A kind of preparation method of ethyl pyruvate

Abstract

The invention provides a method for preparing ethyl pyruvate, which comprises the following specific steps: S1. mixing ethyl lactate and lactate dehydrogenase, adding phosphate buffer, shaking, inactivating the enzyme, and obtaining a mixed solution; S2. adding crude alcohol dehydrogenase enzyme solution to the mixed solution, shaking, inactivating the enzyme, and distilling to obtain ethyl pyruvate. The yield and purity of ethyl pyruvate prepared by two-step dehydrogenation of lactate dehydrogenase and alcohol dehydrogenase are improved, which provides a method for industrial production of ethyl pyruvate.

Description

Technical Field

[0001] The invention belongs to the technical field of organic matter synthesis, and particularly relates to a method for preparing ethyl pyruvate. Background Art

[0002] Ethyl pyruvate, also known as ethyl α-ketopropionate and ethyl 2-oxopropionate, is an extremely important organic synthesis intermediate, which is widely used in medicine (synthetic thiamidine drugs), pesticides (herbicides and thiamidine fungicides produced by DuPont), food (preservatives, preservatives, etc.), cosmetics and other industries. Ethyl pyruvate itself has a special fragrance and can be used in flavors and fragrances. It is also an important raw material for synthetic resins and plastics. Pyruvate ester products can also be used as special solvents in electronic materials. The demand for applications in this area is increasing rapidly at home and abroad, so the artificial synthesis of ethyl pyruvate has developed rapidly.

[0003] There are three main ways to synthesize ethyl pyruvate in traditional ways: 1. Tartaric acid is dehydrated and decarboxylated to prepare pyruvic acid, and then pyruvic acid and ethanol are subjected to esterification reaction to produce ethyl pyruvate. This method has a low yield, and potassium bisulfate is used as a dehydrating agent in the process, which leads to excessive sulfur content in the product; 2. Microbial fermentation method, the product yield is low, and the residual microorganisms cannot be used for downstream drug synthesis, especially for infusion drug products; 3. Ethyl lactate oxidation method, this method uses chloroform as solvent and trichloroisocyanuric acid as oxidant, the production process has high "three wastes" emissions, and the chlorine content in the product ethyl pyruvate exceeds the standard. The reaction systems reported at present are mostly thermal catalytic systems, and organic solvents are used, or precious metals are used as catalysts, or organic oxides are used as oxidants. These methods have problems such as unmild reaction conditions, many side reactions, low conversion rate, low selectivity, and serious pollution, which are not conducive to subsequent practical applications.

[0004] Lactate dehydrogenase is widely present in animal tissues and various microbial cells, and can catalyze the reversible conversion of pyruvate and lactate. Alcohol dehydrogenase (ADH) belongs to the first subclass of oxidoreductase, acting on the -CHOH group, and is based on NAD + , NADP + ADH is a zinc-containing metalloenzyme that uses PQQ as a coenzyme and catalyzes the reversible reaction between primary alcohols and aldehydes. It is an intracellular enzyme and has been widely used in chemical, pharmaceutical, food and other industrial fields. Currently, commercial ADH is mostly derived from animal livers, and the beer yeast discarded by breweries also contains a large amount of alcohol dehydrogenase. Summary of the invention

[0005] Technical problem to be solved: In view of the deficiencies of the prior art, the purpose of the present invention is to provide a method for preparing ethyl pyruvate, which comprises the following specific steps: S1. mixing ethyl lactate and lactate dehydrogenase, adding phosphate buffer, shaking, inactivating the enzyme, and obtaining a mixed solution; S2. adding crude alcohol dehydrogenase enzyme solution to the mixed solution, shaking, inactivating the enzyme, and distilling to obtain ethyl pyruvate. The yield and purity of ethyl pyruvate prepared by two-step dehydrogenation of lactate dehydrogenase and alcohol dehydrogenase are improved, which provides a method for the industrial production of ethyl pyruvate.

[0006] Technical solution: A method for preparing ethyl pyruvate, using a two-step dehydrogenase method, using ethyl lactate as a raw material, and dehydrogenating to prepare ethyl pyruvate;

[0007] The specific steps include:

[0008] S1. Mix ethyl lactate and lactate dehydrogenase, add 30-40 vol.% phosphate buffer with a pH of 6.5-7.0, shake at 35-40 ° C for 2-4h, inactivate the enzyme, and obtain a mixed solution; the amount of lactate dehydrogenase added is 4-7wt.‰;

[0009] S2. Add crude alcohol dehydrogenase enzyme solution to the mixed solution, shake at 30-40° C. for 1.5-2.5 hours, inactivate the enzyme, and distill to obtain ethyl pyruvate; the volume ratio of the mixed solution to the crude alcohol dehydrogenase enzyme solution is (2-3):1.

[0010] Furthermore, the dehydrogenase may also be added after being immobilized.

[0011] Furthermore, the mixed solution in step S1 contains ethyl lactate and ethyl pyruvate.

[0012] Furthermore, the preparation steps of the crude alcohol dehydrogenase solution in step S2 are as follows:

[0013] Step 1: The spent brewer's yeast was concentrated by centrifugation after repeated freezing and thawing, and mixed with a phosphate buffer solution with a pH of 8.5;

[0014] Step 2: Under the condition of less than 4°C, the cells are disrupted by ultrasound-assisted method, centrifuged, and the supernatant is collected;

[0015] Step 3: Add a complex polysaccharide to the supernatant and stir evenly to obtain a crude alcohol dehydrogenase enzyme solution; the complex polysaccharide is raffinose and pullulan, and the mass ratio is (1.5-2.5):1; the addition amount of the complex polysaccharide is 3-4wt.%.

[0016] Furthermore, in step 1, the mass volume ratio of waste brewer's yeast to phosphate buffer is 1 g: (1.5-2) mL.

[0017] Furthermore, in step 2, the power of the ultrasound is 300-350W, and the ultrasound time is 10-15min.

[0018] Furthermore, in step 2, the centrifugal speed is 8000-10000 r / min, and the centrifugal time is 10-15 min.

[0019] Beneficial effects:

[0020] 1. The mixed solution prepared by adding commercial lactate dehydrogenase to ethyl lactate contains ethyl lactate and ethyl pyruvate, and ethyl lactate is not completely converted, so ethyl pyruvate is further dehydrogenated again using alcohol dehydrogenase to prepare ethyl pyruvate. The yield and purity of ethyl pyruvate prepared by the two-step method are both high, and the order of adding the two dehydrogenases cannot be interchanged. This method provides ideas for the industrial production of ethyl pyruvate;

[0021] 2. In the preparation of the crude alcohol dehydrogenase solution, the present invention adds a composite polysaccharide of raffinose and pullulan, which is beneficial to improving the enzyme activity and catalytic activity and promoting the dehydrogenation of ethyl lactate into ethyl pyruvate;

[0022] 3. The present invention uses waste beer yeast as raw material and adopts an ultrasonic-assisted method to extract alcohol dehydrogenase therein, thereby improving waste utilization, broadening the source of alcohol dehydrogenase, and reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the chromatogram of ethyl pyruvate prepared in Example 12;

[0024] Figure 2 is the chromatogram of ethyl pyruvate prepared in Comparative Example 4;

[0025] Figure 3 is the chromatogram of ethyl pyruvate prepared in Comparative Example 5;

[0026] Figure 4 is the chromatogram of ethyl pyruvate prepared in Comparative Example 6;

[0027] Figure 5 is the chromatogram of ethyl pyruvate prepared in Comparative Example 7;

[0028] Figure 6 This is the chromatogram of ethyl pyruvate prepared in Comparative Example 8. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with embodiments, which are explanations of the present invention and are not limited to the following embodiments:

[0030] Example 1

[0031] The preparation steps of crude alcohol dehydrogenase enzyme solution are as follows:

[0032] Step 1: 1 kg of waste beer yeast was concentrated by centrifugation after repeated freezing and thawing, and mixed with 1.5 L of phosphate buffer with a pH of 8.5; Step 2: Under conditions below 4°C, cells were disrupted by an ultrasonic-assisted method, with an ultrasonic power of 300 W, an ultrasonic time of 15 min, and centrifuged at 8000 r / min for 5 min, and the supernatant was collected;

[0033] Step 3: Add 3 wt.% of a mixture of raffinose and pullulan (mass ratio is 1.5:1) to the supernatant and stir evenly to obtain a crude alcohol dehydrogenase enzyme solution.

[0034] Example 2

[0035] The preparation steps of crude alcohol dehydrogenase enzyme solution are as follows:

[0036] Step 1: 1 kg of waste beer yeast was concentrated by centrifugation after repeated freezing and thawing, and mixed with 1.6 L of phosphate buffer with a pH of 8.5; Step 2: Under conditions below 4°C, cells were disrupted by an ultrasonic-assisted method, with an ultrasonic power of 320 W, an ultrasonic time of 14 min, and centrifuged at 8000 r / min for 14 min, and the supernatant was collected;

[0037] Step 3: Add 3 wt.% of a mixture of raffinose and pullulan (mass ratio is 2:1) to the supernatant and stir evenly to obtain a crude alcohol dehydrogenase enzyme solution.

[0038] Example 3

[0039] The preparation steps of crude alcohol dehydrogenase enzyme solution are as follows:

[0040] Step 1: 1 kg of waste beer yeast was concentrated by centrifugation after repeated freezing and thawing, and mixed with 1.8 L of phosphate buffer with a pH of 8.5; Step 2: Under conditions below 4°C, cells were disrupted by an ultrasonic-assisted method, with an ultrasonic power of 340 W, an ultrasonic time of 12 min, and centrifuged at 9000 r / min for 12 min, and the supernatant was collected;

[0041] Step 3: Add 4 wt.% of a mixture of raffinose and pullulan (mass ratio is 2:1) to the supernatant and stir evenly to obtain a crude alcohol dehydrogenase enzyme solution.

[0042] Example 4

[0043] The preparation steps of crude alcohol dehydrogenase enzyme solution are as follows:

[0044] Step 1: 1 kg of waste brewer's yeast was concentrated by centrifugation after repeated freezing and thawing, and mixed with 2 L of phosphate buffer with a pH of 8.5;

[0045] Step 2: Under the condition of below 4°C, the cells were disrupted by ultrasound-assisted method, the ultrasound power was 350W, the ultrasound time was 10min, centrifuged at 10000r / min for 10min, and the supernatant was collected;

[0046] Step 3: Add 4 wt.% of a mixture of raffinose and pullulan (mass ratio is 2.5:1) to the supernatant and stir evenly to obtain a crude alcohol dehydrogenase enzyme solution.

[0047] Comparative Example 1

[0048] The difference between this comparative example and Example 3 is that only raffinose is added, as follows:

[0049] The preparation steps of crude alcohol dehydrogenase enzyme solution are as follows:

[0050] Step 1: 1 kg of waste beer yeast was concentrated by centrifugation after repeated freezing and thawing, and mixed with 1.8 L of phosphate buffer with a pH of 8.5; Step 2: Under conditions below 4°C, cells were disrupted by an ultrasonic-assisted method, with an ultrasonic power of 340 W, an ultrasonic time of 12 min, and centrifuged at 9000 r / min for 12 min, and the supernatant was collected;

[0051] Step 3: Add 4 wt.% raffinose to the supernatant and stir evenly to obtain a crude alcohol dehydrogenase enzyme solution.

[0052] Comparative Example 2

[0053] The difference between this comparative example and Example 3 is that only pullulan is added, as follows:

[0054] The preparation steps of crude alcohol dehydrogenase enzyme solution are as follows:

[0055] Step 1: 1 kg of waste beer yeast was concentrated by centrifugation after repeated freezing and thawing, and mixed with 1.8 L of phosphate buffer with a pH of 8.5; Step 2: Under conditions below 4°C, cells were disrupted by an ultrasonic-assisted method, with an ultrasonic power of 340 W, an ultrasonic time of 12 min, and centrifuged at 9000 r / min for 12 min, and the supernatant was collected;

[0056] Step 3: Add 4 wt.% of pullulan to the supernatant and stir evenly to obtain a crude alcohol dehydrogenase enzyme solution.

[0057] Comparative Example 3

[0058] The difference between this comparative example and Example 3 is that raffinose and pullulan are not added, and the details are as follows:

[0059] The preparation steps of crude alcohol dehydrogenase enzyme solution are as follows:

[0060] Step 1: 1 kg of waste beer yeast was repeatedly frozen and thawed, then centrifuged and concentrated, and mixed with 1.8 L of phosphate buffer with a pH of 8.5; Step 2: Under conditions below 4°C, cells were disrupted using an ultrasonic-assisted method, the ultrasonic power was 340 W, the ultrasonic time was 12 min, and the mixture was centrifuged at 9000 r / min for 12 min, and the supernatant was taken to obtain a crude alcohol dehydrogenase enzyme solution.

[0061] Alcohol dehydrogenase (ADH) enzyme activity assay:

[0062] ADH enzyme activity (U / mL): ADH oxidizes ethanol to dehydrogenate acetaldehyde per minute at 37°C, that is, it reduces NAD + The absorbance change of NADH was measured at 340 nm, and an increase of 0.001 A340nm per minute was defined as 1 activity unit (U).

[0063] Table 1 Enzyme activity of crude alcohol dehydrogenase solution prepared in Examples 1-4

[0064] Example 1 Example 2 Example 3 Example 4 Alcohol dehydrogenase activity (U / mL) 1405 1471 1580 1513

[0065] Table 2 Enzyme activity of crude alcohol dehydrogenase solution prepared in Comparative Examples 1-3

[0066] Comparative Example 1 Comparative Example 2 Comparative Example 3 Alcohol dehydrogenase activity (U / mL) 1327 1289 1205

[0067] As can be seen from Table 1, the crude alcohol dehydrogenase enzyme solution prepared in Examples 1-4 has an enzyme activity of 1405-1580 U / mL. In Table 2, Comparative Examples 1 and 2 respectively added only raffinose and pullulan, and the enzyme activity decreased slightly. Comparative Example 3 did not add complex polysaccharides, and the enzyme activity was only 1205 U / mL. Therefore, the crude alcohol dehydrogenase enzyme solution prepared in Example 3 was selected for the subsequent preparation of ethyl pyruvate.

[0068] Example 5

[0069] A method for preparing ethyl pyruvate comprises the following steps:

[0070] S1. Mix 1L of ethyl lactate and 4g of lactate dehydrogenase, add 30vol.% phosphate buffer with a pH of 6.5, shake at 35°C for 4h, inactivate the enzyme, and obtain a mixed solution;

[0071] S2. Add crude alcohol dehydrogenase solution to the mixed solution at a volume ratio of 2:1, shake at 30°C for 2.5 hours, inactivate the enzyme, and distill to obtain ethyl pyruvate.

[0072] Example 6

[0073] A method for preparing ethyl pyruvate comprises the following steps:

[0074] S1. Mix 1L of ethyl lactate and 5g of lactate dehydrogenase, add 30vol.% phosphate buffer with a pH of 6.5, shake at 35°C for 4h, inactivate the enzyme, and obtain a mixed solution;

[0075] S2. Add crude alcohol dehydrogenase solution to the mixed solution at a volume ratio of 2:1, shake at 30°C for 2.5 hours, inactivate the enzyme, and distill to obtain ethyl pyruvate.

[0076] Example 7

[0077] A method for preparing ethyl pyruvate comprises the following steps:

[0078] S1. Mix 1L of ethyl lactate and 7g of lactate dehydrogenase, add 30vol.% phosphate buffer with a pH of 6.5, shake at 35°C for 4h, inactivate the enzyme, and obtain a mixed solution;

[0079] S2. Add crude alcohol dehydrogenase solution to the mixed solution at a volume ratio of 2:1, shake at 30°C for 2.5 hours, inactivate the enzyme, and distill to obtain ethyl pyruvate.

[0080] Example 8

[0081] A method for preparing ethyl pyruvate comprises the following steps:

[0082] S1. Mix 1L of ethyl lactate and 5g of lactate dehydrogenase, add 30vol.% phosphate buffer with a pH of 6.5, shake at 40°C for 2h, inactivate the enzyme, and obtain a mixed solution;

[0083] S2. Add crude alcohol dehydrogenase solution to the mixed solution at a volume ratio of 2:1, shake at 30°C for 2.5 hours, inactivate the enzyme, and distill to obtain ethyl pyruvate.

[0084] Example 9

[0085] A method for preparing ethyl pyruvate comprises the following steps:

[0086] S1. Mix 1L of ethyl lactate and 5g of lactate dehydrogenase, add 30vol.% phosphate buffer with a pH of 6.5, shake at 40°C for 3h, inactivate the enzyme, and obtain a mixed solution;

[0087] S2. Add crude alcohol dehydrogenase solution to the mixed solution at a volume ratio of 2:1, shake at 30°C for 2.5 hours, inactivate the enzyme, and distill to obtain ethyl pyruvate.

[0088] Example 10

[0089] A method for preparing ethyl pyruvate comprises the following steps:

[0090] S1. Mix 1L of ethyl lactate and 5g of lactate dehydrogenase, add 30vol.% phosphate buffer with a pH of 6.5, shake at 40°C for 4h, inactivate the enzyme, and obtain a mixed solution;

[0091] S2. Add crude alcohol dehydrogenase solution to the mixed solution at a volume ratio of 2:1, shake at 30°C for 2.5 hours, inactivate the enzyme, and distill to obtain ethyl pyruvate.

[0092] Embodiment 11

[0093] A method for preparing ethyl pyruvate comprises the following steps:

[0094] S1. Mix 1L of ethyl lactate and 5g of lactate dehydrogenase, add 30vol.% phosphate buffer with a pH of 6.5, shake at 40°C for 4h, inactivate the enzyme, and obtain a mixed solution;

[0095] S2. Add crude alcohol dehydrogenase solution to the mixed solution at a volume ratio of 2.5:1, shake at 30°C for 2.5 hours, inactivate the enzyme, and distill to obtain ethyl pyruvate.

[0096] Example 12

[0097] A method for preparing ethyl pyruvate comprises the following steps:

[0098] S1. Mix 1L of ethyl lactate and 5g of lactate dehydrogenase, add 30vol.% phosphate buffer with a pH of 6.5, shake at 40°C for 4h, inactivate the enzyme, and obtain a mixed solution;

[0099] S2. Add crude alcohol dehydrogenase solution to the mixed solution at a volume ratio of 3:1, shake at 30°C for 2.5 hours, inactivate the enzyme, and distill to obtain ethyl pyruvate.

[0100] Embodiment 13

[0101] A method for preparing ethyl pyruvate comprises the following steps:

[0102] S1. Mix 1L of ethyl lactate and 5g of lactate dehydrogenase, add 30vol.% phosphate buffer with a pH of 6.5, shake at 40°C for 4h, inactivate the enzyme, and obtain a mixed solution;

[0103] S2. Add crude alcohol dehydrogenase solution to the mixed solution at a volume ratio of 3:1, shake at 40°C for 1.5 hours, inactivate the enzyme, and distill to obtain ethyl pyruvate.

[0104] Embodiment 14

[0105] A method for preparing ethyl pyruvate comprises the following steps:

[0106] S1. Mix 1L of ethyl lactate and 5g of lactate dehydrogenase, add 30vol.% phosphate buffer with a pH of 6.5, shake at 40°C for 4h, inactivate the enzyme, and obtain a mixed solution;

[0107] S2. Add crude alcohol dehydrogenase solution to the mixed solution at a volume ratio of 3:1, shake at 40°C for 2 hours, inactivate the enzyme, and distill to obtain ethyl pyruvate.

[0108] Embodiment 15

[0109] A method for preparing ethyl pyruvate comprises the following steps:

[0110] S1. Mix 1L of ethyl lactate and 5g of lactate dehydrogenase, add 30vol.% phosphate buffer with a pH of 6.5, shake at 40°C for 4h, inactivate the enzyme, and obtain a mixed solution;

[0111] S2. Add crude alcohol dehydrogenase solution to the mixed solution at a volume ratio of 3:1, shake at 40°C for 2.5 hours, inactivate the enzyme, and distill to obtain ethyl pyruvate.

[0112] Comparative Example 4

[0113] The difference between this comparative example and Example 15 is that only lactate dehydrogenase is added, as follows:

[0114] A method for preparing ethyl pyruvate comprises the following steps:

[0115] Mix 1 L of ethyl lactate and 5 g of lactate dehydrogenase, add 30 vol.% phosphate buffer with a pH of 6.5, shake at 40°C for 6.5 h, inactivate the enzyme, and distill to obtain ethyl pyruvate.

[0116] Comparative Example 5

[0117] The difference between this comparative example and Example 15 is that only crude alcohol dehydrogenase enzyme solution is added, which is as follows:

[0118] A method for preparing ethyl pyruvate comprises the following steps:

[0119] Add crude alcohol dehydrogenase solution to ethyl lactate at a volume ratio of 3:1, shake at 40°C for 6.5h, inactivate the enzyme, and distill to obtain ethyl pyruvate.

[0120] Comparative Example 6

[0121] The difference between this comparative example and Example 15 is that the order of exchanging the crude enzyme solutions of lactate dehydrogenase and alcohol dehydrogenase is as follows:

[0122] A method for preparing ethyl pyruvate comprises the following steps:

[0123] S1. Add crude alcohol dehydrogenase solution to ethyl lactate at a volume ratio of 3:1, shake at 40°C for 2.5h, inactivate the enzyme, and obtain a mixed solution;

[0124] S2. Add 5 wt.% lactate dehydrogenase to the mixed solution, add 30 vol.% phosphate buffer with a pH of 6.5, shake at 40°C for 4 hours, inactivate the enzyme, and distill to obtain ethyl pyruvate.

[0125] Comparative Example 7

[0126] The difference between this comparative example and Example 15 is that the crude alcohol dehydrogenase enzyme solution is replaced with malate dehydrogenase, as follows:

[0127] A method for preparing ethyl pyruvate comprises the following steps:

[0128] S1. Mix 1L of ethyl lactate and 5g of lactate dehydrogenase, add 30vol.% phosphate buffer with a pH of 6.5, shake at 40°C for 4h, inactivate the enzyme, and obtain a mixed solution;

[0129] S2. Add malate dehydrogenase to the mixed solution at a mass ratio of 3:1, shake at 40°C for 2.5 hours, inactivate the enzyme, and distill to obtain ethyl pyruvate.

[0130] Comparative Example 8

[0131] The difference between this comparative example and Example 15 is that the crude alcohol dehydrogenase enzyme solution prepared in Comparative Example 3 is as follows:

[0132] A method for preparing ethyl pyruvate comprises the following steps:

[0133] S1. Mix 1L of ethyl lactate and 5g of lactate dehydrogenase, add 30vol.% phosphate buffer with a pH of 6.5, shake at 40°C for 4h, inactivate the enzyme, and obtain a mixed solution;

[0134] S2. Add the crude alcohol dehydrogenase solution prepared in Comparative Example 3 to the mixed solution at a volume ratio of 3:1, shake at 40°C for 2.5h, inactivate the enzyme, and distill to obtain ethyl pyruvate.

[0135] Performance Test:

[0136] (1) Yield and purity

[0137] Table 2 Yield and purity of ethyl pyruvate prepared in each embodiment and comparative example

[0138]

[0139] As shown in Table 2, in the process of preparing ethyl pyruvate in Example 5-15, the yield of ethyl pyruvate in the mixed solution in step S1 is 62.8-67.1%, the yield of ethyl pyruvate prepared in step S2 is 87.5-91.9%, and the purity is 98.7-99.4%. In Comparative Example 4, only lactate dehydrogenase is added, and the dehydrogenation is incomplete. The obtained product also contains a mixture of ethyl lactate and ethyl pyruvate, and the yield and purity are both reduced. In Comparative Example 5, only ethanol is added for dehydrogenation. The yield and purity of crude enzyme liquid of lactate dehydrogenase and alcohol dehydrogenase are also lower than those of the embodiment; Comparative Example 6 exchanges the order of crude enzyme liquid of lactate dehydrogenase and alcohol dehydrogenase, and the yield is 84.9%, and the purity is not significantly different from that of the embodiment; Comparative Example 7 replaces the crude enzyme liquid of alcohol dehydrogenase with malate dehydrogenase, and the yield is 83.8%, which is still slightly lower than the yield and purity of ethyl pyruvate prepared in the embodiment; Comparative Example 8 uses the crude enzyme liquid of alcohol dehydrogenase prepared in Comparative Example 3, and the yield and purity are also slightly lower than those of the embodiment. Therefore, ethyl pyruvate is prepared by a two-step method using dehydrogenase for ethyl lactate, first using lactate dehydrogenase, and then adding crude enzyme liquid of alcohol dehydrogenase, so that ethyl pyruvate with high yield and high purity can be obtained.

[0140] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing ethyl pyruvate, characterized in that: The two-step dehydrogenase method was used to prepare ethyl pyruvate by dehydrogenation of ethyl lactate. The specific steps include: S1. Mix ethyl lactate and lactate dehydrogenase, add 30-40 vol.% phosphate buffer with a pH of 6.5-7.0, shake at 35-40 ° C for 2-4h, inactivate the enzyme, and obtain a mixed solution; the amount of lactate dehydrogenase added is 4-7wt.‰; S2. Add crude alcohol dehydrogenase enzyme solution to the mixed solution, shake at 30-40° C. for 1.5-2.5 hours, inactivate the enzyme, and distill to obtain ethyl pyruvate; the volume ratio of the mixed solution to the crude alcohol dehydrogenase enzyme solution is (2-3):

1.

2. The method for preparing ethyl pyruvate according to claim 1, wherein: The mixed solution in step S1 contains ethyl lactate and ethyl pyruvate.

3. The method for preparing ethyl pyruvate according to claim 1, characterized in that: The preparation steps of the crude alcohol dehydrogenase solution in step S2 are as follows: Step 1: The spent brewer's yeast was concentrated by centrifugation after repeated freezing and thawing, and mixed with a phosphate buffer solution with a pH of 8.5; Step 2: Under the condition of less than 4°C, the cells are disrupted by ultrasound-assisted method, centrifuged, and the supernatant is collected; Step 3: Add a complex polysaccharide to the supernatant and stir evenly to obtain a crude alcohol dehydrogenase enzyme solution; the complex polysaccharide is raffinose and pullulan, and the mass ratio is (1.5-2.5):1; the addition amount of the complex polysaccharide is 3-4wt.%.

4. The method for preparing ethyl pyruvate according to claim 3, wherein: In the step 1, the mass volume ratio of waste brewer's yeast to phosphate buffer is 1 g: (1.5-2) mL.

5. The method for preparing ethyl pyruvate according to claim 3, characterized in that: In step 2, the power of the ultrasound is 300-350W, and the ultrasound time is 10-15min.

6. The method for preparing ethyl pyruvate according to claim 3, characterized in that: In step 2, the centrifugal speed is 8000-10000 r / min, and the centrifugal time is 10-15 min.

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