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Method for producing L-ornithine by immobilized enzyme method

A technology for immobilizing enzymes and ornithine, applied in the direction of microorganism-based methods, biochemical equipment and methods, immobilized on or in inorganic carriers, etc., can solve the unfavorable industrialized continuous production of free cell transformation and reduce arginine Enzyme and substrate contact area, separation and purification of unfavorable products and other issues, to achieve the effect of improving catalytic efficiency, improving conversion efficiency, and optimizing composition

Pending Publication Date: 2022-03-18
XINTAI JIAHE BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, its production process uses free cells to transform the substrate L-arginine, and the transformation solution will contain a small amount of bacterial protein and other impurities, which is not conducive to the separation and purification of the product
Therefore, free cell transformation is not conducive to industrial continuous production, and the utilization rate of enzymes is not high
Aiming at this, patent CN 1661026A, patent CN102286563A have proposed the method for immobilized enzyme to prepare L-ornithine, but, what above-mentioned prior art immobilized arginase adopts immobilized carrier embedding or utilizes self-made cellulose microbe Spherical carrier adsorption, which will inevitably reduce the contact area between arginase and substrate, thereby reducing the catalytic efficiency of the enzyme

Method used

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  • Method for producing L-ornithine by immobilized enzyme method
  • Method for producing L-ornithine by immobilized enzyme method
  • Method for producing L-ornithine by immobilized enzyme method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: Construction of human recombinant arginase I producing bacteria

[0041] 1. Plasmid transformation:

[0042] The pHT304 plasmid (purchased from Youbao Biology) was digested with NdeI and HincII, and then the Pg3 promoter (sequence shown in SEQ ID NO.1) was integrated into the pHT304 plasmid after double digestion to construct the plasmid pHT304-Pg3( figure 1 ).

[0043] The constructed plasmid pHT304-Pg3 is resistant to single ampicillin, has a lactose operon, and its nucleotide sequence is shown in SEQ ID NO.2.

[0044] The purpose of transforming the pHT304 plasmid is: first, to reduce the length of the vector and improve its expression stability in Bacillus subtilis; second, to integrate the Pg3 promoter into the pHT304 plasmid, and the Pg3 promoter only starts after adding IPTG Therefore, after integrating the Pg3 promoter, the strain grows fast in the early stage, and the time required to enter the stable phase is short, thereby shortening the expr...

Embodiment 2

[0063] Embodiment 2: the fermentative production of human recombinant arginase I

[0064] (1) Strain activation:

[0065] Streak-inoculate the human recombinant arginase I producing bacteria constructed in Example 1 onto an LB plate containing 100 μg / ml ampicillin, and culture at 33° C. for 24 hours.

[0066] (2) First-level species cultivation:

[0067] One inoculation loop of bacteria was drawn from the plate and transferred to primary seed medium (LB liquid medium supplemented with 50 ppm ampicillin), and cultured at 33° C., pH 7.0, and rotational speed 200 rpm for 18 hours.

[0068] (3) Secondary species cultivation:

[0069] With 1% (volume fraction) inoculation amount, inoculate primary seed liquid in secondary seed medium (LB liquid medium that adds 50ppm ampicillin), 33 ℃, dissolved oxygen (DO) 20-40%, cultivate to Dilute 100 times OD 600nm Value 0.5.

[0070] (4) Fermentation culture:

[0071] The secondary seed liquid of L-ornithine producing bacteria is inocul...

Embodiment 3

[0075] Embodiment 3: Production of L-ornithine by immobilized enzyme method

[0076] (1) Arginase immobilization treatment:

[0077] Adjust the pH value of the fermented broth after the induction culture in Example 2 to 7.2, and break it through a homogenizer. The mixed and homogenized cells after crushing are passed through a ceramic membrane, and the filtrate is collected; the pore diameter of the ceramic membrane is 100 nm, and the pressure is 0.5 MPa.

[0078] Dilute the filtrate to arginase concentration of 10g / L, add PBS to make the concentration of PBS 50mM, pass through nickel column, flow rate 3m 3 / h.

[0079] Nickel column affinity chromatography conditions: 50mM PBS was used as the binding buffer, and the filtrate was hung on the column.

[0080] (2) L-arginine conversion:

[0081] Substrate solution (containing L-arginine 50g / L, pyridoxal phosphate 1mg / L, dimethyl sulfoxide 0.1mg / L) in 3m 3 The flow rate of / h flows through the nickel column after the column ...

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Abstract

The invention discloses a method for producing L-ornithine by an immobilized enzyme method, which comprises the following steps: (1) inoculating a seed solution of a human recombinant arginase I producing strain into a fermentation culture medium for fermentation culture, cooling, adding IPTG (isopropyl-beta-d-thiogalactoside) into the system, and performing induced culture for 20-28 hours; (2) carrying out bacterium breaking treatment on the culture solution subjected to induced culture, passing through a ceramic membrane, and collecting filtrate; hanging the filtrate on a column; and (3) enabling a substrate solution containing L-arginine to flow through the nickel column with the hung column for biotransformation, and collecting an effluent transformation solution, so as to produce L-ornithine. According to the invention, a novel human recombinant arginase I producing strain is firstly constructed, and a fermentation process is optimized, so that the yield and enzyme activity of the human recombinant arginase I are improved; then carrying out immobilization treatment on the human recombinant arginase I produced by fermentation by adopting a nickel column hanging column; and then a substrate solution is fed for conversion treatment, so that the L-ornithine is produced by the immobilized enzyme method, and the conversion rate is increased.

Description

technical field [0001] The invention relates to the technical field of amino acid production, in particular to a method for producing L-ornithine by an immobilized enzyme method. Background technique [0002] L-ornithine is one of the ubiquitous amino acids in living organisms. In 1877, Jeffer found ornithine in the urine hydrolyzate of birds fed benzoic acid, hence the name. In animals, ornithine is mainly involved in the urea cycle, and L-ornithine, as an intermediate product in the production of urea, participates in the metabolism of amino acids such as citrulline and arginine, especially in the ornithine cycle. , can promote the excretion of ammonia in the body to a large extent for detoxification, so for the liver cells of the human body, L-ornithine is very important. In medicine, L-ornithine also has a wide range of applications, has an important position and broad market prospects in the family of amino acid health products, and directly participates in the biolog...

Claims

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Application Information

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IPC IPC(8): C12P13/10C12N9/78C12N15/75C12N15/55C12N15/66C12N11/14C12N1/21C12N1/38C12R1/125
CPCC12P13/10C12N9/78C12N15/75C12N15/66C12N11/14C12N1/38C12Y305/03001C12N2800/101C12N2800/22C12N2800/80
Inventor 曹华杰谢沛岳明瑞郭永胜
Owner XINTAI JIAHE BIOTECH CO LTD
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