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Fed-batch fermentation preparation of lysine

A technology of fed-feed fermentation and lysine, applied in the field of amino acid fermentation, can solve the problem of no report on enzyme variant research, and achieve the effects of increasing fermentation yield, increasing yield and simplifying operation.

Active Publication Date: 2011-11-09
NINGXIA EPPEN BIOTECH +1
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  • Claims
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AI Technical Summary

Problems solved by technology

Although subunits of wild-type pyridine nucleotide transhydrogenase have been published (see NCBI (http: / / www.ncbi.nlm.nih.gov) protein and gene accession numbers NP416120.1 and AAC74674.1; See Chinese patent ZL94194707), but the research of this enzyme variant has not reported

Method used

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  • Fed-batch fermentation preparation of lysine
  • Fed-batch fermentation preparation of lysine
  • Fed-batch fermentation preparation of lysine

Examples

Experimental program
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Effect test

Embodiment 2

[0032] The activity assay of embodiment 2 escherichia coli and the fermentation experiment of coryneform bacterium

[0033] According to the existing assay method for transhydrogenase activity (see Clarke DM et al. Cloning and expression of the transhydrogenase gene of Escherichia coli.J.Bacteriol., 162:367-373), E.coli transformed with pMS2-cispnt plasmid -cispnt bacterial strain and the negative Top10F' bacterial strain as a control measure the transhydrogenase activity respectively, and the specific enzyme activity of the found E.coli-cispnt bacterial strain is 163% higher than that of the negative Top10F' bacterial strain, much higher than the prior art Enzyme specific activity increase rate with wild-type pyridine nucleotide transhydrogenase expressed in .

[0034] The pMS2-cispnt plasmid is transferred to L-lysine-fermenting coryneform bacteria engineering bacteria (available from the American Type Microorganism Collection (ATCC), product number ATCC 31269) by electropor...

Embodiment 3

[0036] Fed-batch fermentation example 1 of embodiment 3L-lysine

[0037] The conversion of embodiment 2 has the coryneform bacterium engineering bacterium of pMS2-cispnt plasmid with 0.5% inoculum size and inserts 20 cubic meters of fermentors (wherein the substratum formula is: 600 kilograms of glucose, 200 kilograms of sugarcane molasses, 520 kilograms of corn steep liquor , KH 2 PO 4 45 kg, MgSO 4 ·7H 2 O 7 kg, FeSO 4 ·7H 2 O 0.5 kg, MnSO 4 ·7H 2 (0.5 kg, 12 grams of biotin, and 5 grams of folic acid, fixed to 18 cubic meters with water), cultured at 35° C. with saturated aeration for 15 hours to increase the cell density.

[0038] Then, directly inject the nutrient solution of 20 cubic meters of fermentation tanks into 350 cubic meters of fermentation tanks (wherein the medium formula is: 12000 kilograms of glucose, 1000 kilograms of sugarcane molasses, 12000 kilograms of corn steep liquor, KH 2 PO 4 1000 kg, MgSO 4 ·7H 2 O 100 kg, FeSO 4 ·7H 2 O 12 kg, MnSO ...

Embodiment 4

[0039] Fed-batch fermentation example 2 of embodiment 4L-lysine

[0040] Basically the same as in Example 3, the difference is that the culture solution of the 20 cubic meter fermenter is directly injected into the 350 cubic meter fermenter, and cultured at 39° C. for 3 hours with saturated aeration. Then, 1100 kilograms of glucose was fed per hour for 15 hours; after that, 1500 kilograms of glucose and 700 kilograms of ammonium sulfate were fed per hour for continuous fermentation for 55 hours. Thin-layer chromatography detected that 128 g / L of L-lysine was produced.

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Abstract

The invention provides a method for fed-batch fermentation of L-lysine, which comprises the following steps of: introducing engineering bacteria for expressing pyridine nucleotide transhydrogenase variants into a first fermentation tank for culturing, and inoculating the obtained culture solution to a second fermentation tank for culturing; then, continuously feeding sugar in batches to the second fermentation tank; and then, continuously feeding sugar and nitrogen sources in batches to the second fermentation tank.

Description

technical field [0001] The present invention belongs to the field of amino acid fermentation. Specifically, the present invention relates to a method for fed-batch fermentation of L-lysine, which comprises introducing engineering bacteria expressing pyridine nucleotide transhydrogenase variants into a first fermentation tank for cultivation and Inoculate the obtained culture solution in the second fermenter for culture, then continuously add sugar to the second fermenter, and then continuously add sugar and nitrogen source to the second fermenter. In addition, the present invention also provides products produced by the method and the like. 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....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/08C12R1/15
Inventor 马吉银陈崇安孟刚曹洪程耀东刘鑫
Owner NINGXIA EPPEN BIOTECH
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