543 B CN116904543 B CN 116904543B CN 202311146033 A CN202311146033 A CN 202311146033A CN 116904543 B CN116904543 B CN 116904543B
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杨智
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Yunhe Tianjin Biotechnology Co ltd
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Abstract

The invention provides an application of dehydrogenase in synthesizing R-configuration vitronectin and a synthetic method thereof, wherein the step 1 of the method is that D-xylose, an acetoacetate analogue and an alkaline substance are mixed and heated in aqueous solution, and a vitronectin intermediate product is obtained after reaction; step 2, mixing the vitriol intermediate product, dehydrogenase SMADH2, glucose dehydrogenase GDH, coenzyme and glucose in a buffer solution, and reacting to obtain the R-configuration vitriol. The synthesis method of the R-configuration glass color factor adopts dehydrogenase SMADH2 and glucose dehydrogenase GDH to specifically synthesize the glass color factor with a single R configuration, and simultaneously avoids the use of harmful chemicals, so that the whole reaction process is more environment-friendly.

Description

Application of dehydrogenase in synthesis of R-configuration vitronectin and synthesis method
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to application of dehydrogenase in synthesis of R-configuration vitronectin and a synthesis method.
Background
Most of the synthesis routes of the existing chemical method for the vitrein are two-step reactions, the first step is to prepare xyloside from D-xylose, the conversion is smooth, and the difficulty is that the second step is to selectively reduce xyloside. The separation of the pair of diastereomers obtained is difficult due to the poor stereoselectivity of the carbonyl groups of the chemically reduced ketones. Moreover, the existing route often needs to use dangerous reagents and processes such as sodium borohydride, lithium aluminum hydrogen, catalytic hydrogenation and the like, and a large amount of pollutants are introduced.
There are few patents for synthesizing vitrein by enzyme catalysis and no examples for synthesizing a single R configuration.
The Chinese patent publication No. CN111876452A (publication No. 2020, 11/3) discloses a method for preparing vitronectin by an enzyme one-pot method, but the sources of isopropyl alcohol dehydrogenase, vitronectin synthase, carbonyl reductase and the like used in the method are unknown and difficult to be trusted.
Chinese patent publication No. CN 113717997A (publication No. 2022, 2, 8) discloses a method for preparing vitriol factor by using combined enzyme, but the method can only synthesize S vitriol factor.
Disclosure of Invention
In view of the above, the present invention aims to overcome the defects in the prior art, and provides an application of dehydrogenase in synthesizing R-configuration vitronectin and a synthesis method thereof.
In order to achieve the above purpose, the technical scheme of the invention is realized as follows:
the invention provides an application of dehydrogenase SMADH2 in synthesis of R-configuration vitronectin.
The invention also provides a dehydrogenase composition, which comprises dehydrogenase SMADH2 and glucose dehydrogenase GDH.
Further, the mass ratio of the dehydrogenase SMADH2 to the glucose dehydrogenase GDH is 1-3:1.
The invention also provides application of the dehydrogenase composition in synthesis of R-configuration vitronectin.
The invention also provides a synthesis method of the R-configuration glass color factor, which comprises the following steps:
step 1, mixing D-xylose, an acetoacetic ester analogue and an alkaline substance in an aqueous solution, heating, and reacting to obtain a glassy factor intermediate product;
step 2, mixing the vitriol intermediate product, dehydrogenase SMADH2, glucose dehydrogenase GDH, coenzyme and glucose in a buffer solution, and reacting to obtain the R-configuration vitriol.
Further, the temperature of the heating step in the step 1 is 50-56 ℃ and the time is 3-5 hours; the molar ratio of D-xylose, the acetoacetate analogue and the alkaline substance in the step 1 is 1:1-1.3:1.3-1.6; the ethyl acetoacetate analogue is at least one of acetylacetone, ethyl acetoacetate, propyl acetoacetate, isopropyl acetoacetate or butyl acetoacetate; the alkaline substance is at least one of potassium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate or potassium carbonate.
Further, after the reaction step in the step 1 is finished, the intermediate product of the vitriol factor is obtained after the pH value of the solution is regulated, decolorized and filtered; the pH value of the step of adjusting the pH value of the solution is 7.0-8.0; the decoloring step is performed by using activated carbon.
Further, the mass ratio of the vitriol intermediate product to the dehydrogenase SMADH2 in the step 2 is 1:0.075-0.15; the mass ratio of the dehydrogenase SMADH2 to the glucose dehydrogenase GDH is 1-3:1. Preferably, the mass ratio of the vitriol intermediate product to the dehydrogenase SMADH2 in the step 2 is 1:0.1.
Further, the molar ratio of the glass-color factor intermediate product to glucose in the step 2 is 1:1-2; the molar ratio of the glass color factor intermediate product to the coenzyme in the step 2 is 198-794:1, a step of; the coenzyme in the step 2 is NADP.
Preferably, the molar ratio of the glass-color factor intermediate product to glucose in the step 2 is 1:1.5; the molar ratio of the vitriol intermediate product to the coenzyme in the step 2 is 396-794:1, a step of; more preferably, the molar ratio of the glass-color factor intermediate product to the coenzyme in the step 2 is 594:1.
further, the buffer solution in the step 2 is potassium phosphate buffer solution; the pH value of the buffer solution in the step 2 is 6.25-7.0; the concentration of the potassium phosphate buffer solution is 10-100mM; the reaction temperature in the step 2 is 20-40 ℃ and the reaction time is 6-12 hours.
Preferably, the pH value of the buffer solution in the step 2 is 6.75; the concentration of the potassium phosphate buffer solution is 10-50mM; the reaction temperature in the step 2 is 25-35 ℃ and the reaction time is 8-10 hours.
More preferably, the concentration of the potassium phosphate buffer is 25mM; the reaction temperature in the step 2 is 30 ℃ and the time is 10 hours.
Compared with the prior art, the invention has the following advantages:
the synthesis method of the R-configuration glass color factor adopts dehydrogenase SMADH2 and glucose dehydrogenase GDH to specifically synthesize the glass color factor with a single R configuration, and simultaneously avoids the use of harmful chemicals, so that the whole reaction process is more environment-friendly.
Drawings
FIG. 1 is a gel electrophoresis diagram of SMADH2 according to example 1 of the present invention;
FIG. 2 is a gel electrophoresis diagram of GDH according to example 2 of the present invention;
FIG. 3 is a synthetic route diagram of a glassy factor intermediate according to example 3 of the present invention;
FIG. 4 is a liquid chromatogram of a glassy chromogenic intermediate according to example 3 of the present invention;
FIG. 5 is a synthetic route diagram of the R-configuration glass color factor according to example 3 of the present invention;
FIG. 6 is a nuclear magnetic resonance spectrum of R-configuration vitronectin according to example 3 of the present invention;
FIG. 7 is a chiral liquid chromatogram of a commercial vitronectin product according to example 3 of the present invention (in the racemic state (S: R=42:58));
FIG. 8 is a chiral liquid chromatogram of a single R-configuration glass color factor according to example 3 of the present invention.
Detailed Description
Unless defined otherwise, technical terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
The amount of enzyme required for converting 1. Mu. Mol of the substrate at room temperature for 1 minute was U, and the amount of enzyme U/g per gram of cells was calculated based on the cell concentration of the bacterium.
The present invention will be described in detail with reference to examples.
EXAMPLE 1 preparation of dehydrogenase composition
1. The dehydrogenase is SMADH2 derived from Stenotrophomonas maltophilia LH and has NCBI accession number WP_088028380.1. The construction mode of the recombinant enzyme vector and the synthesis mode of the recombinant enzyme are as follows: after sequence optimization, the genes corresponding to the enzymes are synthesized by the company from the order to the department of Optimago (Beijing), and NdeI/BamHI restriction sites are introduced and subcloned into a pET 28a expression vector. Plasmid with correct sequence is transferred by heat shock methodE.coli(BL 21) competent cells were subjected to plate culture (Optimago) and monoclonal miniculture, and the bacteria with correct protein expression were finally subjected to stepwise amplification liquid culture. It specifically comprises single colony transfer into 5ml containing 50Mu M kanamycin in LB medium (37 ℃) and when cells grow to the log phase they are inoculated into 250 ml TB medium containing the same antibiotics and when they grow to the log phase they are transferred to a 5L culture fermenter for cultivation and final protein expression. In 5L fermenter culture, 0.25. 0.25 mM isopropyl-. Beta. -D-thiogalactopyranoside (IPTG) was added at 16℃to induce protein expression for 18 hours when the cell OD was about 20, and finally the cells were collected by high-speed centrifugation (6500 rpm,5 min) to obtain 450g of wet cells over-expressed with the enzyme. A small amount of cells were mixed with potassium phosphate buffer (50 mM, pH 7.0) and then disrupted by an ultrasonic disrupter, and the supernatant after cell wall removal by high-speed centrifugation was subjected to SDS-PAGE gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to detect protein expression, and the results are shown in FIG. 1. Cells with correct protein expression were used for the next catalytic experiment. LB medium consisted of: 1% of tryptone, 0.5% of yeast powder and 1% of NaCl; the TB medium consisted of: 1.2% tryptone, 2.4% yeast powder, 0.232% dipotassium hydrogen phosphate, 1.665% dipotassium hydrogen phosphate and 0.5% glycerol.
2. Glucose dehydrogenase GDH, derived from Priestia megaterium NBRC 15308, accession number CP035094.1. The construction mode of the recombinant enzyme vector and the synthesis mode of the recombinant enzyme are as follows: after sequence optimization, the genes corresponding to the enzymes are synthesized by the company from the order to the department of Optimago (Beijing), and NdeI/BamHI restriction sites are introduced and subcloned into a pET 28a expression vector. Plasmid with correct sequence is transferred by heat shock methodE.coli(BL 21) competent cells were subjected to plate culture (Optimago) and monoclonal miniculture, and the bacteria with correct protein expression were finally subjected to stepwise amplification liquid culture. Specifically, the single colony was transferred to 5ml LB medium (37 ℃) containing 50. Mu.M kanamycin for culture, and when the cells were grown to the logarithmic phase, they were inoculated to 250 ml TB medium containing the same antibiotics, and when the cells were grown to the logarithmic phase, they were transferred to a 5L culture fermenter for culture and final protein expression was performed. In 5L fermenter culture, protein expression was induced at 16℃for 18 hours by adding 0.25. 0.25 mM isopropyl-. Beta. -D-thiogalactopyranoside (IPTG) when the cell OD.apprxeq.20, and finally, cells were collected by high-speed centrifugation (6500 rpm,5 min) to obtain 450g of enzyme over-expression wet cells. A small amount of cells were mixed with potassium phosphate buffer (50 mM, pH 7.0) and then disrupted by an ultrasonic disrupter, and the supernatant after cell wall removal by high-speed centrifugation was subjected to SDS-PAGE gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to detect protein expression, and the results are shown in FIG. 2. Cells with correct protein expression were used for the next catalytic experiment. LB medium consisted of: 1% tryptone, 0.5% yeast powder and 1% NaCl. The TB medium consisted of: 1.2% tryptone, 2.4% yeast powder, 0.232% dipotassium hydrogen phosphate, 1.665% dipotassium hydrogen phosphate and 0.5% glycerol.
The amount of enzyme required for converting 1. Mu. Mol of the substrate at room temperature for one minute was U, and the amount of enzyme contained per g of cell was U/g.
Example 2 enzyme Activity detection
1. Enzyme activity detection system of SMADH 2: 1g of bacterial sludge is taken and mixed with 20ml of potassium phosphate buffer solution with pH7.0 and molar concentration of 50mM, and the cell suspension is broken by an ultrasonic breaker for 400w, 15min and 2s for 4s. Subsequently, the mixture was centrifuged at 10000rpm for 20 minutes at a low temperature (4 ℃) to obtain 100ul of a supernatant, which was diluted to 900ul of pH7.0 and prepared into a crude enzyme solution in 50mM potassium phosphate buffer. The total reaction system was 3ml, including: 50mM,2910ul of potassium phosphate buffer pH7.0, 30ul of 25% of the vitronectin intermediate, 30ul of 20mM of coenzyme NADPH in water and 30ul of crude enzyme solution. After the components are evenly mixed, an ultraviolet spectrophotometer is used for detecting the change of the absorbance at 340 nm. The calculation formula is [ Δax3000/(6.22×30) ] ×200, where Δa is the difference in absorbance range. The enzyme activity obtained in the invention is generally 200U/g-250U/g.
2. Enzymatic activity detection system for GDH: 1g of bacterial sludge is taken and mixed with 20ml of potassium phosphate buffer solution with pH7.0 and molar concentration of 50mM, and the cell suspension is broken by an ultrasonic breaker for 400w, 15min and 2s for 4s. Subsequently, the mixture was centrifuged at 10000rpm for 20 minutes at a low temperature (4 ℃) to obtain 100ul of a supernatant, which was diluted to 900ul of pH7.0 and prepared into a crude enzyme solution in 50mM potassium phosphate buffer. The total reaction system was 3ml, including: 50mM,2910ul of potassium phosphate buffer pH7.0, 30ul of 25% glucose solution, 30ul of 20mM coenzyme NADP aqueous solution and 30ul of crude enzyme solution. After the components are evenly mixed, an ultraviolet spectrophotometer is used for detecting the change of the absorbance at 340 nm. The calculation formula is [ Δax3000/(6.22×30) ] ×200, where Δa is the difference in absorbance range. The enzyme activity obtained in the invention is generally 200U/g-250U/g.
Example 3 Synthesis of a vitronectin of R configuration
1. Firstly, putting 36L of water and 2.4kg of sodium hydroxide into a 100L reaction kettle, controlling the temperature to 25 ℃ and controlling the error to be not more than 3 ℃; then adding 6kg of D-xylose and 4.8kg of acetylacetone to react for 3 hours at the temperature of 53 ℃ and the error of not more than 3 ℃; adding 35kg of 2.5N dilute hydrochloric acid to adjust the pH to 7.0-8.0; adding 1.2kg of active carbon, keeping the temperature at 53 ℃ for decoloring for 1h, filtering the active carbon, and obtaining a filtrate which is a vitronectin intermediate solution required by the second enzyme reaction, wherein the molecular formula of the vitronectin intermediate is C 8 H 14 O 5 The synthetic route of the intermediate of the glass color factor is shown in figure 3, and the liquid chromatogram is shown in figure 4.
2. To 833g of the vitriol intermediate solution (containing 100g of the vitriol intermediate) were added 156g of glucose monohydrate, 25mL of 1M potassium phosphate buffer pH7.0, 0.66g of NADP, the temperature was raised to 25℃and the pH was adjusted to 7.0 with 4M sodium hydroxide, and finally 100ml of 100g/L of SMADH2 enzyme solution and 50ml of 100g/L of GDH enzyme solution were added. And inserting a PH electrode, heating the system to 30 ℃, supplementing 4M sodium hydroxide solution by a peristaltic pump according to the pH falling speed displayed by the electrode to maintain the pH of the system to 6.75, stirring at constant temperature for 10 hours, ending the reaction, and detecting the reaction result by a liquid phase. After the reaction, hydrochloric acid is added to adjust the pH to 2, and sodium hydroxide is added to adjust the pH to neutrality. The synthetic route of the R-configuration glass color factor is shown in fig. 5, the nuclear magnetic spectrum is shown in fig. 6, the chiral liquid chromatography is shown in fig. 7-8, and the configuration ratio of fig. 7 to the production of Shanghai Yun Luo is (S: R) 42:58, wherein a peak with retention time of 11.256min in the figure is an S-configuration glass color factor, a peak with retention time of 12.717min in the figure is an R-configuration glass color factor, and S and R configurations exist in the liquid glass color factor at the same time; FIG. 8 shows that the product of this example was subjected to liquid chromatography and only peaked at a retention time of 12.307min, indicating that there was only a single R-configuration of the glass color factor in the product.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims (6)

1. An application of a dehydrogenase composition in synthesizing R-configuration vitronectin, which is characterized in that: the composition comprises dehydrogenase SMADH2 and glucose dehydrogenase GDH; the mass ratio of the dehydrogenase SMADH2 to the glucose dehydrogenase GDH is 1-3:1;
dehydrogenase SMADH2 is derived from Stenotrophomonas maltophilia LH, which NCBI accession number is WP_088028380.1; glucose dehydrogenase GDH is derived from Priestia megaterium NBRC 15308 and has accession number CP035094.1;
the R-configuration glass color factor is prepared by a synthesis method comprising the following steps:
step 1, mixing D-xylose, an acetoacetic ester analogue and an alkaline substance in an aqueous solution, heating, and reacting to obtain a glassy factor intermediate product;
step 2, mixing the vitriol intermediate product, dehydrogenase SMADH2, glucose dehydrogenase GDH, coenzyme and glucose in a buffer solution, and reacting to obtain the R-configuration vitriol;
the molar ratio of D-xylose, the acetoacetate analogue and the alkaline substance in the step 1 is 1:1-1.3:1.3-1.6;
the mass ratio of the vitriol intermediate product to the dehydrogenase SMADH2 in the step 2 is 1:0.075-0.15;
the molar ratio of the glass color factor intermediate product to glucose in the step 2 is 1:1-2; the molar ratio of the glass color factor intermediate product to the coenzyme in the step 2 is 198-794:1.
2. a synthetic method of R-configuration glass color factor is characterized in that: the method comprises the following steps:
step 1, mixing D-xylose, an acetoacetic ester analogue and an alkaline substance in an aqueous solution, heating, and reacting to obtain a glassy factor intermediate product;
step 2, mixing the vitriol intermediate product, dehydrogenase SMADH2, glucose dehydrogenase GDH, coenzyme and glucose in a buffer solution, and reacting to obtain the R-configuration vitriol;
dehydrogenase SMADH2 is derived from Stenotrophomonas maltophilia LH, which NCBI accession number is WP_088028380.1; glucose dehydrogenase GDH is derived from Priestia megaterium NBRC 15308 and has accession number CP035094.1;
the molar ratio of D-xylose, the acetoacetate analogue and the alkaline substance in the step 1 is 1:1-1.3:1.3-1.6;
the mass ratio of the vitriol intermediate product to the dehydrogenase SMADH2 in the step 2 is 1:0.075-0.15; the mass ratio of the dehydrogenase SMADH2 to the glucose dehydrogenase GDH is 1-3:1;
the molar ratio of the glass color factor intermediate product to glucose in the step 2 is 1:1-2; the molar ratio of the glass color factor intermediate product to the coenzyme in the step 2 is 198-794:1.
3. the method for synthesizing the R-configuration glass color factor according to claim 2, which is characterized in that: the temperature of the heating step in the step 1 is 50-56 ℃ and the time is 3-5 hours; the ethyl acetoacetate analogue is at least one of acetylacetone, ethyl acetoacetate, propyl acetoacetate, isopropyl acetoacetate or butyl acetoacetate; the alkaline substance is at least one of potassium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate or potassium carbonate.
4. The method for synthesizing the R-configuration glass color factor according to claim 2, which is characterized in that: after the reaction step in the step 1 is finished, the intermediate product of the vitreous color factor is obtained after the pH value of the solution is regulated, decolored and filtered; the pH value of the step of adjusting the pH value of the solution is 7.0-8.0; the decoloring step is performed by using activated carbon.
5. The method for synthesizing the R-configuration glass color factor according to claim 2, which is characterized in that: the coenzyme in the step 2 is NADP.
6. The method for synthesizing the R-configuration glass color factor according to claim 2, which is characterized in that: the buffer solution in the step 2 is potassium phosphate buffer solution; the pH value of the buffer solution in the step 2 is 6.25-7.0; the concentration of the potassium phosphate buffer solution is 10-100mM; the reaction temperature in the step 2 is 20-40 ℃ and the reaction time is 6-12 hours.
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CN114540380A (en) * 2022-03-17 2022-05-27 成都格纯生物医药有限公司 Sorbitol dehydrogenase sorDHGo gene, encoding protein and application in preparation of vitreous chromogen
CN115747272A (en) * 2022-11-30 2023-03-07 闽江学院 Synthetic method of vitreous chromogen
CN115896199A (en) * 2022-12-30 2023-04-04 杭州海普沃辉生物医药有限公司 Method for synthesizing high-concentration (S) -configuration vitronectin by double-enzyme coupling

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CN111876452A (en) * 2020-07-01 2020-11-03 江苏瑞蓓丽生物科技有限公司 Method for preparing vitronectin by biological enzyme one-pot method
CN113717997A (en) * 2021-11-04 2021-11-30 深圳瑞德林生物技术有限公司 Enzyme composition and method for synthesizing vitronectin by chemical enzyme method
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