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Alcaligenes and method for preparing D-p-hydroxyphenylglycine by using same

A technology of p-hydroxyphenylglycine and Alcaligenes, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problems of serious environmental pollution, low product yield, complex process, etc., and achieve environmental protection, The effect of simple breeding method and easy preparation

Inactive Publication Date: 2012-04-04
SHANGHAI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of chemical synthesis are: complex process, high cost, and serious environmental pollution
The disadvantages of the enzymatic resolution method are: 1. The yield of the product is low, the highest not exceeding 50%; 2. The racemized acetylated DL-HPG substrate must be synthesized in advance, and the process is complicated
The enzymatic conversion method can theoretically convert 100% of the substrate into amino acids of a single enantiomer, but there are still very few microbial strains that can achieve the above conversion with high yield and high optical purity to prepare D-hydroxyphenylglycine

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Bacterial medium formula is: glycerol 10.0g / L, yeast extract 10.0g / L, KH 2 PO 4 2.0g / L, MgSO 4 0.5g / L, pH 7.0. Take the slant strain of Alcaligenes preserved at 4°C, pick a loop and inoculate it into a 250ml shake flask containing 50ml of culture medium, culture it at 30°C with shaking at 160rpm for 24h, and harvest the cells by centrifugation. The enzyme activity of the fermentation broth is about 10-20U / L, the wet weight of the cell concentration is 5-10g / L, and the enzyme activity of the unit cell is 2U / g wet cell. The cell viability unit is defined as the amount of enzyme required to catalyze the hydrolysis of p-hydroxyphenylhydantoin to produce 1.0 μmol of N-carbamoyl-p-hydroxyphenylglycine per minute under the conditions of 30°C and pH 7.0.

Embodiment 2

[0051] Take the above-mentioned Alcaligenes cells with a wet weight of 1.0g, suspend them in 9.5ml sodium phosphate buffer solution (0.1M, pH 8.5), add 200mg p-hydroxyphenylhydantoin and 0.5ml dimethyl sulfoxide, and the reaction mixture is heated at 30 ℃, 160r / min constant temperature shaking reaction, intermittent sampling with liquid chromatography (chromatographic column is a Chromasil C18 chromatographic column) analysis p-hydroxyphenylhydantoin and N-formyl p-hydroxyphenylglycine content, after 12 hours of reaction, The analytical yield of N-formyl-p-hydroxyphenylglycine was 95%.

Embodiment 3

[0053] Take the above-mentioned Alcaligenes cells with a wet weight of 20.0g, suspend them in 190ml of sodium phosphate buffer solution (0.1M, pH 8.5), add 4g of p-hydroxyphenylhydantoin and 10ml of dimethyl sulfoxide, and the reaction mixture is heated at 30°C, Shake the reaction on a constant temperature shaker at 160r / min, centrifuge the reaction solution for 18-20hr, take the supernatant, slowly add concentrated hydrochloric acid (6mol / L) in an ice-water bath, and adjust the pH value to about pH 3.0. Then, with the freshly prepared 10% NaNO 2 Solution, continue to slowly dropwise add 3hr under the condition of ice-water bath, wherein, concentrated hydrochloric acid and 10% NaNO 2 The volume ratio of the solution is about 4:5. After the dropwise addition was completed, the reaction was continued for 2 hr. The product was purified by domestic 732 cation exchange resin, and the final yield of D-p-hydroxyphenylglycine was 90%, [α] D 20 (C=1, 1 mol / l HCl)=160.5 (literature ...

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Abstract

The invention relates to alcaligenes and an application thereof, in particular to the alcaligenes and a method for preparing D-p-hydroxyphenylglycine by using the same. The prior art for preparing the D-p-hydroxyphenylglycine has the disadvantages that: the process is complicated, the cost is high and the pollution to the environment is serious. The alcaligenes with the strain collection number of CGMCC (China General Microbiological Culture Collection Center) No.3872 is obtained by separating and screening from soil. The method for preparing the D-p-hydroxyphenylglycine comprises the following steps of: taking alcaligenes cells and putting the cells into a potassium phosphate buffer solution; adding racemate hydantoin into the buffer solution to react at a temperature of 20-50 DEG C for 3-96 hours; after reacting a reaction solution with NaNO2, separating and purifying to obtain the D-p-hydroxyphenylglycine. The alcaligenes and the method provided by the invention have the advantages that: the method for breeding the alcaligenes is simple; the effects of the prepared D-p-hydroxyphenylglycine are good and the yield is up to 90%; the cost of the D-p-hydroxyphenylglycine is low; no pollutants are emitted and environmental friendliness is easy to realize.

Description

technical field [0001] The present invention relates to Alcaligenes bacterium and its application, more specifically to a kind of Alcaligenes bacterium and a method for preparing D-p-hydroxyphenylglycine by using the Alcaligenes bacterium. Background technique [0002] D-Hydroxyphenylglycine is an important pharmaceutical raw material for the synthesis of β-lactam antibiotics, such as amoxicillin, cefaclor, ceftrixin, cefradine and other antibiotics. These drugs have killing effects on Gram-positive bacteria, Gram-negative bacteria, Toxoplasma, spirochetes, etc., are widely used in clinical medicine, and play an important role in the treatment of many common diseases. There are two methods for preparing D-p-hydroxyphenylglycine in the prior art: chemical synthesis method and enzymatic method. The disadvantages of chemical synthesis are: complex process, high cost, and serious environmental pollution. Compared with chemical synthesis, enzymatic preparation of D-hydroxypheny...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P13/04C12R1/05
Inventor 陈建波孙婧徐毅
Owner SHANGHAI NORMAL UNIVERSITY
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