Preparation method of D-p-hydroxyphenylglycine

Abstract

The invention relates to the technical field of biosynthase catalysis, and discloses a preparation method of D-p-hydroxyphenylglycine, which comprises the following steps: adding deionized water, DL-p-hydroxyphenylhydantoin and enzyme liquid into an enzyme reaction tank, reacting, filtering by a micro-filtration membrane, evaporating and concentrating, decolorizing by activated carbon, crystallizing and the like to prepare the D-p-hydroxyphenylglycine. According to the method, the D-p-hydroxyphenylglycine is generated through enzymatic catalysis, the D-p-hydroxyphenylglycine is generated through one-pot enzymatic catalysis of the p-hydroxyphenylhydantoin, and by improving the substrate tolerance of the enzyme, the yield of the final product reaches 90% or above, and the purity is high.

Description

Technical Field

[0001] The invention relates to the technical field of biosynthetic enzyme catalysis, in particular to a method for preparing D-p-hydroxyphenylglycine. Background Art

[0002] L-p-hydroxyphenylglycine, also known as D-p-hydroxyphenylglycine, is a pharmaceutical fine chemical with the appearance of white crystalline powder. It is an important side chain for the synthesis of β-lactams and semi-synthetic antibiotics. It can be used for the synthesis of broad-spectrum antibacterial antibiotics such as amoxicillin, cefoperazone, cefadroxil, cefprozil, and ceftriaxone sodium. The preparation methods of D-p-hydroxyphenylglycine can be roughly divided into two categories. One is the chemical synthesis method, that is, anisaldehyde is cyclized and hydrolyzed to produce DL-p-hydroxyphenylglycine, which is then esterified with methanol and split with a sulfonic acid splitting agent to obtain D(-)-p-hydroxyphenylglycine. Most domestic D-p-hydroxyphenylglycine manufacturers currently use the glyoxylic acid process for production, but the splitting agent of this method is expensive, the sulfide odor is heavy, and the environmental pollution is large. The other type is preparation by bio-enzyme catalysis, that is, p-hydroxyphenylhydantoin is synthesized into D-p-hydroxyphenylglycine under the action of D-hydantoinase and D-carbamoylase. Due to the low catalytic efficiency and poor enzyme stability of the enzyme, the product preparation cost of this method has always been higher than the chemical preparation method. Therefore, there is no large-scale production on the market. Summary of the invention

[0003] The technical problem solved by the invention is to provide a method for preparing D-p-hydroxyphenylglycine with high yield.

[0004] The technical solution of the present invention is: a method for preparing D-p-hydroxyphenylglycine:

[0005] Step (1), adding deionized water, DL-hydroxyphenylhydantoin and enzyme solution into an enzyme reaction tank, stirring for reaction, and obtaining D-hydroxyphenylglycine enzyme conversion solution. The enzyme solution is a potassium phosphate buffer solution containing D-hydantoinase and D-carbamoylase, and the mass ratio of D-hydantoinase to D-carbamoylase is 1:2.

[0006] The enzyme solution is a 20% bacterial concentration Escherichia coli engineering bacterial solution (20g Escherichia coli is resuspended in 0.1M potassium phosphate buffer (PH8.0) to 100mL) obtained by homogenizing and crushing

[0007] Step (2), adding a hydrochloric acid solution with a mass fraction of 25-37% to the D-p-hydroxyphenylglycine enzyme conversion solution, adjusting the pH of the conversion solution to 3-4, then filtering through a microfiltration membrane, washing the microfiltration membrane with deionized water, and combining the filtrate to obtain a D-p-hydroxyphenylglycine solution.

[0008] Step (3), evaporating and concentrating the D-p-hydroxyphenylglycine solution in an evaporation tower to obtain a D-p-hydroxyphenylglycine concentrated solution.

[0009] Step (4), adding activated carbon to the D-p-hydroxyphenylglycine concentrate for decolorization, filtering, and obtaining a D-p-hydroxyphenylglycine decolorized solution. When the impurities in the concentrate are obvious, return to step (2) for microfiltration membrane filtration.

[0010] Step (5), adding the D-p-hydroxyphenylglycine decolorizing solution to a crystallization tank, adding a sodium hydroxide solution with a mass concentration of 350-400 g / L, adjusting the pH of the decolorizing solution to 5.5-6, precipitating crystals, centrifuging, collecting the separated solution and crystals, washing the crystals with deionized water, and heating at 100-110° C. for 6-8 hours to obtain D-p-hydroxyphenylglycine.

[0011] Preferably, the reaction temperature in step (1) is 40-45° C., the reaction time is 36-60 h, and the pH of the reaction solution is controlled to be 7-8 during the reaction process.

[0012] Preferably, the mass concentration of D-p-hydroxyphenylglycine in the D-p-hydroxyphenylglycine concentrate in step (3) is 460-520 g / L.

[0013] Preferably, during the decolorization process in step (4), the temperature is 35-45° C. and the time is 60-90 min.

[0014] Preferably, the activated carbon filtered in step (4) is washed with water, and the obtained washing liquid is returned to step (3) for evaporation and concentration.

[0015] Preferably, concentrated hydrochloric acid is added to the separated liquid obtained in step (5) to adjust the pH to 3-4, first pass through a cation exchange column, then pass through an ion anion exchange column, and then return the solution to step (3) for evaporation and concentration.

[0016] Preferably, the washing liquid obtained after washing with deionized water in step (5) is returned to step (3) for evaporation and concentration.

[0017] The technical effect of the present invention is as follows: the present invention generates D-p-hydroxyphenylglycine by enzymatic catalysis, and one-pot enzymatic catalysis generates D-p-hydroxyphenylglycine from p-hydroxyphenylhydantoin, and by improving the substrate tolerance of the enzyme, the yield of the final product reaches more than 90%, and the purity is high. The present invention simultaneously solves the environmental problems caused by the chemical production of D-p-hydroxyphenylglycine, and solves the catalytic efficiency and production cost problems of preparing D-hydroxyphenylglycine by the enzyme catalysis process. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with specific embodiments, which are intended to explain the present invention rather than to limit it.

[0019] Example 1

[0020] Step (1), adding 100L of deionized water, 15kg of DL-p-hydroxyphenylglycine, and 1000mL of enzyme solution into an enzyme reaction tank, wherein the enzyme solution is a potassium phosphate buffer solution containing D-hydantoinase and D-carbamoylase, and the mass ratio of D-hydantoinase to D-carbamoylase is 1:2; heating to 45°C, stirring and reacting for 36h, and controlling the pH of the solution to 8 during the reaction process to obtain D-p-hydroxyphenylglycine enzyme conversion solution.

[0021] Step (2), adding a 30% by mass hydrochloric acid solution to the D-p-hydroxyphenylglycine enzyme conversion solution, adjusting the pH of the conversion solution to 3, then filtering through a microfiltration membrane, washing the microfiltration membrane with deionized water, and combining the filtrate to obtain a D-p-hydroxyphenylglycine solution.

[0022] Step (3), evaporating and concentrating the D-p-hydroxyphenylglycine solution in an evaporation tower to obtain a D-p-hydroxyphenylglycine concentrated solution. The mass concentration of D-p-hydroxyphenylglycine in the concentrated solution is controlled to be 460 g / L.

[0023] Step (4), add activated carbon to the D-p-hydroxyphenylglycine concentrated solution, heat to 35°C, decolorize for 60 minutes, filter, and obtain D-p-hydroxyphenylglycine decolorized solution. Wash the filtered activated carbon with water, and the obtained water washing solution is returned to step (3) for evaporation and concentration.

[0024] Step (5), add the D-p-hydroxyphenylglycine decolorizing solution to a crystallization tank, add a sodium hydroxide solution with a mass concentration of 370g / L, adjust the pH of the decolorizing solution to 6, precipitate crystals, centrifuge, collect the separated solution and crystals, wash the crystals with deionized water, and heat at 105°C for 6h to obtain D-p-hydroxyphenylglycine. Add concentrated hydrochloric acid to the separated solution, adjust the pH to 3.5, first pass through a cation exchange column, then pass through an ion anion exchange column, and then return the solution to step (3) for evaporation and concentration. The washing solution obtained after washing the crystals with deionized water is returned to step (3) for evaporation and concentration.

[0025] Example 2

[0026] Step (1), add 100L of deionized water, 15kg of DL-p-hydroxyphenylglycine, and 1000mL of enzyme solution into an enzyme reaction tank, wherein the enzyme solution is a potassium phosphate buffer solution containing D-hydantoinase and D-carbamoylase, and the mass ratio of D-hydantoinase to D-carbamoylase is 1:2. Heat to 45°C, stir and react for 36h, and control the pH of the solution to 8 during the reaction process to obtain D-p-hydroxyphenylglycine enzyme conversion solution.

[0027] Step (2), adding a 25% by mass hydrochloric acid solution to the D-p-hydroxyphenylglycine enzyme conversion solution, adjusting the pH of the conversion solution to 4, then filtering through a microfiltration membrane, washing the microfiltration membrane with deionized water, and combining the filtrate to obtain a D-p-hydroxyphenylglycine solution.

[0028] Step (3), evaporating and concentrating the D-p-hydroxyphenylglycine solution in an evaporation tower to obtain a D-p-hydroxyphenylglycine concentrated solution. The mass concentration of D-p-hydroxyphenylglycine in the concentrated solution is controlled to be 520 g / L.

[0029] Step (4), add activated carbon to the D-p-hydroxyphenylglycine concentrated solution, heat to 40°C, decolorize for 90 minutes, filter, and obtain D-p-hydroxyphenylglycine decolorized solution. Wash the filtered activated carbon with water, and the obtained water washing solution is returned to step (3) for evaporation and concentration.

[0030] Step (5), add the D-p-hydroxyphenylglycine decolorizing solution to a crystallization tank, add a sodium hydroxide solution with a mass concentration of 400g / L, adjust the pH of the decolorizing solution to 5.5, precipitate crystals, centrifuge, collect the separated solution and crystals, wash the crystals with deionized water, and heat at 110°C for 7h to obtain D-p-hydroxyphenylglycine. Add concentrated hydrochloric acid to the separated solution, adjust the pH to 3, first pass through a cation exchange column, then pass through an ion anion exchange column, and then return the solution to step (3) for evaporation and concentration. The washing solution obtained after the crystals are washed with deionized water is returned to step (3) for evaporation and concentration.

[0031] Example 3

[0032] Step (1), add 100L of deionized water, 15kg of DL-p-hydroxyphenylglycine, and 900mL of enzyme solution into an enzyme reaction tank, wherein the enzyme solution is a potassium phosphate buffer solution containing D-hydantoinase and D-carbamoylase, and the mass ratio of D-hydantoinase to D-carbamoylase is 1:2. Heat to 40°C, stir and react for 48h, and control the pH of the solution to 7.5 during the reaction process to obtain D-p-hydroxyphenylglycine enzyme conversion solution.

[0033] Step (2), adding a 37% hydrochloric acid solution by mass to the D-p-hydroxyphenylglycine enzyme conversion solution, adjusting the pH of the conversion solution to 4, then filtering through a microfiltration membrane, washing the microfiltration membrane with deionized water, and combining the filtrate to obtain a D-p-hydroxyphenylglycine solution.

[0034] Step (3), evaporating and concentrating the D-p-hydroxyphenylglycine solution in an evaporation tower to obtain a D-p-hydroxyphenylglycine concentrated solution. The mass concentration of D-p-hydroxyphenylglycine in the concentrated solution is controlled to be 500 g / L.

[0035] Step (4), add activated carbon to the D-p-hydroxyphenylglycine concentrated solution, heat to 35°C, decolorize for 90 minutes, filter, and obtain D-p-hydroxyphenylglycine decolorized solution. Wash the filtered activated carbon with water, and the obtained water washing solution is returned to step (3) for evaporation and concentration.

[0036] Step (5), add the D-p-hydroxyphenylglycine decolorizing solution to a crystallization tank, add a sodium hydroxide solution with a mass concentration of 400g / L, adjust the pH of the decolorizing solution to 5.5, precipitate crystals, centrifuge, collect the separated solution and crystals, wash the crystals with deionized water, and heat at 110°C for 6h to obtain D-p-hydroxyphenylglycine. Add concentrated hydrochloric acid to the separated solution, adjust the pH to 3, first pass through a cation exchange column, then pass through an ion anion exchange column, and then return the solution to step (3) for evaporation and concentration. The washing solution obtained after washing the crystals with deionized water is returned to step (3) for evaporation and concentration.

[0037] Example 4

[0038] Step (1), add 100L of deionized water, 15kg of DL-p-hydroxyphenylglycine, and 1050mL of enzyme solution into an enzyme reaction tank, wherein the enzyme solution is a potassium phosphate buffer solution containing D-hydantoinase and D-carbamoylase, and the mass ratio of D-hydantoinase to D-carbamoylase is 1:2. Heat to 40°C, stir and react for 60h, and control the pH of the solution to 7 during the reaction process to obtain D-p-hydroxyphenylglycine enzyme conversion solution.

[0039] Step (2), adding a 25% by mass hydrochloric acid solution to the D-p-hydroxyphenylglycine enzyme conversion solution, adjusting the pH of the conversion solution to 4, then filtering through a microfiltration membrane, washing the microfiltration membrane with deionized water, and combining the filtrate to obtain a D-p-hydroxyphenylglycine solution.

[0040] Step (3), evaporating and concentrating the D-p-hydroxyphenylglycine solution in an evaporation tower to obtain a D-p-hydroxyphenylglycine concentrated solution. The mass concentration of D-p-hydroxyphenylglycine in the concentrated solution is controlled to be 480 g / L.

[0041] Step (4), add activated carbon to the D-p-hydroxyphenylglycine concentrated solution, heat to 45°C, decolorize for 60 minutes, filter, and obtain D-p-hydroxyphenylglycine decolorized solution. Wash the filtered activated carbon with water, and the obtained water washing solution is returned to step (3) for evaporation and concentration.

[0042] Step (5), add the D-p-hydroxyphenylglycine decolorizing solution to a crystallization tank, add a sodium hydroxide solution with a mass concentration of 380g / L, adjust the pH of the decolorizing solution to 5.5, precipitate crystals, centrifuge, collect the separated solution and crystals, wash the crystals with deionized water, and heat at 110°C for 6h to obtain D-p-hydroxyphenylglycine. Add concentrated hydrochloric acid to the separated solution, adjust the pH to 4, first pass through a cation exchange column, then pass through an ion anion exchange column, and then return the solution to step (3) for evaporation and concentration. The washing solution obtained after the crystals are washed with deionized water is returned to step (3) for evaporation and concentration.

[0043] Comparative Example 1

[0044] Step (1), add 100L of deionized water, 15kg of DL-p-hydroxyphenylglycine, and 1000mL of enzyme solution into an enzyme reaction tank, wherein the enzyme solution is a potassium phosphate buffer solution containing D-hydantoinase and D-carbamoylase, and the mass ratio of D-hydantoinase to D-carbamoylase is 1:2. Heat to 45°C, stir and react for 36h, and control the pH of the solution to 8 during the reaction process to obtain D-p-hydroxyphenylglycine enzyme conversion solution.

[0045] Step (2), adding a 30% by mass hydrochloric acid solution to the D-p-hydroxyphenylglycine enzyme conversion solution, adjusting the pH of the conversion solution to 3, then filtering through a microfiltration membrane, washing the microfiltration membrane with deionized water, and combining the filtrate to obtain a D-p-hydroxyphenylglycine solution.

[0046] Step (3), evaporating and concentrating the D-p-hydroxyphenylglycine solution in an evaporation tower to obtain a D-p-hydroxyphenylglycine concentrated solution. The mass concentration of D-p-hydroxyphenylglycine in the concentrated solution is controlled to be 460 g / L.

[0047] Step (4), add activated carbon to the D-p-hydroxyphenylglycine concentrated solution, heat to 35°C, decolorize for 60 minutes, filter, and obtain D-p-hydroxyphenylglycine decolorized solution. Wash the filtered activated carbon with water, and the obtained water washing solution is returned to step (3) for evaporation and concentration.

[0048] Step (5), adding the D-p-hydroxyphenylglycine decolorizing solution to a crystallization tank, adding a sodium hydroxide solution with a mass concentration of 370 g / L, adjusting the pH of the decolorizing solution to 6, precipitating crystals, centrifuging, collecting the separated liquid and crystals, washing the crystals with deionized water, and heating at 105° C. for 6 hours to obtain D-p-hydroxyphenylglycine.

[0049] Comparative Example 2

[0050] Step (1), add 100L of deionized water, 15kg of DL-p-hydroxyphenylglycine, and 1000mL of enzyme solution into an enzyme reaction tank, wherein the enzyme solution is a potassium phosphate buffer solution containing D-hydantoinase and D-carbamoylase, and the mass ratio of D-hydantoinase to D-carbamoylase is 1:2. Heat to 45°C, stir and react for 36h, and control the pH of the solution to 8 during the reaction process to obtain D-p-hydroxyphenylglycine enzyme conversion solution.

[0051] Step (2), adding a 30% by mass hydrochloric acid solution to the D-p-hydroxyphenylglycine enzyme conversion solution, adjusting the pH of the conversion solution to 3, then filtering through a microfiltration membrane, washing the microfiltration membrane with deionized water, and combining the filtrate to obtain a D-p-hydroxyphenylglycine solution.

[0052] Step (3), evaporating and concentrating the D-p-hydroxyphenylglycine solution in an evaporation tower to obtain a D-p-hydroxyphenylglycine concentrated solution. The mass concentration of D-p-hydroxyphenylglycine in the concentrated solution is controlled to be 460 g / L.

[0053] Step (4), adding activated carbon to the D-p-hydroxyphenylglycine concentrated solution, heating to 35° C., decolorizing for 60 minutes, filtering, and obtaining a D-p-hydroxyphenylglycine decolorized solution.

[0054] Step (5), adding the D-p-hydroxyphenylglycine decolorizing solution to a crystallization tank, adding a sodium hydroxide solution with a mass concentration of 370 g / L, adjusting the pH of the decolorizing solution to 6, precipitating crystals, centrifuging, collecting the separated liquid and crystals, washing the crystals with deionized water, and heating at 105° C. for 6 hours to obtain D-p-hydroxyphenylglycine.

[0055] Comparative Example 3

[0056] Step (1), adding 100L of deionized water, 15kg of DL-p-hydroxyphenylglycine, and 1000mL of enzyme solution into an enzyme reaction tank, wherein the enzyme solution is a potassium phosphate buffer solution containing D-hydantoinase; heating to 45°C, stirring and reacting for 36h, and controlling the pH of the solution to 8 during the reaction process to obtain D-p-hydroxyphenylglycine enzyme conversion solution.

[0057] Step (2), adding a 30% by mass hydrochloric acid solution to the D-p-hydroxyphenylglycine enzyme conversion solution, adjusting the pH of the conversion solution to 3, then filtering through a microfiltration membrane, washing the microfiltration membrane with deionized water, and combining the filtrate to obtain a D-p-hydroxyphenylglycine solution.

[0058] Step (3), evaporating and concentrating the D-p-hydroxyphenylglycine solution in an evaporation tower to obtain a D-p-hydroxyphenylglycine concentrated solution. The mass concentration of D-p-hydroxyphenylglycine in the concentrated solution is controlled to be 460 g / L.

[0059] Step (4), add activated carbon to the D-p-hydroxyphenylglycine concentrated solution, heat to 35°C, decolorize for 60 minutes, filter, and obtain D-p-hydroxyphenylglycine decolorized solution. Wash the filtered activated carbon with water, and the obtained water washing solution is returned to step (3) for evaporation and concentration.

[0060] Step (5), add the D-p-hydroxyphenylglycine decolorizing solution to a crystallization tank, add a sodium hydroxide solution with a mass concentration of 370g / L, adjust the pH of the decolorizing solution to 6, precipitate crystals, centrifuge, collect the separated solution and crystals, wash the crystals with deionized water, and heat at 105°C for 6h to obtain D-p-hydroxyphenylglycine. Add concentrated hydrochloric acid to the separated solution, adjust the pH to 3.5, first pass through a cation exchange column, then pass through an ion anion exchange column, and then return the solution to step (3) for evaporation and concentration. The washing solution obtained after washing the crystals with deionized water is returned to step (3) for evaporation and concentration.

[0061] Comparative Example 4

[0062] Step (1), adding 100L of deionized water, 15kg of DL-p-hydroxyphenylhydantoin, and 1000mL of enzyme solution into an enzyme reaction tank, wherein the enzyme solution is a potassium phosphate buffer solution containing D-carbamoylase; heating to 45°C, stirring and reacting for 36h, and controlling the pH of the solution to 8 during the reaction process to obtain D-p-hydroxyphenylglycine enzyme conversion solution.

[0063] Step (2), adding a 30% by mass hydrochloric acid solution to the D-p-hydroxyphenylglycine enzyme conversion solution, adjusting the pH of the conversion solution to 3, then filtering through a microfiltration membrane, washing the microfiltration membrane with deionized water, and combining the filtrate to obtain a D-p-hydroxyphenylglycine solution.

[0064] Step (3), evaporating and concentrating the D-p-hydroxyphenylglycine solution in an evaporation tower to obtain a D-p-hydroxyphenylglycine concentrated solution. The mass concentration of D-p-hydroxyphenylglycine in the concentrated solution is controlled to be 460 g / L.

[0065] Step (4), add activated carbon to the D-p-hydroxyphenylglycine concentrated solution, heat to 35°C, decolorize for 60 minutes, filter, and obtain D-p-hydroxyphenylglycine decolorized solution. Wash the filtered activated carbon with water, and the obtained water washing solution is returned to step (3) for evaporation and concentration.

[0066] Step (5), add the D-p-hydroxyphenylglycine decolorizing solution to a crystallization tank, add a sodium hydroxide solution with a mass concentration of 370g / L, adjust the pH of the decolorizing solution to 6, precipitate crystals, centrifuge, collect the separated solution and crystals, wash the crystals with deionized water, and heat at 105°C for 6h to obtain D-p-hydroxyphenylglycine. Add concentrated hydrochloric acid to the separated solution, adjust the pH to 3.5, first pass through a cation exchange column, then pass through an ion anion exchange column, and then return the solution to step (3) for evaporation and concentration. The washing solution obtained after washing the crystals with deionized water is returned to step (3) for evaporation and concentration.

[0067] The obtained D-p-hydroxyphenylglycine was weighed and the yield was calculated, and the purity was tested by liquid chromatography.

[0068] Table 1

[0069]

[0070]

[0071] The technical principle of the present invention is described above in conjunction with specific embodiments. These descriptions are only for explaining the principle of the present invention and cannot be interpreted as limiting the scope of protection of the present invention in any way. Based on the explanations herein, those skilled in the art can associate other specific implementations of the present invention without paying creative labor, and these methods will fall within the scope of protection of the present invention.

Claims

1. A method for preparing D-p-hydroxyphenylglycine, characterized in that: The preparation method is: Step (1), adding deionized water, DL-p-hydroxyphenylglycine, and enzyme solution into an enzyme reaction tank, stirring for reaction, and obtaining D-p-hydroxyphenylglycine enzyme conversion solution; Step (2), adding hydrochloric acid solution to the D-p-hydroxyphenylglycine enzyme conversion solution, filtering through a microfiltration membrane, washing the microfiltration membrane with deionized water, and combining the filtrate to obtain a D-p-hydroxyphenylglycine solution; Step (3), evaporating and concentrating the D-p-hydroxyphenylglycine solution in an evaporation tower to obtain a D-p-hydroxyphenylglycine concentrated solution; Step (4), adding activated carbon to the D-p-hydroxyphenylglycine concentrated solution for decolorization, filtering, and obtaining a D-p-hydroxyphenylglycine decolorized solution; Step (5), adding the D-p-hydroxyphenylglycine decolorizing solution to a crystallization tank, adding a sodium hydroxide solution, precipitating crystals, centrifuging, collecting the separated solution and crystals, washing the crystals with deionized water, and drying to obtain D-p-hydroxyphenylglycine.

2. The method for preparing D-p-hydroxyphenylglycine according to claim 1, characterized in that: The reaction temperature in step (1) is 30-50° C., the reaction time is 36-60 hours, and the pH of the reaction solution is controlled to be 7-8 during the reaction process.

3. The method for preparing D-p-hydroxyphenylglycine according to claim 1, characterized in that: In the step (2), hydrochloric acid solution is added to adjust the pH of the conversion solution to 2-5, and the mass fraction of the hydrochloric acid solution is 20-37%.

4. The method for preparing D-p-hydroxyphenylglycine according to claim 1, characterized in that: The mass concentration of D-p-hydroxyphenylglycine in the D-p-hydroxyphenylglycine concentrated solution in step (3) is 400-550 g / L.

5. The method for preparing D-p-hydroxyphenylglycine according to claim 1, characterized in that: During the decolorization process in step (4), the temperature is 30-50° C. and the time is 60-120 min.

6. The method for preparing D-p-hydroxyphenylglycine according to claim 1, characterized in that: The activated carbon filtered in step (4) is washed with water, and the obtained washing liquid is returned to step (3) for evaporation and concentration.

7. The method for preparing D-p-hydroxyphenylglycine according to claim 1, characterized in that: In the step (5), sodium hydroxide solution is added to adjust the pH of the decolorizing solution to 5-6.5, and the mass concentration of the sodium hydroxide solution is 300-500 g / L.

8. The method for preparing D-p-hydroxyphenylglycine according to claim 1, characterized in that: Concentrated hydrochloric acid is added to the separated liquid obtained in step (5) to adjust the pH to 3-5, and the separated liquid is first passed through a cation exchange column and then through an ion anion exchange column, and then the solution is returned to step (3) for evaporation and concentration.

9. The method for preparing D-p-hydroxyphenylglycine according to claim 1, characterized in that: The washing liquid obtained after washing with deionized water in step (5) is returned to step (3) for evaporation and concentration.

10. The method for preparing D-p-hydroxyphenylglycine according to claim 1, characterized in that: The mass of the enzyme solution is 6-7% of the mass of DL-p-hydroxyphenylhydantoin; the enzyme solution is a potassium phosphate buffer solution containing D-hydantoinase and D-carbamoylase, and the mass ratio of D-hydantoinase to D-carbamoylase is 1:2.

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