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Method for preparing d-arginine hydrochloride and l-ornithine hydrochloride by splitting dl-arginine by microbial enzymatic method

A technology of arginine hydrochloride and ornithine hydrochloride, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the cumbersome separation and extraction process, limited expression enzyme activity level, extraction process cumbersome and other problems, to achieve the effect of simple process steps, less enzyme input and safe operation

Inactive Publication Date: 2011-12-21
天津启仁医药科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] (1) The extraction method is to extract from the membrane components of bacteria and polypeptide antibiotic substances, but the cost of extracting and preparing ornithine by this method is too high, and the extraction process is cumbersome
[0009] (2) The chemical method is divided into organic synthesis method and arginine hydrolysis method. This method uses a large amount of organic solvents, and the obtained DL-type mixture has low recovery rate and difficult separation and purification.
The growth cycle of these strains is generally not less than 24 hours, and due to the limited level of enzyme activity expressed by itself, after adding a certain amount of enzyme source cells to the transformation system, the separation and extraction process after transformation is bound to be relatively cumbersome.

Method used

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  • Method for preparing d-arginine hydrochloride and l-ornithine hydrochloride by splitting dl-arginine by microbial enzymatic method
  • Method for preparing d-arginine hydrochloride and l-ornithine hydrochloride by splitting dl-arginine by microbial enzymatic method
  • Method for preparing d-arginine hydrochloride and l-ornithine hydrochloride by splitting dl-arginine by microbial enzymatic method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, the construction of genetically engineered bacteria Escherichia coli and the expression of recombinant L-arginase

[0038] According to the rat arginase gene sequence recorded in the NCBI database, the upstream primer arg1: 5'-GCGGGATCC ATG AGC TCC AAG CCA AAG CC-3' and the downstream primer arg2: 5'-GCC GTCGAC TTATTT CGG TGG TTT A- 3'. Rat liver total RNA was extracted, and cDNA was prepared by reverse transcription. Using rat liver cDNA as a template, arginase gene was amplified by PCR. The recombinant expression plasmid pET-21a(+)-Arg was constructed by genetic engineering method. The recombinant expression vector pET-21a(+)-Arg was transformed into E.coli BL21(DE3) to obtain the genetic engineering strain Arg5.

[0039] The positive recombinant strain was inoculated into 4 ml of LB medium containing 100 μg / mL of ampicillin for overnight culture, and then inserted into 100 mL of fresh LB medium containing 100 μg / mL of ampicillin at a transfer rate of...

Embodiment 2

[0041] Embodiment 2, fermentation of recombinant L-arginase genetically engineered recombinant bacteria

[0042] The recombinant Escherichia coli in Example 1 was inoculated in 4 mL of LB medium containing 100 μg / mL ampicillin and cultivated overnight as a primary seed liquid; then inserted into 100 mL of LB culture containing 100 μg / mL ampicillin with 1% transfer amount culture medium at 37°C and 200rpm for 8h-12h as the secondary seed liquid; the secondary seed liquid was transferred to a 100L fermenter with 1% inoculum, the fermentation medium was fresh LB medium, and the culture temperature was 37°C. The initial rotation speed is 300rpm, and the initial pH is 7.2. Samples were taken regularly, and when the OD600nm of the bacterial solution reached 1.0-1.2, IPTG induction solution with a final concentration of 1 mM was added for induction. The fermented liquid in the lower tank was centrifuged at 3,000 rpm for 15 minutes at 4°C to collect the bacterial cells to obtain the ...

Embodiment 3

[0043] Embodiment 3, the investigation of spray-drying condition and the preparation of solid enzyme preparation

[0044] To the bacterial suspension obtained in Example 2, add 20 g / L sugars, polymers, polyols, proteins, surfactants, salts, etc. as investigation objects, and after fully dissolving, preheat in a 50°C water bath After 30 minutes, the remaining enzyme activity of the blank sample was regarded as 100%, and the relative remaining enzyme activity was compared. The results are shown in Table 1. Skimmed milk powder, sodium glutamate, and gum arabic all have good protective effects. Among them, skimmed milk powder has the best protective effect. If cost is considered, sodium glutamate or gum arabic can be preferred.

[0045] The influence of table 1 protective agent on temperature stability

[0046]

[0047] Add a certain amount of skimmed milk powder, sodium glutamate or gum arabic to the bacterial suspension with a concentration of 100-200g / L as a protective agen...

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Abstract

A method for preparing D-arginine hydrochloride and L-ornithine hydrochloride by splitting DL-arginine by microbial enzymatic method. The present invention utilizes the fermented bacterium of recombinant E. coli or its spray-dried preparation that highly expresses L-arginase as an enzyme source, uses DL-arginine as a substrate, and splits DL-arginine through an enzymatic reaction to produce D-arginine and L-ornithine. The resulting liquid is converted into L-ornithine monohydrochloride by adding hydrochloric acid, then adding a certain volume of ethanol to precipitate it, and collecting the precipitate to obtain L-ornithine hydrochloride; in the supernatant, D- The purification method of arginine is: adsorption and elution of ion exchange resin, concentration and drying under reduced pressure, to obtain D-arginine hydrochloride; this patent provides a new D-arginine hydrochloride and L- Green production process route of ornithine hydrochloride.

Description

technical field [0001] The invention belongs to the technical field of amino acid production by biotechnology, and relates to a method for producing D-arginine hydrochloride and L-ornithine hydrochloride by decomposing DL-arginine through microbial enzymatic method. Background technique [0002] D-arginine (D-arginine, D-Arg), as a non-protein amino acid, is an important chiral reagent and pharmaceutical intermediate. Studies have shown that D-arginine has antihypertensive effects. As a constituent amino acid of polypeptides, the important physiological functions of D-arginine are also manifested in inhibiting the spread of cancer, treating disorders caused by excessive release of growth hormone, and inhibiting DNA synthesis. and inhibit the proliferation of prostate cancer cells. [0003] The preparation method of D-arginine mainly contains at present: [0004] (1) Chemical method The chemical method is to react DL-arginine with an optically active resolving agent to gene...

Claims

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Application Information

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IPC IPC(8): C12P41/00C12P13/10C12R1/19
Inventor 高智慧刘磊王文芳
Owner 天津启仁医药科技有限公司
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