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Method for preparing highly-purified L-Lysine sulphate through one-time fermentation

A technology of lysine sulfate and lysine, applied in the field of amino acid fermentation

Active Publication Date: 2013-02-13
NINGXIA EPPEN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Through the inventor's long-term research and experiments, it has been found surprisingly that adding a certain proportion of other types of amino acids in the lysine sulfate with poor hygroscopicity resistance will reduce the purity of the lysine sulfate in the product, but It will significantly improve the moisture absorption resistance of lysine sulfate products, and the purity of the product will not affect its effectiveness as a lysine feed additive

Method used

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  • Method for preparing highly-purified L-Lysine sulphate through one-time fermentation
  • Method for preparing highly-purified L-Lysine sulphate through one-time fermentation
  • Method for preparing highly-purified L-Lysine sulphate through one-time fermentation

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Experimental program
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Effect test

Embodiment 1

[0063] The preparation of embodiment 1 RNA polymerase sigma-32 factor gene construct

[0064] According to the amino acid sequence of the protein accession number AAB18436.1 of NCBI (http: / / www.ncbi.nlm.nih.gov), the inventors designed a moderate expression codon (non-expression optimized codon), and obtained The pathway commissioned Shanghai Sangon Biotechnology Co., Ltd. to synthesize the gene encoding RNA polymerase sigma-32 factor and construct it into E. coli expression plasmid pET-20b(+) (available from Novagen, USA, product number Cat.No.69739-3 )middle. The cloning process was carried out according to the "Molecular Cloning Experiment Guide" and the operation guide of the commercial reagents used. The brief process is as follows:

[0065] Synthesize nucleic acid fragments of the RNA polymerase sigma-32 factor gene by an automatic DNA synthesizer, use T4 polynucleotide kinase (purchased from TaKaRa company) to phosphorylate the 5' ends of these nucleic acid fragments, ...

Embodiment 2

[0067] The preparation of the L-lysine sulfate product of embodiment 2 escherichia coli fermentation

[0068] The L-lysine-producing Escherichia coli strain W3110(tyrA) / pCABD2 constructed by the method described in Chinese Patent No. 94194962 was transformed into a pET-sigma plasmid, and a positive clone was obtained after identification (named as W3110(tyrA) / pCABD2 -sigma), the strains W3110(tyrA) / pCABD2 and W3110(tyrA) / pCABD2-sigma were subjected to lysine fermentation experiments with reference to Chinese Patent No. 03120099. In short, the bacterial strain was inserted into the liquid LB medium for shaking culture until the OD500 reached 0.35, and was inserted into the lysine fermentation medium with a 5% inoculum size (the formulation of the medium per liter was: 100g glucose, 60g (NH 4 ) 2 SO 4 , 50g CaCO 3 , 35mL peptone-B (Soy Protein Enzymatic Hydrolysate, purchased from Shanghai Xinran Biotechnology Co., Ltd., the total nitrogen content is not less than 3%), 1g KH ...

Embodiment 3

[0072] The anti-hygroscopicity test of embodiment 3L-lysine product

[0073] Get the L-lysine sulfate product prepared by W3110(tyrA) / pCABD2-sigma (referred to as sigma) of embodiment 2 and the L-lysine sulfate product prepared by W3110(tyrA) / pCABD2 (represented as Control 1).

[0074] In addition, the chemically pure reagents in the following weight ratios were mixed evenly (referred to as control 2): ​​lysine sulfate, 69.9; aspartic acid, 1.54; threonine, 0.94; serine, 0.56; glutamic acid, 2.4; Glycine, 0.92; Alanine, 1.4; Valine, 0.93; Methionine, 0.42; Isoleucine, 0.61; Leucine, 1.27; Tyrosine, 0.67; Phenylalanine, 0.64; amino acid, 0.97; and, arginine, 1.1.

[0075] In addition, the chemically pure reagents in the following weight ratios were mixed evenly (referred to as control 3): lysine sulfate, 69.9; aspartic acid, 1.54; and, glutamic acid, 2.4.

[0076] The above samples were placed at 25°C for 7 days under different relative humidity environments, and then the eq...

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Abstract

The invention provides a method for preparing products containing L-Lysine sulphate by fermenting, wherein a gene which is known to have nothing to do with lysine metabolism is lead into bacteria which produce L-Lysine, and the content of lysine in the L-Lysine sulphate achieves purity of market products. Furthermore, the invention further provides the L-Lysine sulphate produced by the method, and the L-Lysine sulphate can obviously increase moisture absorption resistance ability relative to existing products containing the L-Lysine sulphate, thereby being suitable for long-term preservation.

Description

technical field [0001] The present invention belongs to the field of amino acid fermentation. Specifically, the present invention relates to a method for fermenting and preparing products containing L-lysine sulfate, which includes innovatively introducing a lysine that is generally considered to be related to lysine into L-lysine-producing bacteria. Acid metabolism unrelated genes, and the lysine content in the product has reached the purity of commercially available products. In addition, the invention also provides products and applications produced by the method. Background technique [0002] L-lysine is an important amino acid raw material, which can be used as condiment, food, feed additive, and also as an effective or auxiliary ingredient in health care products and medicines. It is widely used in food industry, feed industry, pharmaceutical industry and other chemical industries. In industry, especially lysine salts containing impurities (such as sulfate, hydrochlor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/04C12P13/08A23K1/16C12R1/19
Inventor 马吉银陈崇安孟刚
Owner NINGXIA EPPEN BIOTECH
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