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Strain producing hydantoinase and carboxamide hydrolase and preparation method and application of strain

A hydantoinase and carbamyl technology, applied in the field of applied microorganisms, can solve the problem of low synthesis efficiency

Inactive Publication Date: 2019-11-01
UNIV OF SCI & TECH OF CHINA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to screen out a strain that produces highly active hydantoinase and N-carbamyl hydrolase for the low synthesis efficiency of the method for synthesizing D-p-hydroxyphenylglycine in the prior art, The hydantoinase and N-carbamyl hydrolase prepared by the strain are used to catalyze the synthesis of D-p-hydroxyphenylglycine from D,L-p-hydroxyphenylhydantoin, and the conversion rate can reach more than 99%, thus providing a new Green, efficient and convenient method for synthesizing D-p-hydroxyphenylglycine

Method used

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  • Strain producing hydantoinase and carboxamide hydrolase and preparation method and application of strain
  • Strain producing hydantoinase and carboxamide hydrolase and preparation method and application of strain
  • Strain producing hydantoinase and carboxamide hydrolase and preparation method and application of strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Determination of enzyme activity

[0106] ①Definition of the activity of hydantoin enzyme with D,L-p-hydroxyphenylhydantoin as the substrate: the activity of 1 unit of hydantoin enzyme is defined as the reduction of 1 μmol D,L-p-hydroxybenzene per minute at pH 8.0, 45°C The amount of enzyme needed for hydantoin.

[0107] ②Definition of the activity of N-carbamyl hydrolase with N-carbamoyl-p-hydroxyphenylglycine as the substrate: the activity of 1 unit hydantoinase is defined as the reduction of 1 μmol N per minute at pH 8.0, 45°C - The amount of enzyme required for carbamoyl-p-hydroxyphenylglycine.

[0108] The determination method is as follows:

[0109] By analyzing the content of D-p-hydroxyphenylglycine and N-carbamoyl-p-hydroxyphenylglycine formed in the standard reaction, the activity of hydantoinase and N-carbamoylhydrolase in the soluble multi-enzyme extract was determined.

[0110] Standard reaction system for measuring hydantoin enzyme activity: D,L-p-hydro...

Embodiment 2

[0113] Study on Enzymatic Properties of Multi-enzyme Extract

[0114] Optimum pH and pH Stability for Enzymatic Reactions

[0115] Take 0.1M buffer (pH range 7.0-10.4, pH 7.0-8.0 (0.1M Na 2 PO 4 ), pH8.0-9.0 (0.1M Tris-HCl), pH 9.0-10.4 (0.1M K 2 CO 3 )), reaction system: D, L-p-hydroxyphenylhydantoin (10g / L), dilute the enzyme solution, set up three parallels, and measure the generation of D-p-hydroxyphenylglycine and N-ammonia under the conditions of each pH value at 45°C Formyl p-hydroxyphenylglycine capacity. The highest production of D-p-hydroxyphenylglycine was taken as 100%, and the relative yield was plotted against pH.

[0116] Place the diluted enzyme solution in buffer solutions of different pH (pH range 7.0-10.4) at 45°C for 2 hours, set up three parallels for each, and measure the ability and N-carbamoyl-p-hydroxyphenylglycine, the highest D-p-hydroxyphenylglycine produced by the untreated enzyme was defined as 100%, and the relative yield was plotted agains...

Embodiment 3

[0121] In a 300L enzyme-catalyzed reactor, add 210kg of anaerobic water, then put in 32-38kg of D,L-p-hydroxyphenylhydantoin, stir at 65rpm, add wet enzyme preparation with an enzyme activity of 0.62U / ml for about 15kg, heat, control the temperature at 32-44°C, take samples at intervals of 3 hours and use high-pressure liquid chromatography to detect and reflect the situation. After the reaction is carried out for 6-8 hours, D-hydroxyphenylglycine crystals are precipitated, and D-hydroxyphenylglycine is specifically formed. The results of real-time monitoring conversion rate of phenylglycine are attached Figure 7 shown. When the N-carbamoyl-p-hydroxyphenylglycine in the reaction system is below 0.25%, the reaction is basically completed, and the conversion rate is above 99%. The product is obtained after partial refining of the crude crystal and the solution, and the total yield is 85%.

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Abstract

The invention relates to a novel microorganism namely pseudomonas monteilii CM strain producing hydantoinase and N-carboxamide hydrolase at the same time, a preparation method of the strain, and an application of the strain to synthesis of D(-)-4-hydroxyphenylglycine. The invention screens out a strain which can produce high-activity hydantoinase and N-carboxamide hydrolase. The hydantoinase and the N-carboxamide hydrolase prepared from the strain are used for catalyzing D, L-4-hydroxyphenyl hydantoin for synthesis of the D(-)-4-hydroxyphenylglycine, the conversion rate can reach 99% or above,and a new green high-efficiency convenient method for synthesizing the D(-)-4-hydroxyphenylglycine is provided.

Description

technical field [0001] The invention belongs to the technical field of applied microorganisms, and in particular relates to a new microorganism Pseudomonas monsonii CM strain that simultaneously produces hydantoinase and N-carbamyl hydrolase, a preparation method of the strain and the synthesis of D- Application of p-hydroxyphenylglycine. Background technique [0002] D-Hydroxyphenylglycine is an important intermediate for the synthesis of β-lactam semi-synthetic broad-spectrum antibiotics (such as: penicillins, cephalosporins, etc.), and is also an important intermediate for the synthesis of antibacterial and antiviral drugs and artificial sweeteners body. China is a big country in the production and demand of antibiotics, and the Chinese pharmaceutical industry has focused on the development of semi-synthetic penicillins and semi-synthetic cephalosporins. Therefore, the research on the new process of D-hydroxyphenylglycine has important practical significance. [0003] T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/80C12N9/86C12P13/04C12R1/38
CPCC12N9/86C12Y305/02002C12N9/80C12Y305/01087C12P13/04C12R2001/38C12N1/205
Inventor 傅尧李向杰徐海刘孝龙汪哲仑
Owner UNIV OF SCI & TECH OF CHINA
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