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Strain capable of producing carboxyethylhydantoinase and application thereof

A technology for the production of carboxyethyl hydantoin and bacteria, applied in the fields of bioengineering and enzyme engineering, can solve the problem that carboxyethyl hydantoin enzyme is blank, and achieve the effect of simple fermentation method and good application prospects

Inactive Publication Date: 2013-08-14
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although carboxyethylhydantoinase belongs to the same category as hydantoinase in the form of catalytic reaction, due to various reasons, the research on carboxyethylhydantoinase at home and abroad is almost blank at present.

Method used

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  • Strain capable of producing carboxyethylhydantoinase and application thereof
  • Strain capable of producing carboxyethylhydantoinase and application thereof
  • Strain capable of producing carboxyethylhydantoinase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: This experiment illustrates the screening process of natural strains producing carboxyethylhydantoinase.

[0024] Medium:

[0025] A. Broth medium (g / L): beef extract 3, peptone 10, NaCl5, agar 20, pH=7.2

[0026] B. Enrichment medium (g / L): carboxyethyl hydantoin 5, glucose 3, peptone 3, potassium dihydrogen phosphate 5, magnesium sulfate 0.5, ferrous sulfate 0.055, manganese sulfate 0.0045, pH7.0

[0027] C. Screening medium (g / L): add 20g L to the enrichment medium -1 agar powder

[0028] D. Seed medium (g / L): glucose 15, peptone 15, sodium chloride 3, yeast extract 5, potassium dihydrogen phosphate 2,

[0029] Magnesium sulfate 0.25, pH=7.0-7.2

[0030] E. Fermentation medium (g / L): glucose 10, peptone 10, yeast extract 10, sodium chloride 3, potassium dihydrogen phosphate 2, magnesium sulfate 0.25, cobalt chloride 0.05, carboxyethylhydantoin 2, pH= 7.0

[0031] All media were prepared with deionized water, 0.1MPa, sterilized at 121°C for 15 minutes...

Embodiment 2

[0036] Example 2: This experiment illustrates the identification of the biological properties of the carboxyethylhydantoinase-producing strain CW221.

[0037] Biological properties of carboxyethylhydantoinase-producing bacteria CW221: under high-power microscope observation, the cells are mainly oval, short rod-shaped, and the aspect ratio is about 2-3. There are also approximately spherical cells, and the cells are arranged irregularly. Exist alone, no clusters or clusters of cells exist. Gram-negative bacteria; the colonies grown on solid medium plates for 24 hours are round, with a diameter of about 1mm, smooth, milky white, opaque, with uneven edges and a slight protrusion in the middle; cultured on a slant, the colonies grow along the lines , the edge is not smooth and has small thorns; when cultured in shake flasks, the culture medium is milky white and opaque. Physiological and biochemical characteristics show that the bacteria is obligate aerobic, can not use glucose ...

Embodiment 3

[0039] Example 3: This experiment illustrates the purification procedure of carboxyethylhydantoinase.

[0040] Buffer A: 0.02mol / L Tris-HCl, pH9.0,

[0041] Weigh 15 g of fermented bacteria (see the fermentation method of the screening process in Example 1 for details) and dissolve in 60 ml of buffer A to prepare a bacterial suspension. After ultrasonication (ultrasonic time 3s, intermittent time 5s, power 400W, protection temperature 25°C, whole process 10min, repeat 3 times), centrifuge at 10000r / min for 30min at 4°C, and collect the supernatant. After 20%, 40%, 60%, 80% saturation ammonium sulfate graded precipitation. The precipitate was collected by centrifugation, dissolved in buffer A, transferred to a dialysis bag, dialyzed with the same buffer at 4°C, and then concentrated with polyethylene glycol-20000. Take the concentrated sample and put it on the DEAESepharose F.F anion exchange column, use buffer A and 1mol / L NaCl for linear gradient elution, and collect the co...

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Abstract

The invention relates to a strain capable of producing carboxyethylhydantoinase. The strain capable of producing carboxyethylhydantoinase is characterized in that classification name of the strain is alcaligenes faecalis subsp.faecalis, and the strain is preserved in China General Microbiological Culture Center on May 13th, 2013 with the preservation number of CGMCC No.7585. The invention also discloses an application of the strain capable of producing carboxyethylhydantoinase. Activity of carboxyethylhydantoinase produced by the strain alcaligenes faecalis subsp.faecalis is 0.064U / ml, the activity of purified carboxyethylhydantoinase can be 0.61U / ml, and enzyme activity of a constructed genetically engineered bacterium is 3.06U / ml, and a fermentation method of recombinant bacteria is simple. Conversion rate of substrate is high, the conversion rate of the substrate after reaction is carried out for 2 hours is 95%, the conversion rate of the substrate after reaction is carried out for 4 hours is 99.98%, and the strain has a better application prospect in catalyzing synthesis of carboxyethylhydantoinase.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and enzyme engineering, and specifically relates to a carboxyethyl hydantoin enzyme production strain and its application. Background technique [0002] The catabolism pathways of L-histidine mainly include the following five pathways: 1. Decarboxylation to histamine, further oxidation to imidazole acetaldehyde and imidazole acetic acid; 2. Deamination to urocanic acid, further degradation to glutamic acid, entering Glutamate metabolism pathway; 3. Transamination to form imidazole pyruvate, which is then reduced to imidazole lactic acid or imidazole acetic acid; 4. Methylation to form 1- or 3-methylhistidine; 5. Combination with β-propion Amino acid condensation reaction to form carnosine or anserine. The second pathway is the most basic pathway of L-histidine metabolism, which is relatively conservative in various species from prokaryotes to eukaryotes. L-glutamic acid is obtained in sta...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/86C12N15/55C12N15/70C12N1/21C12P13/14C12R1/05
Inventor 曹飞薄薇周佳栋姚琴李晓婉文斌斌汤智群
Owner NANJING UNIV OF TECH
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