CN101906408B - Enzyme preparation for synthesizing D-aryl glycine through biocatalysis and preparation method and application thereof - Google Patents

Abstract

The invention relates to an enzyme preparation for synthesizing D-aryl glycine through biocatalysis, which consists of activated cells of arthrobacter aurescens producing hydantoinase and activated cells of agrobacterium radiobacter producing carbamoylase. The invention also relates to a preparation method and application of the enzyme preparation for synthesizing the D-aryl glycine through the biocatalysis. The enzyme preparation can be used for synthesizing the D-aryl glycine or derivatives thereof through the catalysis of heterogeneous hydrolysis of hydantoin. Through the enzyme preparation, the production cost is reduced greatly and the yield of products is improved.

Description

Enzyme preparation for biocatalytic synthesis of D-arylglycine and its preparation method and application

technical field

The invention belongs to the field of microbial fermentation, and in particular relates to an enzyme preparation for biocatalytically synthesizing D-arylglycine, a preparation method and application thereof.

Background technique

D-aryl amino acids (e.g., D-arylglycine) are a very important class of unnatural amino acids, which can be used in drugs (e.g., antagonists), peptides, and sweeteners due to their configuration opposite to natural amino acids In recent years, with the further development of its application value, the demand of the market is growing rapidly.

The traditional preparation method of D-aryl amino acid (such as D-arylglycine) includes chemical synthesis and resolution, but it needs to waste half of the enantiomers, resulting in low production efficiency, low production capacity, energy Disadvantages such as high consumption.

In the biocatalytic D-aryl hydantoin hydrolysis method, the traditional technology is the "one bacterium, two enzymes" method, as described in Chinese patents or patent applications 01105347, 200710191968, etc. However, a microorganism used in this technology has to take care of two kinds of enzymes, it is difficult to tolerate high concentrations of substrates and maintain continuous enzyme catalysis in industrial production, so it is difficult to add saturated concentrations of substrates to achieve non-uniformity. Phase enzyme catalysis. Therefore, even if these patent applications are authorized, the patent rights have been abandoned because they are not suitable for actual industrial production.

Chinese patent applications 200610048209.0 and 200710062118.7 disclose a method for synthesizing D-arylglycine or its derivatives by catalyzing the hydrolysis of 5-arylhydantoin by heterogeneous enzymes, using the "two bacteria and two enzymes" method to replace the traditional "one bacteria Two-enzyme method, wherein Arthrobacter aurescens LZ98 or its mutant strain UV-LZ98 is used to produce Hydantoinase and Agrobacterium radiobacter LZ99 or its mutant strain UV-LZ99 is used to produce Carbamidohydrolase, thereby producing an enzyme preparation with certain enzyme activity to enable the corresponding synthesis method to be realized. However, the purchase cost of the two bacterial strains used to prepare the enzyme preparations used in this method is relatively high, especially LZ99 and the mutant strain UV-LZ99 are not only expensive, but also the carbamidohydrolase produced is not stable enough for long-term fermentation on an industrial scale , The lack of fermentation durability greatly limits the industrial application of the patented heterogeneous enzyme catalyzed hydrolysis of 5-arylhydantoin to synthesize D-arylglycine.

Contents of the invention

In order to overcome the problems in the prior art, the present invention provides an enzyme preparation for the biocatalytic synthesis of D-arylglycine and its preparation method and application. The enzyme preparation in the method for preparing D-arylglycine by Genehydine greatly reduces the production cost, and the method is simple and easy for industrial application.

The present invention adopts following technical scheme to realize:

The first aspect of the present invention is an enzyme preparation for biocatalytically synthesizing D-arylglycine, which consists of activated cells of Arthrobacter aureus producing hydantoinase and activated cells of Agrobacterium radiata producing carbamoylase.

The enzyme preparation for the biocatalytic synthesis of D-arylglycine is characterized in that the initial enzyme activity of the hydantoinase produced by the Arthrobacter aureus is greater than 0.32 U, and the carbamoyl hydrolysis produced by the Agrobacterium radialis The initial activity of the enzyme is greater than 10U.

In the enzyme preparation for the biocatalytic synthesis of D-arylglycine, the initial hydantoinase activity of Arthrobacter aureus is 0.32U-0.45U.

In the enzyme preparation for biocatalyzing the synthesis of D-arylglycine, the Arthrobacter aureus is the activated cell of Arthrobacter aureus 3256, and the Agrobacterium radialis is the activated cell of Agrobacterium radialis 3255.

In a second aspect of the present invention, the preparation method of the enzyme preparation for the biocatalytic synthesis of D-arylglycine comprises the following steps:

a. inoculate the Arthrobacter aureus producing hydantoinase in the liquid culture medium containing corn steep liquor, molasses, aqueous sodium hydroxide solution, sodium chloride, 2-thiouracil, magnesium sulfate, manganese sulfate and diammonium hydrogen phosphate, Stir and activate the culture at 30-34°C. Among them, the stirring speed is 60rpm, the activation time is 12-18h, preferably 14-16h; the air pressure is 0.18-0.23Mpa, preferably 0.2Mpa; the air flow is 20-26L/h; the reactor pressure is 0.04-0.06Mpa, It is preferably 0.05Mpa; then the culture fluid is flocculated with chitosan aqueous solution, and the solid enzyme preparation 1 is harvested by filtration;

The liquid medium contains 360-460 parts by weight of corn steep liquor, 380-480 parts by weight of molasses, 15-25 parts by weight of aqueous sodium hydroxide solution with a concentration of 25-35% (w/w), and 40-60 parts by weight of sodium chloride. 27-37 parts by weight of 2-thiouracil, 8-10 parts by weight of magnesium sulfate, 1.2-1.8 parts by weight of manganese sulfate and 3-4 parts by weight of diammonium hydrogen phosphate, and add water to make the volume to 8000 parts by weight;

b. Inoculate Agrobacterium radiata producing carbamoylase in a liquid medium containing corn steep liquor, glucose, peptone, sodium chloride, manganese sulfate, diammonium hydrogen phosphate and arabinose, and stir at 32-38°C Activation culture, wherein the stirring speed is 60rpm; the activation time is 15-22h, preferably 16-18h, most preferably 17h; the air pressure is 0.18-0.23Mpa, preferably 0.2Mpa; the air flow is 18-22L/h; The pressure is 0.04～0.06Mpa, preferably 0.05Mpa; adding sodium hydroxide to the medium to adjust the pH is 6.5～7.8, preferably 6.8～7.3, most preferably 7.0, and then the culture solution is treated with chitosan aqueous solution Flocculation, filtering and harvesting solid enzyme preparation 2;

The liquid medium contains 200-400 parts by weight of corn steep liquor, 180-260 parts by weight of glucose, 80-100 parts by weight of peptone, 20-30 parts by weight of 25g sodium chloride, 1.6-2.0 parts by weight of manganese sulfate, diammonium hydrogen phosphate 6.8 to 7.6 parts by weight and 12 to 16 parts by weight of arabinose; adding water to the liquid medium to make up to 8000 parts by weight;

c. Mix the solid enzyme preparation 1 prepared in step a and the solid enzyme preparation 2 prepared in step b to prepare an enzyme preparation for biocatalytically synthesizing D-arylglycine. Wherein the weight ratio of the solid enzyme preparation 1 and the solid enzyme preparation 2 varies with the substrate, usually the ratio of the two is controlled to be 1～5:1～50, and the preferred ratio is 1～5: 1-20, the more preferred ratio is 1-2:1-5.

In the third aspect of the present invention, the enzyme preparation for biocatalytically synthesizing D-arylglycine is used in the method for enzymatically catalyzing heterogeneous hydrolysis of hydantoin to synthesize D-arylglycine.

The method for the synthesis of D-arylglycine by the enzyme-catalyzed heterogeneous hydrolysis of hydantoin comprises the following steps:

①In the enzyme-catalyzed reaction tank, add water and 5-arylhydantoin, and adjust the pH value to 6.5-7.8, and the addition of arylhydantoin accounts for 5-21% of the total weight of water and 5-arylhydantoin ( w/w), to obtain a mixed solution;

In step ①, the materials thus proportioned will form a suspension, that is, the solution is in a supersaturated state, and there are solid raw materials of 5-arylhydantoin.

② Add the enzyme preparation for the biocatalytic synthesis of D-arylglycine according to any one of claims 1 to 5 into the enzyme-catalyzed reaction tank under stirring conditions, and stir the reaction at 25-40°C until 5-arylglycine cannot be detected. Aryl hydantoin, get D-aryl glycine;

The enzyme preparation for the biocatalytic synthesis of D-arylglycine accounts for 1-10% of the total weight of the mixed solution in step ①, preferably 2-8%; the reaction time is 8-66 hours;

③ Separating the D-arylglycine solid through a 200-mesh sieve, and obtaining D-arylglycine crystals through recrystallization;

In step ③, due to the presence of thalline and tiny solid product crystals, it is preferably separated by a 200 mesh sieve, and the intercepted solid is larger D-arylglycine crystals (crude product), thallus and broken small The granular solid product enters the membrane separation system through the mesh.

④ Extract the D-arylglycine dissolved in the enzyme-catalyzed reaction solution by ultrafiltration with a molecular weight cut-off of 5,000 to 50,000 and evaporation concentration, and then recrystallize to obtain D-arylglycine crystals.

Since the solution obtained after the separation of D-arylglycine crystals is saturated D-arylglycine, in order to further increase the yield, step ④ was adopted. That is, the liquid that can flow through the sieve in step ③ is filtered through a membrane to remove bacteria, and the filtrate is concentrated to obtain D-arylglycine crystals, which are recrystallized to obtain D-arylglycine crystals.

Preferably, the method step ④ of the heterogeneous enzyme catalyzed hydrolysis of hydantoin to synthesize D-arylglycine can also adopt the following steps:

First pass through the ultrafiltration membrane to filter and sieve to separate the part that passes through the sieve holes, and then obtain the solution in which D-arylglycine is dissolved in the enzyme-catalyzed reaction tank, and the membrane dialysate is evaporated and concentrated to obtain another part of the crude product of D-arylglycine , and then recrystallized to obtain D-arylglycine crystals.

The D-arylglycine in the method for the synthesis of D-arylglycine by the heterogeneous enzyme catalyzed hydrolysis hydantoin is D-phenylglycine, D-p-hydroxyphenylglycine, D-fluorophenylglycine, D-chlorophenylglycine , D-bromophenylglycine, D-iodophenylglycine, D-methoxyphenylglycine, D-pyridine series glycine, D-naphthalene series glycine, D-quinoline series glycine D-pyrimidine glycine, D-pyrazine glycine, D -pyridazine glycine, D-indole glycine, D-thiophene glycine, D-furan glycine, D-pyrrole glycine, D-pyrazole glycine, D-imidazole glycine, D-thiazole glycine, D-oxazole glycine and D- One of the isoxazole glycine.

Further, the D-arylglycine in the method for preparing D-arylglycine by catalyzing the hydrolysis of hydantoin by heterogeneous enzymes is D-phenylglycine; the preparation method of D-phenylglycine is:

①In the enzyme-catalyzed reaction tank, add 30kg of water and 2.8kg of 5-phenylhydantoin, and adjust the pH value to 6.8;

2. Add 2.8kg of the enzyme preparation for biocatalytic synthesis of D-arylglycine according to any one of claims 1-5 to the enzyme catalytic reaction tank under stirring conditions, and stir and react at 38°C until 5-arylglycine cannot be detected. Phenylhydantoin, and the content of the intermediate product N-carbamoylglycine is less than or equal to 0.25% (w/w), indicating that the reaction is basically completed and D-phenylglycine is obtained;

③ Separation and precipitation of D-phenylglycine solid, after recrystallization, D-phenylglycine crystal product is obtained;

④ Extract the D-phenylglycine dissolved in the enzyme-catalyzed reaction solution through ultrafiltration membrane filtration and evaporation concentration to obtain another part, namely the D-phenylglycine product.

In the enzyme preparation for biocatalytically synthesizing D-arylglycine, both the Arthrobacter aureus and the Agrobacterium radiata are live bacteria, preferably activated cultured bacteria. Therefore, it is preferred that the enzyme preparation for the biocatalytic synthesis of D-arylglycine provided by the first aspect of the present invention is composed of the activated cells of Arthrobacter aureus producing hydantoinase and the activated cells of Agrobacterium radiata producing carbamoylase . In the present invention, "activated cells" means that the bacterial cells have been activated and cultured, so that the cells have the corresponding enzymatic activities of purified enzymes, namely hydantoinase activity and carbamoylase activity. Therefore, the enzyme preparation for biologically catalyzing the synthesis of D-arylglycine can maintain long-term enzyme activity during industrial production. Although the enzyme preparation for the biocatalytic synthesis of D-arylglycine is insoluble (including bacteria), according to the long-term observation of the inventors, the D-aryl The crude crystal particles of glycine are much larger than the bacteria and its fragments, so the crude crystal particles can be easily extracted by sieving and separation without introducing too much bacteria impurities to affect the purity, and will not lose the crude Crystal particles lead to a decrease in yield.

In the first aspect of the present invention, the initial hydantoinase activity of Arthrobacter aureus is greater than 0.30U, the initial carbamoylase activity of Agrobacterium radiatus is greater than 9U, preferably the initial hydantoinase activity is greater than 0.32U, also The preferred initial carbamoylase is greater than 10U, the optimal initial carbamoylase is 9.5U-15U, and the most optimal initial carbamoylase is 10U-12U. Due to the industrial production process, the activity of the enzyme will gradually decrease. Therefore, the bacteria in the first aspect of the present invention need to have a certain catalytic ability, and the catalytic ability needs to be quantitatively controllable in industrial production. In this way, the present invention uses the initial activity measured for the bacteria after activation and before feeding as an index of catalytic ability, and its specific measurement method can be carried out according to the methods listed in the specific examples of the present invention. The inventors have found that the catalytic efficiency and stability of Agrobacterium radiatus have been changed to produce carbamoylase, and the accumulation of intermediate products is less. Therefore, the Arthrobacter aureus producing hydantoinase does not need to be too good. Due to enzyme activity, it can be preferably 0.32 to 0.45 U. In this way, commercially available less expensive Arthrobacter aureus, such as Arthrobacter aureus 3256, can be used. In addition, since the screening of carbamoylase-producing Agrobacterium radiatus is easier, the expensive LZ series strains can be used instead of the cheaper commercially available Agrobacterium radiatus, such as Agrobacterium radiatus 3255. In a specific embodiment of the present invention, the preferred enzyme preparation is composed of Agrobacterium radialis 3255 and Arthrobacter aureus 3256, and the more preferred enzyme preparation is composed of Agrobacterium radialis 3255 activated cells and Arthrobacter aureus 3256 activated cells.

In order to further improve the deficiencies in the prior art, we have found through hard experiments that when using properly activated and cultured strain cells to directly prepare the enzyme preparation for the biocatalytic synthesis of D-arylglycine of the present invention (i.e. using the "two" of the present invention Bacteria" method), even if the more conventional carbamylase (Carbamylase) is used to replace the carbamylase in the existing "two bacteria and two enzymes" method, it can also meet the requirements of heterogeneous enzyme catalyzed hydrolysis of 5- Arylhydantoin needs to synthesize D-arylglycine, so expensive strains such as mutant bacteria UV-LZ99 can be used to extract the corresponding carbamidohydrolase, and finally formulated as a biocatalytic synthesis of D-arylglycine Enzyme. What is even more unexpected is that when the method of the present invention is used to activate the strain cells and prepare the enzyme preparation for the biocatalytic synthesis of D-arylglycine, even the hydantoinase production strain can use more conventional and cheaper strains. The use of the above strains reduces the cost and does not affect the stability and durability of the industrial-scale enzyme-catalyzed heterogeneous hydrolysis of 5-arylhydantoin to synthesize D-arylglycine, and the original heterogeneous phase hydrolysis can also be used directly. Enzyme-catalyzed equipment and fermentation processes do not require investment in new equipment costs, and there is no need to retrain technicians on the production line to master other fermentation processes.

The beneficial effect that the present invention has compared with prior art is:

① Compared with the application of the original "two bacteria and two enzymes" method in the synthesis of D-arylglycine by the heterogeneous enzyme catalyzed hydrolysis of 5-arylhydantoin, the present invention uses the enzyme and the activation and preparation method instead, The invention has the advantages of fast reaction speed, energy saving, high yield, stable product quality and the like.

②The "two bacteria" method does not need the step of extracting enzymes, and the activation process of enzymes (bacteria) and the cultivation and amplification process of bacteria cells are combined into one, which simplifies the actual production steps and reduces the production cost.

③ The cost of strains is low, and there is no need for a variety of expensive strains to cooperate (even if the activity of Hydantoinase is low, it will not affect the final fermentation efficiency), and the new activation and preparation methods make the enzyme preparations obtained after preparation in production It is durable and stable, and does not need to monitor the enzyme activity during the long-term reaction process and replenish it in a timely manner like the "two enzymes and two bacteria" method. It can be easily scaled up to industrial-scale production, and is especially suitable for industrial production.

④ Strong substrate adaptability, no need for the existing "two bacteria and two enzymes" method, D-hydroxyphenylglycine can use the enzyme produced by the fermentation of Agrobacterium radiata LZ99, while other D-arylglycine requires additional The screened enzymes of Agrobacterium radialis UV-LZ99.

⑤ Directly using cells instead of soluble enzymes can avoid the steps of mixed fermentation and enzyme extraction, and only needs to activate the strains. For the problem of protein impurities that have long plagued crystal purity, we have found that cell bodies and heterogeneous phases The size of the crude product crystallized by the method is different, and it can be simply separated with a commercially available 200-mesh sieve, which is very convenient for industrial production.

Detailed ways

The present invention will be described in detail below in conjunction with specific embodiments.

Embodiment one

1. Preparation of hydantoin enzyme preparation

In a 10L fermenter, add 416g corn steep liquor, 428g molasses, 20mL 30% (w/w) sodium hydroxide aqueous solution, 50g sodium chloride, 32g 2-thiouracil, 10g magnesium sulfate, 15g manganese sulfate, 3.6g Diammonium hydrogen phosphate and water until the volume reaches 8L, sterilize at 0.1-0.12Mpa pressure, 120-125°C for 0.5h, and when the temperature is lowered to 30.5°C, Arthrobacter aureus 3256 that has been cultured in a 250mL shake flask in advance (purchased from Shijiazhuang Zhongtian Biotechnology Co., Ltd.) were inoculated in fermenters. Subsequently, under the premise of an air pressure of 0.2Mpa and an air flow rate of 20-26L/h, the pressure of the reactor was controlled to be 0.05Mpa and the temperature was 33°C, and the fermentation was stirred for 15h. Then, the fermented liquid was flocculated with 1% (w/w) chitosan 400g, and the hydantoinase-containing cells were precipitated, and the wet hydantoinase preparation was filtered under reduced pressure, which was activated Arthrobacter aureus 3256 cells.

At the same time, another 1 mL of fermentation broth was taken to measure the enzymatic activity of hydantoinase: centrifuge 1 mL of fermentation broth at 4800 r/min for 30 min, discard the supernatant and resuspend the obtained cells in normal saline to make the total volume reach 1 mL, without extracting the enzyme Add 9 mL of phosphate buffer solution (pH 8.0) containing 2.2% (w/w) p-hydroxyphenylhydantoin directly to the cell suspension, shake slowly at 38°C for 30 min, then add 0.1 mL of 6N hydrochloric acid and mix well The reaction was terminated, followed by centrifugation for 30 min. Take the supernatant, and measure the concentration of D-N-carbamoyl-p-hydroxyphenylglycine therein by HPLC. The enzyme activity unit (U) of 1 hydantinase is defined as the amount of D-N-carbamoyl-p-hydroxyphenylglycine that can be produced by cells contained in 1 mL of fermentation broth per 1 min. According to the results of three fermentations, hydantoin enzyme activities were 0.37U, 0.42U and 0.39U respectively, indicating that cells with high enzyme activity could be directly and stably activated by this method.

2. Preparation of carbamoylase preparations

In a 10L fermenter, add 360g corn steep liquor, 220g glucose, 90g peptone, 25g sodium chloride, 1.8g manganese sulfate, 7.2g diammonium hydrogen phosphate, 14g arabinose and water to a volume of 8L, and adjust the pH with sodium hydroxide is 6.8. Sterilize at 0.1-0.12Mpa pressure, 12.-125°C for 0.5h, cool down to 30.5°C, pre-cultivated Agrobacterium radialis 3255 in a 250mL shake flask (purchased from Shijiazhuang Zhongtian Biotechnology Co., Ltd. company) inoculated in the fermenter. Then, under the premise of a pressure of 0.2Mpa and an air flow rate of 18-22L/h, the pressure of the reactor was controlled at 0.05Mpa and the temperature was 35°C, and the fermentation was stirred for 17h. Then, the fermented liquid was flocculated with 1% (w/w) chitosan 400g to precipitate the cells, and the wet carbamoylase preparation obtained by filtration under reduced pressure was the activated Agrobacterium radialis 3255 cells

At the same time, another 1 mL of fermentation broth was taken to measure the enzyme activity of carbamoyl hydrolase: 1 mL of fermentation broth was centrifuged at 4800 r/min for 30 min, the supernatant was discarded and the resulting cells were resuspended in normal saline to make the total volume reach 1 mL. To extract the enzyme, directly add 9 mL of phosphate buffer solution (pH 8.0) containing 2.2% (w/w) D-N-carbamoyl-p-hydroxyphenylglycine to the cell suspension, shake slowly at 38°C for 30 min, and then add Mix 0.1mL 6N hydrochloric acid to stop the reaction, and then centrifuge for 30min. Take the supernatant, and measure the concentration of D-p-hydroxyphenylglycine therein by HPLC. The enzyme activity unit (U) of 1 carbamoylase is defined as the amount of D-p-hydroxyphenylglycine that can be produced by cells contained in 1 mL of fermentation broth per 1 min. According to the results of three fermentations, the enzyme activities of carbamoylase were 10.1U, 11.7U and 10.6U respectively, indicating that cells with high enzyme activity could be stably activated directly by this method.

3. Enzyme preparation for biocatalytic synthesis of D-arylglycine

The wet hydantoin enzyme preparation prepared above and the wet carbamoylase preparation are mixed evenly to obtain a double enzyme preparation—an enzyme preparation for biocatalytically synthesizing D-arylglycine.

Embodiment two,

Application of Enzyme Preparation for Biocatalytic Synthesis of D-Arylglycine--Catalyzed Reaction Example

1. Preparation of D-p-phenylglycine

In a 50L enzyme-catalyzed reaction tank, first add 30kg of water, then put in 2.8kg of 5-phenylhydantoin, add hydrochloric acid or sulfuric acid to adjust the pH to 6.8, and then add the prepared by Example 1 step 1 under stirring conditions 1.5 kg of wet hydantoin enzyme preparation and 1.5 kg of wet carbamoylase enzyme preparation prepared in Step 2 of Example 1 were mixed uniformly, the temperature was controlled at 38° C., and the reaction was stirred.

When the reaction lasted for about 4 hours, D-phenylglycine crystals were precipitated, during which time samples were taken and the reaction was detected by high-performance liquid chromatography. The concentration of D-phenylglycine in the reaction solution was basically constant at about 1.6%. When the substrate 5-phenylhydantoin can hardly be detected by liquid chromatography, and the content of the intermediate N-carbamoylglycine is lower than 0.25%, the reaction is stopped. The conversion rate of dihydantoin is above 99.6%.

The material in the 50L enzyme-catalyzed reaction tank was passed through a 200-mesh sieve to separate the solid product from the tiny bacterial cells and the solution, and obtain crude crystals of D-phenylglycine. Using water as a solvent, 1.48kg of D-phenylglycine can be obtained through acid-base conversion and recrystallization, which is the first part of the product; the liquid part obtained by screening and separation is further extracted: first, the liquid is filtered through an ultrafiltration membrane with a molecular weight cut-off of 5000, The filtrate was concentrated by evaporation under reduced pressure, during which D-phenylglycine crystallized out. Collect D-phenylglycine crystals by filtration, and then use the same recrystallization method as the first part of the product to obtain the second part of product 0.51 kg. The two parts of D-phenylglycine were combined to obtain a product with a purity of more than 99.8% and a mass of 1.99 kg, with a total yield of 83%.

2. Preparation of D-phenylglycine derivatives

Using the same preparation method and parameters as the preparation of 1D-p-phenylglycine above, 4.5 kg of 5-p-hydroxyphenylhydantoin was used instead of 2.8 kg of 5-phenylhydantoin to finally obtain D-p-hydroxybenzene with a purity of more than 99.7%. Glycine crystals 3.1kg.

Using the same preparation method and parameters as the preparation of 1D-p-phenylglycine above, 3.2 kg of 5-p-methoxyphenylhydantoin was used instead of 2.8 kg of 5-phenylhydantoin to finally obtain D-p-phenylglycine with a purity of more than 99.8%. Methoxyphenylglycine crystal 2.3kg.

Using the same preparation method and parameters as the preparation of 1D-p-phenylglycine above, 3.8 kg of 5-p-fluorophenylhydantoin was used instead of 2.8 kg of 5-phenylhydantoin to finally obtain D-p-fluorobenzene with a purity of more than 99.8%. Glycine crystals 2.6kg.

Using the same preparation method and parameters as the preparation of 1D-p-phenylglycine above, 3.5 kg of 5-p-chlorophenylhydantoin was used instead of 2.8 kg of 5-phenylhydantoin to finally obtain D-p-chlorobenzene with a purity of more than 99.8%. Glycine crystals 2.4kg.

Using the same preparation method and parameters as the preparation of 1D-p-phenylglycine above, 4.1 kg of 5-p-bromodihydantoin was used instead of 2.8 kg of 5-phenylhydantoin to finally obtain crystalline D-p-bromine with a purity of more than 99.8%. Phenylglycine crystals 2.9kg.

Using the same preparation method and parameters as in Example 2, 2.8 kg of 5-phenylhydantoin was used instead of 2.8 kg of 5-phenylhydantoin to finally obtain 2.1 kg of D-iodophenylglycine crystals with a purity of more than 99.7%. .

3. Preparation of D-3-pyridine glycine

In a 50L enzyme-catalyzed reaction tank, first add 32kg of water, drop 3.8kg of 5-(3-pyridyl)hydantoin under stirring conditions, add hydrochloric acid or sulfuric acid to adjust the pH to 6.3, and then add 1 Prepared 1.8 kg of wet hydantoin enzyme preparation and 3.0 kg of wet carbamoylase enzyme preparation prepared in 2 of Example 1, mixed uniformly, and stirred and reacted at 33°C.

When the reaction was carried out to about 6h, D-3-pyridine glycine crystals began to separate out, and then samples were taken regularly and the reaction was detected by high performance liquid chromatography, and the substrate 5-(3-pyridyl) seawater was hardly detected by liquid chromatography. Therefore, and when the content of the intermediate N-carbamoyl-3-pyridine glycine is lower than 0.20% (w/w), the reaction is stopped. The conversion rate of 5-(3-pyridyl)hydantoin is above 99.6%.

The reaction mixture is sieved through 200 mesh to separate the solid product from the bacteria and the solution, and the crude crystal of D-3-pyridine glycine is obtained. Using water as the solvent, D-3-pyridine can be obtained by recrystallization through acid-base conversion Glycine 1.87kg, this is the first part of the product; the liquid obtained through the sieve is further extracted: firstly, the liquid is filtered through an ultrafiltration membrane with a molecular weight cut-off of 10,000, and the filtrate is evaporated and concentrated under reduced pressure, during which there is D-3-pyridine Glycine crystallized out. Collect the crystals of D-3-pyridineglycine by filtration, and then use the same recrystallization method as the first part of the product to obtain the second part of product 0.81kg. The two parts of D-3-pyridineglycine were combined to obtain a product with a purity of more than 99.2% and a mass of 2.68 kg, with a total yield of 82%.

4. Preparation of D-2-naphthylglycine

In a 50L enzyme-catalyzed reaction tank, first add 37kg of water, drop in 3.8kg of 5-(2-naphthyl)hydantoin under stirring conditions, add hydrochloric acid or sulfuric acid to adjust the pH to 7.1, and then add the Prepared 1.5 kg of wet hydantoin enzyme preparation and 2.3 kg of wet carbamoylase enzyme preparation prepared in 2 of Example 1, mixed uniformly, and stirred and reacted at 38°C.

When the reaction was carried out to about 7h, D-2-naphthylglycine crystals began to separate out, and then samples were taken regularly and the reaction was detected by high-performance liquid chromatography. The liquid chromatography was almost unable to detect the substrate 5-(2-naphthyl) seawater When the corresponding intermediate is reached, stop the reaction. The conversion rate of 5-(2-naphthyl)hydantoin is about 99.0%.

Pass the reaction mixture through a 200-mesh sieve to separate the solid product from the bacterial cells and the solution, and obtain the crude crystals of D-2-naphthylglycine. Using water as a solvent, recrystallize through acid-base conversion to obtain D-2-naphthylglycine 1.90kg, this is the first part of the product; part of the liquid obtained through the sieve is further extracted: first, the liquid is filtered through an ultrafiltration membrane with a molecular weight cut-off of 10,000, and the filtrate is evaporated and concentrated under reduced pressure, during which there is D-2-naphthylglycine Crystallized out. Collect D-2-naphthylglycine crystals by filtration, and then use the same recrystallization method as the first part of the product to obtain the second part of product 0.63kg. The two parts of D-2-naphthylglycine were combined to obtain a product with a purity of more than 98.8% and a mass of 2.53 kg, with a total yield of 75%.

5. Preparation of D-2-quinoline glycine

In a 50L enzyme-catalyzed reaction tank, first add 36kg of water, put 4.2kg of 5-(2-quinolyl)hydantoin under stirring conditions, adjust the pH to 6.5, and then add the wet solution prepared by 1 in Example 1 1.5 kg of the hydantoin enzyme preparation and 3.2 kg of the wet carbamoylase enzyme preparation prepared in 2 in Example 1 were mixed uniformly, and stirred and reacted at 37° C.

When the reaction was carried out to about 6h, D-2-quinoline glycine crystals began to separate out, and then samples were taken regularly and the reaction was detected by high performance liquid chromatography, and the substrate 5-(2-quinolyl) was hardly detected by liquid chromatography. ) Hydantoin, and when the content of its intermediate N-carbamoyl-D-quinoline glycine in the reaction solution is lower than 0.3% (w/w), the reaction is stopped. The conversion rate of 5-(2-quinolyl)hydantoin is about 99.5%.

The reaction mixture was passed through a 200-mesh sieve to separate the solid product from the bacterial cells and the solution to obtain crude crystals of D-2-quinolineglycine. Using water as a solvent, 2.55kg of D-2-quinolineglycine was obtained through acid-base conversion and recrystallization, which is the first part of the product; the liquid obtained through the sieve was further extracted: first, the liquid was passed through an ultrafiltration membrane with a molecular weight cut-off of 20,000 After filtering, the filtrate was evaporated and concentrated under reduced pressure, during which D-2-quinolineglycine crystals were precipitated, and the D-2-quinolineglycine crystals were collected by filtration. Using the same recrystallization method as the first part of the product, 0.63kg of the second part of the product can be obtained. After passing the test, the two parts of D-2-quinolineglycine are combined to obtain a total purity of more than 98.5% and a quality of 3.18kg. Product, total yield 85%.

Claims (7)

1. The enzyme preparation of biocatalytic synthesis of D-arylglycine is characterized in that it is composed of the activated cells of Arthrobacter aureus producing hydantoinase and the activated cells of Agrobacterium radiata producing carbamoylase; The preparation method of the enzyme preparation of described biocatalytic synthesis D-arylglycine, it comprises the following steps: a. inoculate the Arthrobacter aureus producing hydantoinase in the liquid culture medium containing corn steep liquor, molasses, aqueous sodium hydroxide solution, sodium chloride, 2-thiouracil, magnesium sulfate, manganese sulfate and diammonium hydrogen phosphate, Stir and activate culture at 30-34°C, wherein the stirring speed is 60rpm; the activation time is 12-18h; the air pressure is 0.18-0.23Mpa; the air flow rate is 20-26L/h; the pressure of the reactor is 0.04-0.06Mpa; The culture solution is flocculated with water-soluble chitosan, and the solid enzyme preparation 1 is harvested by filtration; The liquid medium contains 360-460 parts by weight of corn steep liquor, 380-480 parts by weight of molasses, 15-25 parts by weight of aqueous sodium hydroxide solution with a mass concentration of 25-35%, 40-60 parts by weight of sodium chloride, 2- 27-37 parts by weight of thiouracil, 8-10 parts by weight of magnesium sulfate, 1.2-1.8 parts by weight of manganese sulfate, and 3-4 parts by weight of diammonium hydrogen phosphate, and add water to make up to 8000 parts by weight; b. Inoculate Agrobacterium radiata producing carbamoylase in a liquid medium containing corn steep liquor, glucose, peptone, sodium chloride, manganese sulfate, diammonium hydrogen phosphate and arabinose, and stir at 32-38°C Activation culture, wherein the stirring speed is 60rpm; the activation time is 15-22h; the air pressure is 0.18-0.23Mpa; the air flow rate is 18-22L/h; the pressure of the reactor is 0.04-0.06Mpa; hydrogen is added to the medium Sodium oxide adjusts the pH to 6.5-7.8, then flocculates the culture solution with water-soluble chitosan, and filters to harvest the solid enzyme preparation 2; The liquid medium contains 200-400 parts by weight of corn steep liquor, 180-260 parts by weight of glucose, 80-100 parts by weight of peptone, 20-30 parts by weight of 25g sodium chloride, 1.6-2.0 parts by weight of manganese sulfate, diammonium hydrogen phosphate 6.8 to 7.6 parts by weight, and 12 to 16 parts by weight of arabinose; and adding water to the liquid medium to make up to 8000 parts by weight; c. Mix the solid enzyme preparation 1 prepared in step a with the solid enzyme preparation 2 prepared in step b to prepare an enzyme preparation for biocatalytically synthesizing D-arylglycine, wherein the solid enzyme preparation 1 and the solid enzyme preparation The weight ratio of preparation 2 is 1-5:1-50. the 2. the enzyme preparation of biocatalytic synthesis D-arylglycine as claimed in claim 1, is characterized in that, the initial enzymatic activity of the hydantoin enzyme that described Arthrobacter aureus produces is greater than 0.32U, and described Agrobacterium radialis The initial activity of the carbamoylase produced was greater than 10U. the 3. The enzyme preparation for biocatalytically synthesizing D-arylglycine according to claim 1, characterized in that the initial hydantoinase activity of Arthrobacter aureus is 0.32U-0.45U. the 4. the enzyme preparation of biocatalytic synthesis D-arylglycine as claimed in claim 1, it is characterized in that, described Arthrobacter aureus is Arthrobacter aureus 3256 activated cell, and described Agrobacterium radialis is Agrobacterium radialis 3255 activate cells. the 5. the enzyme preparation of biocatalytic synthesis D-arylglycine as claimed in claim 1, is characterized in that, described enzyme preparation is used for enzyme catalyzed heterogeneous phase hydrolysis hydantoin synthetic D-arylglycine; The method for the synthesis of D-arylglycine by the enzyme-catalyzed heterogeneous hydrolysis hydantoin comprises the following steps: ①Add water and 5-arylhydantoin to the enzyme-catalyzed reaction tank, and adjust the pH value to 6.5-7.8. The ratio of the amount of 5-arylhydantoin to the total mass of water and 5-arylhydantoin is 5～21%, get mixed solution; ②Add the enzyme preparation according to any one of claims 1 to 5 into the enzyme-catalyzed reaction tank under stirring conditions, and stir the reaction at 25-40°C until 5-arylhydantoin cannot be detected to obtain D- Arylglycine; the enzyme preparation accounts for 1-10% of the total weight of the mixed solution in step ①, and the reaction time is 8-66 hours; ③ Separating and precipitating the D-arylglycine solid through a 200-mesh sieve, and obtaining D-arylglycine crystals through recrystallization. the 6. the enzyme preparation of biocatalytic synthesis D-arylglycine as claimed in claim 1, is characterized in that, described D-arylglycine is D-phenylglycine, D-p-hydroxyphenylglycine, D-fluorophenylglycine , D-chlorophenylglycine, D-bromophenylglycine, D-iodophenylglycine, D-methoxyphenylglycine, D-pyridine series glycine, D-naphthalene series glycine, D-quinoline series glycine D-pyrimidine glycine, D -Pyrazine glycine, D-pyridazine glycine, D-indole glycine, D-thiophene glycine, D-furan glycine, D-pyrrole glycine, D-pyrazole glycine, D-imidazole glycine, D-thiazole glycine, D- One of oxazole glycine and D-isoxazole glycine. the 7. the enzyme preparation of biocatalytic synthesis D-arylglycine as claimed in claim 6, is characterized in that, described D-arylglycine is D-phenylglycine, and the preparation method of described D-phenylglycine is: ①In the enzyme-catalyzed reaction tank, add 30kg of water and 2.8kg of 5-phenylhydantoin, and adjust the pH value to 6.8; 2. Add 2.8kg of the enzyme preparation for biocatalytic synthesis of D-arylglycine according to any one of claims 1-5 to the enzyme catalytic reaction tank under stirring conditions, and stir and react at 38°C until 5-arylglycine cannot be detected. Phenylhydantoin, and the content of the intermediate product N-carbamoylphenylglycine is less than or equal to 0.25% by mass ratio to obtain D-phenylglycine; ③ Separation and precipitation of D-phenylglycine solid, after recrystallization, D-phenylglycine crystals were obtained; ④ Extract D-phenylglycine partially dissolved in the solution in the enzyme-catalyzed reaction tank through ultrafiltration membrane filtration and evaporation concentration to obtain D-phenylglycine. the